Designing Heart Performance by Gene Transfer

Transcription

Physiol Rev 88: 1567–1651, 2008;
doi:10.1152/physrev.00039.2007.
Designing Heart Performance by Gene Transfer
JENNIFER DAVIS, MARGARET V. WESTFALL, DEWAYNE TOWNSEND, MICHAEL BLANKINSHIP,
TODD J. HERRON, GUADALUPE GUERRERO-SERNA, WANG WANG, ERIC DEVANEY,
AND JOSEPH M. METZGER
Department of Integrative Biology and Physiology, University of Minnesota Medical School,
Minneapolis, Minnesota
Downloaded from http://physrev.physiology.org/ by 10.220.33.1 on October 14, 2016
I. Introduction
A. Perspective
B. Scope of review
II. Cardiac Gene Transfer Tools and Principles
A. Viral vectors
B. Nonviral vectors
C. In vivo vector delivery techniques
D. In vitro gene transfer/acute genetic engineering
III. Ca2⫹ Handling Proteins
A. Regulators of Ca2⫹ release from the SR
B. Regulators of cytoplasmic Ca2⫹ removal
C. Ca2⫹ binding proteins that modulate cardiac performance
IV. Sarcomeric Targets and Templates
A. Protein turnover and stoichiometry
B. Thin filament proteins, isoforms, mutants, and chimeras
C. Thick filament proteins
V. Cytoskeletal Proteins
A. Dystrophin and dystrophin-associated proteins
B. Intermediate filaments (desmin)
C. Microtubules
VI. Cardiac Signaling Pathways
A. Gene transfer influencing the ␤-adrenergic signaling pathway
B. Gene transfer of Ca2⫹/calmodulin kinase
C. Gene transfer and PKC signaling
D. Gene transfer of protein phosphatases
E. Gene transfer and MAPK signaling
F. Myocardial nitric oxide synthase signaling and contractile function
G. Other signaling proteins of interest
VII. Future Directions
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Davis J, Westfall MV, Townsend D, Blankinship M, Herron TJ, Guerrero-Serna G, Wang W, Devaney E,
Metzger JM. Designing Heart Performance by Gene Transfer. Physiol Rev 88: 1567-1651, 2008; doi:10.1152/
physrev.00039.2007.—The birth of molecular cardiology can be traced to the development and implementation of
high-fidelity genetic approaches for manipulating the heart. Recombinant viral vector-based technology offers a
highly effective approach to genetically engineer cardiac muscle in vitro and in vivo. This review highlights
discoveries made in cardiac muscle physiology through the use of targeted viral-mediated genetic modification. Here
the history of cardiac gene transfer technology and the strengths and limitations of viral and nonviral vectors for
gene delivery are reviewed. A comprehensive account is given of the application of gene transfer technology for
studying key cardiac muscle targets including Ca2⫹ handling, the sarcomere, the cytoskeleton, and signaling
molecules and their posttranslational modifications. The primary objective of this review is to provide a thorough
analysis of gene transfer studies for understanding cardiac physiology in health and disease. By comparing results
obtained from gene transfer with those obtained from transgenesis and biophysical and biochemical methodologies,
this review provides a global view of cardiac structure-function with an eye towards future areas of research. The
data presented here serve as a basis for discovery of new therapeutic targets for remediation of acquired and
inherited cardiac diseases.
www.prv.org
0031-9333/08 $18.00 Copyright © 2008 the American Physiological Society
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DAVIS ET AL.
I. INTRODUCTION
B. Scope of Review
A. Perspective
FIG. 1. Cardiac muscle subcellular
organization. Key elements of cardiac
muscle structure and function: from the
organ to the molecular level with emphasis on intracellular Ca2⫹ handling and the
constituents of the contractile apparatus,
the cardiac sarcomere (circular inset).
The adult myocardium is comprised primarily of striated muscle cells (cardiac
myocytes) organized as a functional syncytium such that a single stimulus causes
the entire myocardium to synchronously
depolarize and contract. Within a cardiac
myocyte, sarcomeres are arranged in series and in parallel providing optimal
contractile architecture. Surrounding
each myofibril is a highly organized sarcoplasmic reticulum (SR, green) and
transverse tubule (T-tubules, blue) network that contains the elements responsible for the electrochemical coupling
from action potential to Ca2⫹ to force
generation. The dynamic interplay of
these elements forms the basis of excitation-contraction (EC coupling) in cardiac
myocytes.
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Heart disease is the leading cause of combined
morbidity and mortality in the western world. Globally,
it is estimated that over the course of the next three
decades heart disease will be the leading cause of death
worldwide, including both high- and low-income countries (970). The past years have seen tremendous insights into the molecular underpinnings of cardiac performance leading to clinically relevant therapeutics to
treat heart disease. Nonetheless, the growing burden of
cardiovascular disease in this country and throughout
the world necessitates continued vigor directed at the
mechanistic basis of heart disease with the goal of
identifying new therapeutic targets and implementing
effective treatment modalities. The elucidation of the
human genome, together with a growing appreciation
of the complexities of cardiac gene expression and
proteome, lends hope that new discoveries will be
forthcoming in treating acquired and inherited diseases
of the heart. Cardiac gene transfer presents a unique
strategy to design cardiac performance by tailoring
specific physiological outcomes in the heart.
This review is focused on the application of recombinant viral vector systems as gene delivery vehicles to
the normal and diseased heart. One could argue that the
birth of molecular cardiology was ushered in during the
early 1990s by the development and implementation of
genetic strategies for targeted engineering of gene expression in cardiac muscle. Since that time, there have been a
number of excellent reviews focused on the application of
transgenesis and ES cell gene targeting in mammals relating to the heart (151, 400, 517, 746, 999). This review
concentrates on another avenue of gene-based engineering by featuring emergent viral vector technologies. First,
the origins and applications of gene transfer technologies
for the heart are reviewed, with an emphasis on the
strengths and limitations of recombinant viral vector and
nonviral systems for cardiac gene delivery. Next, cardiac
muscle targets and the applications of vector technology
to the heart are highlighted by discussing gene transfer of
key elements of cardiac excitation-contraction (EC) coupling, with an emphasis on intracellular Ca2⫹ handling and
contractile/regulatory proteins of the sarcomere (Fig. 1). In
concert, new discoveries in the cytoskeletal matrix as
applied to inherited and acquired cardiac disease are
discussed with an eye towards new targets for acute and
long-term genetic engineering in the heart. Finally, we
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CARDIAC MUSCLE GENE TRANSFER
review vector-based approaches for modifying essential
components of the cell signaling network in the heart.
Recent advances in gene transfer of cardiac membrane
channels and biological pacemakers are addressed elsewhere (17, 184, 185, 524).
Collectively, the goal of this review is to provide the
reader with a comprehensive analysis of the state of the
art in cardiac gene transfer with the aim of identifying and
evaluating new targets and opportunities directed at genebased remediation of cardiac disease in vivo.
With the continued advancement in gene transfer
technologies, genetic manipulation of cardiac muscle
both in vivo and in vitro has gained tremendous momentum as an important experimental and therapeutic reagent. Gene transfer, either through permanent modification of the mammalian genome or expression of a transgene in somatic cells, is a powerful experimental tool for
resolving basic science questions as well as discovering
the primary etiologies and mechanistic basis for disease
pathogenesis. Transgenic animal models have been essential for understanding the effects of gene expression on
organ function in a physiologically relevant and integrative context. The confounding influence, however, of
complex compensatory (mal)adaptations may make data
interpretation from transgenic animal models difficult.
Specifically, it is challenging to ascertain whether a functional defect is a direct or adaptive manifestation of a
given gene product. Thus results can represent a combination of primary and secondary outcomes. Acute gene
transfer has the potential to alleviate these issues. For
instance, in vitro gene transfer to isolated adult cardiac
myocytes utilizes a stably differentiated primary cardiac
muscle cell and offers an experimental system devoid of
complex systemic and environmental interactions. In addition to experimental advantages, direct gene transfer
technologies constitute a viable therapeutic modality for
remediation of acquired and inherited cardiac diseases.
TABLE
1.
A. Viral Vectors
1. Retroviral vectors
Retroviral vectors are derived from a variety of wildtype retroviruses. Moloney murine leukemia virus (MLV)
and lentivirus are two common examples, but employing
less common retroviruses, such as foamy virus, for gene
transfer is also gaining in popularity (39, 555). Despite
many differences, these vectors all share common traits.
One is the storage of genetic information in the form of
single-stranded RNA that is reverse-transcribed into double-stranded DNA (provirus) once the virus enters the
host cell (636). A vital characteristic of retroviral infection
is its integration as double-stranded DNA into the host’s
genome (636). Unlike some retroviruses, lentivirus can
efficiently transduce nondividing cells such as cardiac
myocytes (633, 1009), and therefore, this section will
focus on the use of lentiviral vectors (503). As lentivirus
integrates into the host genome, lifetime transduction
could potentially be achieved following a single transduction event. This is not without risk, however, as any
integrating vector holds the potential for serious insertional mutagenesis events. Like many integrating vectors,
lentivirus shows preference for inserting into active chromatin (123, 162, 472).
Viral vectors for cardiac gene transfer
Viral Vector
Adeno-associated
virus (AAV)
Adenovirus
Gutted adenovirus
Lentivirus
Capsid Type
Packaging Size, kb
Proteinaceous
⬃4.5
Proteinaceous
Proteinaceous
Enveloped proteinaceous
⬃8–10
⬃36
⬃8–10
Delivery Method
Direct injection, local vascular isolation,
systemic vascular delivery
Direct injection, local vascular isolation
Direct injection, local vascular isolation
Direct injection
Toxicity
⫹
⫹⫹⫹
⫹⫹
⫹⫹
Summary of four commonly used cardiac gene transfer vectors reviewed in this article. Capsid structure, average vector packaging size,
delivery methods, and relative toxicity are described. Toxicity is rated in a relative “⫹” scale, where the number of “⫹” signs represents increasing
toxicity. Adenovirus refers to first- and second-generation vectors. The packaging size of lentivirus vectors is not an absolute limit, though larger
inserts package with progressively less efficiency.
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II. CARDIAC GENE TRANSFER TOOLS
AND PRINCIPLES
The field of gene therapy and gene transfer in general
has utilized an array of unique vector systems. As a thorough review of all of these systems could easily fill a
textbook, this section only examines commonly used
gene transfer vectors (Table 1) for myocardial and isolated myocyte transduction and briefly describes their
major advantages and drawbacks. The field of immunology, as it pertains to delivered transgenes, could also
constitute an entire review by itself. As such, discussions
of immunology here are focused only on responses to a
particular gene transfer vector. Gene transfer vectors are
generally classified as viral or nonviral based, and this
distinction provides the framework for the following discussion.
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2. Adenoviral vectors
Adenovirus-based vectors (Ad) have been a workhorse for a variety of gene transfer studies spanning several decades. Despite its limitations, this experimental
approach will likely continue to be a valuable tool in the
future. Along with AAV vectors, Ad vectors represent one
of the most efficient means of both in vivo and in vitro
cardiac transduction. Adenovirus has been an invaluable
reagent for cardiac muscle gene transfer (170, 245, 294,
439, 813, 858, 966).
Adenovirus is a member of the Adenoviridae class of
viruses. It is a double-stranded DNA virus (dsDNA) with a
36-kb genome capped with inverted terminal repeats
(ITRs). The ITRs function as origins of viral genome
replication. The genome encodes dozens of protein products divided into early and late transcriptional events
using a variety of space-efficient internal promoters and
splice sites. Adenovirus has a nonenveloped icosahedron
capsid (60 –90 nm in diameter) surrounding the dsDNA
virion (119, 267). Its proteinaceous capsid consists of
many different proteins with functions ranging from providing structure to docking and infection. The 12 vertices
each contain a penton base and fibrous “spike” that is
used to attach the virus to coxsackievirus (CAR) or adenovirus receptors on the host cell membrane (Fig. 2) (46).
Once attached, the adenovirus is endocytosed and transported to the endosome where the virion is thought to
escape through a pH-dependent endoplasmolysis (291,
971). These events occur over an estimated 15–20 min
time span (291, 971), and subsequently the dsDNA migrates to the nucleus for transcription (466).
There are more than 50 infectious adenoviral serotypes, with some being causative agents of human diseases like conjunctivitis and the “common cold.” Engineered replication-deficient serotypes 2 and 5 are the
most commonly used biomedical reagents. Adenoviral
vectors are produced in several varieties. The early generation vectors (sometimes called first- or second-generation Ad vectors) contain deletions in key areas of their
genome. These deletions render the adenovirus replication deficient in nonpermissible cells and provide a substantial cloning capacity (⬃7 kb, Table 1) for exogenous
expression cassettes. Vector DNA is transfected into permissive packaging cells (commonly HEK 293), which are
eventually lysed as a result of viral production. The viral
lysate is plaque purified, and a pure clone is used to seed
more packaging cells. The viral lysate/cell debris is collected, and the Ad vector is purified/concentrated using a
variety of methods such as centrifugation or chromatography.
Gutted adenovirus has large portions of the adenoviral genome deleted, yielding vectors with a much larger
cloning capacity (30 – 40 kb, Table 1) relative to Ad and
AAV (35, 196, 264, 265, 807). Gutted adenovirus can be
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Lentivirus is a retrovirus related to the HIV virus
(443). It contains a proteinaceous capsid surrounded by
an envelope derived from the host plasma membrane.
Production of the vector is an ever-evolving field, with
improvements being made in production efficiency, purity, and safety (481). Production generally involves either
a stably transduced cell line, which buds off lentivirus
vectors into the supernatant and is collected and concentrated, or a plasmid cotransfection system that introduces
a genome coding plasmid and helper virus into cells (103,
418, 515, 529). The plasmid cotransfection system is widely
used to produce lentiviral vectors and has undergone a
number of generations of development. Each generation
was aimed at increasing growth efficiency while reducing
the chance of generating replication-competent viruses. Currently, lentiviral vectors cannot be grown to titers on par
with adenoviral or adeno-associated viral vectors (AAV)
(294, 529, 807). Also, unlike vectors such as AAV, highly
purified, large-scale, lentiviral vector production is extremely challenging. Lentivirus vectors are mostly concentrated but with some concomitant impurities. However, technologies such as high-performance liquid chromatography (HPLC) offer the possibility of truly purifying
vector stocks (796). A final disadvantage is that lentiviral
vectors are less stable than other vectors and are more
difficult to manipulate due to this lability (529).
Currently, relatively few studies have been reported
using lentivirus vectors to transduce the myocardium in
vivo (71, 238). Although these vectors are capable of
transducing 80 –100% of cardiac myocytes in vitro (71,
588, 779, 780), in vivo efficiencies rarely achieve a transduction efficiency of above 30% (71, 238). For cardiac
expression, direct injection is by far the most efficient
means of delivery, with little expression seen after vascular delivery (996). However, in cardiac transplant rejection studies, this relatively low level of expression has
yielded significant results (1008). The immunology/toxicology of lentivirus vectors when used for cardiac delivery is not yet well understood, but data will undoubtedly
be available in the future.
Lentiviral vectors represent an appealing method for
cardiac transduction. They offer the ability to stably transduce nondividing cardiac myocytes, a potentially useful
experimental/therapeutic characteristic. As the use of lentiviral vectors to transduce the myocardium is a relatively
young field, many questions remain unanswered. Future studies will have to focus on increasing transduction efficiencies, determining optimal administration
methods, and elucidating the host immune response
following vector administration/transduction. Advances in production and purification of these vectors
will also expand the potential applications and ease of
use of this vector.
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grown concurrently with a helper virus (which resembles
first-generation Ad vectors) to provide critical functions
in trans. After production, the gutted vector is separated
from the helper virus by density using equilibrium centrifugation. Commonly, the packaging signal of the helper
virus is flanked by recombinase targets resulting in excision of the packaging signal in the presence of the proper
recombinase. The recombinase is expressed in the packaging cells, thus limiting the amount of packaged helper
that needs to be removed by differential centrifugation.
Adenovirus represents some of the largest viral vectors
available for cardiac gene transfer. Most gene transfer to
adult cardiac myocytes has been performed with secondand third-generation serotype 5 adenovirus (439, 965).
Adenoviral vectors are extremely efficient at transducing the myocardium. This is true with a variety of
injection methods and experimental animal models. After
direct injection into the myocardium (Fig. 3, Table 2),
60 – 80% of the exposed cardiac cells can be transduced
(170, 307, 439, 568, 858). Peak expression is generally seen
within 3 days and is typically very strong. In addition to its
use as an experimental tool, adenoviral delivery has been
used to improve cardiac function in a number of studies
and as such makes this vector a potential candidate for
clinical applications (583, 801, 858, 968). Injection of the
vector directly into the intravascular space results in poor
transduction of the myocardium, and large amounts of Ad
vector are acutely toxic, especially to the liver (720).
However, when the cardiac circulation is isolated, either
during transplantation studies or during heart-lung bypass, global transduction efficiencies of 30 –50% can be
achieved (83, 84, 187, 188, 801, 845). These techniques
frequently make use of elevated hydrostatic pressure and
permeabilizing adjuvants (187, 188). In vitro adenoviral
transduction is even more efficient, and a preferred gene
delivery vector for cultured cardiac myocytes will be
discussed in subsequent sections. Reporter assays in
which adenoviral delivery of chloramphenicol acetylPhysiol Rev • VOL
transferase (CAT), green fluorescent protein (GFP), or
Lac Z (␤-galactosidase) genes to isolated adult cardiac
myocytes have shown a transduction efficiency approaching 100%, which occurs rapidly between days 1–2 in culture (132, 424, 439, 490, 765, 965).
Despite its many advantages, Ad vectors have some
serious limitations. Adenovirus elicits a potent cellular
immune response (416, 991). Cells transduced with early
generation Ad vectors are cleared rapidly from the body.
This potent immune response is against both capsid and
viral proteins that are encoded by the residual adenoviral
DNA present in the vector. Gutted adenoviral vectors
were developed to circumvent this problem as these vectors are devoid of most viral genes except for the ITRs
and packaging signal (35, 172, 333, 807). Although these
gutted-Ad vectors demonstrate lower immunogenicity,
they still trigger a cellular immune response presumably
due to residual helper virus contamination or the vector
capsid itself (172, 333, 334). However, even with this
immune response, expression can be detected longer than
18 mo after administration. The various serotypes or chimeras of Ad vectors continue to be extremely useful
cardiac gene transfer reagents, but their inherent immunogenicity/ toxicity will likely limit them to experimental/
therapeutic protocols that do not require long-term expression and can tolerate a potential immune response.
3. AAV
Recombinant AAVs are generating considerable interest in the field of cardiac gene transfer (294, 390, 668,
893, 997). AAV is a Dependovirus member of Parvoviridae (63, 821) with a particle size of ⬃20 nm. As a Dependovirus, AAV is incapable of replicating in host cells
under most physiological circumstances and requires
coinfection with a helper virus for replication. Adenovirus
or herpesvirus is the most frequently used helper virus,
but others including human papillimo virus have also
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FIG. 2. Adenoviral-mediated gene transfer to
cardiac myocytes. Adenovirons (serotype 2 and 5)
for gene transfer have a double-stranded DNA genome that is rendered replication incompetent by
deleting several viral transcriptional elements required for replication (E1, E3 or E2, E4). The 36-kb
adenoviral genome is packaged in an icosahedral
protein capsid that contains a penton base and fiber
knob at each vertex. The fiber knob of the adenovirus binds to coxsackievirus/adenovirus receptors
(CAR) permitting entry into the cardiac myocyte by
endocytosis. Once internalized, the virion DNA
leaves the endosome and translocates to the nucleus where the double-stranded DNA can be transcribed and translated by the myocyte’s own machinery into the recombinant protein of interest
(circular inset).
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DAVIS ET AL.
been effective helper viruses (63). The AAV capsid is a
nonenveloped proteinaceous capsid made of three proteins termed VP1, VP2, and VP3, and the capsid seems to
be devoid of most posttranslational modifications. AAV
has a single-stranded DNA (ssDNA) genome where both
Watson and Crick strands appear to be packaged equally.
The AAV genome is relatively small (⬃4.5 kb, Table 1).
Several promoters and alternate splicing control the expression of the capsid and Rep proteins. Rep proteins are
TABLE
2.
critical elements involved in genome replication, integration, and packaging. The AAV genome also contains two
ITRs, which are important for genome packaging, replication, and stability (47, 516, 836, 842, 984). Several distinct serotypes of AAV have been identified (Table 3),
with serotype 2 being the most commonly used for gene
transfer vectors. (For reviews on AAV, see Ref. 443.)
AAV has several features that make it an appealing
cardiac gene transfer vector. AAV vectors have a favor-
Recombinant viral vector delivery methods for the heart in vivo
Delivery Method
Advantages
Direct injection
Restricted transduction,
increased safety
Systemic
Global cardiac transduction,
transduction of noncardiac
tissue, ease of administration
Global cardiac transduction, low
chance of noncardiac tissue
transduction
Local vascular
Disadvantages
Vectors
Difficult to achieve global cardiac transduction,
requires physical disruption of the heart,
may require visualization/access to the heart
(thoracotomy)
Requires large concentration of vector, chance
of increased toxicity, potential transduction
of noncardiac tissue
Can require toxic adjuvants, requires greater
technical expertise
AAV, adenovirus, gutted
adenovirus, lentivirus
AAV
AAV, adenovirus, gutted
adenovirus
A summary of the three primary viral vector delivery methods reviewed in this article and their respective advantages/disadvantages as it
pertains to myocardial gene transfer in vivo. Included are the common viral vectors used with each delivery method. AAV, adeno-associated virus.
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FIG. 3. In vivo viral vector delivery techniques. A: systemic delivery involves injection of the viral vectors into the venous space for delivery
to the heart and throughout the circulation, including the coronary circulation. The vector (green) should gain access to most cardiac myocytes via
the heart’s capillary network. B: coronary delivery involves open-chest surgery in which the heart circulation (inflow, outflow, or both) is isolated
with clamping (cross-clamp) or balloon angioplasty. The vector is introduced in this case under high pressure and allowed to dwell in the coronary
circulation. C: direct injection involves injection of the vector by a syringe or similar device directly into the heart musculature. The vector gains
access to the myocardial cells through the interstitial space and enters by vector-specific mechanisms and is more local in terms of transduction
(shown in green).
TABLE
3.
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Tissue tropism of identified adeno-associated virus serotypes
Tissue
Reference Nos.
AAV9,
AAV1,
AAV1,
AAV2
AAV5,
AAV2
AAV4
AAV8, AAV6, AAV1, AAV7, AAV5, AAV2
AAV7, AAV6, AAV8, AAV9
AAV2, AAV5
AAV5,
AAV5
AAV3
AAV9,
AAV9,
AAV8,
AAV2,
AAV4
AAV1, AAV4, AAV2
390, 431, 672, 893, 944
62, 108, 254, 255, 982
112, 298
112, 645
12, 91, 157, 248, 938
157
157, 501
512, 949, 979
949
502
75, 254, 321, 1000
254, 255, 627, 885
114, 507, 936, 945
75, 866
AAV6, AAV5
AAV8, AAV6, AAV2, AAV1
AAV1, AAV2, AAV6
AAV9 (neonatal)
Summary of the transduction of tissues by various adeno-associated virus (AAV) serotypes. Several tissues can be transduced by multiple AAV
serotypes albeit with varying efficiencies. Tissue transduction efficiency is not only dependent on AAV serotype but on delivery method, vector titers,
and promoters.
able safety profile relative to other vectors (Table 1, Refs.
21, 294, 522) as no AAV serotype or Dependovirus has
been implicated as a causative agent in human disease
(59). Early work suggested that AAV vectors may be able
to circumvent an immune reaction, but recent studies
have now demonstrated that AAV vectors can elicit both
a humoral and cellular immune response in a variety of
animal models (116, 294, 432, 522, 579, 604). This immune
response is generally less robust than those triggered by
other viral vectors like adenovirus, making AAV a preferred vector for in vivo gene transfer. While data from
human clinical trials demonstrated a clear immune response against AAV-mediated gene transfer (432, 522,
604), the response was mild, and investigations into transient immune suppression to overcome this difficulty are
underway.
Integration of gene transfer vectors into the host
genome is a safety concern that can be seen as both a
benefit and a potential limitation. Integrating gene transfer vectors offer the possibility of permanent transduction
avoiding the need for repeat gene delivery. Integrating
vectors, aside from AAV, have occasionally caused human
disease through insertional mutagenesis (309, 310, 800).
For AAV vectors, this phenomenon must be interpreted in
light of several facts. Classically, wild-type AAV integrates
preferentially into two specific sites in the human genome, perhaps guided by the homology between the ITRs
and genomic sequence. This integration is mediated by
Rep proteins (785, 935). As the Rep proteins are deleted in
AAV vectors, the rate of integration is much lower than
that of the wild-type virus. Also, much of the site specificity of integration appears to be lost in recombinant AAV
vectors (700). Recombinant AAV vectors typically have a
low rate and random pattern of integration and appear to
favor integration into transcriptionally active and open
chromatin (628 – 630). Even in the absence of integration,
Physiol Rev • VOL
expression from AAV vectors can persist in striated musculature for many years (21).
AAV vectors can be produced to high purity and titer
(63). Early methods used adenovirus as a helper virus for
AAV vector production, but adenoviral contamination
was often found in these preparations. Current protocols
now employ a helper-free plasmid cotransfection system
to avoid an Ad contamination (299, 300, 983). Here a
plasmid containing the AAV genome is transfected into
cells, frequently HEK293 cells, along with a plasmid containing helper genes to function in trans. Vector purification and concentration are dependent on the specific
capsid serotype. Capsids from some serotypes, such as
serotypes 2 and 6, bind heparin avidly enough to be
purified over a heparin sulfate-Sepharose column (124,
321, 716). Vector production of all AAV serotypes can use
serial centrifugation methods. Of note, the capsids of
most serotypes can cross-package the ITRs from genomes
of other virus serotypes (commonly serotype 2) in a process termed pseudotyping (63, 716). This phenomenon
permits easy manipulation of the vector capsid, with altered production and tissue tropism characteristics (Table 3) without having to clone new vector genomes. In the
last several years methods using baculovirus expression
to produce AAV vectors have also been described (831,
910, 911).
One of the main disadvantages of AAV vectors is their
relatively limited cloning capacity (Table 1). The entire
expression cassette of interest, including the open reading frame and transcriptional regulatory sequence, must
not exceed ⬃4.5 kb, allowing for 300 bp of ITR sequence
(836). This cloning capacity severely limits the potential
for gene transfer cassettes. Strategies to overcome this
limitation include cotransduction with two vectors containing one-half of an expression cassette. The two genomes then rearrange in vivo to yield a larger, intact
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Cardiac muscle
Skeletal muscle
Vascular endothelium
Vascular smooth muscle
CNS
Midbrain
Ependyma / astrocytes
Eye
Retina
Photoreceptors
Cochlear hair cells
Lung
Liver
Pancreas
Kidney
AAV Serotype
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in cardiac transduction. This does not preclude these
vectors, however, as viable options for cardiac gene transfer in future studies.
B. Nonviral Vectors
1. Naked DNA
Pure DNA carrying an expression cassette is perhaps
one of the most basic transfer vectors. In this situation,
clonal DNA that is generally derived from a bacterial
plasmid (pDNA) is introduced to the tissue of interest.
Through means not completely understood, the cells take
up the pDNA, transport it to the nucleus, and express the
exogenous gene. This type of striated musculature transduction has been known for approximately two decades
(974, 975).
pDNA vectors have many potential advantages.
pDNA can be readily produced in large amounts to very
high purity through a variety of methods available commercially and for the laboratory environment. Common
laboratory methods for purifying pDNA include alkaline
lysis and phenol-chloroform extraction and DEA-dextran
(diethylaminoethyl) binding columns. Huge fermentors are
commercially available to produce and HPLC purify gram
quantities of extremely pure plasmid under good manufacturing practice (GMP) conditions. Because there are no true
“infection particles” with pDNA, the resultant material can
also be stored easily for long periods of time without loss
of potency. Additionally, recombinant manipulation of the
pDNA is much easier. Unlike recombinant viral genomes,
expression cassette size is much less of a concern. Although smaller genomes are generally propagated more
efficiently, bacterial strains can easily maintain plasmids
of 30 kb and above. This allows for the cloning of extremely large expression cassettes with larger portions of
native regulatory sequence compared with viral vectors.
This is advantageous in terms of having more natural
transcriptional activity and possible tissue-specific expression patterns, a challenge with viral vectors such as
AAV.
pDNA vectors are also attractive gene transfer reagents because they lack a viral proteinaceous or membranous component. This contributes to both the stability
and small immune response associated with pDNA vectors. Because organisms have evolved ways of neutralizing viral transduction events, virally delivered genes can
elicit very potent humoral and cellular immune responses
against the genetic vectors. Overall pDNA does not tend
to elicit a potent immune reaction, but “bacterial” DNA
sequences are recognized by the body and can spark a
small immune reaction. In a potentially related protective
mechanism, a DNA sequence that is covalently attached
to bacterial sequences can be silenced in vivo. In a series
of experiments, delivery of circular DNA with excised
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expression cassette (261, 262, 469). Also, many large
genes have been modified into “micro” versions that still
retain functionality, yet meet the size requirements for
AAV vectors (329, 893, 997). Initial reports using this
approach have been very encouraging, although a broad
range of transduction efficiencies have been reported.
Another concern is that the general human population has
an antibody titer to AAV. This titer generally has a reasonable amount of cross-reactivity with several other serotypes (116, 322), raising the possibility that a neutralizing titer may limit AAV’s therapeutic potential in vivo
(522).
AAV vectors have been widely used for cardiac gene
transfer. Serotypes 1, 6, 7, 8, and 9 (Table 3) appear to be
the most efficient for transduction, although many of the
newer less described serotypes may also efficiently transduce myocardial tissue (293, 294, 672, 844). Depending on
the exact capsid serotype and delivery method, AAV vectors have transduced nearly 100% of cardiac tissues (294,
893). Various studies utilizing intravenous delivery demonstrate global cardiac transduction, an exciting finding
in terms of therapeutic strategies as well as experimental
manipulations (294, 390, 893). More focused delivery to
the heart by direct injection results in strong local expression of the gene product (997). Using AAV vectors, expression levels do not peak as fast as with adenovirus and
generally take between 10 days and 2 mo to reach their
peak. Once present, AAV expression can last for years
(21, 63).
AAV vectors are currently one of the most promising
vectors for genetic manipulation of the heart in vivo.
Studies using AAV for cardiac transduction have been
very successful in delivering a variety of genes to several
mammalian species including mice, rats, rabbits, dogs,
pigs, and nonhuman primates (21, 422, 668, 672, 893).
Additionally, several clinical trials have demonstrated
clear human transduction with AAV vectors (432, 522,
604). Studies in heart kinetics and energetics, transplant
rejection, and structural abnormalities have all been successfully performed using AAV vectors (113, 293, 778).
These include several very promising therapeutic reports
where a dramatic reduction of disease morbidity and
mortality was demonstrated in several animal models of
muscular dystrophy (293, 294, 893). The future use of AAV
for clinical application will likely require further investigation of the immune response to AAV, development of
smaller regulatory sequences for use in AAV vectors, and
elucidation of the intracellular handling of the AAV capsid
and genome, a complicated and poorly understood topic
not covered in this review.
Finally, other viral-based vectors such as EpsteinBarr, foamy, and simian virus 40 (SV40) (555, 843, 890)
have also been reported for use in gene transfer studies
but have not been included in this discussion for reasons
of space, frequency of use, and a lack of a rich literature
C. In Vivo Vector Delivery Techniques
Delivery of foreign expression cassettes to the myocardium has a range of possible experimental and therapeutic applications. Regardless of the experimental or
therapeutic objective, these genetic manipulations will
likely rely on efficient transduction of the myocardium.
Transduction efficiency requires the marriage of safe, efficient, and producible gene transfer vectors with a delivery system that is technically efficient, feasible, and well
tolerated. The following two sections describe the current
state of vector delivery technologies used for in vivo
transduction of the myocardium.
In general, the ideal gene delivery method should be
technically simple, inexpensive, safe, and only transduce
specified regions of the targeted tissue. At present, delivery techniques meeting all of these criteria do not exist.
Current delivery methods can be classified into two broad
categories: direct delivery to the myocardium and intravascular systemic delivery (Fig. 3, Table 2). As so many
specific methods have been developed for vector delivery
within each category, the following discussion will highlight the basic permutations of each delivery method.
While this section is separated into vectors and delivery
methods for the purpose of discussion, the pairing of
vector and delivery system can have specific advantages
and disadvantages as they pertain to transduction efficiency. For instance, intravascular delivery is often combined with AAV vectors (Table 2), while AAV is rarely if
ever delivered by electroporation.
1. Direct myocardial injection
Direct injection of the gene transfer vector to the
myocardium is perhaps conceptually the most obvious
approach. This method has been extensively used with
most available gene transfer vectors and involves the
introduction of an injectant directly into the heart musPhysiol Rev • VOL
culature by a syringe or similar device (858) (Fig. 3). The
vector gains direct access to the myocardial cells through
the interstitial space and enters via the specific vector’s
entry mechanisms. This method has been utilized in studies addressing a variety of disease models including ischemic heart disease, heart failure, and muscular dystrophies. Additionally, several different animal models ranging from common laboratory animals (rodents and rabbits) to larger mammals (canines and pigs) are amenable
to this type of manipulation. The exact method of injection varies by the experimental system and objectives.
Overall, direct injection has been associated with
excellent survival rates. Injections can be done blind by
targeting the heart through the chest wall or trans-diaphragm from the abdominal cavity. While this method
may seem rather unreliable, it has been used with effectiveness by a number of investigators, especially in small
animals (858). The injection can also be done through the
chest wall, under the guidance of ultrasound technology.
Ultrasound guidance improves the accuracy of direct injection by allowing some visualization of the heart itself.
Despite the technical simplicity of the direct injection
method, it is difficult to reproducibly transduce the same
area of the heart to similar magnitudes between animals
and studies, even with ultrasound guidance. Direct injection can also be done following surgeries that expose the
heart, permitting direct visualization for delivering injectant to the target tissue (858). This technique has been
used both on beating hearts and after a transient cardiac
arrest with similar effectiveness. Either ultrasound guidance or injection based on coronary vascular anatomy
permits reasonably comprehensive transduction of the
vascular walls of rodents (320). Although this necessitates
a surgery and all of the associated complications, it
heightens investigator confidence in the site of injection(s).
With either blind or visualized direct injection technique, one major limitation is the targeted musculature
must be physically large enough and easily accessible for
the procedure. Thus, in most rodent studies, the sites of
injection to nonseptal walls of the ventricles are limited. It
is possible that in larger animals with thicker atrial walls
the technique could also be applied to atrial tissue. Another limitation of this delivery route is the poor accessibility of the septal wall, trabeculae, and papillary muscles,
but ultrasound guidance has been used to improve gene
transfer to the septal wall by direct injection.
Transduction efficiency not only depends on the
method of direct injection but also appears to be highly
dependent on the vector and vector dosage. The use of
adenovirus and AAV vectors with direct injection has
resulted in strong transduction of much of the ventricular
tissue (858). In contrast, injection with retrovirus-based
vectors, such as lentivirus, results in comparatively lower
expression and amount of transduced cardiac tissue (71,
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bacterial sequence resulted in prolonged expression of
the transgene.
There are many potential benefits of using pDNA
vectors to transduce the myocardium, but at least two
major obstacles remain: low efficiencies of transduction
and persistence of transduction. Despite attempts with a
variety of administration techniques, pDNA vectors have
very low cardiac transduction efficiency compared with
their viral counterparts (975). Nonetheless, pDNA vectors
are still being explored for cardiac transduction due to
their many advantageous qualities, and they are still
considered useful experimental and therapeutic reagents
for studies that do not require global and persistent expression. Continued investigation into pDNA vectors may
result in gains in both transduction efficiencies and persistence of expression.
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Physiol Rev • VOL
Direct injection of vectors to the myocardium is a
useful technique due to its relative simplicity and application to a variety of vectors, animal models, and experimental systems. It is, however, limited by transduction
patterns and the requirement for directly visualizing the
heart through surgery to achieve significant levels of
transduction. For experimental and therapeutic manipulations that only require transduction of a limited amount
of the myocardium (e.g., focal revascularization), direct
injection remains an attractive technique. It is likely that
any experiment or therapy requiring global transduction
of the myocardium will be unable to use direct injection
methods.
2. Intravascular delivery methods
The vasculature offers an attractive portal for delivering gene transfer vectors as most cells, including those
in the myocardium, lie in close proximity to capillary
beds. Vectors placed in the venous space travel to the
heart and throughout the circulation, including the coronary circulation (Fig. 3, Table 2). By using the heart’s vast
capillary network in this way, gene tranfer vectors gain
access to a majority of the heart’s myocytes. However, a
number of imposing roadblocks complicate this scenario.
The blood itself may contain neutralizing antibodies to the
vector of choice, especially when they are viral-based.
The blood also contains proteins, like albumin and platelets, which may absorb or inactivate the vector. There are
also physical barriers to transduction including the endocardial cells and the capillary endothelium. Once out of
the vascular lumen, the vector must also cross the ECMfilled interstitial space to transduce the target cell. The
lung also tends to act as a sponge for many gene transfer
vectors (833– 835), and any vectors placed in the venous
space have to pass through the lung before reaching the
left heart for ejection into the systemic circulation. Finally, there is the response of the host itself. Large intravascular amounts of a foreign material, especially from
viral delivery, can lead to organ toxicity and/or an allergic
reaction (720). Despite early difficulties, many groups
have developed several vascular delivery methods that
can result in high-efficiency transduction.
Historically, direct injection of pDNA and adenovirus
vectors into the bloodstream has resulted in poor transduction of the myocardium, although some tissues such
as the liver transduced well (383, 743, 884). With intravenous injection of pDNA, transduction of the heart was
sparse. Viral vectors, such as adenovirus, are not tolerated well when intravenously injected, and they have
inefficient expression. These results likely reflect the difficulty in overcoming the physical barriers to transduction
at levels of vector that are readily prepared and tolerated
by the experimental animal. Recently this approach has
been revisited using a high-pressure/high-volume tech-
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779, 780, 1009). The delivery of naked or complexed
pDNA with this technique results in patchy and lower
intensity expression throughout the injected wall relative
to the transduction efficiency of viral-based vectors.
One permutation of the direct injection method is to
inject the vector into the pericardial space rather than the
myocardium itself. The injection of adenoviral vectors
into the pericardial space resulted in moderate to weak
expression in 30 – 40% of the rodent heart 6 wk after
injection (247). As might be expected, the pattern of
expression appeared to have a graded intensity with the
strongest areas of expression neighboring the pericardial
space. In this case, transduction was observed after including enzymes such as collagenase in the injectant to
disrupt the extracellular matrix and to allow for more
diffuse dissemination of the vector.
The vast majority of studies have introduced the
injectant with a syringe of some type; however, vectors
have also been introduced using “gene gun” technology
(537, 645, 907, 908). In this instance, the vector consists of
gold particles complexed with pDNA. Transduction involves surgically exposing the heart of the animal, commonly a rat, and bombarding the cardiac musculature
with the gold particles under gas pressure with a device
such as the Helios Gene Gun. Although this technique is
not dependent on biologically active particles as when
using virally derived vectors, it has several limitations.
These studies have reported a reasonably high death rate
from the gene gun procedure that seems related mostly to
complications from the thoracotomy rather than the use
of the gene gun itself. Heart expression levels with this
technique are not robust (537, 645, 907, 908). The transduced cells are limited to a rather superficial layer of the
myocardium, and expression within this layer is not
strong. Given the nature of the technique, it is unlikely
that many deeper cells could be transduced without killing the more superficial muscle. Presently, this approach
is not significantly advantageous relative to direct injection with viral or nonviral vectors.
Most injectants contain the vector and a physiological salt solution, but some studies have reported the
addition of enzymes like proteases or hyaluronidase to
degrade the extracellular matrix (ECM) (247, 462, 694,
695). The idea behind adding enzymes to the injectant is
to facilitate vector diffusion through the tissue by degrading the ECM, thus yielding greater transduction efficiency.
In fact, such techniques have improved the transduction
efficiency of direct injections targeted for tumors and the
pericardial space. Whether these compounds have a place
in direct injection of the cardiac musculature is unclear as
degradation of the cardiac ECM is fraught with potential
complications and needs careful control. Adjuvants have
a clearer role in intravascular delivery to the heart, which
will be described shortly.
Physiol Rev • VOL
appropriate equipment are also required. Despite these
drawbacks, percutaneous catheters represent an attractive and efficient means of cardiac vector gene delivery.
A method with similar goals to the percutaneous
catheters has been termed cardiac isolation or crossclamping (83, 84, 292, 318). This is a widely varied method
with many individual techniques. In general, the animal is
subjected to open-chest surgery where the heart circulation (inflow, outflow, or both) is isolated with clamping or
balloon angioplasty. The animal is placed on heart-lung
bypass with induced cardioplegia. Frequently, the blood
is washed out and replaced with a buffer containing permeabilizing agents such as adenosine, histamine, and papaverine. The vector is then introduced under pressure
and commonly allowed to have a dwell time of up to 15
min. Vectors are most frequently an adenovirus, though
AAV and plasmid vectors have been used. The cardioplegia is reversed, and the animal is taken off heart-lung
bypass (83, 84). In another variation, the heart is excised
and manipulated in situ before transplant (662). This technique is comparable to the catheter-based method, it is
highly invasive and has surgical risks, that can include
infection or aortic dissection, commensurate with any
open chest manipulation of the heart. Obviously, this
technique also requires tremendous surgical expertise
and an operating suite capable of supporting the procedures. One potential advantage of this technique is that it
can limit exposure of the vector and potentially toxic
adjuvants, such as papaverine, to the heart only (83, 84).
Thus systemic toxicity and transduction of noncardiac
tissues may be avoided.
Recently, some promising results were reported with
AAV vectors. AAV vectors pseudotyped with capsid proteins from some of the less commonly used serotypes,
such as 1, 6, 8, and 9 among others, are known to transduce muscle cells much more efficiently than the commonly used serotype 2 capsid (Table 3). Several reports
have been published demonstrating global cardiac transduction, with both marker and therapeutic genes, in mice
after a single injection of AAV into the tail vein (Fig. 3)
(262, 293, 294, 390, 893, 944, 1014). Surprisingly, tail vein
injection does not require permeabilizing adjuvants, but at
suboptimal vector doses the permeabilizing agent vascular endothelial growth factor (VEGF) has been advantageous (294). Potential drawbacks of tail vein injection
include the requirement of high vector doses and the
possibility of vector expression in noncardiac tissues.
This technique is capable of transducing other tissues,
such as skeletal muscle, although the development of
cardiac specific expression cassettes may be able to overcome this possible problem (783). Nonetheless, cardiac
restricted expression has been achieved (893). Despite
the use of high AAV doses, tail vein injection was tolerated in mice and did not result in early morbidity, obvious
toxicity, or immune responses directed at the vector cap-
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nique sometimes called hydroporation (359, 974, 1001). In
this technique a viral or nonviral vector is delivered in a
very high volume of injectant (approximately blood volume in some cases) resulting in high vascular pressures.
Frequently, this is restricted to a specific limb by occlusion of the local vasculature but has been applied bodywide to rodents. Large volumes and pressures of injectant
are thought to physically disrupt barriers formed by the
endothelium and ECM, which in turn permit greater escape of the vector from the vascular lumen and wider
dissemination throughout the tissue. This may result in a
mild and temporary disruption of the plasma membranes
themselves, which would further aid transduction. This
high pressure/volume technique can result in moderatehigh transduction of the skeletal but not the cardiac musculature (292). Also, it is unclear how this technique
would be tolerated by various animals and how easily it
can be adapted to the heart.
In light of the initial disappointing cardiac transduction obtained after the administration of vectors to the
vascular space, many groups began developing intravascular delivery techniques aimed at overcoming these barriers to cardiac transduction. These techniques mainly
focused on increasing local cardiac vector dose and/or
dwell time and increasing the permeability of the cardiac
microvasculature. For instance, percutaneous catheters
have been used to deliver vectors directly to the heart
(346, 373) as this technique should increase the local
vector dosage in the heart, thereby increasing transduction efficiency. Catheters have been used to deliver a
variety of vectors (pDNA, adenovirus, and AAV) to several
areas of the heart including the right atrium and in the
root of the aorta just above the sinuses for access to the
coronary arteries. A guiding modality such as ultrasound
or fluoroscopy is often used (377). The technique has also
proven effective in a range of animal models including
rodents, canines, and sheep (84, 377, 658). Some studies
have used balloon catheters to occlude the vascular outflow of the heart in an attempt to further increase dwell
time and pressure of the injectant (346). Adjuvants such
as nitroprusside, substance P, adenosine, and histamine
have been added to the injectant to increase microvascular permeability to aid in vector extravasation (186, 188,
294, 504, 505). Overall, these techniques have resulted in
impressive levels of myocardial transduction. Additionally techniques that use permeabilizing agents and viral
vectors tend to be highly efficient. Recently, this technique has been combined with others such as sonoporation to increase the efficiency of complexed DNA transduction. This technique is mildly invasive, requiring vascular catheters that are associated with risks such as
bleeding and infection. Some of the adjuvants used are
toxic at high levels and can expose multiple tissues to the
gene transfer vector, resulting in nonspecific transduction. Expertise in catheter manipulation and access to
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D. In Vitro Gene Transfer/Acute
Genetic Engineering
A complementary approach to in vivo gene transfer
and transgenic animal models is gene transfer to isolated
cardiac myocytes in vitro (Figs. 2 and 4). Acute gene
transfer in vitro offers a powerful experimental approach
for understanding the direct effects of a genetic manipulation on cardiac myocyte structure and function. Traditional transfection methods have been partially successful in understanding the mechanisms of cardiac growth
and differentiation in neonatal and fetal cardiac myocytes
as these myocytes are easily cultured and are amenable to
transfection by foreign DNA (306, 674, 676, 886, 900).
Extrapolation of these data to the adult cardiac myocyte
has been difficult due to several key differences between
neonatal and adult cardiac myocytes that include features
like morphological and sarcomeric organization, contractile and Ca2⫹ handling protein isoform expression, signaling molecule and transcription factor expression, and the
dynamic versus quiescient nature of the culturing system
(397, 624, 803). Compared with neonatal myocytes, acute
genetic modification of adult cardiac myocytes is more challenging, because differentiated cardiac myocytes are more
difficult to maintain in primary culture, and they are not
amenable to traditional transfection techniques (DEAdextran, electroporation, Ca2⫹ phosphate, and lipofection; Refs. 424, 439, 765). Alternative methods, like direct
injection of foreign DNA into the myocardium, are equally
inefficient (⬍0.02%) as the foreign DNA tends to localize
to the injection site (93, 94, 494). Thus the aforementioned
approaches are unsuitable for comprehensively manipulating cardiac gene expression in a controlled environment.
The development and implementation of viral vectors and improved serum-free culturing methods were
Physiol Rev • VOL
critical for overcoming these limitations. To date, the
greatest success for in vitro gene transfer has come from
recombinant adenoviral vectors, which will be one focus
of this and subsequent sections. Adenoviral vectors have
some distinct advantages over other viral vectors for genetically engineering postmitotic and terminally differentiated cell types like the adult cardiac myocyte. In contrast to other viral vectors, adenovirus can uniformly and
efficiently transduce nonreplicating cells. Additionally,
adenoviral DNA remains episomal, thereby eliminating
any confounding influences of integration site and mutagenesis within the host cell’s genome (11, 466). Importantly, adenovirus can be grown to high titers (1010–1012
pfu/ml) and can transduce myocytes in vitro with little to
no toxicity (11, 466).
The first successful reports of adenoviral gene transfer to isolated adult cardiac myocytes were published in
the early 1990s (424, 439). Isolated adult rodent cardiac
myocytes cultured in serum-free conditions were adenovirally transduced with various reporter gene constructs
driven by powerful, constitutively active promoters. In
both studies nearly 100% of the adult cardiac myocytes
were uniformly transduced, which is in stark contrast to
the low transduction efficiency seen with pDNA transfection to neonatal cells (424, 439). Kass-Eisler et al. (424)
also performed direct injection of the adenoviral reporter
gene construct into the myocardium and found a 5,000fold increase in transduction efficiency relative to direct
pDNA injection. Viral-based reporter gene activity was first
measured 4 h posttransduction (424), and there was a dosedependent increase in reporter activity which was assessed
across time in culture (424, 439). These studies showed
great promise for adenoviral gene transfer as a tool for
understanding cardiac function, but questions still remained about transgene stability, the effects of adenovirus on myocyte morphology, contractile protein expression, myocyte contractile function, and the stability of
cardiac myocytes in serum-free primary culture. Rust
et al. (765) answered several of these questions by reporting culturing methods in which adult cardiac myocytes
were stable and retained their differentiated state for ⬃1
wk in serum-free culture conditions (765). In cardiac
myocyte primary culture, adenovirus-mediated gene expression achieved nearly 100% transduction efficiency
(765). Furthermore, adenoviral transduction did not affect the normal rod-shaped adult cardiac myocyte morphology, contractile protein isoform expression, or the
isometric tension-pCa relationship for the duration of the
culturing period providing evidence that the adult cardiac
myocyte phenotype is truly retained (765).
1. Cardiac myocyte culturing systems
Isolating adult cardiac myocytes for study in shortterm primary culture has a reputation for being challeng-
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sid itself (294). At present, tail vein injection represents
the only method available to transduce virtually every
cardiac myocyte in small rodents, such as mice and hamsters. Future studies are needed to demonstrate if this
delivery method can be efficiently translated to larger
animals. One canine has been manipulated by systemic
injection with a suboptimal vector dose (based on mouse
data). Encouragingly, this canine demonstrated ⬃60% cardiac transduction, similar to the mouse model, with very
little expression in other tissues (61). Another limitation
is that systemic delivery currently achieves high expression levels only with AAV vectors, which have a limited
packaging capacity (Table 1). Similar experiments with
other vectors, such as adenovirus, fail to transduce the
myocardium to the same extent. These intravascular delivery methods represent the best current technology for
achieving global cardiac transduction.
Physiol Rev • VOL
cannulated, the hearts/myocardial tissue undergoes retrograde perfusion on a modified Langendorff apparatus
(Fig. 4) such that a low Ca2⫹ enzymatic solution travels
from the aorta through the coronaries for global exposure
of the myocardium to the enzyme mixture. Most enzymatic solutions contain collagenase, hyaluronidase, protease, or a combination of these enzymes. Enzymatic
perfusion is followed by gentle mechanical digestion and
a slow titration of Ca2⫹ to bring the concentration back
up to physiological levels. For some species, such as
mouse or human hearts, Ca2⫹ titration is performed in the
presence of an EC coupling inhibitor like 2,3-butanedione
monoxime (BDM) as these myocytes tend to be ultrasensitive to extracellular Ca2⫹ after isolation.
There are numerous publications documenting the
successful isolation and use of adenoviral gene transfer to
cardiac myocytes from failing and nonfailing rodents
(158, 319, 355, 378, 564, 764, 956 –967), rabbits (109, 156,
355, 810, 878), canines (361), felines (166, 527), and humans (160, 168, 169). In all cases, adenoviral gene transfer
is reported to be highly efficient and efficacious. Adenoviral gene transfer to myocytes isolated from a variety of
species is complementary to making multiple transgenic
lines in both rodent and larger mammals without the
added cost, time, or larger mammal limitations to transgenesis. In addition, the availability of adenoviral gene
transfer is tremendously important as intact Ca2⫹ handling is a critical component of both physiological and
pathophysiological processes. Rodent EC coupling is
quite different from that of larger mammals (rabbit, dog,
and human; Ref. 49), which can impact the extrapolation
of results from rodent models to the human.
Acute genetic engineering has become a valuable
experimental approach for elucidating the physiological
role of normal and disease-related Ca2⫹ handling, myofilament, cytoskeletal, and signaling proteins in cardiac
muscle. The following sections report the physiological
insights gained from a rich and growing body of literature
involving vector-mediated gene transfer to cardiac myocytes in vitro and in vivo.
III. Ca2ⴙ HANDLING PROTEINS
Proper intracellular Ca2⫹ handling is essential for the
normal beat-to-beat function of cardiac muscle. The cardiac myocyte is designed for highly orchestrated changes
in cytosolic [Ca2⫹] during EC coupling (Fig. 5) in the heart
(50). In the healthy heart, a transient increase in intracellular Ca2⫹ concentration ([Ca2⫹]i) is the initial driving
force for mechanical contraction, and Ca2⫹ removal initiates relaxation. The intracellular Ca2⫹ movement is
tightly controlled by proteins associated with the sarcoplasmic reticulum (SR) and sarcolemmal membrane (Fig.
5). At the molecular level, EC coupling is a process
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ing as they can be unstable in the presence of physiological extracellular Ca2⫹ and they readily dedifferentiate in
the presence of serum (928). Nonetheless, cell culture
experiments are important for uncovering the primary
molecular and cellular effects of an experimental manipulation in a controlled environment. It is debatable
whether there are any true immortal cardiac cell lines that
retain both the genetic and protein profile, and morphological and contractile characteristics of bonafide adult
cardiac myocytes. For instance, the SV40 large T transformed atrial cells (125) and ventricular tumor cells (770)
possess some adult cardiac myocyte features but are
largely inadequate both structurally and functionally compared with isolated adult cardiac myocytes. For this reason, many laboratories have become quite adept at cardiac myocyte isolation for primary culture and gene transfer (231, 317, 319, 347, 606, 956 –967, 1012). Historically,
the rat has been the preferred species for cardiac myocyte
culture mainly because of availability and size (582). Isolation and culturing methods have now been expanded to
include mouse and larger mammals such as rabbit, cat,
dog, and human (109, 156, 160, 169, 355, 361). For contractile structure-function studies, it is imperative that
cultured cardiac myocytes meet the following criteria:
1) be tolerant of physiologic Ca2⫹ (1.2–1.8 mM), 2) retain
a functional metabolic system, 3) create a homogeneous
population of myocytes absent of contaminating and proliferating cells, 4) maintain their ultrastructure, cellular
morphology and Ca2⫹ handling systems, and 5) remain
quiescent and stable in culture. These criteria ensure that
myocyte preparations are repeatable and yield a standard
for “healthy” and physiologically relevant myocytes.
Early reports of Ca2⫹-tolerant isolated cardiac myocytes were published more than 30 years ago (397). There
were two main cardiac myocyte culturing approaches,
one requiring serum and the other termed the “cell reattached” method which used serum-free conditions. Serum-based cardiac myocyte culturing protocols were successful and could maintain cells for weeks and even
months (125, 384, 396, 397), but the serum contained
enough growth and miscellaneous factors that within
days these cultured myocytes “dedifferentiated” and lost
the adult myocyte phenotype. In contrast, serum-free culture conditions permitted adult cardiac myocytes to retain their highly differentiated rod-shaped morphology,
myofilament and metabolic ultrastructure, and intact
Ca2⫹ handling and transverse-tubule density (581, 693,
765). Many of the current protocols have been derived
from the original work on serum-free rat myocyte culturing methods of Haworth et al. (343, 344) and Jacobson
and Piper (397). Although isolation and culturing procedures differ slightly between laboratories and across species, they generally require certain fundamental elements
described below. Once the heart is excised or tissue
fragments obtained (as in human heart isolation) and
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FIG. 4. Enzymatic isolation and acute gene transfer of adult cardiac
myocytes in vitro. Hearts are excised and retrograde perfused with
enzymatic solution containing a combination of collagenase, hyaluronidase, and/or protease on a modified Langendorff apparatus. Once adult
cardiac myocytes are isolated, they are plated on laminin-coated coverslips and transduced in serum-free conditions with viral vectors for
subsequent studies of myocyte structure and function.
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whereby a small amount of Ca2⫹ enters through voltagegated dihydropyridine receptor (DHPR) or L-type Ca2⫹
channels to trigger large-scale Ca2⫹ release from the ryanodine receptor (RyR), the Ca2⫹ release channel located
on the SR membrane. This process is known as Ca2⫹induced Ca2⫹ release (CICR, Fig. 5). The released Ca2⫹,
which rises from ⬃100 nM at diastole to ⬃500 nM to 1 ␮M
during systole binds to troponin C and induces myofilament activation initiating cross-bridge cycling. The removal of Ca2⫹ from the cytoplasm during relaxation is
carried out by the ATP-dependent Ca2⫹ pump, SERCA2a,
and the sarcolemmal sodium-calcium exchanger (NCX)
(50) (Fig. 5).
Impaired EC coupling is a prominent feature of the
diseased and failing heart. Inherited mutations in key
Ca2⫹ handling proteins have been associated with cardiomyopathy (602). In some cases, altered Ca2⫹ cycling precedes the observed depression of mechanical performance. In acquired heart failure (HF), SR Ca2⫹ content is
typically decreased due to the following HF-related maladaptations: upregulation of NCX, reduction in SERCA2a
content/activity, decreased SERCA2a/PLN ratio, and increased RyR open probability which produces spontaneous Ca2⫹ leak (450, 517, 577, 681, 682). Decreased SR
Ca2⫹ content contributes to reduced EC coupling gain
and diminished Ca2⫹ transients, which negatively affect
cardiac contractility. Reduced SERCA2a activity and insufficient energy supply impair Ca2⫹ reuptake resulting in
Ca2⫹ transient remodeling. Furthermore, delayed Ca2⫹
recycling can result in elevated diastolic Ca2⫹ concentration that contributes to heightened arrhythmogenic potential. Additionally, elevated cytosolic Ca2⫹ concentration has been implicated as a stimulus for maladaptive
growth and other morphological changes associated with
HF (57, 337, 690).
High-resolution biophysical and genetic approaches
including transgenesis and acute gene transfer have been
used to elucidate the mechanistic role of important Ca2⫹
cycling modulators in the heart under normal and diseased conditions (49, 55, 121, 140, 451, 517, 952). Acute
genetic engineering provides a means for directly studying gene dosage effects. Titration of the transgene expression to obtain “physiological” versus “pharmacological”
levels of expression is an important consideration when
interpreting the functional outcomes of genetically manipulating Ca2⫹ handling proteins. Acute gene transfer
studies are not limited to overexpression strategies as
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dominant negative mutants or delivery of knockdown
molecules offer alternative approaches for understanding
the direct role of Ca2⫹ handling proteins in adult cardiac
myocyte function. This section focuses on acute genetic
engineering approaches to modulate Ca2⫹ cycling and
cardiac function. This will be accomplished by highlighting the following three components of EC coupling:
1) molecules involved in Ca2⫹ release from the SR, 2)
molecules involved in Ca2⫹ sequestration, and 3) additional Ca2⫹ binding proteins that alter cardiac performance.
A. Regulators of Ca2ⴙ Release From the SR
RyR (RYR2 isoform) is the Ca2⫹ release channel in
cardiac muscle SR. RyR is a homotetramer composed of
four ⬃565-kDa monomeric subunits (667). RyR is extensively regulated via several associated proteins including
protein kinase A (PKA), anchoring protein (mAKAP), protein phosphatases (PP1 and PP2A), sorcin, calmodulin,
S100 proteins, and FKBP12.6 (Calstabin2) (531). RyR is
also a substrate for posttranslational modification, and its
functionality is altered by several small molecules including Ca2⫹, ATP, and Mg2⫹ (551). In the junctional SR, RyR
interacts with calsequestrin (CSQ), triadin, and junctin
Physiol Rev • VOL
(610). The interactions between these proteins contribute
to the CICR mechanism of EC coupling (Fig. 5). RyR
structure-function has not yet been studied using acute
genetic engineering techniques. The large open reading
frame of RyR raises challenges for viral-mediated gene
transfer strategies (Table 1). Additional challenges to
studying the physiological role of RyR through gene transfer technology includes finding physiologically relevant
doses of RyR subunit overexpression and the incorporation of exogenous RyR subunits into the SR membrane.
Nonetheless, several laboratories have addressed the molecular mechanisms of CICR and RyR function through modulating the RyR’s regulatory proteins (FKBP12.6, CSQ, junctin, and triadin) as reviewed in the following sections.
1. FKBP12.6/calstabin2
FKBP12.6 is a cis-trans isomerase protein that binds
tightly to the cardiac ryanodine receptor (RYR2). It is a
12.6-kDa protein that is thought to bind to each subunit of
the channel in a 1:1 ratio on the cytosolic side of RyR (478,
951). FKBP12.6 is considered a RyR channel stabilizer in
the closed state during diastole, and it appears to faciltitate the functional coupling of RyR channels (534, 981).
Elegant biophysical, transgenesis, and acute gene transfer
strategies have yielded important insights into the physi-
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2⫹
FIG. 5. Cardiac muscle excitation-contraction coupling. Illustration of the dynamic interplay between an action potential, intracellular Ca
fluxes, Ca2⫹ handling proteins, and the sarcomere in a process known as excitation-contraction coupling. A single action potential initiates
extracellular Ca2⫹ entry through voltage-gated Ca2⫹ channels (DHPR). This Ca2⫹ entry triggers the release of a large amount of Ca2⫹ from the
intracellular organelle, the sarcoplasmic reticulum (SR), through the ryanodine receptor (RyR). The rise in cytosolic Ca2⫹ reaches a [Ca2⫹]i sufficient
for Ca2⫹ to bind to the myofilament regulatory protein, troponin, which initiates cross-bridge cycling and force generation (systole). Myocyte
relaxation (diastole) requires the removal of cytosolic Ca2⫹ via two primary mechanisms: sequestration of Ca2⫹ into the SR by the Ca2⫹-ATPase
pump (SERCA2a, pink) or Ca2⫹ extrusion by the sodium-calcium exchanger (NCX) or sarcolemma Ca2⫹-ATPase pump. SERCA2a activity is tightly
regulated by the phospho-protein phospholamban (PLN).
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relative to FKBP12.6 in adult cardiac myocytes, although
ultimately both FKBP proteins caused an increase in SR
Ca2⫹ content.
2. Junctional SR proteins: CSQ, triadin, junctin,
and histidine-rich Ca2⫹-binding protein
RyR has several binding partners located on the luminal side of the channel within the SR (Fig. 5). Calsequestrin (CSQ, ⬃46 kDa) is the most abundant SR Ca2⫹
binding protein. It binds ⬃20 Ca2⫹/molecule (cardiac isoform) with moderate affinity (Kd ⫽ 1–100 ␮M), and it
actively participates in Ca2⫹ cycling by regulating the SR’s
luminal Ca2⫹ concentration (825). CSQ’s binding partners
triadin (isoform 1, ⬃35 kDa) and junctin (⬃26 kDa) act
together to tether CSQ to the RyR complex, permitting a
physical coupling of CSQ to RyR (1003). The interaction
between these proteins is thought to play a vital role in
CICR. In addition, histidine-rich Ca2⫹-binding protein
(HRC; 170 kDa) is a moderate-affinity, high-capacity SR
Ca2⫹ binding protein that interacts with SR luminal proteins (e.g., triadin) and is considered a secondary intra-SR
Ca2⫹ storage source other than CSQ.
A) CSQ. Physiological insights into CSQ’s role in cardiac muscle function have been obtained from transgenic
mouse (414, 792) and acute genetic manipulation studies
(571, 877), but the focus here will be placed primarily on
results obtained from acute gene transfer. Acute two- to
fourfold overexpression of CSQ in adult rat and rabbit
cardiac myocytes directly increased SR Ca2⫹ content as
assessed by rapid caffeine application (571, 877). Rat
cardiac myocytes acutely overexpressing CSQ had increased amplitude and duration of Ca2⫹ sparks and waves
in the absence of changes in the frequency of these
Ca2⫹ release events (458, 877), providing evidence that
CSQ overexpression delays the closure of RyR. Imperatoxin A, a pharmacological activator of local RyR-mediated Ca2⫹ release events, was also used to assess the
direct effects of CSQ overexpression on RyR refractory
period (877). Results from this experiment demonstrated that imperatoxin A-induced Ca2⫹ spark frequency was reduced with CSQ overexpression in rat
cardiac myocytes, suggesting that CSQ influences the
RyR’s Ca2⫹-dependent refractory period through its
buffering of luminal Ca2⫹ in the SR (877). Targeted
knockdown of CSQ by adenoviral delivery of antisense
CSQ to isolated adult rat cardiac myocytes caused the
opposite effects of CSQ overexpression (458, 877) in
which SR Ca2⫹ content and Ca2⫹ current (ICa)-triggered
Ca2⫹ transient amplitude were significantly reduced.
CSQ knockdown also reduced the periodicity of Ca2⫹
sparks as well as increased the probability of Ca2⫹
wave propagation (458, 877). The combined approach
of acute CSQ overexpression and knockdown provides
evidence that CSQ plays a vital role in determining SR
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ological role of FKBP12.6 (478, 531, 952). Adenoviralmediated gene transfer of FKBP12.6 in both rat and rabbit
cardiac myocytes revealed consistent gains in cardiac
myocyte function. A five- to sixfold overexpression of
FKBP12.6 in rat and rabbit myocytes directly reduced SR
Ca2⫹ leak and Ca2⫹ spark frequency and amplitude (280,
513, 710), corroborating biophysical evidence for
FKBP12.6’s role in stabilizing channel gating during diastole (534, 981). The reduction in SR Ca2⫹ leak in turn
elevated SR Ca2⫹ load and increased myocyte fractional
shortening (280, 710). Unique to the rat myocyte was the
finding that FKBP12.6 overexpression hastened Ca2⫹
transient decay (280, 710). FKBP12.6 gene transfer and
overexpression appears to synchronize RyR channel
opening during systole, which in turn stabilizes the RyR
and reduces random RyR openings and Ca2⫹ leak during
diastole. It should be noted that the effects of RyR phosphorylation on FKBP12.6-RyR interactions and the subsequent functional outcome still requires further clarification as there is some discrepancy in the literature (376,
410, 534, 727, 837, 951). Nonetheless, FKBP12.6 represents an excellent candidate molecule for further gene
transfer studies that could be used to explore the role of
FKBP12.6 in heart failure, in posttranslational modification of the RyR, and as a potential therapeutic agent.
A related FKBP isoform, FKBP12.0, is also expressed
in the heart, and in some mammals the concentration of
FKBP12.0 is higher than FKBP12.6 (409). Both FKBP12.0
and 12.6 have highly homologous structure and function,
yet their affinities for the cardiac RyR (RYR2) differ significantly (409, 710). FKBP12.0 has a high affinity for the
skeletal muscle RyR (RYR1) and can interact with RYR2,
but FKBP12.6 has a much higher affinity and seemingly
preferential interaction with the cardiac RYR2 (409, 710).
Interestingly, knocking out FKBP12.0 in a transgenic
mouse is lethal early in development due to severe congenital cardiac defects in the absence of skeletal muscle
pathology (819). This mouse model implicates a key role
for FKBP12.0 in the early functioning myocardium, but
the physiological function of FKBP12.0 versus FKBP12.6
in the adult heart remains unclear. Recently, FKBP12.0
was acutely overexpressed in isolated adult rabbit cardiac
myocytes by adenoviral gene transfer (809). A threefold
FKBP12.0 overexpression increased SR Ca2⫹ content similar to FKBP12.6 (809). This acute gene transfer model
also uncovered several functional differences between
FKBP isoforms. Overexpression of FKBP12.0 (809) had
opposing effects on Ca2⫹ spark amplitude, duration, and
frequency relative to acutely transduced FKBP12.6 myocytes (513, 710). Additionally, FKBP12.0 reduced the sensitivity of the cardiac RyR to the CICR mechanism causing
a decrease in EC coupling gain (809), a result not seen in
FKBP12.6 transduced rabbit myocytes (513, 710). These
acute gene transfer studies suggest that FKBP12.0 has
distinct and possibly reciprocal effects on RyR function
Physiol Rev • VOL
the CSQ mutant, D307H, in adult rat cardiac myocytes had
a dominant negative effect to decrease SR Ca2⫹ content
and ICa-induced Ca2⫹ transient amplitude (925). These
results demonstrated that the D307H mutant directly decreases the sensitivity of the SR to CICR mechanisms.
Additionally, isoproterenol and escalations in pacing frequency induced extra nonrhythmic Ca2⫹ transients, a cellular mimetic of delayed afterdepolarizations (DAD) that
are characteristic of CPVT. In contrast, threefold overexpression of the R33Q CSQ mutant in isolated rat cardiac
myocytes did not affect SR Ca2⫹ content and had a dominant effect to increase Ca2⫹ transient amplitude in response to a triggering ICa (876). The R33Q mutant also
increased spontaneous Ca2⫹ spark and wave frequency,
suggesting this mutant enhances Ca2⫹ leak and thus RyR
activity (925). The physiological consequences of expressing D307H versus R33Q mutant CSQ were clearly different, yet both resulted in CPVT. In the case of the D307H
mutant, the arrhythmic phenotype was ascribed to the
inability of this mutant to bind Ca2⫹, thus causing a
misregulation of Ca2⫹ release (876) similar to the results
from acute CSQ knockdown (877). The R33Q CSQ mutant, however, had Ca2⫹ binding properties similar to wild
type, but the heightened Ca2⫹ leak was attributed to
altered CSQ-RyR interactions that caused overactivation
of RyR (876).
B) JUNCTIN. Junctin has been identified as an important tethering component in CSQ’s interaction with
RyR. To date, there is only one report of acute gene
manipulation of junctin in the heart (260). Canine junctin was adenovirally delivered to isolated adult cardiac
myocytes. An acute twofold overexpression of junctin
directly decreased SR Ca2⫹ content as well as decreased the Ca2⫹ transient amplitude but did not alter
cellular fractional shortening (260). Overexpression of
junctin also increased myocyte contractility and accelerated relaxation kinetics (260). The mechanistic basis
for this disconnect between contractile function and
Ca2⫹ transient parameters in junctin overexpressing
myocytes remains unclear. It is possible that junctin
may affect additional aspects of EC coupling beyond
that of Ca2⫹ release. Interestingly, complementary
transgenic mouse models in which pharmacological
doses of junctin were achieved also demonstrated reduced SR Ca2⫹ content, but the Ca2⫹ transient amplitude was preserved (369, 1002). The differences between acute and long-term genetic engineering in this
case were likely due to the compensatory changes in
Ca2⫹ handling proteins that were detected in the transgenic mouse models (369, 1002). Additionally, junctin
overexpressing mouse models demonstrated remodeling of the SR and t-tubule system with 10-fold junctin
overexpression (1002) and hypertrophy and histopathology with 30-fold overexpression (369).
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Ca2⫹ storage capacity and in modulating RyR function
through its influence on SR luminal [Ca2⫹] (458, 877).
The effects of acute CSQ overexpression on EC coupling appeared species dependent, as differences in
CICR and Ca2⫹ wave and spark generation varied between rodents and larger mammals. In contrast to rat
myocytes overexpressing CSQ, Ca2⫹ transient amplitude, when triggered by ICa, was reduced in rabbit
myocytes overexpressing CSQ (571, 877). Additional
species-dependent differences were seen with Ca2⫹
spark measurements in which CSQ overexpression did
not alter the amplitude, duration, or frequency of Ca2⫹
sparks in rabbit myocytes. These discrepancies on the
direct effects of CSQ on EC coupling may be attributed
to several factors including 1) species-dependent differences in ICa (49), 2) gene dosage effects in which rat
had 4-fold overexpression versus rabbit myocyte transduction which had 1.5-fold overexpression, or 3) the
difference in CSQ backbone used for mutagenesis as
the rat studies utilized the canine CSQ cDNA while the
rabbit studies used rabbit CSQ sequence.
In comparing complementary genetic models of
acute in vitro and chronic in vivo overexpression of CSQ,
data from both models suggest that CSQ is a vital mediator of Ca2⫹ storage in the SR. Functional findings in
transgenic mouse models (414, 792), however, differed
from acute gene transfer studies as 10- to 20-fold overexpression of CSQ caused a reduction in SR Ca2⫹ release,
which attenuated CICR mechanisms. These transgenic
mice also showed signs of cardiac hypertrophy (414) and
a transition to the fetal gene program (792). Interpreting
the results from CSQ overexpression in transgenic mouse
models compared with those of adenoviral mediated
acute gene transfer is challenging as the transgenic mouse
models had adaptive responses to the transgene that resulted in hypertrophy, altered cellular morphology, and
changes in the expression of Ca2⫹ release and Ca2⫹ reuptake proteins. All of these alterations could contribute
to the difference in functional outcomes measured in
acute versus long-term gene transfer models. Additionally, the pharmacological levels of CSQ overexpression
obtained in the transgenic models relative to the lower
level of CSQ overexpression obtained with acute adenoviral gene transfer likely contribute to the differential
findings and should be considered when performing a
comparative analysis between models.
CSQ dysregulation has been associated with arrhythmic disorders. Seven different allelic variants in the CSQ
locus have been linked to inherited forms of catecholaminergic polymorphic ventricular tachycardia (CPVT), a disorder that is associated with stress-induced sudden cardiac
death (http://www.fsm.it/cardmoc/, inherited arrhythmias
database). To date, only two of the mutant CSQ alleles,
D307H and R33Q, have been studied using acute genetic
engineering (876, 925). Acute, fourfold overexpression of
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induced an upregulation of triadin and junctin in the
absence of changes in other SR Ca2⫹ release (CSQ and
RyR) and Ca2⫹ reuptake (SERCA2a, PLN) proteins (223).
The increased protein levels of triadin and junctin contribute to the difficulty of understanding HRC’s direct role
in regulating both Ca2⫹ storage and release. It is likely
that the combined effects of the changes in these luminal
SR proteins are affecting myocyte physiology reported
here. Interestingly, acute gene transfer with triadin and
junctin, when expressed at levels similar to those measured in acutely engineered HRC myocytes, had opposite
effects on SR Ca2⫹ load and Ca2⫹ release (260, 875).
These findings further underscore the complex interactions between HRC, triadin, and junctin and their effects
on myocyte physiology. While HRC affects SR Ca2⫹ content, similar to that demonstrated with the acute expression of CSQ (877), HRC has divergent effects on SR
Ca2⫹ release, suggesting that CSQ and HRC are modulating RyR Ca2⫹ release via different mechanisms. To
date, the direct effects of HRC still remain unresolved,
possibly necessitating the use of other acute genetic
engineering approaches including adenoviral-mediated
HRC downregulation or dominant negative mutagenesis strategies.
Interestingly, HRC null and overexpression transgenic
mouse models have been generated and showed an increase
in triadin expression with no change in SR load (297, 398).
These findings confound our understanding of HRC’s role in
cardiac muscle function as both downregulation and overexpression of HRC produced similar effects on SR Ca2⫹
storage. Results from the HRC overexpression mouse model
(297) show important differences compared with results
from acute gene transfer (223). In contrast to the acute HRC
overexpression in rat myocytes, transgenic mice had slow
Ca2⫹ reuptake by SERCA2a and slow Ca2⫹ extrusion by
NCX. This in turn slowed the Ca2⫹ transient decay rate but
not cellular contractile function (297). Chronic HRC overexpression also caused the development of cardiac hypertrophy and an age-dependent transition to congestive heart
failure (297). Remodeling of the heart was not apparent in
transgenic lines with less than fourfold overexpression, suggesting that high levels of HRC are not tolerated at the
organismal level (297). Perhaps gene dosage is partially
responsible for the disparity between the complementary
overexpression models as are the compensatory changes in
other Ca2⫹ handling proteins including SERCA and NCX
that occurred with chronic overexpression of HRC (297).
B. Regulators of Cytoplasmic Ca2ⴙ Removal
1. SERCA2A and phospholamban
Cardiac cytosolic Ca2⫹ content is highly regulated via
transport proteins that either sequester Ca2⫹ into the SR
or extrude Ca2⫹ across the sarcolemma. The mechanisms
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C) TRIADIN. There are three cardiac triadin isoforms,
triadins 1, 2 and 3, with the dominant isoform being
triadin 1 (304). Triadin and junctin are the products of
different genes but have highly homologous sequences
and structures that are postulated to play redundant
roles in regulating Ca2⫹ release through RyR. The anchoring role of triadin, like junctin, has been fairly well
defined, but to date, triadin’s functional role in the Ca2⫹
release process has remained elusive. To address these
issues, adenoviral-mediated acute overexpression of
triadin in cultured adult rat ventricular myocytes revealed a direct ability of triadin to activate RyR and
promote Ca2⫹ release (875). With threefold triadin
overexpression, Ca2⫹ release from RyR was enhanced
as Ca2⫹ spark frequency increased, but spark amplitude
was lowered. Triadin overexpression also increased
RyR open probability during single-channel recording
and increased Ca2⫹ transient amplitude at smaller trigger ICa (875). Consequently, SR Ca2⫹ content was decreased due to heightened spontaneous Ca2⫹ release at
rest. Transduced myocytes were also arrhythmogenic
when stimulated in the presence of isoproterenol (875).
Triadin’s direct activation of RyR may be through its
interaction with CSQ and RyR, since a truncated triadin
mutation lacking the domain important for CSQ interaction showed no effect on RyR Ca2⫹ release (875). An
acute genetic approach showed consistent results supporting a direct regulatory role of triadin on RyR. The
discrepancy between results from acute versus longterm triadin overexpression in transgenic mice (438)
was postulated to involve compensatory adaptations by
other key Ca2⫹ handling proteins, culture-related
changes of myocyte structure and function, the level of
overexpression, and/or location of these overexpressed
triadin molecules (600). The transgenic mouse models
have also shown that pharmacological doses of triadin
can negatively affect EC coupling and lead to maladaptive cardiac hypertrophy (438).
D) HRC. HRC is a luminal SR binding protein that has
a histidine-rich repeat region located at the center of the
molecule and is responsible for both Ca2⫹ binding and
interactions with other junctional proteins. The Ca2⫹
binding ability of HRC and its interaction with junctional
proteins suggest a potentially important physiological
role of HRC in cardiac function. Adenoviral-mediated
acute gene transfer of HRC demonstrated a direct and
significant inhibitory effect on the Ca2⫹ transient and
myocyte contractility when overexpressed at low levels
of ⬃1.7-fold (223). In this study, HRC overexpression
slowed Ca2⫹ transient decay and as a consequence myocyte relaxation (223). Additionally, HRC overexpression
increased SR Ca2⫹ load but blunted Ca2⫹ transient amplitude and fractional shortening, suggesting that HRC not
only affects SR Ca2⫹ storage but also modulates Ca2⫹
release (223). Unexpectedly, acute HRC overexpression
Physiol Rev • VOL
take and SERCA2a activity (263, 337, 478) which become
diminished in the failing heart. The reduction in Ca2⫹
reuptake in the failing heart is attributed to reduced expression of SERCA2a or a decreased pump activity, which
can also be associated with an increase in the PLN/
SERCA2a ratio (18, 559). Acute genetic engineering strategies have been used to increase SERCA2a expression
and/or activity to restore cardiac function in models of
heart failure. Studies employing adenoviral gene transfer
of SERCA2a have shown that overexpression of SERCA2a
can significantly enhance Ca2⫹ release, hasten relaxation,
and decrease diastolic [Ca2⫹] (167, 169, 268, 317, 319, 559,
583, 801). Acute overexpression of PLN increases the
PLN/SERCA2a ratio, which functionally results in decreased Ca2⫹ transient amplitude, prolonged Ca2⫹ transient decay time, and increased diastolic [Ca2⫹], characteristics that are similar to myocytes from failing hearts
(320). Overexpressing SERCA2a can rescue the “failing
myocyte” phenotype that is created when the PLN/
SERCA2a ratio is increased (168, 169, 317, 319, 583, 801).
Importantly, aberrant Ca2⫹ cycling and contractile deficits characteristic of failing human myocytes can be corrected by restoring the level of SERCA2a expression. This
functional correction of the failing myocyte by SERCA2a
gene transfer is manifest in increased shortening and
relaxation velocity, heightened peak systolic Ca2⫹, and
lower diastolic [Ca2⫹], in addition to a corrected cell
shortening-frequency response (169).
Hirsch et al. (361) demonstrated the effects of adenoviral-mediated overexpression of SERCA2a in cardiac
myocytes isolated from a canine model of diastolic heart
failure. This model was generated by a descending thoracic aortic coarctation resulting in left ventricular (LV)
pressure overload over a year. Cardiac myocytes isolated
from canines with diastolic dysfunction were transduced
with SERCA2a Ad5 vectors. Acute expression of
SERCA2a enhanced relaxation in the failing canine myocytes. In this model, SERCA2a overexpression unexpectedly resulted in a loss of isoproterenol-mediated inotropy
during cardiac myocyte contractile measurements in vitro
(361). The mechanism of this effect is unknown but is
postulated to be due to a diminished PLN/SERCA2a ratio
in SERCA2a transduced myocytes. Considering that cardiac reserve is critical to global cardiac performance, a
full understanding of the effects of SERCA2a overexpression on ␤-adrenergic molecular inotropy is important to
ascertain.
In vivo adenoviral gene transfer of SERCA2a has
been performed in several animal models of heart failure.
In a rat model of heart failure induced by aortic banding,
Miyamoto et al. (583) used intracoronary gene delivery of
SERCA2a at the time of transition from compensated
hypertrophy to heart failure. SERCA2a expression restored systolic and diastolic function in this model (583).
In a subsequent study, the same group demonstrated that
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for extruding Ca2⫹ from the cytosol in mammalian cardiac muscle include the following: SERCA2a, sarcolemmal NCX, mitochondrial Ca2⫹ uniporter, and sarcolemmal
Ca2⫹-ATPase (50). SERCA2a sequesters cytosolic Ca2⫹
into the SR by transporting two Ca2⫹ per molecule of
hydrolyzed ATP against a steep Ca2⫹ gradient. There are
five SERCA isoforms that are encoded by three genes,
with SERCA2a being the predominant isoform expressed
in the cardiac muscle (263). SERCA2a is regulated by the
closely associated phosphoprotein, phospholamban (PLN),
although sarcolipin, described below, can also contribute
to SERCA regulation (55, 539). PLN, a small pentameric
protein complex comprised of 6-kDa monomers (55), dynamically regulates SERCA2a function on a beat-to-beat
basis by its phosphorylation state. It is the interplay between kinases and phosphatases that determines PLN’s
phosphorylation status (539). In the unphosphorylated
state, PLN lowers the affinity of SERCA2a for Ca2⫹,
thereby inhibiting Ca2⫹ transport (25, 451). Phosphorylation of PLN by PKA at Ser-16 or by CAMKII at Thr-17
reverses the PLN-mediated inhibition of SERCA2a (see
also sect. VI) (539). PLN phosphorylation is an important
contributor to the hastening of myocardial relaxation during ␤-adrenergic stimulation as this PLN modification increases Ca2⫹ reuptake into the SR (950). PLN protein
levels are generally unchanged in failing human hearts;
however, a reduction in the extent of PKA-mediated
Ser-16 phosphorylation of PLN and/or an increase in PLN
to SERCA2a ratio are often noted in heart failure. Consequently, SERCA2a function is decreased in heart failure
(517). Thus both SERCA2a and PLN are considered attractive gene therapy candidates for improving Ca2⫹ handling in heart failure patients.
PLN phosphorylation status is contingent on the activity of PP1 (106, 539), the phosphatase involved in counteracting PKA-mediated phosphorylation of PLN. PP1 dephosphorylates PLN which in turn inhibits SERCA2a activity. An additional level of regulation is due to the
actions of inhibitor 1 (I-1) protein (539). When activated
by PKA, I-1 negatively controls the phosphatase activity of
PP1 (539). The resulting I-1-dependent inhibition of PP1
maximizes the phosphorylation state of PLN, thereby increasing SERCA2a function. I-1 plays a vital role in the
positive inotropic effects of ␤-adrenergic stimulation as it
assists in maximizing PKA activity in a cardiac myocyte
(678, 850). The roles of PP1 and I-1 have been studied
using acute gene transfer and are fully reviewed in
section VI.
Important insights into the physiological role of
SERCA2a and PLN in cardiac muscle have come from
transgenic mouse models and are reviewed elsewhere
(517, 558). This section highlights experimental results
obtained through the use of acute genetic engineering.
Several of these gene transfer studies have been performed with the eventual goal of restoring SR Ca2⫹ up-
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therapy for progressive dilated cardiomyopathy (DCM)
and associated heart failure.
Several acute PLN gene knock-down strategies have
also been used to alter SERCA2a function. Adenoviral
gene transfer of antisense RNA directed towards PLN in
neonatal adult rat (206, 347) and human failing cardiac
myocytes (168) demonstrated decreased PLN expression
and consequently increased rates of Ca2⫹ transient decay
and enhanced myocyte contractility. In another knockdown approach, AAV-mediated gene transfer of a short
hairpin RNA (shRNA) to neonatal rat myocytes silenced
PLN but did not significantly influence the levels of other
Ca2⫹ cycling or cellular PKA targets (227). This silencing
of PLN significantly increased basal SR Ca2⫹ uptake, but
reduced PKA-directed Ca2⫹ uptake for up to 7 days after
gene transfer. While delivery of antisense PLN significantly decreased PLN levels in neonatal rat myocytes, it
failed to decrease PLN levels in adult cardiac myocytes
(347), causing some disagreement about using an antisense PLN approach to knockdown PLN. In a separate
study, the expression of PLN was significantly decreased
after adenoviral delivery of antisense PLN to failing human myocytes (168).
Viral-mediated gene delivery of an antibody targeted
to the cytoplasmic portion of PLN has also been used to
acutely alter SERCA2a activity (557). Expression of this
antibody did not affect the expression or localization of
PLN and affected the Ca2⫹ transient similar to phosphorylated PLN when expressed in adult mouse myocytes
(557). In vivo gene delivery of this antibody also acutely
improved basal contractility and relaxation in mice with
diabetic cardiomyopathy. However, ␤-adrenergic modulation of contractile function was muted in myocytes expressing this antibody, which may ultimately be detrimental for long-term survival. Although the increases in basal
Ca2⫹ uptake observed with knockdown strategies are
beneficial in terms of improved relaxation rates, the loss
of adrenergic modulation may also be important for contractile function in failing hearts. In a subsequent study,
silencing PLN by adenovirally delivering an antibody directed towards PLN’s cytoplasmic domain restored contractile function and Ca2⫹ handling both at the myocyte
and organ level in cardiomyopathic hamsters (178). Gene
transfer of PLN antibodies represents a new approach for
modifying PLN-SERCA interactions and provides a novel
therapeutic strategy for improving failing cardiac myocyte contractile function.
2. Sarcolipin
Sarcolipin (SLN) is a 31-amino acid SR membrane
protein (23, 25, 660) with close structural similarity to
PLN (660). The SLN and PLN sequences diverge significantly at the NH2 and COOH termini (55). SLN does not
contain the Ser-16 or Thr-17 phosphorylation sites but has
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in vivo SERCA2a gene transfer normalized SERCA2a expression levels in the failing myocardium which in turn
improved cardiac function, energetics, and survivability
(170). Appropriate titration of SERCA2a expression in
failing cardiac muscle can restore the normal stoichiometry between PLN and SERCA, which prevents cytosolic
Ca2⫹ overload and left ventricular dysfunction. Taken
together, these studies implicate SERCA2a gene transfer
as a potential treatment for contractile dysfunction in
failing hearts.
PLN gene knockdown and targeted mutant gene delivery have also been used successfully to modulate
SERCA2a activity and consequently myocardial physiology (206, 227, 347, 1018). Acute expression of a dominant
negative PLN construct, K3E/R14E, significantly increased fractional shortening and hastened Ca2⫹ transient decay and relaxation times in isolated rabbit ventricular myocytes (347). Gene transfer of PLN K3E/R14E
to myocytes isolated from a rabbit HF model directly
increased SR Ca2⫹ content, which in turn corrected the
contractile dysfunction in the failing rabbit myocytes
(1018). A V49A PLN mutant also acted as a dominant
negative form of PLN to enhance myocyte contractility
and relaxation (577). Both sets of dominant negative PLN
variants identified critical sites within PLN that have dominant functional consequences over native PLN to reverse
its normal inhibition of SERCA2a activity.
An alternative gene transfer strategy to enhance
SERCA2a activity involves using a constitutively phosphorylated PLN mimetic developed by substituting the
serine residue at codon 16 with glutamic acid (PLN S16E)
(372). Acute adenoviral delivery of this PLN phosphomimetic to neonatal rat cardiac myocytes increased contractility and had positive lusitropic effects in the absence of
any ␤-adrenergic stimulation (372), suggesting that the
PLN S16E enhances Ca2⫹ cycling and thus contractile
function relative to baseline. The efficacy of PLN S16E to
halt heart failure progression was also examined using in
vivo transcoronary delivery of recombinant AAV serotype
2 to BIO14.6 cardiomyopathic hamsters (372). Transcoronary delivery of an AAV2 reporter construct had 79%
transduction efficiency, and the transgene was stable for
at least 7 mo (372). In this model, PLN S16E increased SR
Ca2⫹ cycling and slowed the loss of systolic and diastolic
function characteristic of the cardiomyopathic hamster
(372). Additionally, PLN S16E prevented thinning of the
posterior LV wall (372). In a complementary rodent heart
failure model, AAV in vivo delivery of PLN S16E blocked
the transition to ventricular dilation observed in the nontransduced infarcted rodent model (394). Hemodynamically, PLN S16E improved LV contractility and diastolic
function as well as lowered end diastolic pressures and
prevented the transition to LV dilation in this HF rat
model (394). Together, these studies highlight the potential for in vivo AAV-mediated delivery of PLN S16E as a
Physiol Rev • VOL
3. NCX
The NCX transports 3 Na⫹ for 1 Ca2⫹ and is an
important regulator of Ca2⫹ homeostasis and contractility
in cardiac myocytes, especially in larger mammals. NCX
can operate in both Ca2⫹ efflux (forward) and Ca2⫹ influx
(reverse) modes, depending on the internal and external
concentration of both Na⫹ and Ca2⫹ and membrane potential (53). During diastole, SERCA2a and NCX both
contribute to cytosolic Ca2⫹ removal. The competition
between SERCA2a and NCX for Ca2⫹ consequently determines the Ca2⫹ content of the SR per cardiac cycle (49).
NCX has also been postulated as an important component
in heart failure as several animal models of hypertrophy
and heart failure have shown elevated NCX expression
and/or activity (53, 824), a characteristic that has also
been reported in tissue from failing human hearts (53,
824). There are, however, conflicting reports of heart
failure-induced decreases or unchanged NCX content.
Hassenfuss and Pieske (337) found that in failing human
heart samples the ratio of NCX to SERCA protein expression consistently increased two- to fourfold relative to
nonfailing hearts (824).
Insights into NCX function under normal and diseased conditions have been gained through acute gene
transfer studies. Adenoviral-mediated gene transfer of
NCX to isolated cardiac myocytes has shown somewhat
conflicting results. Several studies have demonstrated
that gene transfer of NCX causes contractile dysfunction
in rabbit ventricular myocytes due to a reduced SR Ca2⫹
load (719, 798, 799). Bölck et al. (69) reported that NCX
gene transfer enhanced both systolic Ca2⫹ amplitude and
myocyte fractional shortening in rat cardiac myocytes at
low stimulation rates, but these functional parameters
became reduced at higher pacing frequencies. In a recent
study, Münch et al. (611) investigated the functional consequences of NCX overexpression using both in vitro and
in vivo adenoviral gene transfer to nonfailing and failing
rabbit hearts. With in vitro adenoviral gene transfer, NCX
overexpression in isolated nonfailing cardiac myocytes
depressed contractility at all pacing frequencies, while
NCX overexpression in failing myocytes had an even
greater frequency-independent reduction in contractility.
In contrast, in vivo NCX overexpression in failing rabbit
hearts improved contractility and contractile reserve as
the hearts had increased responsiveness to ␤-adrenergic
stimulation and, therefore, enhanced inotropic and lusitropic responses (611). Long-term NCX overexpression in
nonfailing rabbits in vivo had only minor effects on myocardial contractility and led to myocardial hypertrophy, in
accordance with previous findings in transgenic mice (8,
736, 879, 992). The mixed results obtained from rat versus
rabbit NCX gene transfer models might be attributed to
the higher intracellular Na⫹ concentration in rodent cardiac myocytes, which would favor reverse-mode NCX
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the potential for phosphorylation at Thr-5 (55). The sequence variation in the COOH terminus may be responsible for the differential regulatory function of the proteins
at high [Ca2⫹] (24, 921). SLN is predominantly expressed
in the atria and fast skeletal muscle, and it can be found
in the ventricle but at much lower concentrations than
PLN (25, 55). Similar to PLN, SLN inhibits SERCA activity
in the basal state, but the mechanism of reversal of SLNmediated inhibition by ␤-adrenergic activation is unclear
(23, 24).
SLN expression is highly species dependent. In rodents, both SLN mRNA and protein are expressed in the
atria but are almost negligible in the ventricle. In contrast,
in larger mammals including humans, SLN mRNA is
highly expressed in fast-twitch skeletal muscle compared
with atria and ventricle (920). SLN expression is developmentally regulated and has a pathophysiological component as patients with atrial arrhythmias have low levels of
SLN transcript (578, 920).
Adenoviral (31) or transgenic (25, 30) overexpression
of SLN has been performed to identify the role of SLN in
cardiac physiology. Isolated adult rat cardiac myocytes
acutely engineered with SLN had decreased myocyte
shortening in the absence of any changes in Ca2⫹ transient amplitude relative to control myocytes (31). Acute
SLN overexpression also slowed myocyte relaxation and
Ca2⫹ transient decay time, suggesting that SLN depresses
SERCA2a activity in ventricular myocytes (31). Immunofluorescence assays showed an overlap of vector-derived
SLN with the native SERCA2a and PLN providing evidence of SLN colocalization with SERCA2a and PLN (31).
This was further supported in PLN pull-down assays with
SLN (31). In two different complementary transgenic
mouse models (25, 30), chronic SLN overexpression in
the ventricle caused a loss of function similar to the
acute gene transfer model in which a reduction in rate
of shortening and relaxation was observed at the organ
(25) and myocyte (30) level. One SLN transgenic mouse
model revealed diminished Ca2⫹ transient and myocyte
shortening amplitude (30), which was ascribed to SLNdependent changes in SERCA2a’s Ca2⫹ affinity. Stimulating ␤-adrenergic pathways corrected the loss in contractility reported in SLN overexpressing mice (25, 30)
and in PLN knockouts with SLN overexpression (289).
These findings suggest that SLN inhibition of SERCA
may be reversed by ␤-adrenergic signaling, with the
caveat that compensatory changes in SLN mouse models may also be contributing to the isoproterenoldependent enhancement of contractility and Ca2⫹ dynamics. These studies provide evidence that SLN is
another potential therapeutic target, although additional work is required to identify the in vivo mechanism of SLN-mediated SERCA2a inhibition.
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that rodent Ca2⫹ extrusion relies heavily on SR reuptake
whereas NCX plays a greater role in larger mammals (50),
further study in larger animal models is required to fully
understand the physiological and pathophysiological role
of NCX.
4. Phospholemman
Phospholemman (PLM) is a 72-amino acid transmembrane protein localized to the sarcolemma of cardiac and
skeletal muscle. It is a member of the recently discovered
“FKYD” family that regulates ion transport (854) and is a
substrate for ␣- and ␤-adrenergic signaling molecules
PKA and protein kinase C (PKC) (496, 709). PLM associates with Na⫹-K⫹-ATPase pump (NKA) and can regulate
the pump’s affinity for Na⫹ (143). Several studies have
now demonstrated that PLM acts on NKA in a manner
similar to phospholamban’s regulation of SERCA2a, such
that phosphorylation of PLM by PKA increases NKA pump
activity through a mechanism of PLM-NKA disinhibition
which concomitantly increases the pump’s affinity for
Na⫹ (176, 251, 822). Sodium homeostasis is critical under
both normal cardiac function and during heart failure.
There is evidence that PLM expression is decreased relative to NKA and that a higher percentage of PLM is
phosphorylated in failing human and rabbit hearts (74). In
contrast, PLM expression was elevated in a rat model of
heart failure (115, 808, 1006). Together these studies suggested an important role for PLM in both healthy and
diseased cardiac myocytes.
Song et al. (832) virally transduced isolated adult
cardiac myocytes with PLM which resulted in PLM overexpression (⬃42% greater than than wild-type) within 3
days after gene transfer. Overexpression of PLM increased maximal myocyte shortening and Ca2⫹ transient
amplitudes at low [Ca2⫹]o (0.6 mM). These parameters
were blunted at higher [Ca2⫹]o (5.0 mM) and had minimal
affects at physiological [Ca2⫹] (832). This same model
was also used to assess the direct effects of phospholemman overexpression on SR Ca2⫹ content, NCX current,
and action potential generation (1006). In this study, 3.5fold overexpression of PLM at 3 days after gene transfer
caused similar contractile changes at high and low
[Ca2⫹]o as demonstrated earlier (832). These contractile
abnormalities, however, could be corrected by coexpressing the PLM with NCX (1006). Here, SR Ca2⫹ content in
PLM overexpressing myocytes was unchanged, but the
Ca2⫹ decay rate was significantly slower, suggesting that
NCX and PLM are functionally interacting. Additionally,
the myocyte action potential and resting membrane potential were not significantly altered, but the reversemode NCX current was lower at higher clamped voltages.
Taken together, these studies suggest that PLM directly
affects cardiac myocyte function, perhaps through
NCX. Recent evidence from in vitro pull-down assays
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action (51). Elevated intracellular Na⫹ concentrations
have been found in human (692) and rabbit (177) failing
hearts, in which case overexpression of NCX may be
beneficial for heart failure by improving SR Ca2⫹ load;
however, this benefit may be offset by heightened arrhythmogenicity.
As NCX is the primary mechanism of Ca2⫹ efflux
from cardiac myocytes, it has been postulated that loss of
this extrusion mechanism should cause significant Ca2⫹
overload and thus contribute to heart failure (52, 54, 379,
948). Several laboratories have demonstrated that global
NCX1 knockout in mice results in embryonic lethality
(117, 452, 934). Interestingly, a recently engineered cardiac specific NCX1 knockout mouse survived well into
adulthood with no signs of cardiomyopathy (354, 703–705,
737, 738). Pharmacological inhibitors of NCX function
have been utilized, but the results are more difficult to
interpret owing to drug specificity issues (739). To better
understand NCX’s role in cardiac myocyte physiology in
the absence of complex influences from adaptive responses in transgenic mice or the pleiotropic effects of
pharmacological inhibitors, transient knockdown strategies have been employed to acutely suppress NCX expression and activity. The initial reports of NCX knockdown
used an antisense oligonucleotide strategy in which naked DNA was delivered to embryonic (863) or neonatal
rat cardiac myocytes (497, 827). These studies showed
that naked DNA delivery of antisense NCX is a viable
strategy for knocking down NCX expression in neonatal
(497, 827) but not adult rat cardiac myocytes (379, 859).
To improve efficiency, Tadros et al. (859) used adenoviral
delivery of the antisense NCX and achieved a 30 and 66%
knockdown at 3 and 6 days after gene transfer, respectively. Under physiological conditions (37°C, 1.1 mM
[Ca2⫹]o, 1–3 Hz pacing frequency), a 30% knockdown of
NCX in adult rat cardiac myocytes resulted in heightened
diastolic [Ca2⫹]i in the absence of action potential remodeling or changes in myocyte contractility/Ca2⫹ transient
amplitude during caffeine contracture. NCX knockdown
tempered the contractile and Ca2⫹ transient response to
varying extracellular [Ca2⫹] in cardiac myocytes, showing
the importance of extracellular [Ca2⫹] in addition to NCX
density on Ca2⫹ efflux. Higher levels of NCX knockdown
have been achieved with adenoviral delivery of interference RNA (RNAi) to cultured neonatal rat myocytes
(379). This strategy was highly efficient and elicited ⬎90%
knockdown of NCX expression. NCX knockdown in this
system depressed the Ca2⫹ transient amplitude and relaxation rate in addition to action potential remodeling.
Taken together, these studies have demonstrated that
acute genetic engineering strategies can be used to knockdown NCX protein expression. These studies, however,
have not entirely resolved the direct effects of NCX
knockdown on adult cardiac myocyte function as only
rodent models have been tested at this point. Considering
C. Ca2ⴙ Binding Proteins That Modulate
Cardiac Performance
1. Parvalbumin
Parvalbumin (Parv) is a cytosolic divalent cation
buffer that is a member of the EF-hand Ca2⫹ binding
protein family (199). Parv contains two high-affinity Ca2⫹
binding sites (Kpvca ⫽ 107–109 M⫺1) that competitively
bind Mg2⫹ but with much lower Mg2⫹ affinity (Kpvmg ⫽
103–105 M⫺1) (199). Parv is expressed in several tissues
including fast skeletal muscle but is not normally found in
the mammalian heart (199). In fast skeletal muscle, Parv
is thought to act as a delayed Ca2⫹ buffer that hastens
relaxation in a dose-dependent manner (352, 374).
Although not naturally occurring in the heart, there is
evidence of successful human Parv gene transfer in cardiac myocytes. The effects of ectopic Parv gene transfer
on cardiac performance have been pursued both in vitro
and in vivo. Parv gene transfer in isolated rodent cardiac
myocytes caused a significant increase in Ca2⫹ sequestration rate and myocyte relaxation performance under
basal conditions (931). The same acute Parv gene transfer
procedure rescued diastolic function in myocytes obtained from hypothyroid rats, a model of diastolic heart
failure (931). Diastolic dysfunction is also a clinical feature of hypertrophic cardiomyopathy (HCM). As such a
cellular mimetic of HCM was developed by acutely expressing an HCM-linked mutant ␣-tropomyosin in isolated
cardiac myocytes. Notably, cotransduction of Parv corrected the slow relaxation, a characteristic of most HCM
myocyte models, by increasing the rate of Ca2⫹ removal
(139). In addition, acute Parv gene transfer successfully
reversed the slow relaxation kinetics of senescent rodent
cardiac myocytes (378). In vitro gene transfer of Parv or
SERCA2a in myocytes from a canine model of diastolic
heart failure showed comparative efficacy in hastening
myocyte relaxation (361). In considering Parv as a potenPhysiol Rev • VOL
tial therapeutic for the treatment of diastolic heart failure,
a notable feature of Parv-transduced myocytes is their
retained capacity for responding to ␤-adrenergic stimulation, a result not obtained with SERCA2a overexpression
(361). These studies indicate that Parv gene transfer may
offer unique potential as a primary treatment for diastolic
dysfunction in failing hearts.
The effects of Parv in single myocytes have been
translated to organ-level function. In vivo gene transfer of
Parv via direct intramyocardial injection achieved significant Parv expression (858). The Parv expressing hearts
showed accelerated relaxation rates as measured by a
working heart preparation as well as by in vivo micromanometry and echocardiography (858). In vivo adenovirusmediated Parv gene transfer in aged rats showed a reduced expression efficiency (568); nonetheless, Parv still
redressed the slow relaxation in these aged hearts (568).
A model of Parv’s role in cardiac myocyte performance has been proposed (140) (Fig. 6). Briefly, Parv
binds Mg2⫹ during late diastole due to the high [Mg2⫹]
relative to [Ca2⫹] (Fig. 6A). The voltage-dependent increase in cytosolic [Ca2⫹] (1 ␮M) triggers an exchange of
bound Mg2⫹ to Ca2⫹ at Parv’s metal binding sites (Fig.
6B). This process results in delayed Ca2⫹ binding by Parv
through early diastole because the unbinding of Mg2⫹ is
relatively slow. As intracellular [Ca2⫹] declines to near
resting levels in mid to late diastole, Ca2⫹ dissociates
from Parv (Fig. 6, C and D). Therefore, significant
amounts of Ca2⫹ can be transiently buffered by Parv
during early relaxation, which in turn accelerates relaxation in Parv-transduced cardiac myocytes relative to
controls.
A limitation of wild-type Parv is that high doses can
lead to excessive Ca2⫹ buffering during systole, which
negatively affects fractional shortening and Ca2⫹ transient amplitude (361). Experimental data as well as mathematical modeling have been carried out to determine the
optimal concentration range of Parv for expression in
cardiac myocytes. This range was set at ⬃0.01– 0.10 mM
for wild-type Parv (141). This critical concentration range
limits the buffering of Ca2⫹ to the relaxation phase.
Therefore, peak contraction and Ca2⫹ transient amplitude
remain unaffected while diastolic relaxation and Ca2⫹
transient decay times are accelerated. Recent studies using Parv isoforms suggest that experimentally modified
Parv constructs may be developed to limit the deleterious aspects of Parv function (i.e., depressing contractility) while maintaining enhanced relaxation performance (755).
2. Sorcin
Sorcin, soluble resistance-related calcium-binding
protein, is a highly conserved 22-kDa protein that has five
EF-hand Ca2⫹ binding domains (519). Sorcin is expressed
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and cotransfection of PLM and NCX into HEK cells suggests there is a direct physical interaction between these
proteins (941). There are a few limitations to the acute
gene transfer studies presented here that may be confounding our understanding of PLM’s role in cardiac myocyte physiology. For example, PLM is a target for ␣- and
␤-adrenergic posttranslational modification which regulates NKA. In these published acute gene transfer studies,
there is no control for phosphorylation status of PLM.
Acute gene transfer of targeted amino acid substitutions
to create phosphomimetics or permanently inactive PLM
could yield more insight into the regulatory role of PLM in
cardiac function. Furthermore, species differences in
Ca2⫹ and Na⫹ homeostasis, particularly with respect to
the role of NCX, may result in differential speciesdependent roles for PLM.
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FIG. 6. Model of parvalbumin’s effects on cardiac myocyte function.
A: late diastole in which [Ca2⫹]i is low and parvalbumin (PARV) is
binding Mg2⫹ with the myofilaments in a relaxed state. B: systole in
which [Ca2⫹]i is rapidly rising. The heightened [Ca2⫹]i causes Mg2⫹ to
slowly dissociate from PARV. During this time, Ca2⫹ is binding to the
myofilament proteins, which initiates cross-bridge cycling and thus muscle contraction. C: late systole and early diastole in which PARV competes with other intracellular buffers for Ca2⫹. Ca2⫹ binds to PARV,
which facilitates a faster decay of the Ca2⫹ transient and subsequently
accelerates sarcomeric relaxation. D: mid to late diastole in which
[Ca2⫹]i is low causing PARV to exchange bound Ca2⫹ for Mg2⫹, while
Ca2⫹ is being sequestered into sarcoplasmic reticulum and cross-bridge
cycling is sterically blocked.
in several tissues, including the heart (561). Sorcin is
localized to the t tubules and SR where it can interact with
the RyR in cardiac myocytes (508, 561, 563). Increases in
intracellular [Ca2⫹] initiate a Ca2⫹-dependent movement
of Sorcin from the cytosol to the SR (563), where it can
interact with specific target proteins including RyR (561),
L-type Ca2⫹ channel (DHPR) (562), and SERCA2a (536).
In lipid bilayer preparations, sorcin directly alters the
open probability of single RyR units inhibiting local Ca2⫹
release (225, 508). This effect is abrogated upon sorcin
phosphorylation by PKA (508). Sorcin also plays a role in
the diseased heart, as sorcin expression is altered
in several heart failure models (536, 664) and mislocalized
in spontaneously hypertensive rats (560).
Both acute viral-based genetic engineering (242, 536,
810, 846) and mouse transgenesis (560) have been used to
elucidate the physiological role of sorcin. Gene transfer of
sorcin to isolated adult rat (242, 536) and mouse (846)
cardiac myocytes caused a gain in contractility with a
3.5-fold increase in sorcin expression. In isolated rodent
myocytes, sorcin overexpression increased fractional
shortening, Ca2⫹ transient amplitude, and SR Ca2⫹ content (242, 536, 846). Sorcin’s role as an acute positive
inotrope was also confirmed at the organ level using in
vivo adenoviral gene delivery to adult rodent hearts. In
vivo, sorcin acutely hastened the rate of pressure development and relaxation and significantly enhanced systolic
pressure (242, 846). In contrast, acute overexpression of
sorcin in vitro (810) had the opposite effect on cellular
function relative to in vivo gene transfer to the rodent
myocardium. In rabbit myocytes, sorcin significantly
blunted shortening and Ca2⫹ transient amplitudes (810).
Additionally, sorcin overexpression in the rabbit model
had reduced SR Ca2⫹ content and Ca2⫹ spark amplitude
and duration, suggesting that sorcin has a negative effect
on EC coupling in rabbit myocytes (810). The discrepancy
between these acute gene transfer models could be related to the following factors: 1) the different levels of
sorcin overexpression in rabbit versus rodent myocytes
and 2) the enhanced contribution of NCX to Ca2⫹ handling in rabbit versus rodent myocytes (50). For instance,
in rabbit myocytes overexpressing sorcin, there was an
NCX-dependent slowing of Ca2⫹ decay time (810). In
contrast, rodent myocytes that acutely overexpressed sorcin had enhanced Ca2⫹ transient decay time (536, 846)
and SERCA2a activity (536), providing evidence of species-dependent differences in sorcin function. Notably,
pharmacological levels of sorcin expression (20-fold increase) in a transgenic mouse model caused significant
reductions in cardiac contractility and relaxation properties in the absence of cardiac hypertrophy or morphological remodeling (560), highlighting the importance of sorcin dosage and time of exposure to functional outcome at
the organ level.
3. S100 proteins
S100 proteins constitute a highly conserved 21⫹
member family of EF-hand Ca2⫹-binding proteins (351,
525). S100 proteins are small in size (10 –12 kDa) and have
similar structural features that include a conventional
COOH-terminal divalent cation binding domain and a
lower affinity NH2-terminal unconventional EF-hand
metal binding domain (525). The S100 isoforms differ in
terms of their metal binding affinities, dimerization capacity, and posttranslational modifications (525). S100 isoforms are differentially expressed in a tissue-dependent
manner (351, 525). In cardiac muscle, S100A1 is the predominant isoform, but S100A4, S100A6, and S100B have
also been identified in the heart (315, 425). In general, the
binding of Ca2⫹ to an S100 protein exposes a hydrophobic
region of the molecule that is available to interact/modulate various intracellular target proteins (525). These
Ca2⫹-dependent protein-protein interactions are important for various molecular processes including contraction, cell cycle regulation, cell growth, and secretion.
Physiol Rev • VOL
Several of the S100 proteins can also interact with target
proteins independent of binding Ca2⫹ (525).
Gene transfer studies performed in vitro and in vivo
have contributed greatly to understanding the role of
S100A1 protein on cardiac function in the healthy and
diseased heart (607). Acute adenoviral-mediated gene
transfer of S100A1 to healthy isolated rabbit (605) and rat
(731) adult ventricular cardiac myocytes and engineered
cardiac tissue (605, 732) caused a positive inotropic response in which cell shortening and Ca2⫹ transient amplitudes were significantly enhanced. A fivefold overexpression of S100A1 directly hastened the time to peak
Ca2⫹ and Ca2⫹ transient decay velocity, which in turn
increased myocyte shortening and relaxation kinetics
(605). This gain in inotropy was attributed to heightened
Ca2⫹ reuptake by SERCA2a (605). Although S100A1 is
downregulated in heart failure (730), S100A1 knockout
(194), and cardiac-specific S100A1 overexpressing (608)
transgenic mouse hearts have normal morphology and
tissue histology. Similar to the acute gene transfer studies,
a fourfold overexpression of S100A1 resulted in increased
contractility and Ca2⫹ transient amplitudes at both the
cellular and organ level under basal and ␤-adrenergic
stimulated conditions despite the Ca2⫹ transient kinetics
remaining unchanged (608). The gain in inotropy was
ascribed to an increased SR Ca2⫹ load that occurred
without detectable changes in the expression of several
key Ca2⫹ handling proteins (608). The increased SR Ca2⫹
content could be due to several factors including S100A1’s
interaction with RyR to decrease Ca2⫹ leak (927) and the
enhanced Ca2⫹ uptake by SERCA2a when S100A1 is overexpressed (605). Target proteins of S100A1 have been
reported to include titin (986), SERCA2a (435, 436), and
RyR (435, 436, 608, 895, 927), which are all molecular
elements that regulate intracellular Ca2⫹ homeostasis
and/or cardiac myocyte contractility. Interestingly,
S100A1 knockout mice appear to have normal cardiac
function under basal conditions but have impaired cardiac contractility when challenged with hemodynamic
stressors like ␤-adrenergic stimulation (194). Infarcted
S100A1 knockout mice also have a hastened transition to
heart failure, low survivability, and significant loss in
contractile function relative to infarcted wild-type mice
and nontransgenic littermates (609). In contrast, S100A1
overexpressing mice have improved mortality, normal
cardiac function, and protection from hypertrophic remodeling after infarct (609). Taken together, these complementary genetic models highlight a potential role for
S100A1 in the compromised heart and in the contractile
responsiveness to ␤-adrenergic signaling.
S100A1 is considered an attractive prospect for heart
failure gene therapy because of its positive inotropic qualities under baseline conditions. As S100A1 protein levels
are substantially reduced during heart failure, S100A1’s
capabilities as a therapeutic transgene have been studied
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The functional role of PKA-dependent posttranslational modification of sorcin is still unclear. Valdivia et al.
(912) suggest that sorcin itself might be influenced by
␤-adrenergic stimuli, since PKA phosphorylation of sorcin
diminishes its inhibitory effect on RyRs (508). In contrast,
Frank et al. (242) used ␤-adrenergic agonists and blockade with acute sorcin overexpression in rat cardiac myocytes and found that sorcin-mediated gains in contractile
function occurred independent of ␤-adrenergic stimulation. These data suggest that unmodified sorcin may be
regulating RyR function. Interestingly, PKA-mediated hyperphosphorylation of sorcin was identified in animal
models of heart failure (536). PKA-mediated phosphorylation enhanced sorcin’s Ca2⫹ sensitivity, which in turn
may modulate the translocation of this protein to the SR
during pathophysiological conditions in the heart.
Structural and genetic studies have suggested causality between a naturally occurring sorcin mutant (F112L)
and inherited hypertension and hypertrophic cardiomyopathy (552, 595, 912). Currently, the sorcin F112L mutant
has not been studied using acute gene transfer, but a
transgenic mouse model with 20-fold overexpression of
this mutation has a dilated phenotype (131). Surprisingly,
isolated cardiac myocytes from sorcin F122L mice have
increased SR Ca2⫹ load, which enhanced both Ca2⫹ transient and shortening amplitudes (131). These results are
opposite to the results obtained with similar levels of
wild-type sorcin overexpression (560). The gene dosage
effects of sorcin F112L on cardiac function remain unclear and require further experiments to determine the
primary dosage effects of sorcin variants on cardiac myocyte physiology.
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shown in vitro and in vivo that the induction of S100B
expression is a compensatory response to myocardial
stress (901, 902). They demonstrated that the expression
of S100B limited norepinephrine-induced cardiac hypertrophy structurally and genetically. It appears that S100B
expression could be a component of the myocyte response to trophic stimulation that serves as a negativefeedback mechanism to limit cellular growth and associated alterations in gene expression. It is still unclear,
however, whether S100B expression contributes directly
to the pathogenesis of the failing heart.
Unlike S100B, S100A6 protein (calcyclin) is also normally expressed in cardiac muscle as well as in a wide
distribution of tissues (463). S100A6 expression in the
heart is upregulated in rodent infarct models and in neonatal myocytes that are exposed to hypertrophic agents
(901). The precise function of S100A6 in the adult heart,
however, is unclear at present and could be readily studied using gene transfer strategies.
IV. SARCOMERIC TARGETS AND TEMPLATES
The sarcomere is the primary functional unit of striated muscle (Figs. 1 and 7). It is arranged as a hexagonal
array of overlapping thick and thin myofilaments (575).
The thin filament (Fig. 7) consists of 1-␮m-long filaments
of polymerized actin monomers, along with troponin (Tn)
FIG. 7. Three-dimensional schematic of the cardiac sarcomere. Thin filaments of polymerized actin monomers and regulatory proteins troponin
and tropomyosin form a hexagonal lattice surrounding the myosin containing thick filament. Projections from the myosin rod contain the motor
domain which cyclically interacts with actin in an ATP-dependent process of cross-bridge cycling. Inset is a schematic depicting the secondary
structure of troponin subunits cTnC (Ca2⫹ binding subunit, yellow), cTnT (tropomyosin binding subunit, purple), and cTnI (inhibitory subunit, red).
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in the failing rodent myocardium both in vitro (606) and in
vivo (606, 697, 698) using either adenoviral or adenoassociated (AAV, serotype 6) viral gene delivery. These
studies utilized a cryothermic approach to induce myocardial damage leading to progressive heart failure over a
12-wk time span. In vitro adenoviral S100A1 delivery to
isolated failing cardiac myocytes returned S100A1 expression to nonfailing myocyte levels and significantly enhanced failing myocyte contractility, Ca2⫹ transient amplitude, and SR Ca2⫹ content (606). S100A1 delivery to
failing cardiac myocytes in vitro partially restored
SERCA2a ATPase activity, decreased diastolic Ca2⫹ leak,
and returned intracellular [Na⫹] back to nonfailing cardiac myocyte concentrations (606). After infarct, in vivo
S100A1 delivery by adenovirus (606, 698) and AAV (697)
had comparable rescuing effects on both organ level and
myocyte contractility under baseline conditions and during ␤-adrenergic stimulation despite having inhomogeneous expression across the myocardium. Additionally,
increased S100A1 expression after infarct reversed
changes in gene expression classically observed during
heart failure (606, 697, 698). Collectively, these reports
suggest that S100A1 gene transfer could correct aberrant
Ca2⫹ cycling and contractility common to heart failure,
thereby improving the performance of the diseased heart.
S100B expression, which is normally undetectable in
healthy myocardium, is induced in the diseased heart
(419, 675, 902). Tsoporis and co-workers (901, 902) have
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A. Protein Turnover and Stoichiometry
The human heart faithfully generates 3 billion contractile cycles over an average life span. During this time,
the heart must regularly replace “old” sarcomeric proteins
with new ones (Fig. 8). It is extraordinary that mechanisms are in place to accomplish high-fidelity myofilament
replacement while preserving sarcomeric stoichiometry
and while maintaining full cardiac functionality. The sarcomere is a highly ordered three-dimensional lattice, and
in the living adult cardiac myocyte this complex architecture has to be maintained as the myocyte contracts. In
this context, sarcomere maintenance reflects a state of
dynamic equilibrium in which newly synthesized myofilament proteins incorporate into the contractile apparatus
as the old myofilament proteins are replaced (Fig. 8).
Insights into the process of myofilament protein dynamics
have been achieved using multiple approaches. One approach has been to study myofilament assembly during
development (27, 203, 205). These seminal studies uncovered the mechanistic basis for the de novo synthesis of
myofibrils or what has been termed myofibrillogenesis.
With the use of cultured embryonic cardiac myocytes and
high-resolution imaging techniques, the formation of nascent myofibrils could be temporally and spatially visualized. Several studies used isoform-specific antibodies (27,
180, 495, 741, 942), transfection (150), and/or microinjection of recombinant fluorophore-tagged myofilament proteins to observe the time course of nascent myofibril
formation and the highly ordered assembly of new myofilament proteins in the developing sarcomere (788, 789,
929). These studies and others also documented the precise regulation of myofilament protein gene expression
Physiol Rev • VOL
FIG. 8. Model of myofilament turnover and the principles of sarcomeric gene transfer. A: the sarcomere is a three-dimensional lattice with
highly order architecture that is maintained as the myocyte contracts
and generates force. Sarcomeric maintenance is modeled as a dynamic
equilibrium in which “old” myofilaments turnover and are replaced by
the precise incorporation of newly synthesized myofilament proteins
into the contractile apparatus (represented by altered colors). This
stoichiometric replacement occurs in the adult cardiac sarcomere
within a short time frame. B: principles of adenoviral-mediated troponin
gene transfer. Viral gene transfer strategies utilize a strong constitutively
active promoter (e.g., CMV) to drive expression of recombinant myofilament protein, in this case troponin. The CMV promoter out-competes
the endogenous myofilament protein message, so as troponin turns over
in a 3-day time window the “old” native troponin (blue circles) is
degraded and replaced with “new” vector-derived troponins (red circles)
in the absence of changes in myofilament stoichiometry. Bottom panels
illustrate that gene transfer of troponin/myofilaments results in a timedependent stoichiometric replacement of old troponin/myofilaments
with new ones.
during myofibrillogenesis (27, 203, 205, 495, 659, 741, 797,
914). Collectively, these studies demonstrated that during
myofibrillogenesis sarcomere assembly is extremely dynamic as it involves orchestrated changes in myofilament
gene expression, cyclical patterns of protein degradation
and synthesis, and new protein incorporation.
De novo synthesis of myofibrils and sarcomere assembly in embryonic striated muscle differs both temporally and spatially from the process of sarcomeric maintenance in the postmitotic adult cardiac myocyte. Sarcomeric maintenance involves the continual replacement of
old or damaged sarcomeric proteins in the midst of the
unceasing cardiac cycle. There are several reports documenting the turnover of myofilament proteins. Martin
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and tropomyosin (Tm) (282, 888). The thick filament (Fig.
7) consists of the molecular motor myosin (MyHC), its
regulatory and essential light chains, and other accessory
proteins (i.e., titin and myosin binding protein C) (725).
Cardiac muscle contraction occurs when chemical energy
in the form of ATP is directly converted into mechanical
work, such that actin and myosin interact to generate
force and movement via cross-bridge cycling. The forcegenerating capacity of the sarcomere is ultimately responsible for myocardial performance. This process is regulated by the thin filament regulatory machinery, consisting of Tn and Tm, and the activating ligand Ca2⫹ (282,
888). The highly orchestrated events of Ca2⫹ binding to
Tn during systole permits cross-bridge cycling and thus
cardiac contraction (Fig. 5). As cytosolic Ca2⫹ is lowered,
the thin filament regulatory system sterically blocks
cross-bridge cycling, which results in cardiac relaxation
(282, 888). Owing to the sarcomere’s central role in cardiac performance, it has been an attractive target for
genetic engineering by gene transfer technology.
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stood; however, structural constraints and free energy
barriers are likely influential variables. The mechanism
of Tn turnover as a unit also remains in question. One
hypothesis is that Tn stays bound to actin as newly
synthesized subunits replace endogenous components.
Alternatively, it is possible that the entire “old” Tn
complex dissociates, reassembles with new Tn constituents (i.e., TnI, TnT, and TnC), and then reattaches to
actin-Tm. Studies of sarcomeric maintenance with
acute gene transfer have been performed primarily in
rodent models, but more recently the overexpression of
sarcomeric genes and stoichiometric replacement in
larger mammals including rabbit (355, 786) and human
(160) has now been demonstrated.
The conservation of myofilament protein stoichiometry in adult striated muscle highlights sarcomeric proteins as unique targets for genetic manipulation. Sarcomeric proteins manipulated by acute gene transfer include isoforms, mutants, and chimeric Tm, TnT, and TnI
(564, 565, 764, 956 –967) as well as MyHC (355). A model
system of replacement, as demonstrated by several cardiac myofilament proteins, is well suited for elucidating
the fundamental contributions of the myofilaments to
cardiac muscle physiology using genetic engineering
strategies. With the advances in gene transfer technology,
genetic manipulation of myofilament proteins has been
used to study function both in vitro and in vivo. Two
genetic approaches, transgenic animals and gene transfer to isolated adult cardiac myocytes, have served as
the predominant methods for studying myofilament
function in intact myocytes. Both model systems offer
unique and complementary opportunities for advancement in understanding cardiac muscle function. Transgenic animal models have been critically important for
understanding the role of sarcomeric proteins in myocardial function under normal and diseased conditions
in vivo, and these models and functional outcomes have
been extensively reviewed (151, 400, 744, 745, 999).
The strategy governing adenoviral gene transfer of
myofilament proteins to isolated adult cardiac myocytes involves a competition between endogenous and
vector-derived gene expression. Gene transfer utilizes
the host cell’s own transcriptional/translational machinery to express and incorporate the engineered gene
product into the sarcomere (Fig. 2). As the native myofilament proteins turnover, they are replaced one-toone by vector-derived myofilament proteins, resulting
in a time-dependent and progressive increase in the
percent replacement of native myofilament protein
(Fig. 8B). For specific sarcomeric proteins, near full
replacement can be achieved by 6 –7 days in primary
culture, particularly for those myofilament proteins
with shorter half-lives.
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et al. (532) used radiolabeled leucine to estimate the
turnover rates of myofilament proteins in the rodent heart
in vivo. The half-lives of the myofilament proteins varied
from 3 to 10 days, with troponin I (TnI) and troponin T
(TnT) ⬃3.2–3.5 days, troponin C (TnC) ⬃5.3 days, Tm
⬃5.5 days, myosin heavy chain (MyHC) ⬃5.5 days, actin
⬃10 days, and the myosin light chains (MLC) ⬃7–9 days
(Fig. 8A). Studies using microinjection of recombinant
epitope-tagged myofilament proteins in adult cardiac
myocytes or permeabilized dedifferentiated muscle fibers
soaked in labeled sarcomeric proteins further demonstrated that exogenous myofilament proteins can rapidly
(on a minute time scale) incorporate and localize appropriately into the sarcomere (183, 388, 511, 788). More
recent studies using gene transfer strategies that rely on
the myocyte’s native transcriptional/translational machinery have been highly instructive in ascertaining the mechanisms of sarcomeric maintenance in adult cardiac myocytes. Michele et al. (565) used adenoviral gene transfer
of recombinant epitope-tagged tropomyosin (␣Tm) and
cardiac troponin I (cTnI) into isolated adult rat cardiac
myocytes and high-resolution confocal imaging to examine sarcomere assembly in a physiologically intact
adult cardiac myocyte. They found that newly synthesized epitope-tagged thin filament proteins (cTnI and
␣Tm) stoichiometrically incorporate into the adult sarcomere in a time-dependent manner (565). The timedependent stoichiometric replacement revealed itself
through the increase in epitope-tagged ␣Tm and cTnI
with a concomitant decrease in the targeted native
sarcomeric protein, leaving total thin filament protein
expression unchanged as quantified by Western blot
and confocal microscopy. This result was interpreted
as the native “old” thin filament protein turning over
and being replaced by the newly synthesized ␣Tm and
cTnI (Fig. 8B). Notably, the rate of incorporation correlated to the previously determined biochemical halflives of the thin filament (532). This study also demonstrated that ␣Tm has an ordered stoichiometric replacement, where newly synthesized Tms have targeted
replacement at the pointed end of the thin filament
(565). In contrast, TnI replacement was stochastic
(565). These results, in conjunction with previous work
(532, 533), suggested a model of Tm and Tn maintenance in which newly synthesized Tms and TnIs incorporate into the thin filament in an ordered (Tm) and
stochastic (TnI) fashion, directly replacing the “old”
Tms/Tns (Fig. 8B) (565). Transgenic mouse models corroborate this model of stoichiometric myofilament replacement as transgenic animals also maintain myofilament protein stoichiometry despite an overabundance
of sarcomeric transgene transcripts (151, 258, 403, 567,
621, 735, 872, 874, 903, 999).
The mechanism that governs ordered stoichiometric replacement in the sarcomere is not well under-
B. Thin Filament Proteins, Isoforms, Mutants,
and Chimeras
1. Cardiac TnI
Cardiac TnI was the first myofilament protein targeted by acute gene transfer technology. The recent crystal structure determination in conjunction with NMR, biochemical, and functional studies has modeled cTnI as a
molecular switch within the sarcomere (Fig. 7) that regulates Ca2⫹-dependent cross-bridge cycling (484, 556, 830,
867). During diastole, myocyte intracellular Ca2⫹ is low
and cTnI binds tightly to actin, inhibiting strong actinmyosin interactions. The elevation of intracellular Ca2⫹
initiates systole by weakening the cTnI-actin interaction,
causing a conformational change in cTnI that promotes
strong cTnI-cTnC interaction and in turn permits active
cross-bridge cycling.
Cardiac TnI (cTnI) plays a fundamental role in cardiac function under both physiological and pathophysiological conditions. cTnI is also a prominent player in the
heart’s developmental transition from an embryonic
stage, where the dominant TnI isoform expressed is slow
skeletal troponin I (ssTnI), to the adult heart where the
predominant isoform is cTnI (729, 774). During physiological stress, cTnI can also be posttranslationally modified
in response to ␤-adrenergic signaling and changes in pH
(556). Pathophysiologically, cTnI has been implicated as a
dominant player in both acquired and inherited cardiomyopathies (278, 279, 638), and it has been identified as a site
for proteolytic cleavage resulting in an acute loss of function known as myocardial stunning (617).
2. Gene transfer of TnI isoforms
Westfall et al. (966) first determined the direct functional effects of two TnI isoforms expressed in the heart
using adenoviral gene transfer of ssTnI into isolated adult
cardiac myocytes. With near full replacement of native
cTnI by the ssTnI isoform, there was a concomitant gain
in myofilament Ca2⫹ sensitivity of tension and altered
cooperativity demonstrating that TnI isoforms directly
influence sarcomeric function. Cardiac myocytes transduced with ssTnI also showed a gain in function during a
bout of acute acidosis (pH 6.2) where myofilament Ca2⫹
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sensitivity of tension was preserved in ssTnI myocytes
relative to the marked drop in sensitivity sustained by
control cTnI myocytes. In confirmation, an ssTnI transgenic mouse model also demonstrated a gain in myofilament Ca2⫹ and pH sensitivity that resulted in delayed
relaxation and Ca2⫹ transient decay rates in unloaded
isolated myocytes (228). At the organ level, expression of
the ssTnI isoform in the adult heart caused diastolic dysfunction (228).
The TnI isoform specific differences in Ca2⫹ and pH
sensitivity suggested there were unique domains within
each TnI isoform influencing sarcomeric function. To establish the relative contributions of TnI isoform specific
domains on myofilament function, chimeras of the cTnI
and ssTnI isoforms were engineered and expressed via
adenoviral gene transfer in adult cardiac myocytes (956,
957). The N-slow/card-C chimera was developed by engineering 68 amino acids from the NH2 terminus of ssTnI
onto the 110 COOH-terminal domain of cTnI. The other
chimera, N-card/slow-C, was designed by joining the first
100 amino acids of cTnI with the last 120 amino acids of
ssTnI. Gene transfer of N-slow/card-C chimera desensitized the myofilaments to Ca2⫹ in tension-pCa assays. In
contrast, N-card/slow-C myocytes had a dramatic increase in myofilament Ca2⫹ sensitivity of tension. With
these TnI variants expressed at near full replacement, the
following myofilament Ca2⫹ sensitivity of tension hierarchy was established: N-slow/card-C ⬍ cTnI ⬍ ssTnI ⬍⬍
N-card/slow-C. Chimeric TnI studies revealed the importance of TnI’s COOH-terminal domain to myofilament
function and unexpectedly showed an additive effect of
cTnI’s unique NH2 terminal (N-card/slow-C chimera) beyond that of ssTnI alone. Additionally, cTnI’s NH2-terminal domain proved to be critical to normal cTnI function
as the N-slow/card-C chimera caused a loss of function.
Together, these results showed that the NH2-terminal domain of cTnI is an additional and important contributor to
myofilament Ca2⫹ sensitivity, a result that was not predicted in earlier biochemical studies (721, 896, 915).
Complementary ssTnI and TnI chimera gene transfer
approaches underscore TnI’s prominent role in affecting
both myofilament Ca2⫹ and pH sensitivity. Tension-pCa
measurements at pH 6.2 showed a loss of Ca2⫹-activated
tension generation in N-slow/card-C myocytes similar to
wild-type cardiac myocytes. In contrast, N-card/slow-C
myocytes had a tempered response to acidosis similar to
ssTnI myocytes. These data show that the COOH-terminal
region of TnI contains the pH-sensitive domain that induces a TnI isoform-dependent change in myofilament
Ca2⫹ sensitivity during acidosis.
Gene transfer of ssTnI variants with targeted amino
acid substitutions to adult cardiac myocytes identified a
single amino acid that fully converted the pH sensitivity
observed with ssTnI to the cTnI phenotype (160, 959,
961). Acute gene transfer experiments showed that stoi-
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The use of acute genetic engineering is an attractive
approach for studying the role of thin filament proteins on
cardiac myocyte structure and function. Several thin filament proteins have been studied via targeted acute gene
transfer technology. Mouse transgenesis and biochemical
reconstitution protocols have also tremendously improved our understanding of thin filament regulation (278,
279, 871, 999), but this section will focus on key observations and insights gained from acute genetic engineering
of the thin filament by gene transfer.
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topathology and myocyte disarray (403, 786). Both ssTnI
and R146G mutant cTnI caused similar gains in myofilament Ca2⫹ sensitivity, but at the organ level these two TnI
alleles had very different effects on cardiac morphology
and life span. The mechanistic basis for these divergent
outcomes, in lieu of similar gains in Ca2⫹ sensitivity,
remains unknown. Gene transfer of R146G cTnI to adult
rat cardiac myocytes showed that this HCM linked mutant
cTnI behaves quite differently from ssTnI or A164H in the
context of the intact myocyte. R146G mutant cTnI had
poor incorporation efficiency into the sarcomere relative
to wild-type cTnI, cTnI flag, and ssTnI (960). R146G cTnI
also yielded a lower total replacement (⬃45%), but it still
caused a gain in myofilament Ca2⫹ sensitivity of tension at
neutral pH. When the analogous mutation was engineered
into the ssTnI backbone (R115G), Ca2⫹-activated tension
was similar to R146G cTnI and ssTnI. Thus the R115G
ssTnI mutation did not additively increase myofilament
Ca2⫹ sensitivity beyond that of ssTnI alone, suggesting
that the R146G and ssTnI may affect Ca2⫹ sensitivity via
the same mechanism. Tension-pCa relationships were
measured during acidosis (pH 6.2) and showed that, unlike ssTnI, the R146G mutant cTnI and R115G ssTnI responded similarly without having a protective functional
effect during acidosis. R115G ssTnI attenuated the protective pH effect of ssTnI, making it more similar to
R146G cTnI mutant. Collectively, these results suggest
that a combination of increased Ca2⫹ sensitivity in conjunction with increased pH sensitivity may be responsible
for the overt organ level histopathology associated with
some HCM cases but not seen in other Ca2⫹-sensitive
models.
The R146G cTnI mutation is only one of a number of
cardiomyopathy-linked mutations identified in cTnI
which now total over 20 (278, 279, 638). Recently, six new
mutations in cTnI were identified and linked to a highly
malignant and clinically distinct disease entity, restrictive
cardiomyopathy (RCM) (590). The six identified RCMlinked mutant TNNI3 alleles result in the following amino
acid substitutions L145Q, R146W, A172T, K179E, D191G,
and R193H, which occupy several of cTnI’s critical functional domains. Interestingly, mutations at cTnI codon
R146 can result in HCM (R146G or R146Q) or RCM
(R146W). These shared domains suggest that the location
of the mutation is important to the functional outcome,
but they do not address the phenotypic divergence seen at
the organ level. Biochemical reconstitution studies
showed that RCM-linked mutations in cTnI hypersensitize
the myofilaments to Ca2⫹ relative to the already sensitized
HCM counterparts (277, 445, 998). Acute gene transfer
experiments demonstrated that RCM-linked mutant
R193H cTnI myocytes are hypersensitive to Ca2⫹ (158).
Unexpectedly, R193H mutant cTnI also disinhibited the
thin filament at resting Ca2⫹ concentrations and caused
an acute cellular remodeling from normal rod-shaped
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chiometric replacement of native cTnI with a ssTnI variant (ssTnIQAE: R125Q, H132A, V134E) converted ssTnI to
the cTnI phenotype during Ca2⫹-activated tension measurements at neutral and acidic pH in permeabilized myocytes (959, 961, 964). A single codon substitution,
ssTnIH132A, fully converted ssTnI to the cTnI phenotype
(160, 959, 961). In subsequent studies using biochemical
exchange, Dargis et al. (155) reported results from the
converse experiment where His was substituted for an
Ala at codon 162 in human cTnI sequence. This substitution caused a conversion of the cTnI acidic phenotype to
that of ssTnI in ATPase assays. This result was also
demonstrated by targeted adenoviral gene transfer of the
A164H rat cTnI variant in intact adult cardiac myocytes
(160). Not detected in solution biochemical studies (155)
was the gain in Ca2⫹-activated tension at neutral pH that
was observed in A164H myocytes from both acute gene
transfer and the A164H transgenic mouse model (160).
These results highlight the importance of studying function
through multiple approaches and in the context of the intact
adult cardiac myocyte.
Gene transfer of the cTnI A164H variant was also
shown to correct contractile dysfunction associated with
isolated failing human myocytes, raising the prospect that
this phenotype conversion may have protective effects in
chronic heart failure. A164H transgenic mice showed that
a single amino acid substitutions in cTnI can dramatically
alter function, as these mice achieved better myocardial
performance with greater energetic economy during
bouts of ischemia and postmyocardial infarction (160).
Collectively, these studies show that a unique histidine
residue in ssTnI is responsible for TnI isoform differences
in myofilament Ca2⫹ and pH sensitivity. Complementary
gene transfer approaches have aided in understanding
isoform-specific functional outcomes, and they highlight
the mechanistic influence of sarcomeric molecules on
myocardial performance under physiological and pathophysiological conditions (964).
Acute gene transfer with ssTnI, TnI chimeras, and the
A164H variant of cTnI demonstrate the functional importance of cTnI’s COOH-terminal region in determining cardiac myofilament Ca2⫹ sensitivity. The A164H transgenic
mouse did not show histopathology or myocyte disarray
at the organ level (160, 228). This is interesting given that
other Ca2⫹-sensitizing TnI molecules can cause disease,
including inherited HCM. Several publications have linked
HCM alleles to mutant sarcomeric gene products that act
as dominant alleles by causing a gain of myofilament Ca2⫹
sensitivity (258, 278, 279, 403, 811, 871, 960). Mutations in
the cTnI gene, TNNI3, cosegregated with clinical cases of
inherited cardiomyopathy. The HCM-linked cTnI mutation R146G increased Ca2⫹-activated tension generation
in detergent-skinned papillary muscles from the R146G
transgenic mice and rabbits (403, 786). Unlike A164H
mice, the R146G transgenic animals developed overt his-
3. TnI phosphorylation by PKA
Studies using gene transfer of TnI isoforms and chimeras constructed from the fetal, slow skeletal isoform
and the adult, cardiac isoform provided insight into the
functional TnI domains that contribute to the decrease in
myofilament Ca2⫹ sensitivity observed with PKA phosphorylation (967). PKA phosphorylates the Ser-23/24 cluster, which is located within the 32-amino acid extension
of cTnI, but gene transfer studies demonstrated that it
does not phosphorylate ssTnI (967). In permeabilized
myocytes, PKA significantly decreases myofilament Ca2⫹
sensitivity of tension (967). However, expression of ssTnI
or a TnI chimera lacking the 32-amino acid cTnI extension
prevented this rightward shift in Ca2⫹ sensitivity. Phosphorylation of the TnI chimera with the 32-amino acid
extension and the COOH terminus of ssTnI resulted in a
shift that was comparable to cTnI. Together, these results
indicated that the functional domain responsible for the
phosphorylation-dependent Ca2⫹ shift is located within
the NH2 terminus of cTnI. This conclusion is further
supported by a recent study that used both viral-based
gene transfer and transgenic mouse models in which tandem serine codons (23/24) in cTnI’s NH2 terminus were
converted to aspartic acid residues to mimic PKA-mediated cTnI phosphorylation. Myocytes from both genetic
models had decreased myofilament Ca2⫹ sensitivity and
significantly faster relaxation times relative to controls,
while the addition of isoproterenol only minimally hastened relaxation beyond that of the cTnI phosphomimetic
(993). This study underscores cTnI’s key contribution to
cardiac myocyte relaxation during ␤-adrenergic stimulation.
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4. Cardiac TnT
As a component of the troponin complex, TnT holds
both a structural and functional role in Ca2⫹-mediated
regulation of cross-bridge cycling (Fig. 7). The TnT molecule can be separated into two distinct functional domains: the NH2 terminus, which is responsible for anchoring the entire troponin complex to tropomyosin and mediating the cooperative thin filament transitions, while the
COOH terminus plays an important role in the Ca2⫹dependent interactions between TnI and TnC (282,
444).
Cardiac TnT (cTnT) was the second thin filament
regulatory protein to be manipulated by gene transfer
technology. The impetus for studying cTnT with a gene
transfer strategy was likely motivated by the percentage
(⬃15%) of reported inherited cardiomyopathy cases that
have been attributed to mutations in the gene that encodes for cTnT, TnnT2 (946). Gene transfer strategies
have been critical for understanding cTnT’s role both
physiologically and during the HCM pathogenic processes
(528, 765, 855, 947). The first acute gene transfer study
utilized Ca2⫹ phosphate transfection of embryonic quail
skeletal myotubes to express recombinant wild-type cTnT
and several HCM-linked mutant cTnTs: I79N, R92Q,
⌬E160, and a truncated cTnT resulting from a premature
splice donor site in intron 15 (855, 947). This model
system had high transfection efficiency, but at least 10% of
the myotube’s sarcomeres were structurally dysfunctional, which could result from the dynamic nature of
avian myotubes in culture. Functionally, all cTnT mutants
decreased maximum Ca2⫹-activated tension and tended
to desensitize the myofilaments to Ca2⫹ in tension-pCa
assays (855, 947).
Adenoviral gene transfer of these cTnT mutants into
stable, intact, adult cardiac myocytes was also used to
study HCM-linked mutations (528, 764, 765). Marian et al.
(528) transduced feline cardiac myocytes with the human
cTnT mutant R92Q and showed a dose-dependent loss of
contractile function starting 48 h after gene transfer. This
group also performed direct injection of these recombinant adenoviruses into adult rabbit hearts. Due to the
wide range in transduction efficiency (2– 60%), they were
unable to show any myofibrillar morphology changes and
thus did not examine function. Rust et al. (764) transduced isolated adult rat cardiac myocytes with recombinant adenoviral vectors harboring TnT mutants R92Q and
I79N mutations. These HCM-linked mutations caused a
significant desensitization of the myofilaments to Ca2⫹ in
the absence of a concomitant change in maximum tension
generation. Acute gene transfer results (764) are in apparent conflict with those obtained from quail myotube system (855, 947). Adult cardiac myocyte primary culture and
avian embryonic skeletal myotubes are very different cellular systems in terms of the dynamic nature and sarco-
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myocyte morphology to a “short-squat” phenotype. This
acute cellular remodeling occurred with the progressive
stoichiometric incorporation of RCM-linked mutant cTnI
into the cardiac sarcomere and could not be explained by
altered Ca2⫹ sensitivity alone.
The 193 codon in cTnI is not only the site of an
RCM-linked de novo point mutation but also a site of
proteolytic cleavage during myocardial infarction (MI).
MI can cause an acute but reversible disease state called
myocardial stunning. The pathogenesis of myocardial
stunning still remains elusive. It has been investigated
with a transgenic mouse model (618) and biochemical
reconstitution studies (239) but with conflicting results.
The “stunned” transgenic mouse model had a desensitization of the myofilaments to Ca2⫹-activated tension (618),
while reconstitution studies found the opposite effect on
ATPase activity (239). These divergent results highlight
the importance of delineating the primary versus secondary effects of the stunned cTnI variant for understanding
the direct effects of truncated cTnI on myocyte function.
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5. Cardiac TnC
TnC is the Ca2⫹ sensor of the thin filament regulatory
system (Fig. 7). The crystal structure of TnC in both the
Ca2⫹-bound and unbound state has been elucidated (224,
282, 888), and the atomic structure of Tn’s core domain
has recently been published (867). The crystal structure in
addition to NMR and fluorescence resonance energy
transfer (FRET) has identified TnC as a dumbbell-shaped
protein that contains a central ␣-helical core with a globular domain on either end. There are two isoforms of TnC,
cardiac or slow TnC (cTnC) and fast skeletal TnC (sTnC),
which are the products of two different genes, TnnC1 and
TnnC2. TnC is highly conserved across isoforms and
vertebrate species (⬎70% similarity). Cardiac/slow TnC
(cTnC) is exclusively expressed in cardiac and slow skeletal muscles while sTnC is expressed exclusively in fast
skeletal muscles. Structurally, cTnC and sTnC differ at the
low-affinity NH2-terminal Ca2⫹ binding sites (I/II). Three
divalent cation binding sites are dispersed throughout the
globular domains of cTnC with one low-affinity binding
site (II) in the NH2 terminus and two high-affinity Ca2⫹/
Mg2⫹ binding sites (III/IV) in the COOH-terminal region.
At rest, when cytosolic [Ca2⫹] is low, Mg2⫹ binds the
high-affinity divalent cation sites III/IV until Mg2⫹ becomes displaced by Ca2⫹ during the systolic rise in intracellular Ca2⫹. It is the binding of Ca2⫹ to site II, however,
that initiates the cascade of structural events permissive
of cross-bridge cycling (282, 444, 888).
Most of what is known about TnC isoform structure
and function has come from solution biochemistry and
replacement studies (224, 282, 888). In 1988, the first
cloning and mutagenesis of avian sTnC cDNA was used by
Reinach and Karlsson to examine structure function relationships in sTnC (728). This opened the door for further
mutagenesis studies in both sTnC and cTnC from differPhysiol Rev • VOL
ent species to elucidate the mechanism of Ca2⫹-induced
regulation of contraction (28, 29, 286, 483, 715, 815, 816).
Currently, there are no published reports describing the
use of acute genetic engineering for understanding TnC’s
role in cardiac physiology. Work by Edwards et al. (202a)
demonstrated that adenoviral-mediated acute gene transfer of TnC variants to isolated adult cardiac myocytes
caused direct changes in cardiac myocyte function. This
study sets the stage for using gene transfer as an effective
method for understanding TnC’s physiological role in cardiac muscle. Mutations in cTnC have been linked to both
HCM and DCM (56, 364, 591, 711, 802). Critical to understanding the pathogenic process of HCM and DCM is
determining the primary effects of disease-linked mutant
TnCs on cardiac muscle physiology, an aim that would
benefit from acute genetic engineering technology. One
potential limitation is the slower turnover rate of cTnC
(⬃5 days) versus cTnI or cTnT (⬃3 days) (532). This
could constrain experimental outcomes due to limits in
the amount of exogenous TnC replacement that can be
achieved within the time frame of myocyte viability in
serum-free primary culture. Nonetheless, elucidating the
role of TnC in an intact myocyte and at the myocyte and
whole organ level is still an under researched area that
could be pursued through acute genetic engineering strategies.
6. Tropomyosin
Tropomyosin (Tm) is an extended coiled-coil peptide
that spans seven actin monomers in the thin filament (Fig.
7). Tm interacts with neighboring Tm molecules in a head
to tail fashion, providing stability to overlapping tropomyosins and promoting binding to sarcomeric actin.
Aside from binding actin, ␣Tm is tethered to Tn via TnT.
During diastole, when [Ca2⫹] is low, Tm blocks strong
cross-bridge binding sites on actin. As cytosolic [Ca2⫹]
increases during systole, Tm moves from a closed to an
open position, exposing sites on actin for stereospecific
myosin binding. Thus Tm can be modeled as a relay
switch that promotes strong cross-bridge binding in response to the Ca2⫹ signal (282, 283).
There are four isoforms of Tm (␣, ␤, ␥, and ␦), and
␣Tm is the predominant adult cardiac isoform. There is a
developmental transition from 20% ␤Tm expression in the
embryonic heart to less than ⬃10% in a newborn, and it
becomes almost negligible in the adult myocardium (622).
Tropomyosin can exist as either a homo- or heterodimer
in the adult heart (␣␣, ␣␤, or ␤␤ but is mostly limited to
␣␣ homodimers). Tropomyosin has been the subject of a
range of structure-function studies that utilized elegant
biochemical methodologies (282, 283, 353, 360, 362, 550,
888, 889). A better understanding of Tm’s role under both
physiological and pathophysiological conditions in intact
cardiac myocytes has been gained through the use of gene
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meric components characteristic of skeletal myotubes.
Despite conflicting results, these studies have shown that
disease-linked mutant cTnTs play a direct role in determining the myofilament’s Ca2⫹ sensitivity and contractile
function.
Mutations in the gene that encodes for slow skeletal
muscle TnT, TNNT1, have been linked to nemaline myopathy (276). The pathogenic process associated with
nemaline myopathy (described below) is widely unknown
and is another potential gene transfer target. Diseaselinked single point mutations in TnT can result in a loss or
gain of sarcomeric function showing the relative importance of TnT to the regulation of cross-bridge cycling.
Many TnT mutants have been studied using biochemical
and reconstitution preparations, which goes beyond the
scope of this review but are no less important in understanding TnT’s role in thin filament function (276, 279,
871).
Physiol Rev • VOL
tant difference between acute and long-term genetic strategies and the contribution of background strain effects on
organ-level phenotype. With transgenic models alone, it
would be difficult to differentiate the direct effects of the
E180G mutation in Tm as there were changes in myofilament Ca2⫹ sensitivity, Ca2⫹ handling function, and hypertrophic remodeling. However, coupling acute gene transfer with a transgenic mouse model pinpoints the primary
versus adaptive changes that result from the transgene
expression.
The D175N transgenic mouse model was also engineered (FVB/N strain) producing several lines with varying replacement. To see any changes in working heart
performance or myofilament Ca2⫹ sensitivity, assays had
to be performed on lines with ⬎40% replacement. In
higher replacement lines, the D175N Tm mutation increased myofilament Ca2⫹ sensitivity of tension and
slowed myocardium relaxation times (218, 623). Contrasting the D175N transgenic mouse results with those of
acute gene transfer illustrates the importance of the
amount of replacement achieved by the transgene. Acute
gene transfer of D175N mutant Tm yielded 40% replacement with no change in myofilament Ca2⫹ sensitivity. The
ceiling for percent replacement of native Tm with mutant
Tm is set by the endogenous turnover of myofilament
proteins and the time-dependent viability for myocytes in
serum-free primary culture. Therefore, a transgenic
mouse model in this case permits higher levels of replacement because it is not constrained by primary culture
conditions. The different functional phenotypes reported
for the D175N mouse model and acute gene transfer
myocytes could be explained in part by the heightened
percent Tm replacement reported in the D175N transgenic mouse. This underscores the importance of gene
dosage effects on cardiac function, which is particularly
relevant in inherited cardiomyopathy cases where replacement in human heterozygotes is estimated to be at or
near 50% (811).
Notably, the development of E180G and D175N rat
transgenic models (955) yielded different results from
those described previously in mouse (567, 623) or acute
gene transfer studies (564). Extremely low levels of
E180G or D175N Tm replacement had no effect on myofilament Ca2⫹ sensitivity or relaxation function in E180G
rat myocytes, while D175N Tm desensitized the myofilaments to Ca2⫹ and accelerated relaxation rates (955). The
divergent transgenic rat phenotype could be due to expressing a human ␣Tm sequence in the rat (955), but
Michele et al. (564) used the human ␣Tm cDNA as the
backbone for mutagenesis in the previously described
acute gene transfer studies which closely paralleled the
work done in transgenic mouse models. Alternative explanations are the potential differential contributions of
rat versus mouse genetic modifiers and/or low levels of
replacement obtained in the transgenic rat.
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transfer technology. Over the past 10 years, several studies have successfully used acute genetic engineering to
elucidate the direct effects of diseased linked mutant Tm
on cardiac myocyte function.
Mutations in ␣Tm (TPM1) have been linked to inherited cardiomyopathy and nemaline myopathy (skeletal
muscle ␣Tm gene, TPM3). Michele et al. (564) used acute
adenoviral gene transfer of HCM-linked Tm mutations
D175N, E180G, K70T, and A63V to isolated adult rat cardiac myocytes to directly elucidate the structure-function
effects of these mutant Tms. The incorporation of mutant
Tm closely followed the turnover of native Tm, yielding
40% stoichiometric replacement. Isometric tension-pCa
assays revealed that E180G, K70T, and A63V increased
myofilament Ca2⫹ sensitivity and had no effect on maximal tension generation. The D175N Tm mutant had no
effect on Ca2⫹-activated tension even when myocytes
were exposed to higher viral titers. The heightened Ca2⫹
sensitivity of E180G and A63V ␣Tm mutations slowed
relaxation in unloaded intact myocytes (566). The D175N
and E180G mutations are in a region thought to be important for Tm-TnT interactions (969), while the NH2
terminus mutations K70T and A63V are in a region important for actin binding (360). Despite these distinct regional differences, three of the mutations had similar
effects on myofilament Ca2⫹ sensitivity, suggesting that
these mutations cause similar changes to Tm structure.
K70T, A63V, and E180G are located in regions of the
heptad repeat structure of Tm where charge is critical for
maintaining salt bridge interactions that stabilize the Tm
dimer heptad positions e and g (546, 841). It was postulated that the loss of charge caused by K70T and E180G
would destabilize the coiled-coil Tm structure causing an
increase in myofilament Ca2⫹ sensitivity. Biochemical reconstitution preparations in conjunction with circular dichroism analysis have shown a loss of thermal stability in
Tm NH2 terminus with K70T and A63V mutated Tm, confirming the loss of stability created by these mutations
(353).
The E180G and D175N mutant Tms have been expressed in transgenic mice (567, 623, 706, 707) and more
recently in rats (955). The direct effects of the E180G Tm
mutation in transgenic mice closely paralleled that of
acute gene transfer, but the compensatory changes varied
between E180G transgenic mouse models. Compensatory
changes in Ca2⫹ handling proteins in myocytes from the
E180G-FVB/N strain mouse model contributed to a
marked change in diastolic function (623, 706, 707). Additionally, E180G Tm, when expressed on an FVB/N
mouse genetic background, resulted in marked histopathology and hypertrophy closely mimicking HCM (706). In
contrast, E180G Tm when expressed on C57BL/6 background did not show altered myocyte morphology or any
histopathology (567). These reported compensatory adaptations in E180G transgenic mice underscore the impor-
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7. Actin
Actin is a globular protein that spontaneously polymerizes and forms F-actin, the backbone of the thin filament (Fig. 7). F-actin is visualized as a double-stranded
helical filament and is a structurally polar molecule with
a barbed end at the Z-line and one pointed end at the
middle of the sarcomere (699). F-actin contains strong
and weak myosin binding sites that are exposed when Tm
Physiol Rev • VOL
slides from a blocked/closed to an open position in response to cyclical [Ca2⫹]i fluctuations (282, 283). The
polar arrangement of actin is critical as it directs myosin
heads to slide actin filaments in the appropriate direction,
resulting in myocyte shortening (913).
Much of what is known about actin’s functional role
in cardiac muscle physiology has come from various biophysical and biochemical techniques including X-ray diffraction, cryoelectron microscopy, and in vitro motility
(367, 417, 457, 510, 546, 547, 572–574, 724, 814). The
physiological role of actin isoforms and disease-linked
actin mutants is a field in its infancy. Acute genetic engineering involving actin may be limited by its endogenous
half-life of ⬃10 days. Actin is the slowest myofilament
protein to turnover, which is the biggest hurdle to overcome within the 1-wk window of viable myocytes in serum-free cell culture. This temporal constraint may not
provide ample time for incorporation and a measurable
functional phenotype. Additionally, it is not well known
whether actin will replace and incorporate in the same
manner that has been described for other thin filament
proteins given the tight regulation of its length by capping
proteins and tropomodulin, as well as its interaction with
Z-line proteins.
Further understanding of actin’s role in cardiac muscle function will likely come from assessing the direct
functional effects of diseased-linked mutations in the actin gene. Missense mutations in alpha-cardiac actin gene
(ACTC, nine identified to date) and ␣-skeletal muscle
actin gene (ACTA1) have been linked to cases of DCM,
HCM (589, 592, 665), and NM (386, 387, 654, 656, 657;
described above), respectively. The clinical phenotype
associated with ACTC mutations ranges from asymptomatic to severe cardiac dysfunction. Mutations identified in
actin within Z-line anchoring domains (e.g., R312H and
E361G) are associated with DCM. Other actin mutations
within myosin binding domains (e.g., A331P and A295S)
are associated with HCM. One actin HCM mutation
(E99K) has been evaluated in vitro by a series of biochemical assays, which demonstrated a weakened interaction
with myosin and reduced force production (73). Several
other actin mutations have been expressed in vitro and
found to demonstrate variable defects in protein folding
which correlated with impaired incorporation into the
cytoskeleton of mammalian (noncardiac) cell lines (919).
To date, there are no published studies using acute gene
transfer of cardiac or skeletal muscle actin variants. Unpulished results have shown that four representative cardiac actin HCM and DCM mutations can be expressed in
isolated adult rat cardiac myocytes with efficient sarcomeric incorporation by gene transfer (177a), presenting
proof of principle that acute gene transfer of actin is a
feasible approach for assessing actin’s role in cardiac
muscle function.
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Mutations in Tm alleles are also causal for nemaline
myopathy (NM). NM is considered a clinically heterogeneous group of congenital skeletal muscle myopathies
with a histopathological marker of nemaline rod formation in skeletal muscle fibers. With few exceptions, the
clinical NM phenotype is characterized by generalized
muscle weakness and respiratory insufficiency with early
morbidity (386, 387, 654, 656, 657). Identified NM mutations include the slow skeletal muscle gene TPM3, which
is ⬃90% homologous to the cardiac ␣Tm gene TPM1, as
well as slow skeletal TnT and ␣-skeletal actin. It is notable
that mutations in genes that are nearly identical in both
sequence and protein structure could result in such divergent clinical presentation as seen in HCM versus NM. To
understand the divergent pathogenic processes, Michele
et al. (566) used adenoviral gene transfer of the NM-linked
M9R Tm and HCM-linked A63V mutant Tm to isolated
adult cardiac myocytes. The functional effects of the NM
M9R mutation were indistinguishable from wild-type
myocytes at core body temperature (37°C) (566). The
M9R mutant’s phenotype became apparent at lower temperatures (30°C) that have been reported in vivo temperatures for limb muscles. At these temperatures the M9R
mutant myocytes had increased relaxation speeds relative
to wild-type. This is opposite of the slowed relaxation
measured in the A63V HCM mutant myocytes (566). Biochemical studies showed that the M9R mutant decreases
␣Tm’s affinity for actin with no significant changes in
Ca2⫹-activated ATPase activity relative to wild-type ␣Tm
(362). These results were obtained at room temperature,
which may have affected the Ca2⫹ sensitivity of the myofilaments. In comparison, a transgenic mouse overexpressing the M9R mutant Tm in skeletal muscle resulted
in histopathology and muscle weakness similar to the
human NM phenotype (137). In situ measurements of
force and power from the gastrocnemius muscle of M9R
Tm transgenic mice were indistinguishable from wildtype skeletal muscles (161). Transgenic mouse models of
NM are very difficult to use for extrapolation to the human condition as few murine muscles are considered
purely slow twitch. Furthermore, these studies did not
exclusively examine slow-twitch muscles like the soleus
or measure mutant protein expression, which adds to the
difficulty in extrapolating these results to the human disease condition.
8. Capping proteins and molecular rulers
Actin capping proteins such as CapZ and tropomodulin (Tmod) contribute to the actin monomer dynamics at
the ends of the thin filament (499, 500) and regulate the
length uniformity of actin filaments within the sarcomere
(241). Specifically, CapZ appears to nucleate the developing actin filament at the barbed end within the Z-line
during myocyte fibrillogenesis. The capping of actin by
Tmod at the pointed end is a critical regulator of thin
filament assembly, length regulation, and function (793).
The importance of these capping proteins has been demonstrated by several transgenic knock-out models that
had early morbidity (122, 246, 332), and with overexpression models that had a DCM phenotype (853). The giant
molecular ruler protein nebulin is another important regulator of actin filament length albeit in skeletal muscle
(371, 543). Its role in cardiac muscle is not well understood. Compared with skeletal muscle, in which there are
approximately two nebulin molecules per thin filament,
nebulin is expressed at significantly substoichiometric
levels in cardiac muscle. Despite these low levels, knockdown of nebulin in neonatal rat cardiac myocytes using
RNAi transfection caused dramatic elongation of thin filaments from the pointed end without affecting the barbed
end (544), suggesting a potential role of nebulin in thin
filament assembly.
Given the complexity of actin dynamics and observed effects in transgenic mouse models, maintenance
of thin filament length clearly requires precise regulation.
To date, the precise role of actin capping proteins and
molecular rulers in adult cardiac myofibrillar assembly
and myocyte function are not fully resolved. Much of
what is known about myofibrillar assembly comes from
direct injection or plasmid transfection of highly dynamic
cell culture systems (i.e., chick embryonic myocytes, neonatal myocytes, and serum-induced differentiating myoPhysiol Rev • VOL
cytes) (233, 240, 295). Concerns have been raised about
microinjection techniques as they result in high levels of
physical trauma to cultured myocytes (820). Additionally,
most myofibrillar assembly studies have focused on morphology and histological analysis with very little analysis
of the resulting functional effects of modulating capping
proteins. Studying myofilament assembly and the functional role of capping proteins in the adult cardiac myocyte presents another opportunity for the use of acute
genetic engineering. The major limitation of an acute
genetic engineering approach will again rely on the turnover time of the capping proteins, which is thought to be
fairly long (499). It is well known that the stoichiometry of
capping proteins to actin is critically important (241), and
studies suggest that, unlike other myofilament proteins,
capping proteins can be overexpressed (852). At this time
there has only been one report of the use of adenoviral
gene transfer for studying the role of capping protein,
Tmod, in embryonic cardiac myocytes from chicken
(852). Adenoviral delivery of sense or antisense Tmod to
this culture system was highly efficient, and significant
overexpression/knockdown of the Tmod protein was
seen by 24 h after gene transfer (852). In this setting,
Tmod overexpression yielded shortened thin filaments,
while Tmod downregulation caused longer thin filaments.
This study showed that Tmod is important for maintaining
actin stability and provides a basis for using genetic engineering as an approach for studying capping protein
function.
C. Thick Filament Proteins
The thick filament is predominantly composed of the
molecular motor protein myosin (Fig. 7) that cyclically
interacts with actin to produce force and sarcomere
shortening. Cardiac myosin is a hexamer consisting of
two myosin heavy chains (200 kDa each, discussed
above), two essential light chains (light chain 1, MLC-1),
and two regulatory light chains (light chain 2, MLC-2)
(797). The thick filament also contains other myosin binding proteins such as C, H, X, and M proteins and the large
elastic protein titin. Regulation of cardiac muscle contraction involves complex interactions between Ca2⫹ and
proteins of the thin and thick filaments. While much is
known about the contribution of Ca2⫹ and the thin filament to regulation of contractile function, much less is
known about modulation by thick filament accessory proteins and their posttranslational modification. Acute gene
transfer has not been extensively employed for studying
thick filament proteins, but it offers an intriguing approach for directly assessing the thick filament’s role in
cardiac muscle physiology under normal and diseased
conditions. Notably, the genes that encode for myosin
heavy chain, myosin light chain, and the myosin binding
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Myofilament turnover in the adult myocyte is another
area that might benefit from the use of acute genetic
engineering. Actin thin filament length is highly uniform in
muscle sarcomeres (⬃1 ␮m) (750). Isolated myofibrils are
very stable in vitro, and fluorescently labeled actin monomers can be incorporated only after removal of the capping proteins CapZ or tropomodulin (500). Despite these
observations, in vivo thin filament maintenance is a dynamic process. In living cells, fluorescent actin rapidly
incorporates into thin filaments, and monomer exchange
is relatively quick compared with protein turnover, suggesting that thin filament capping does not restrict monomer addition (499). Injection of fluorescently labeled actin
into thin filaments of living myocytes has been reported
by several groups (183, 270, 336, 545, 817), but the site
(pointed end, barbed end, or both) of actin incorporation
remains debatable (499).
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DAVIS ET AL.
1. Cardiac myosin
Cardiac myosin is the most abundant protein in cardiac muscle and is the primary consumer of cellular energy (ATP). Two structurally and functionally distinct
isoforms of myosin, ␣- and ␤-myosin heavy chain (MyHC),
result from two separate genes (MYH6 and MYH7, respectively) that are differentially expressed in cardiac muscle.
The transcription of each gene is independently controlled but coordinately regulated (313). Cardiac myosin
isoforms can form homodimers or heterodimers and have
been electrophoretically separated leading to the following nomenclature: myosin V1 (␣-␣ homodimer), V2 (␣-␤
heterodimer), and V3 (␤-␤ homodimer) (366, 701).
Despite sharing ⬎90% amino acid homology, cardiac
myosin isoforms are functionally quite distinct. For example, ␤-MyHC, the “slow” molecular motor, hydrolyzes
ATP three to seven times slower than ␣-MyHC (330, 918).
The “fast” motor, ␣-MyHC, represents ⬎90% of the total
myosin expressed in the normal adult rodent heart (235,
356, 553). Expression of the slow ␤-MyHC motor increases relative to ␣-MyHC in rodent models of cardiovascular disease including diabetes (762), hypothyroidism, cardiac hypertrophy (553), and aging (99, 236, 932).
Therefore, increased ␤-MyHC motor expression is one
biomarker of cardiac disease in rodents. The adult cardiac
ventricles of healthy larger mammals have a different
myosin isoform profile where ⬃90% of the total myosin
content consists of ␤-MyHC (26, 284, 498, 585).
In humans, mutations in each cardiac myosin gene
can cause familial cardiomyopathy (100, 226, 811), and
alterations in myosin isoform expression are associated
with heart failure (514, 585, 632). The most commonly
affected sarcomeric protein in inherited cardiomyopathy
patients is ␤-MyHC. In the heart, mutations in the ␤-MyHC
gene MYH7 have been associated with both HCM and
Physiol Rev • VOL
DCM (638, 811). Although the human heart expresses
predominantly ␤-MyHC, mutations of the ␣-MyHC gene
MYH6 have recently been linked to either HCM or DCM
(100). The clinical importance of ␤-MyHC mutations is
further underscored by the finding that ␤-MyHC mutations have been linked to skeletal muscle myopathies
including Laing distal myopathy (554), myosin storage
myopathy (861), and hyaline body myopathy (68). The
clinical importance of cardiac myosin makes it an attractive target for acute gene transfer strategies; however,
only two studies to date have utilized such technology for
studying cardiac myosin structure and function.
The first HCM-causing myosin mutation was discovered in the motor domain of the myosin molecule (258).
This missense mutation results in an arginine to glutamine
substitution at amino acid position 403 (R403Q), which is
located in the actin-binding interface (723). Since the
discovery of this myosin mutation, much effort has been
devoted to determining how it affects myosin motor function and triggers cardiomyopathy (60, 146, 147, 671, 856,
905, 987). Extensive studies performed on a transgenic
mouse model of the R403Q mutation demonstrated delayed cardiac relaxation and slowed chamber filling with
a progressive transition to hypertrophy and myocyte disarray common to HCM (258, 259). In these studies, the
mutation was engineered in the ␣-MyHC gene, which
differs from the human HCM-linked mutation that occurs
in the ␤-MyHC gene (258). The primary effects of the
R403Q substitution in ␤-MyHC on myosin motor and cardiac function are therefore important to determine.
A recombinant adenovirus has been used to study the
structural effects of mutant ␤-MyHC expression on sarcomere assembly and myofibrillar organization (527).
Marian et al. (527) used acute gene transfer to study the
effects of the HCM-associated R403Q mutation of human
MYH7 on cardiac sarcomere structure. This study used
acute gene transfer in adult feline cardiac myocytes in
vitro, which had an efficiency ⬎95%. In the adult feline
cardiac myocytes, expression of mutant and normal
␤-MyHC by viral gene transfer was determined by reverse
transcription-PCR. Electron micrographs and fluorescent
imaging only showed subtle alterations of sarcomere
structure in all experimental groups. The R403Q mutant
resulted in sarcomeric disarray in 50% of the myocytes
that were examined, while no sarcomere disruption was
observed in either control (no virus) or wild-type ␤-MyHC
transduced myocytes. This result was found in both electron micrographs and in experiments using indirect immunofluorescence and fluorescent imaging. The physiological consequence of long-term expression R403Q
␤-MyHC mutation was also examined using a transgenic
rabbit model (526). R403Q transgenic rabbits with ⬃40%
␤-MyHC replacement had significant septal and posterior
wall hypertrophy with myocyte disarray and heightened
collagen content without changes in fractional shortening
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proteins have identified mutant alleles that are causative
for inherited cardiomyopathy. In addition, there are
known changes in myosin and myosin light chain isoform
expression during heart failure (26, 585, 794). Detailed
transgenic and biophysical studies have been performed
on thick filament mutant alleles (151, 258, 456, 723, 724,
905, 999); however, understanding the direct effects of
these mutant proteins on intact cardiac muscle function is
incomplete.
A potential limitation of utilizing acute gene transfer
for modulating the thick filament is the turnover rate of
thick filament proteins. Some of the longest myofilament
half-lives are related to constituents of the thick filament.
The half-life of myosin heavy chain is estimated at ⬃5.5
days, and the light chains are even longer (⬃7–9 days)
(533), which could limit the amount of replacement or
dose-response assessment garnered through acute gene
transfer in serum-free myocyte cultures.
2. The myosin essential (MLC-1/ELC) and regulatory
(MLC-2/ RLC) light chains
Ventricular muscle myosin is comprised of two
unique accessory proteins, the essential (MLC-1) and regulatory (MLC-2) light chains. Both MLCs are bound to the
neck region of myosin where they are thought to modulate myosin motor function. MLC-1 is a 17-kDa protein
product of the MYL3 gene. It contains six functional
domains, an actin binding site, a proline-rich region, and
four helix-loop-helix regions and belongs to the superfamily of EF-hand Ca2⫹ binding proteins (899). The regulatory
light chain, MLC-2, is a 22-kDa protein product of a different gene, MYL2. MLC-2 has several important structural regions including a phosphorylation site on serine-15
and four EF-hand domains (149). Like MLC-1, it also
belongs to the superfamily of EF-hand Ca2⫹ binding proteins. Mutations in the myosin light chains have been
linked to cardiomyopathy (226, 638). Collectively, these
data suggest that both light chains play a critical role in
normal cardiac physiology, particularly in cross-bridge
kinetics.
To date, there are no reports of utilizing acute genetic
engineering for understanding the role of myosin light
chains in cardiac function. However, three different transgenic mouse models that overexpressed either the ventricular isoform of RLC (RLC2v) or ELC (ELC1v) in the
atria or the atrial isoform of ELC (ELC1a) in the ventricle
Physiol Rev • VOL
showed that protein stoichiometry is tightly conserved
despite overexpressing light-chain transcripts (230, 402,
670). Furthermore, multiple transgenic lines have been
generated with varying levels of replacement that ranged
from 0 to 95%. Taken together, these data show that like
other myofilament proteins, MLCs have tightly regulated
stoichiometric replacement and offer titratable gene dosing. Permeabilized fiber studies from these mice showed a
change in cardiac fiber function such that the incorporation of either ELC1v or RLC2v into atrial fibers caused
decreased unloaded shortening velocity (787). Interestingly, myosin light-chain isoform switching, where the
ventricular isoforms convert to the atrial light chains, has
been documented in various human heart failure cases
such as in congestive heart failure or in DCM (26, 601,
794), suggesting important and divergent physiological
roles in cardiac muscle for the ventricular versus atrial
myosin light chains. The precise role of the different
cardiac muscle MLC isoforms in cardiac function is not
fully known. A potential limitation to MLC gene transfer is
the long turnover time of the light chains. The half-lives of
the MLCs have been estimated at 7–9 days, which may
limit the amount of replacement one can achieve with
recombinant light chains delivered to myocytes in primary culture (532, 533). Modest levels of replacement
(⬃30%) in the transgenic mouse model with ECL1a overexpression in the ventricle resulted in a change in function (230), suggesting that the high levels of replacement
may not be necessary to affect cardiac myocyte physiology.
3. Thick filament accessory proteins:
C-protein (MyBP-C)
Myosin binding protein C (MyBP-C) is a myofilament
protein of the intracellular immunoglobulin/ fibronectin
superfamily that is located in the “C-zone” of the A-band
in seven to nine transverse stripes spaced 43 nm apart
(142, 661, 757). Distinct genes encode for the three isoforms of MyBP-C that are expressed in adult mammalian
striated muscle and include fast skeletal, slow skeletal,
and cardiac MyBP-C. MYBP3 is the gene that encodes for
the cardiac isoform (cMyBP-C) (257), and it is expressed
exclusively in cardiac muscle throughout development
(256). Cardiac MyBP-C is organized similarly to the skeletal isoforms with 10 globular domains (C1-C10) and a
highly conserved linker region, the MyBP-C motif (257).
Cardiac MyBP-C has several unique features including an
extra IgI-like NH2-terminal domain (termed C0), phosphorylation sites within the MyBP-C motif, and a 28amino acid insertion within the C5 domain. All isoforms
of MyBP-C interact with the light meromyosin (LMM)
region of the myosin rod, the region that forms the thick
filament backbone (599). The COOH-terminal C10 domain
contains the LMM binding site of all MyBP-C isoforms (14,
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(526). Additionally, R403Q rabbits had markedly reduced
survival rates (526). These results suggest that the primary effect of this mutant myosin is to disrupt sarcomere
assembly and myofilament organization and perhaps is a
constituent in the pathogenic process leading to HCM.
Most studies that have examined the effect of myosin
isoform switching on cardiac muscle function have used
hyper- or hypothyroid animals (235, 356) and transgenic
mice (455, 873) or rabbits (401). It is difficult to determine
the direct effect of myosin isoform shifts in these models
because the phenotype represents the combined effects
of myosin isoform and the compensatory response to the
hormonal or genetic manipulation. Recently, a recombinant adenovirus was used to determine the functional
consequence of increased relative ␤-MyHC expression in
rodent cardiac myocytes (355). ␤-MyHC gene transfer
demonstrated nearly 100% efficiency at the level of the
isolated adult cardiac myocytes. Functionally, adenoviral
gene transfer of ␤-MyHC attenuated sarcomere shortening compared with control myocytes (AdGFP or no viral
treatment) while Ca2⫹ transients were unaltered. These
results suggest that ␤-MyHC expression represents a
Ca2⫹-independent negative inotrope among the cardiac
myofilament proteins and demonstrate the feasibility of
using recombinant adenoviruses to study structure-function relationships of the heart’s molecular motor.
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phospholamban and other Ca2⫹ handling proteins,
cMyBP-C is phosphorylated in response to ␤-adrenergic
stimulation (e.g., isoproterenol) by PKA, thereby suggesting a role for cMyBP-C in the regulation of cardiac muscle
contractility. Studies using gene transfer to investigate the
contribution of posttranslationally modified MyBP-C on
contractile function and/or studies to develop therapeutic
strategies utilizing the phosphorylated versions of this
protein have not been published. However, MyBP-C
knockout and phosphorylation mimetic transgenic mouse
models have been used to examine the role of MyBP-C
phosphorylation as it pertains to the regulation of cardiac
muscle contractility. Knockout models suggest that PKAmediated phosphorylation of MyBP-C contributes to both
the magnitude of desensitization of the myofilaments to
Ca2⫹ (102) and enhanced cross-bridge cycling during
␤-adrenergic stimulation (345, 357, 434). A phosphomimetic transgenic mouse was designed using aspartic acid
substitutions and showed that constitutive phosphorylation of MyBP-C conferred cardioprotection during ischemia-reperfusion injury (772). The precise mechanism
whereby cMyBP-C and its phosphorylation status affect
cardiac contractility and cardioprotective behavior during
ischemia is unclear, but acute genetic engineering of
cMyBP-C may offer a new tool to further uncover the role
of MyBP-C phosphorylation in the fine-tuning of cardiac
muscle contractility.
4. Thick filament accessory proteins: titin
Titin is the largest mammalian protein currently identified. There are several titin isoforms ranging from 2.9 to
3.7 MDa in size that are all splice variants of the same
gene, TTN. The titin gene is made up of 363 exons (33)
and has a cDNA of ⬃82 kb (465). Elegant biophysical
studies have shown titin’s structural and functional role in
cardiac muscle (290). Titin is largely responsible for the
viscoelastic characteristic of striated muscle cells. Structurally, titin plays a key role in the maintenance of thick
filament length and construction of Z and M-lines (296).
Titin connects the Z-line to the M-line and has been modeled as a molecular spring as its structure contains an
extensible region, the Ig-segment (immunoglobulin-like
domain), and PEVK region (rich in proline, glutamate,
valine, and lysine) that generates passive tension as sarcomeres become stretched. The relationship between
passive tension and resting sarcomere length turns hyperbolic beyond that of optimal length. The different titin
isoforms confer distinct variations in passive stiffness as
demonstrated by the cardiac N2B isoform which elicits
higher levels of passive tension per increase in sarcomere
length relative to the N2A isoform due to N2B’s shorter
extensible region (104, 244, 465, 898). The heightened
stiffness of the N2B isoform is thought to promote rapid
return to diastolic filling during the cardiac cycle. Further-
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663). In addition to binding the myosin rod, MyBP-C isoforms also bind to the thick filament region of the massive
structural protein titin (252, 449). The interactions between MyBP-C, myosin, and titin likely contribute to the
highly ordered structure of the sarcomere.
The precise role of cMyBP-C in cardiac function still
remains unclear, but there is evidence suggesting that
MyBP-C contributes to thick filament formation and sarcomere assembly (252, 449) and plays a regulatory role in
cardiac muscle contraction. MyBP-C’s role in cardiac
function is still widely unknown but is suspected to contribute to ␤-adrenergic-mediated gains in function (105,
357, 771, 772, 839, 840), myofilament Ca2⫹ sensitivity (102,
331, 365, 542), and length-dependent changes in myofilament Ca2⫹ sensitivity (105, 358). Additionally, MyBP-C
must play a critical role in normal cardiac function as
20 –30% of inherited HCM cases are attributed to mutations in the gene that encodes for MyBP-C (226, 638). To
date, the role of MyBP-C in cardiac muscle function has
not been studied using acute genetic engineering. Despite
being a fairly sizable gene (⬃21 kb) with 35 exons, the
MyBP-C transcript is ⬃4.5 kb (257) and can be accommodated by common adenoviral vectors (Table 1). Gene
transfer of MyBP-C may be limited by its turnover time,
which at present is unknown, and acute gene transfer may
be further complicated by its stoichiometry. To date, it is
still debatable whether MyBP-C stoichiometry is maintained. This debate is largely fueled by conflicting evidence from both human and transgenic mouse models
that have various levels of MyBP-C expression. For instance, a clinical study reported no detectable levels of
MyBP-C in HCM patients with MyBP-C mutant alleles
(760), suggesting that the mutated MyBP-C is unstable
with a pathogenic mechanism leaning towards one of
haploinsufficiency. A heterozygous MyBP-C null mouse
model also lends some support to the mechanism of
haploinsufficiency as a knockout of one MyBP-C allele
resulted in a slight but significant decrease in total
MyBP-C content (102). In contrast, the development of
transgenic mouse models that have two- to eightfold overexpression of either wild-type MyBP-C or an HCM-linked
truncated MyBP-C demonstrated that MyBP-C stoichiometry is conserved and that the truncated MyBP-C is indeed
stable (990). This result is further supported by ES cellderived transgenic mouse models with various COOHterminal truncations that retained total MyBP-C stoichiometry (542).
The cardiac isoform of MyBP-C is the only isoform
that contains phosphorylation sites. Phosphorylation of
MyBP-C prevents myosin S2 and MyBP-C binding (301).
The phosphorylation state of various C-protein sites alters
thick filament structure and function. Ser-273, Ser-282,
and Ser-302 of cMyBP-C are key substrates for PKA (237,
972), Ca2⫹/calmodulin-dependent kinase CAM kinase;
Ref. 335), and PKC (924). Importantly, in addition to cTnI,
more, titin can be posttranslationally modified and may
directly interact with Ca2⫹ (290). Titin’s large size presents the biggest limitation to overcome when trying to
utilize gene transfer for titin manipulation. Perhaps the
use of titin fragments or truncations for acute gene transfer may be a way of overcoming the packaging size limitations but with the caveat that truncated titins cannot
entirely extrapolate to physiological titin function. Gutted
adenoviral vectors may also be a promising candidate for
titin gene delivery. Furthermore, titin’s turnover time is
unknown and may be rather lengthy, offering an additional limitation for using acute gene transfer in vitro.
Cardiac myocytes have an extensive nonsarcomeric
cytoskeleton network (Fig. 1). This noncontractile cytoskeleton consists of microtubules and lamins supporting the nucleus of the cardiac myocyte (773, 823). Intermediate filaments, primarily desmin, radiate from the
Z-line and interact with neighboring sarcomeres, mitochondria, the nucleus, and the sarcolemma (Fig. 9). Near
the plasma membrane these desmin filaments bind to the
submembranous actin network (679). This submembranous actin lattice serves as an anchoring point for the
mechanical and signaling complexes that form the cos-
tamere (Fig. 9) of cardiac myocytes (784). The costamere
is thought to play a critical role in mechanotransduction
and force transmission (65, 214, 784). Mediating these
functions of the costamere are the proteins associated
with the integrin heterodimer and dystrophin (327, 784).
Cardiac muscle cytoskeletal proteins are critically important for normal physiology and are often affected in disease states (204, 891). For many of these proteins, only a
partial picture of their role in cardiac pathophysiology is
currently available. Despite this incomplete understanding of their function, it is clear that this is an important
group of molecules in the development of heart disease.
The utilization of gene transfer technologies has been an
important factor in understanding cardiac diseases
caused by defective cytoskeletal proteins. Furthermore,
gene transfer has promise as a therapeutic agent for the
treatment of cytoskeletal-based cardiac disease.
A. Dystrophin and Dystrophin-Associated Proteins
Dystrophin and its associated proteins have been
linked to many forms of inherited muscular dystrophy,
several of which present as cardiomyopathies. The nidus
of this protein complex is dystrophin (Fig. 9), a 427-kDa
protein that binds to the submembranous actin lattice and
bridges the gap to the membrane. At the membrane, dystrophin interacts with the integral membrane protein
␤-dystroglycan, which binds to the laminin receptor
␣-dystroglycan, sarcospan, and the sarcoglycan complex
(Fig. 9). This latter group of membrane proteins consists
of ␣-, ␤-, ␥-, and ␦-sarcoglycan. In addition to these interactions with membrane-bound proteins, dystrophin binds
to a variety of adaptor and signaling proteins. The following sections will discuss how gene transfer has been used
to understand and treat diseases resulting from the disruption of the dystrophin glycoprotein complex.
1. Dystrophin
FIG. 9. Schematic of the cardiac cytoskeletal network. The 427-kDa
protein dystrophin links submembranous actin networks which connect
to the sarcomeres via desmin to the transmembrane dystroglycan complex (DGC). The DGC interacts with the laminin-2 receptor and the
extracellular matrix. The sarcoglycan complex and sarcospan are associated with the DGC.
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The dystrophin protein is responsible for Duchenne
and Becker forms of muscular dystrophy (209). In the
more severe Duchenne muscular dystrophy, the dystrophin protein is completely absent from all tissues in the
body, while in the milder Becker muscular dystrophy, a
truncated, but partially functional, dystrophin protein is
expressed in striated muscles. The dystrophin gene spans
2.4 million base pairs of the short arm of the X-chromosome. The resulting 14-kb transcript consists of 79 exons
and produces a protein with 3,685 amino acids and a
molecular mass of 427 kDa, making it one of the largest
known proteins (363). Dystrophin can be divided into four
distinct functional regions (2). The NH2-terminal domain
contains an actin binding region demonstrating homology
with the actin binding proteins ␣-actinin and ␤-spectrin. A
second actin binding domain is located in the central rod
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V. CYTOSKELETAL PROTEINS
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the 5⬘-end of the dystrophin gene (892). These patients
have normal levels of dystrophin expressed in skeletal
muscles, but no dystrophin present in the heart (570, 615,
892). Many of these patients have disruptions in the promoter driving dystrophin expression in muscle. In the
heart, the inactivation of this promoter results in the
absence of dystrophin (37, 38). In contrast, there is an
upregulation of alternative dystrophin isoforms in skeletal muscle which functionally replace the muscle dystrophin (614, 631). In Becker muscular dystrophy (437, 460),
truncated dystrophin molecules resulting from in-frame
mutations are expressed (597). These truncated dystrophin molecules have varying levels of function, which are
not associated solely with the size of the deletion (43).
Information gained from analyzing genotype-phenotype
relationships in Becker muscular dystrophy patients provided critical information for the design of functional
truncated dystrophin molecules that are amenable to
gene transfer.
2. Dystrophin gene transfer
The vast majority of studies utilizing dystrophin gene
transfer have been centered on replacing dystrophin in
skeletal muscle for the treatment of Duchenne muscular
dystrophy. Many of these advances are also critical to
implementing dystrophin gene transfer within the heart.
The most straightforward gene transfer approach is the
direct injection of full-length dystrophin pDNA (Fig. 3).
Unfortunately, this method results in extremely low levels
of dystrophin expression in skeletal muscle (7). Although
inclusion of additional transfection agents may improve
the efficiency of transduction (897, 988), the levels
achieved are likely too low to have physiological/therapeutic significance. The low efficiency of these methods
raises the question of what are therapeutic levels of dystrophin. Studies involving female carriers of mutated dystrophin alleles suggested that levels slightly greater than
50% of normal are protective against skeletal muscle disease (127, 538), although other studies of X-linked DCM
patients report that levels as low as 30% of normal dystrophin expression can prevent significant skeletal muscle disease (637). The therapeutic threshold in the heart is
complicated by the inability to obtain cardiac biopsies to
determine the level of expression and by variability in the
manifestation of cardiac disease in these patients (288,
370, 580, 649).
The disappointing results from direct plasmid injections underscored the need for a more efficient method of
gene delivery. Viral-based gene delivery vectors have been
shown to be the most effective and efficient methods of
introducing exogenous DNA into living cells (see sect.
IIA). The only current viral vector capable of delivering
full-length dystrophin is gutted adenoviral vectors (Table
1) (314, 447, 459). Direct injection of this vector into
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domain (766). These actin binding domains do not bind to
sarcomeric ␣-actin in vivo, but rather interact with Gactin present just below the membrane (326). It has been
proposed that this submembranous actin network is connected to the sarcomeres by an interaction with desmin
(97). The bulk of dystrophin consists of a central rod
domain that contains 24 spectrin-like repeats, each of
which is ⬃100 amino acids in length. Interspersed within
these repeats are hinge regions that are believed to provide flexibility to the molecule. Following this rod domain
is a cystine-rich domain which interacts with the transmembrane protein, ␤-dystroglycan, through a WW domain. ␤-Dystroglycan forms a heterodimer with the laminin-2 receptor ␣-dystroglycan (Fig. 9) (215, 216). The
sarcoglycan complex (see below) and sarcospan are associated with the dystroglycan heterodimer. In skeletal
muscle, both the dystroglycan and sarcoglycan complex
expression require functional dystrophin. In contrast,
most of these proteins are present at relatively normal
levels in the dystrophin-deficient heart (316, 893). The
fourth domain is the COOH-terminal region containing an
␣-dystrobrevin binding domain. Both this dystrophin domain and ␣-dystrobrevin each contains syntrophin binding sites, and syntrophin contains a PDZ domain that may
function to bring signaling molecules (such as nitric oxide
synthase) into close proximity of the dystroglycan and
sarcoglycan complexes (287).
One of dystrophin’s proposed functions is as a stabilizer of the plasma membrane through its interactions
with the intracellular cytoskeleton and extracellular matrix (153, 686). This function is particularly evident during
lengthening contractions, where the extracellular matrix
and intracellular cytoskeleton are moving in opposite directions (171). Another important function ascribed in
part to dystrophin is the transmission of sarcomeric force
to the extracellular matrix at the level of the costamere.
Evidence supporting this function has been derived
largely from the reduced force generation of both skeletal
and cardiac muscle tissues from dystrophin null mice
(171, 404, 790, 994). A third proposed function of dystrophin is related to its ability to form the nidus for the
interaction and localization of many signaling molecules
(287), although in skeletal muscle the contribution of this
activity to the overall function of dystrophin appears to be
limited (718).
Duchenne muscular dystrophy is an X-linked disorder characterized by progressive skeletal muscle weakness (209). In addition to this overt skeletal phenotype,
patients are afflicted with an insidious cardiomyopathy
(209). Heart disease in Duchenne muscular dystrophy is
characterized by both conduction, structural, and contractile abnormalities. End-stage heart disease in this case
most frequently results in DCM and significant fibrosis,
especially within the posterobasal region (232). An Xlinked form of DCM has been linked to alterations within
Physiol Rev • VOL
(174). In many patients with Duchenne muscular dystrophy, skipping a few exons yielded a highly functional
dystrophin molecule. This approach has been shown to be
effective in the heart and shows therapeutic promise for a
significant subset of Duchenne muscular dystrophy patients.
3. Sarcoglycans
Shortly after its initial characterization, dystrophin
was found to interact with a group of membrane-bound
glycoproteins (217). These proteins formed a complex,
which functionally links dystrophin to the extracellular
matrix by binding to laminin (215, 216, 381). This complex
of dystrophin-associated proteins consists of two groups
of proteins: the dystroglycan and sarcoglycan complexes
(995). The laminin binding component of the dystroglycan
complex consists of ␣- and ␤-dystroglycan (Fig. 9), which
are cleaved components of a common precursor (381).
The sarcoglycan complex (Fig. 9) consists of four subunits: ␣-, ␤-, ␥-, and ␦-sarcoglycan (587, 643). All four of
these genes have been implicated in various forms
of limb-girdle muscular dystrophy (LGMD), and many of
them have significant cardiac phenotypes.
4. ␣-Sarcoglycan
␣-Sarcoglycan (␣-SG) is a 387-amino acid protein
with a single transmembrane domain and a large extracellular NH2-terminal domain. Extensive glycosylation of
two conserved asparagine residues yields a final molecular mass of 50 kDa (747). Mutations resulting in the loss of
␣-SG cause LGMD-2D (101, 749). Interestingly, the absence of ␣-SG results in the concurrent loss of the other
members of the sarcoglycan complex (195). ␣-SG deficiency results in severe muscular dystrophy that is essentially localized to skeletal muscle, although there are instances of cardiac disease (195, 691). Gene transfer of
␣-SG in the skeletal muscle of ␣-SG-deficient mice was
shown to fully restore the entire sarcoglycan complex (13,
193). Interestingly, the overexpression of ␣-SG following
gene transfer caused significant cellular toxicity. The
toxic effects of ␣-SG were independent of an immune
response and seemingly secondary to alterations in the
assembly of the sarcoglycan complex in the presence of
excess ␣-SG (193).
5. ␤-Sarcoglycan
␤-Sarcoglycan (␤-SG) has a similar structure to that
of ␣-SG, with a single transmembrane domain and the
majority of the protein present in the extracellular space.
␤-SG is glycosylated at three conserved asparagine residues, resulting in an increase in molecular mass from 35
kDa to a final weight of 43 kDa (72, 493). The clinical
importance of this new protein became evident as genetic
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skeletal muscle resulted in an inefficient transfer of fulllength dystrophin to muscle fibers local to the site of
injection (172, 264, 535), although even these modest
levels of transduction improved muscle function (172,
535). Clearance of transduced cells is a significant problem with adenoviral-based vector systems (see sect. IIA2),
but expression can be prolonged by performing injections
in neonates, which do not develop a strong immune response to the vector (110, 126, 196).
One of the most promising gene therapy vectors for
striated muscle is recombinant AAV. Significantly truncated dystrophin molecules that retain functionality were
first described in patients with Becker muscular dystrophy (212), and additional studies mapped the critical domains required for dystrophin function (144, 329, 687, 718,
777). These studies culminated in several functional dystrophin constructs small enough for AAV delivery (219,
329, 893, 937). Most of these truncated dystrophin constructs improved muscle pathology and membrane integrity following intramuscular injection into skeletal muscle. Only one of these constructs has been accessed to
any degree in the myocardium (293, 893, 997). Dystrophindeficient hearts expressing this truncated “micro-dystrophin” showed a significant improvement in ventricular
geometry (893). The expression of micro-dystrophin protected dystrophin-deficient mice from acute heart pump
failure during a dobutamine stress test (893). This same
construct also significantly extended the life span of mice
deficient in both dystrophin and utrophin, a dystrophin
homolog upregulated in the dystrophin-deficient mouse
(293). Despite these significant improvements in global
cardiac function, detailed physiological assessment of
this truncated dystrophin molecule revealed that deficits
remain, including a reduced transmission of force by
striated muscle expressing micro-dystrophin (293, 893).
These studies indicate that there is room for improvement
in the design of these micro-dystrophins.
While truncated dystrophin molecules are widely expressed in the heart when delivered by a single AAV (293,
294, 893), they are not fully functional. Other approaches
are being explored, including the use of two AAVs, each
containing half of a fully-functional dystrophin protein
with splice sites engineered between them. After transduction of the cell, the AAV genomes concatamerize.
Following transcription of these AAV genomes, the resulting RNA is processed at the engineered splice sites. The
result is a dystrophin molecule that is larger and more
functional than the micro-dystrophin delivered by a single
AAV (469). The efficiency of this splicing event, however,
appears somewhat less than single vector alone (262,
469). Further studies are necessary to determine the functional effects of this “trans-splicing” approach. Another
promising use of cardiac gene transfer in the treatment of
Duchenne muscular dystrophy is the use of AAV to introduce small RNA molecules that modulate splicing activity
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6. ␥-Sarcoglycan
␥-Sarcoglycan (␥-SG) is a glycosylated protein with a
molecular mass of 35 kDa. Mutations within this gene
have been implicated in LGMD-2C (549, 646). In contrast
to other sarcoglycanopathies, the sarcoglycan complex is
not completely lost in the absence of ␥-SG (145, 549).
Clinically, LGMD-2C is characterized as a severe form of
muscular dystrophy, with significant cardiac disease (44,
45). Consistent with this clinical presentation, mice lacking ␥-SG develop severe skeletal and cardiac pathology
(312). Gene transfer of ␥-SG into skeletal muscle was
shown to largely reconstitute the expression of ␥-SG and
corrected the skeletal muscle pathology (138). Interestingly, similar to ␣-SG, overexpression of ␥-SG resulted in
significant pathology, presumably secondary to disruption
of normal assembly of the sarcoglycan complex (1016).
7. ␦-Sarcoglycan
Shortly after the first description of the dystrophinassociated proteins, it was found that the cardiomyopathic hamster was lacking the sarcoglycan complex
(748). The absence of mutations within the ␣-, ␤-, or ␥-SG
genes strongly suggested a fourth gene may cause this
disease in the hamster (548). To uncover this putative
fourth sarcoglycan, a homology screen found a transcript
similar to ␥-SG (643). ␦-SG is a 35-kDa glycoprotein with
a structure similar to ␥-SG (643). This gene was quickly
linked to LGMD-2F (641) and to the cardiomyopathic
hamster (642, 777). Similar to the other disorders involving sarcoglycans, patients with mutations in ␦-SG often
have severe skeletal muscle disease (643). In contrast to
the other sarcoglycanopathies, mutations within ␦-SG
have been linked to cardiac disease in the absence of
skeletal muscle disease (904). Mice lacking ␦-SG also have
significant cardiac and skeletal muscle pathology (136,
311). Intriguingly, the cardiac damage initiated by exerPhysiol Rev • VOL
cise in the ␦-SG null mice was prevented by pretreatment
with vascular relaxing agents, suggesting an important
pathophysiological role of vascular smooth muscle (129,
136).
Like the other sarcoglycans, ␦-SG gene transfer into
skeletal muscle largely corrected the muscular dystrophy
present in the ␦-SG-deficient hamster (368). Similarly,
direct injection of myocardium with Sendai virus-coated
proteoliposomes or AAV containing ␦-SG resulted in reconstitution of the entire sarcoglycan complex (427– 429).
The levels of expression obtained through direct injection
sufficiently improved global cardiac function and increased the life span of the cardiomyopathic hamster
(428, 429). Global cardiac transduction, through the infusion of gene therapy vectors into the coronary circulation,
yielded results similar to that obtained by direct injections
(385). Systemic intravascular administration of AAV containing ␦-SG resulted in wide-spread expression of ␦-SG in both
skeletal and cardiac muscle (1014) and reconstituted the
sarcoglycan complex, correcting pathological changes in
striated muscle. ␦-SG gene transfer also improved cardiac
function and significantly extended the life span of treated
hamsters compared with those receiving no gene therapy
(1014).
B. Intermediate Filaments (desmin)
The most prominent intermediate filament in cardiac
muscle is desmin (Fig. 9), which forms coiled dimers that
self-assemble into homo- and heteropolymers with a number of proteins including nestin and synemin to form
filaments ⬃10 nm in diameter (249). This process is facilitated by the heat shock protein ␣B-crystallin (639). The
lattice of desmin filaments extends from the Z-line interacting with a variety of cellular structures including the
nucleus and mitochondria. In addition to interactions
with these organelles, many desmin filaments connect
adjacent Z-lines, functioning to keep neighboring sarcomeres registered. Desmin filaments emanating from Zlines at the periphery of the myocyte extend towards the
sarcolemma. Near the membrane, these intermediate filaments interact with the proteins that make up the costameres. These proteins, including the complex of proteins associated with the integrins and dystrophin, are
localized in the region of the Z-line via an interaction with
desmin (576). Desmin filaments are also prominent in
Purkinje fibers and at the intercalated disc (442, 887).
A variety of inherited cardiomyopathies have been
defined as desmin-related myopathies and are characterized by the deposition of desmin aggregates within cardiac and/or skeletal muscle. These disorders can be categorized into two groups: primary mutations within the
desmin gene and those within desmin-associated proteins
(96, 679). The latter group consists of mutations of ␣B-
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studies linked ␤-SG to LGMD-2E (72, 493). Clinically,
␤-SG deficiency is characterized by significant skeletal
muscle disease (72, 493). Cardiomyopathy has also been
reported in patients with LGMD-2E (36). Mice in which
␤-SG has been ablated also mirror the condition in humans, with significant pathology present in both skeletal
and cardiac muscle (19, 197). ␤-SG functions as a nidus
for the entire sarcoglycan complex, and the absence of
␤-SG causes a complete loss of other sarcoglycan complex members (197, 647). In contrast to ␣-SG, ␤-SG is also
required for the formation of the sarcoglycan complex in
vascular smooth muscle, which may in part explain the
greater cardiac involvement associated with ␤-SG deficiency (197). Gene transfer of ␤-SG was shown to restore
the expression of the entire sarcoglycan complex and improve pathology (193, 197). Unlike ␣-SG, overexpression of
␤-SG did not result in significant cellular toxicity (193).
1609
ated protein 4 (MAP4) and ␤1-tubulin, but not ␤4-tubulin,
which is the predominate isoform in the heart (634).
Adenoviral-mediated overexpression of these proteins in
normal adult cardiac myocytes revealed that MAP4 is
sufficient for these myocytes to develop microtubular
structure similar to that observed in myocytes isolated
from pressure-overloaded hearts. In contrast, adenoviralmediated expression of either ␤4- or ␤1-tubulin had no
significant effect on microtubule assembly (864). The increased density of microtubules present within pressureoverloaded myocytes resulted in a significant increase in
the viscous load placed on the myofilaments. This increased load significantly reduced the efficiency of the
contracting cardiac myocyte and clearly contributed to
the poor contractile performance of pressure-overloaded
hearts (860). Microtubules play an important role in the
pathophysiology of several models of heart failure secondary to pressure overload, but the importance of microtubules in other models of heart failure remains unclear (130, 163). The potential of microtubules to be manipulated by gene transfer technologies introduces the
possibility of experimental or therapeutic modulation of
microtubules to improve our understanding of these cytoskeletal elements in the failing heart.
C. Microtubules
VI. CARDIAC SIGNALING PATHWAYS
Microtubules are hollow filaments consisting of ␣and ␤-heterodimers of tubulin. Both tubulin subtypes are
GTP-binding protein; however, only the GTP bound to
␤-tubulin is hydrolyzed during polymerization. Microtubules are dynamic structures that run longitudinally
across the myocyte and are concentrated in the perinuclear regions (275). Under normal conditions, microtubules have small effects on the structural properties of
cardiac myocytes (135, 644). They appear to function
primarily as a means of organizing particles, vesicles,
and organelles within the myocyte. Microtubules serve
as the tracks for the complementary motor proteins
kinesin and dynein. These proteins contribute to properly distributing macromolecules and organelles within
the cell. In addition to this role in subcellular organization, microtubules also have critical roles in signal
transduction within the myocyte (95).
Microtubular accumulation is implicated in the
pathophysiology of ischemic cardiomyopathy, depression
of function with hypertrophy, and diabetic cardiomyopathy (95). This effect is particularly apparent with the
cardiac dysfunction associated with pressure overload
hypertrophy. The importance of microtubule accumulation is evidenced by the remediation of the contractile
deficit by agents that depolymerize microtubules (134).
This accumulation of polymerized microtubules is associated with increased expression of microtubule associ-
Gene transfer has been a valuable approach for
studying the effects of signaling pathways on myocyte
function and for identifying the cellular targets within a
signaling cascade. This approach also is helpful for determining whether a signaling pathway acutely influences
cardiac function in a setting that is independent of load
and possible compensatory adaptations that may develop
in transgenic animals. Gene transfer offers the ability to
study the temporal and dose-dependent modulation of contractile function by a signaling pathway, which may be difficult and/or expensive to follow in transgenic animals. This
approach could be useful for studying the relationship between expression of a specific signaling protein in cardiac
pathophysiology and heart failure. The goal of this portion of
the review is to focus on information gained about signaling
pathways and their influence on contractile function using
gene transfer. While gene transfer studies of signaling via
transcription factors are important, studies described here
focus primarily on the influence of signaling pathways on
ion transport, Ca2⫹ cycling, and myofilament proteins, as
well as contractile function.
A potential limitation of studies using in vivo adenoviral-mediated gene transfer is the difficulty in achieving
long-term, homogeneous expression at the organ level.
However, a new generation of vectors appears to be
overcoming issues of expression duration and homogeneous expression (954). Heterogeneous and/or transient
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crystallin (392, 926). These mutations appear to result in
defective assembly and subsequent aggregation of the
desmin filaments (76). Expression of mutated forms of
␣B-crystallin in adult cardiac myocytes was detrimental to
contractile function (521). The myofilaments remained
intact in the presence of the mutated ␣B-crystallin, but
contraction was attenuated. Further studies found a disruption of mitochondrial structure and function in myocytes expressing a mutated ␣B-crystallin (521).
Mutations within desmin can result in pathology in
skeletal muscle, cardiac muscle, or both. Most of these
mutations occur in the central ␣-helical domain of
desmin. Many of these mutated proteins are unable to
form filaments in vitro (34), and all result in the formation
of desmin aggregates in diseased tissue (272, 285, 613,
826). Desmin mutations have been linked to both dilated
(482, 584) and restrictive cardiomyopathies (20, 712, 930).
Gene transfer of mutated desmin into neonatal cardiac
myocytes disrupted the sarcomeric banding pattern (341).
These studies demonstrate that alterations in desmin filament assembly have a dominant effect on cellular sarcomeric assembly, an observation consistent with the
dominant mode of inheritance that characterizes desminrelated cardiomyopathies.
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DAVIS ET AL.
A. Gene Transfer Influencing the ␤-Adrenergic
Signaling Pathway
1. ␤-Adrenergic receptors and G proteins
One of the most studied and best understood signaling pathways involved in modulating cardiac contractile
function is the ␤-adrenergic receptor (␤-AR) pathway (48,
506, 696, 883). Catecholamine binding to the ␤1-AR activates adenylyl cyclase via G␣s to increase cAMP production, which in turn activates PKA (Fig. 10). The PKA
catalytic subunit phosphorylates multiple targets, including proteins located within the sarcolemma, SR, and sarcomeres (Fig. 10) to increase cardiac contractility and
relaxation rate. This basic signaling cascade is well understood based on a variety of experimental approaches
including biochemical, transgenic, and knockout models
(506, 883). While decreases in ␤-AR signaling were noted
during heart failure in the 1980s (79), ␤-AR agonists paradoxically increased mortality in failing hearts (342, 669,
818). Gene transfer studies have been important for providing new insight into the pathway of ␤-AR cycling during desensitization/downregulation and the role of ␤-AR
cycling during heart failure (340, 635, 669). Gene transfer
approaches have also provided fundamental knowledge
about adenylyl cyclase and A-kinase anchoring proteins
(AKAPs) (324, 761). These latter proteins serve as scaffolding proteins that contribute to macromolecular assembly of activated PKA with phosphorylation targets
FIG. 10. Model of protein kinase A (PKA)-mediated ␤-adrenergic signaling in cardiac myocytes. ␤-Adrenergic-1 receptors (␤1AR) are G
protein-coupled receptors that when stimulated initiate adenylyl cyclase (AC)/cAMP-dependent activation of PKA. PKA has several intracellular
targets in the cardiac myocyte including voltage-gated Ca2⫹ channels (DHPR), ryanodine receptor (RyR), phospholamban (PLN), and the
myofilament proteins cardiac troponin I (cTnI) and myosin binding protein C (MyBP-C). Inhibitory protein 1 (I-1) and phosphodiesterase (PDE) are
also substrates of PKA-mediated phosphorylation. ␤-Adrenergic-2 receptors (␤2AR) are also G protein-coupled receptors that when activated can
initiate the phosphatidylinositol 3-kinase (PI3K) and ERK signaling cascades or activate inhibitory protein 1 (I-1), which decreases protein
phosphatase 1 (PP1) activity. PP1 is a negative regulator of PKA activity and dephosphorylates PKA targets like PLN.
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expression may be a desirable goal for studies of signaling
cascades, which more likely operate under spatial and/or
temporal activation. Overexpression of signaling proteins
may be successful if targeted expression below pharmacological levels is achieved in the intact heart.
Many of the signaling pathways discussed below are
mediated via G protein-coupled receptors. The focus of
this section is on interactions between signaling proteins
involved in modulating Ca2⫹ cycling and myofilament
proteins and their overall influence on cardiac myocyte
contractile function. Importantly, signaling pathways can
exert both acute and chronic influences on contractile
function. The signaling section will begin with reviewing the
␤-adrenergic signaling pathway as it pertains to the contributions to the field gleaned from gene transfer studies.
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failure, which may influence signaling via ␤2 receptors
(208). Atrioventricular viral-mediated gene transfer of
wild-type G␣i or constitutively active G␣i (Q205L) significantly improved heart rate during persistent atrial fibrillation in a pig model (41, 186). Gene transfer of G␣i did
not significantly influence baseline isometric force in rabbit trabeculae or shortening in isolated myocytes (405).
However, expression of G␣i blunted the response to ␤-AR
activation by isoproterenol in both the trabecular and
myocyte preparations, and this blockade was prevented
with pertussis toxin. Overall, these results indicate that
signaling via G␣i acts as a brake on the ␤-adrenergic
contractile response, and it may be important for the
beneficial effects of ␤2 receptor gene therapy.
Another strategy along these same lines has been to
increase other G protein-coupled receptors, including
parathyroid hormone related peptide (PTHrp) and vasopressin2 (V2) receptors, using viral-mediated gene transfer (474). Like ␤2-ARs, PTHrp receptors are coupled to Gs
and Gi and downstream activation of phospholipase C,
while V2 receptors are strongly linked only to Gs (58, 474,
806). Neither receptor, however, is expressed endogenously in cardiac myocytes. The increased expression of
V2 receptors had no significant effect on basal contractile
function but significantly improved the response to agonists. In contrast, gene transfer of PTHrp receptors increased basal contractile velocity in failing and nonfailing
myocytes, but there was no further response to agonists.
At present, it is unclear whether therapies with these
receptors significantly improve contractile function at the
cellular and/or organ level under pathophysiological conditions.
2. Cycling of ␤-ARs
G protein-coupled receptor kinases (GRKs) are important for the rapid modulation of ␤-AR density on the
myocyte cell surface (752). The most prominent of these
GRKs, ␤ARK1 (or GRK2), is elevated during pathophysiological conditions (339), such as ischemia (480), heart
failure (192, 201, 752, 909), and coronary artery bypass
(rabbits) (881). ␤ARK1 is responsible for phosphorylation
of agonist-occupied ␤-ARs, which leads to uncoupling of
the receptor from downstream signaling pathways (446).
Adenoviral delivery of the ␤ARK1 inhibitor ␤ARK-1ct,
which is constructed from the COOH terminus of ␤ARK1
and acts as a dominant negative, significantly attenuated
the left ventricular dysfunction in hearts undergoing coronary artery bypass and after myocardial ischemia (813,
881, 968). Viral delivery of this inhibitor to myocytes from
spontaneously hypertensive rats in heart failure also significantly improved ␤-AR-mediated cAMP production,
peak shortening, and the rates of rat myocyte contraction
as well as improved signaling in failing rabbit myocytes
(9, 201). While this inhibitor restored ␤ARK1 levels in two
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(761). In addition, this approach has been instrumental in
identifying the relative contribution of individual targets
to the inotropic and lusitropic effects of ␤-adrenergic
signaling in cardiac myocytes (178, 207, 967). Most importantly, studies on the ␤-AR signaling pathway have provided insight into the paradoxical observation that ␤-AR
agonists increase mortality by demonstrating that ␤-agonists activate a cascade of pathophysiological events in
addition to the downstream increase in contractile function. By addressing the events that directly influence contractile function, viral-mediated delivery systems have begun to provide therapeutic strategies for treating heart
failure using the ␤-AR signaling pathway (201, 635, 881).
An example of therapeutic strategies designed to
improve cardiac function involves viral-mediated gene
delivery of ␤2-adrenergic receptors (192, 202, 540, 882).
While the highest proportion of ␤-ARs in the heart are
␤1-receptors, the proportion of ␤2-receptors increases
with heart failure, as ␤1-ARs are downregulated during
chronic stimulation (754, 828, 1010). Adenoviral gene
transfer of two different naturally occurring ␤1-AR polymorphisms to isolated rodent cardiac myocytes had significant chronotropic effects, and one of the ␤1-AR variants greatly enhanced the sensitivity of the myocytes to a
common ␤-blocker used to treat heart failure (751). Like
␤1-ARs, ␤2-receptors are also coupled to PKA activation
(85). Signaling via ␤2-ARs, however, is more complex than
␤1 (883), because the ␤2 receptor is coupled to both G␣s
and G␣i. G␣i subunits negatively influence contractile
function (980) (Fig. 10). In addition, ␤2-receptors show
significant differences in their spatial activation of PKA
compared with ␤1-ARs (15, 883). Complementary transgenic models with moderate overexpression of ␤2-ARs
improved contractile function without significant hypertrophy (753). Gene transfer of ␤-ARs has also been used
as a therapy to treat failing hearts (412, 464, 569, 882).
Gene transfer of ␤2-AR during cardiopulmonary bypass in
neonatal pigs increased total ␤-AR density and improved
left ventricular function in response to ␤-adrenergic stimulation (412, 413). Increases in these receptors after gene
transfer improved ventricular function at baseline and
with ␤-adrenergic stimulation in loaded and unloaded
rabbit hearts (540, 812) as well as in response to higher
preloads (464). The beneficial effects of ␤2-AR overexpression are clearly dose-dependent, and high levels of
overexpression are not considered beneficial (492). ␤2ARs signal via multiple downstream pathways. Gene
transfer of ␤1-AR or ␤2-AR into double knockout mice
indicated that the protective effects of ␤2-AR are likely
mediated via G␣i and the downstream activation of phosphoinositide 3-kinase (PI3K) and Akt (477, 1015) or via
functionally discrete signaling pools (506, 828).
Gene transfer approaches have also been used to
understand the contribution of G␣i to contractile function. Increased expression of G␣i was observed with heart
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DAVIS ET AL.
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ops in cardiac myocytes or in associated neurons after
gene transfer, and whether or not this strategy may be
appropriate for future therapy to treat heart failure.
3. Downstream ␤-AR signaling
A) ADENYLYL CYCLASE. Gene transfer strategies are providing significant strides toward understanding signaling
downstream from the ␤-AR. Viral-mediated gene transfer
of adenylyl cyclase VI (AC-VI) was first studied in neonatal rat myocytes, which selectively increased cAMP production in response to ␤-AR activation but not to other
agonists coupled to adenylyl cyclase (666). Gene transfer
also increased the cardiac response to ␤-adrenergic activation 12–14 days after recombinant viral delivery in normal, ischemic, and failing hearts (324, 467, 468, 759, 865).
Further work demonstrated that AC-VI delivery increased
the baseline rate of contraction (dP/dtmax), and ␤-adrenergic responses were maintained after gene transfer into
mice (758). Importantly, gene transfer of adenylyl cyclase
into pigs experiencing pacing-induced heart failure improved contractile function and decreased ventricular dilatation, as well as improved cAMP production and reduced indicators of hypertrophy. These findings also
agree with results obtained after transgenic expression of
AC-VI, which improved mortality and ventricular function
in mice with ischemic heart failure (865). Many of the
beneficial effects of AC-VI can be explained by the resulting increase in cAMP production. However, increased
cAMP is associated with arrhythmias, which were not
observed in the transgenic model. Thus the improved
mortality observed in AC-VI transgenic mice may be due
to 1) the close proximity of this isoform to the sarcolemma and 2) cAMP-dependent effects of AC-VI (324,
479). It remains unclear whether gene delivery of AC-VI is
capable of reversing LV morphological and functional
remodeling, but this gene is garnering consideration for
clinical heart failure therapy (324).
B) GS-COUPLED RECEPTORS. Gene transfer has been used
for upstream activation of adenylyl cyclase using receptors for arginine vasopressin (AVP). Increased AVP expression is associated with congestive heart failure and is
linked to a poor prognosis (274). The vasopressin 1 (V1)
receptor is expressed in the heart while V2 receptors are
expressed in renal collecting ducts but not in cardiac
myocytes. The V1 receptor is coupled to phospholipase C
(PLC)-␤ while V2 is coupled to Gs and adenylyl cyclase.
Viral-mediated gene transfer of V2 in adult rat ventricular
myocytes resulted in dose-dependent expression, an increase in cAMP formation, and an increased amplitude of
contraction in isolated myocytes that was blocked by a
V2-specific vasopressin antagonist (473). Coronary-based
adenoviral gene delivery to the myocardium also improved fractional shortening and the rate of contraction in
response to V2 stimulation (953). However, it remains to
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dilated cardiomyopathy models (200, 328), it did not consistently prevent the progression of cardiomyopathy or
premature mortality. These results suggest that ␤ARK-1
plays a critical role in modulating ␤-AR density on the
sarcolemma, but the animal model, type of cardiac pathophysiology, and timing of gene delivery may be critical for
the future development of therapeutic treatments for human heart failure. As described below, events and/or
other signaling pathways may also contribute to the final
progression to heart failure.
More recently, gene transfer of the ␤ARK1ct has been
compared with truncated phosducin, another GRK inhibitor (489). Both inhibitors bind to G␤␥ subunits, yet only
␤ARK-1 contributes to ␤-AR cycling and improves cAMP
accumulation in myocytes from failing rabbit hearts (489).
Gene transfer of either inhibitor improved the left ventricular contractile response to isoproterenol, and it improved fractional shortening and LV end-diastolic dimension in rapidly paced, failing hearts. These results suggest
that the beneficial influence of GRK inhibitors on failing
hearts maybe due, at least in part, to the inhibition of G␤␥
subunits.
Proteins associated with ␤ARK1 in the myocyte have
also been targets for gene transfer and treatment of heart
failure. PI3K binds to ␤ARK1 in the cytosol, is targeted to
␤-ARs during agonist activation of ␤-ARs, and regulates
␤-AR internalization (625). Overexpression of the kinase
domain, PIK, competitively displaces PI3K from ␤ARK1
and prevents ␤-AR internalization and desensitization.
Transient PIK expression decreased PI3K localization
with ␤-ARs in sarcoma cells, and viral-mediated gene
transfer of PIK into myocytes from failing pig hearts
restored the isoproterenol-mediated enhancement of
peak shortening and contraction and relaxation rates toward the nonfailing phenotype (683). The loss of ␤-ARs,
increased norepinephrine (NE) levels, and/or chronic
␤-AR activation that result from heart failure are ultimately maladaptive (640, 683). Future studies may focus
on gene therapy utilizing modified proteins associated
with the ␤-AR cycling pathway, such as ␤-arrestin (477)
and PDE4 (375, 488), which serve as important proteins in
scaffolding and/or desensitization of ␤2-ARs.
In addition to ␤-ARs and associated proteins, studies
have also focused on clearance of hormones involved in
␤-AR signaling, especially NE. Accumulation of NE during
hyperstimulation of ␤-AR was postulated to be a major
cause of structural and functional impairment during
heart failure (726). Efforts to improve local NE clearance
using gene transfer of uptake-1 into rabbit hearts prior to
pacing-induced heart failure acutely improved NE uptake,
␤1-AR receptor, and SERCA2 expression, as well as diastolic and systolic contractile function, over a 2-wk time
period (612). Gene transfer of uptake-1 had no significant
influence on contractile function in nonfailing hearts. It is
currently unclear whether the uptake-1 expression devel-
peutic strategies utilizing the phosphorylated versions of
these proteins have not been addressed in detail using
viral-mediated gene transfer approaches. In the case of
Ca2⫹ channels, as well as other ion channels or transporters, there are multiple subunits. Thus cellular or organdirected gene transfer and overexpression may not necessarily result in appropriate channel subunit organization. Experiments with modifications of MyBP-C on the
PKA-targeted Ser283 (593) also may be difficult to perform, as the turnover of MyBP-C is not clear and may be
too long for the typical 1–7 day time frame for myocyte
contractile assays in vitro.
5. Heat shock proteins, p20
Proteomic analysis of the ␤-AR response to isoproterenol has revealed phosphorylation of the p20/pHSPB6
heat shock protein. This small heat shock protein is an
␣-crystallin and is typically present in the cytosol of cardiac myocytes (120, 476). Phosphorylation of p20 results
in its colocalization with actin (222), although recent
studies suggest that p20 does not bind directly to actin
(90). It has been postulated that the p20 phosphorylation
state determines the translocation and the localization
pattern. Viral-mediated gene transfer of p20 into adult rat
myocytes increased peak shortening and Ca2⫹ transient
amplitudes within 2 days after gene transfer without significantly influencing resting length, relaxation, or Ca2⫹
decay (120). Although the cellular basis for the increase in
contractility and Ca2⫹ transient remains unknown, p20
localization with actin and its ability to bind PP1 (221)
would suggest that this small heat shock protein may have
direct influences on proteins within the Ca2⫹ cycling cascade and/or the myofilaments. It should be noted that
gene transfer has been used to study the protective effect
of other heat shock proteins against stresses such as heat
and/or ischemia in cardiac myocytes (471). However, to
date, gene transfer of these other heat shock proteins has
not focused on the direct influence on contractile function.
6. Protein phosphatase 1
PKA modulates PP1 activity via phosphorylation of
inhibitor proteins. The role played by PP1, the inhibitor
proteins, and modulation of these proteins by PKA and
PKC is discussed in more detail below.
4. Myosin binding protein C
B. Gene Transfer of Ca2ⴙ/Calmodulin Kinase
The ␤-AR pathway phosphorylates a number of other
proteins, including the sarcolemmal L-type Ca2⫹ channel
(DHPR) and sarcomeric MyBP-C. Studies using gene
transfer to investigate the contribution of these proteins
in contractile function and/or studies to develop thera-
The Ca2⫹/calmodulin-dependent protein kinase II␦
(CAMKII␦) isoform phosphorylates several proteins involved in EC coupling, including the RyR and PLN. Increased CAMKII activity is observed during heart failure,
and overexpression of the cytosolic variant CAMKII␦c in
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be determined whether gene delivery of V2 improves
cardiac performance in failing hearts. In addition, the
long-term benefits of this approach are controversial, as
blockade of both V1 and V2 is also predicted to improve
function in individuals with congestive heart failure (273).
This general approach has also been utilized to determine
whether delivery of other noncardiac Gs-coupled receptors to cardiac myocytes acts as a novel therapeutic strategy for bypassing the ␤-AR and boosting contractile function (474).
C) AKAPS. Several experiments on downstream signaling within the ␤-AR pathway have focused on AKAPs.
AKAPs serve as cellular scaffolding for PKA by tethering
the type II regulatory subunit (RII), as well as target
proteins, and phosphatase/phosphodiesterases. At least
13 different AKAPs have been identified in myocardium,
and the localization and function of these AKAPs was
recently reviewed in detail by Reuhr et al. (761). Gene
transfer and expression of Ht-31, a peptide derived from
human thyroid AKAP with a similar binding affinity as
AKAP for the RII domain of PKA, resulted in redistribution of the RII subunits within isolated myocytes (231).
This change in PKA localization enhanced ␤-AR-mediated
peak shortening as well as the rate of shortening and
relengthening compared with controls (231). Paradoxically, the level of myofilament protein phosphorylation
(e.g., cTnI, MyBP-C) in response to ␤-AR stimulation was
significantly reduced in these myocytes compared with
controls. Results from these studies provided insight into
the temporal and spatial targeting of AKAPS within cardiac myocytes (231, 763). In addition, this approach has
provided a better understanding of the role played by
each AKAP in myocyte hypertrophy (182). Future studies
are needed to investigate the influence of AKAPs on contractile function under pathophysiological conditions, as
modified AKAPs could one day be used to treat heart
failure.
D) END-TARGET PROTEINS. The end targets for phosphorylation have also been studied using gene transfer, and
this approach has contributed significantly towards understanding ␤-AR signaling. These studies have focused
primarily on Ca2⫹ cycling and sarcomeric proteins that
serve as targets for ␤-AR signaling, with direct influences
on contractile function. A high proportion of these studies
have focused on phospholamban and sarcomeric troponin I, which have been described above (see sects. III and
IV). Below are studies of other downstream targets.
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C. Gene Transfer and PKC Signaling
Activation of PKC plays a key role in mediating signaling via multiple receptors (e.g., angiotensin II, endothelin, ␣-adrenergic receptors; Fig. 11) (152, 211, 453, 598,
688, 880, 940) coupled to G proteins (e.g., primarily Gq, Gi)
followed by PLC activation. PLC breaks down phosphoinositide bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is translocated
to the SR (Fig. 11), and binding to its receptor contributes
to ⬃5% of SR Ca2⫹ release (399, 603). Recently, FRET was
used by labeling the binding domain of the IP3 receptor
with cyan fluorescent protein, while target protein sequences were labeled with yellow. This biosensor approach demonstrated the spatiotemporal distribution of
IP3 in neonatal cardiac myocytes (733). SR Ca2⫹ release
increases in response to low levels of IP3 (433, 655), and
future gene transfer approaches may prove valuable for
imaging, Ca2⫹ cycling, and/or contractile function studies
in adult cardiac myocytes.
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A much larger body of literature has focused on
direct and indirect modulation of cardiac function by PKC
using gene transfer approaches (556, 958). Divergent contractile responses are observed in response to PKC activation, with reports of both increases and decreases in
contractile performance. The multiple agonists that activate PKC, the presence of multiple PLC and PKC isoforms, downstream activation of other signaling pathways, and phosphorylation of multiple end-target proteins
(10, 152, 426, 453, 651) add to the complexity of this
signaling pathway. Gene transfer studies have helped define and map this pathway in cardiac myocytes, which has
improved our understanding of its direct influence on
contractile function. The remainder of this section focuses on insights gained from gene transfer studies.
1. Signaling upstream from PKC
A) RECEPTORS AND PLC. Gene transfer of receptors that
activate the PKC pathway has been utilized in several
studies (Fig. 11). These studies have focused on the relative contribution of receptor isoforms to hypertrophy mediated via PKC and/or the ability of a specific isoform to
counteract the hypertrophic phenotype produced by a
dominant receptor isoform (243). With this emphasis, the
acute and chronic influence of receptor isoform expression on cardiac contractile function has not yet been as
thoroughly studied by gene transfer. For example, two
major isoforms of the angiotensin II (ANG II) receptor
(AT-1 and AT-2) are expressed in adult myocytes (82,
349). Adenoviral-mediated gene transfer of AT-1 and AT-2
each produced hypertrophy in neonatal rat myocytes, and
direct lentiviral-mediated cardiac delivery of AT-2 reduced the hypertrophic response to chronic ANG II delivery (148, 220). The importance of AT receptors in the
hypertrophic process is clear from these studies, but analysis of cardiac myocyte and/or cardiac contractile function would add to our understanding of these receptors.
Gene transfer of proteins/enzymes involved in hormone metabolism has been used to demonstrate the importance of cardiac PKC-related paracrine and autocrine
mediators. One example is the use of angiotensin converting enzyme 2 (ACE2), which catalyzes the production of
the potent vasodilator angiotensin 1-7 (ANG1-7) (229).
ANG1–7 works via a non-PKC pathway (269) and counteracts the influences of the PKC-linked AT-1 receptor on
cardiac hypertrophy (179, 717, 976). Lentiviral-mediated
gene transfer of ACE2 produced strong cardiac expression and renal expression, albeit to a lesser extent. Functionally, ACE2 delivery also reduced hypertension and
increased left ventricular end-diastolic and end-systolic
dimensions in spontaneously hypertensive but not normotensive rats (179). Although the mechanism responsible
for these beneficial effects is currently not clear, results
from studies like these suggest that approaches designed
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transgenic animals causes heart failure (50, 518). Viral
delivery and overexpression of wild-type CAMKII␦c in
rabbit myocytes altered Ca2⫹ cycling, but overexpression
did not directly alter other proteins such as NCX or
SERCA, changes that are commonly associated with heart
failure (448). The major influences on Ca2⫹ cycling proteins included phosphorylation of RyR, without changes
in the association of RyR with FKBP12.6. This phosphorylation in rabbit myocytes was associated with increased
SR fractional release of Ca2⫹, increased diastolic leak of
Ca2⫹ and reduced SR Ca2⫹ content. Despite the decrease
in SR Ca2⫹ content, the cellular Ca2⫹ transient and shortening amplitude were maintained due to the increase in
peak DHPR current and increased SR fractional Ca2⫹
release. Overexpression also enhanced the frequency-dependent acceleration of relaxation, presumably due to its
phosphorylation of PLN at Ser-17 and the resulting increase in Ca2⫹ uptake by SERCA2. In contrast, gene transfer of wild-type, constitutively active, and dominant negative CAMKII␦c into isolated rat myocytes produced quite
different results (989). Here, increased SR Ca2⫹ content
and reduced Ca2⫹ release in response to elevated Ca2⫹
were observed in myocytes overexpressing wild-type or
constitutively active CAMKII␦c, and these results were
interpreted to indicate that CAMKII␦c acts as a negativefeedback modulator of RyR. The differences between the
studies with rabbit and rat myocytes have been attributed
to species differences in Ca2⫹ handling between rat and
rabbit hearts (989). Future gene transfer studies will be
necessary to better understand the role played by
CAMKII␦ and may ultimately be critical for defining the
direct versus compensatory adaptations that develop in
transgenic mice expressing CAMKII␦.
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to reduce the influence of PKC-linked signaling hormones
on contractile function could be beneficial during heart
failure.
Other steps in the PKC signaling pathway (Fig. 11)
studied using gene transfer include PLC, heat shock proteins, and DAG kinase. PLC is critical for the production
of DAG, which is critical for PKC activation/translocation
in response to mediators such as endothelin, ANG II, and
␣-adrenergic agents. Overexpression of PLC isoforms in
neonatal cardiac myocytes was demonstrated after viralbased gene transfer (22). Heat shock proteins can also
modulate PKC (128), and viral-mediated gene transfer has
been used to investigate the influence of Hsp70 and Hsp90
on PKC-␦ and -␧ expression in neonatal cardiac myocytes
(128). DAG kinases breakdown DAG, and the breakdown
of DAG in response to overexpression of DAG kinase ␨
inhibits endothelin-1-induced activation of PKC-␧ and
downstream signaling (mitogen-activated protein kinase,
MAPK) of hypertrophy in neonatal cardiac myocytes
(862).
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B) PKC ISOFORMS. The PKC family consists of at least 12
different isoforms, and the characteristics of the 3 subclasses have been reviewed in detail (173, 190, 619, 838).
Adult cardiac myocytes in most mammalian species express four major isoforms, including classical PKC-␣,
novel class PKC-␦ and -␧, and to a lesser extent atypical
PKC-␨ (213, 713, 714). Several pathophysiological conditions and heart failure in humans are associated with
increased expression of PKC-␣ and -␦, plus the appearance of PKC-␤, another classical isoform (77, 648). Comparable changes in these PKC isoforms were observed in
rat models of pressure overload (42, 191). The gene transfer studies described below have significantly contributed
to our understanding of the role played by many of these
isoforms in modulating contractile function.
I) PKC-␣. Classical and novel PKC isoforms are activated by PLC-dependent DAG production or addition of
phorbol esters (Fig. 11). The activity of classical isoforms,
such as PKC-␣, also depend on increased cytosolic Ca2⫹
(173). Early studies demonstrated that peptide inhibitors
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FIG. 11. Model of protein kinase C (PKC) signaling directly involved in modulating contractile performance in cardiac myocytes. Several
agonists activate this signaling cascade. Signaling via endothelin A receptors (ETA) and the angiotensin II receptor (ATIIR) are examples of receptors
signaling through G proteins and their subsequent activation of phospholipase C (PLC). The next step in this cascade leads to the production of
diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) from the phosphoinositol PIP2 by phospholipase C (PLC). DAG then activates PKC, as
does synthetic phorbol esters. In nonfailing hearts, the three most predominant PKC isoforms are classical PKC-␣ and novel PKC-␦ and -␧. Heart
failure is associated with upregulation of PKC-␣ and -␦, along with the production of PKC-␤. Activated PKC is translocated to target proteins, and
this activation/translocation process is modulated by receptors for activated C kinase (RACKS), receptors for inhibition of C kinase (RICKS), and
substrates that interact with C kinase (STICKS). A comprehensive understanding of the specific physiological, pathophysiological, and pharmacological targets for each PKC isoform, as well as the potential for cross-talk among isoforms, remains an active area for investigation. One example
of a PKC-␣ targeting pathway shown here was identified with the help of gene transfer studies. PKC-␣ phosphorylates inhibitor-1 (I-1) and in turn
activates protein phosphatase 1 (PP1). A major target for PP1 is phosphorylated phospholamban (PLN). PP1-dependent reduction in PLN
phosphorylation slows Ca2⫹ uptake by SERCA2a and significantly decreases contractile function. Other PKC targets, including the myofilaments,
directly influence cardiac contractile function upon PKC modification and are also included in this model. Targets involving more indirect and/or
transcriptional effects of PKC signaling are not shown.
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cytes from transgenic mice (78, 868, 933). The differing
functional effects may be due to the expression and activity level of PKC-␤ as well as the duration and developmental pattern of expression. In future studies, gene
transfer could provide valuable feedback about the direct
influence of PKC-␤2 on contractile function in isolated,
intact failing and nonfailing cardiac myocytes.
At the subcellular level, incubation of permeabilized
mouse myocytes with PKC-␤2 increased myofilament
Ca2⫹ sensitivity of force, which was blunted in myocytes
expressing a cTnI with Ala substitutions at phosphorylation sites (939). Gene delivery and expression of PKC-␤ to
neonatal myocytes also increased ␤MyHC and MLC-2 expression (423, 939). Signaling downstream from PKC-␤2
has also been studied using gene transfer. These studies
suggest that p90 ribosomal S6 kinase (p90RSK) is a key
downstream effector of PKC-␤ activated in response to
reactive oxygen species, and this kinase is involved in the
phosphorylation of cTnI at Ser-23/24 (393).
III) PKC-␦. Multiple studies have investigated the
modulation of contractile function using myocardial gene
delivery of PKC-␦. In isolated rat myocytes, basal contractile function was not significantly changed after gene
transfer of GFP-tagged PKC-␦ (420). However, phorbol
ester activation of PKC resulted in a transient negative
inotropic effect that was followed by a sustained positive
inotropic effect after gene transfer of PKC␦ into myocytes
(420). Typically, similar doses of phorbol esters produce a
sustained PKC-dependent negative inotropic effect on
contraction (98, 308). The sustained increase in peak
myocyte contractile function observed after PKC-␦ gene
transfer was pH dependent, and this enhanced contractile
function was prevented by the PKC inhibitor bis-indolylmaleimide (bis-1). PKC-␦ gene transfer did not change the
rates of contraction or relaxation. Together, these results
suggest gene transfer of PKC-␦ produces a positive inotropic response to phorbol esters.
The role of PKC-␦ in response to other agonists
and/or cellular stimuli is less clear. For example, the PKC
agonist ET-1 increased PKC-␦ translocation (420, 530), yet
expression of dominant negative PKC-␦ did not alter the
inotropic response to ET-1 (421). In contrast, in vivo gene
delivery studies demonstrated that protein delivery of a
PKC-␦-selective inhibitor (␦V1-1), using the viral protein
TAT for intracellular protein delivery (805), restored cardiac function more quickly after acute ischemia followed
by reperfusion both in vivo and in vitro (389, 391). Further
work is needed to determine whether PKC-␦ activation
has divergent physiological versus pathophysiological influences on function due to cellular localization.
A cellular gene transfer approach has also been utilized to gain insight into the PKC-␦-mediated signaling
cascade responsible for any changes in contractile function. Perinuclear staining of activated PKC-␦ was observed in isolated myocytes, and there was evidence it
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directed to classical PKCs prevented phorbol ester-mediated increases in Ca2⫹ current (1007). Classical PKC isoforms were also found to influence hypertrophy in neonatal cells (80). Gene transfer of PKC-␣ stimulated hypertrophy through downstream activation of MAPK and
extracellular regulated kinase 1/2 (ERK1/2). This hypertrophy was also linked to signaling proteins upstream
from ERK1/2, including Rho GTPases (673). These results
raised questions about the role of PKC-␣ in the development of hypertrophy related to pathophysiological events
and its potential role in modulating contractile function in
adult hearts. Gene transfer into adult rat cardiac myocytes demonstrated that increased PKC-␣ expression significantly decreased the amplitude of shortening and rate
of contraction, without significantly influencing resting
sarcomere length or relaxation (81). Increased PP1 activity was an important contributor to these functional
changes, as described below. Reductions in PKC-␣ activity using dominant negative PKC-␣ (dnPKC␣) produced
the opposite effect, with significant increases in peak
shortening and shortening velocity. Transgenic PKC-␣
overexpression and knockdown of PKC-␣ in mice produced comparable outcomes at the organ system level.
The Ca2⫹ transient amplitude and SR Ca2⫹ release also
increased in myocytes isolated from these mice (81).
PKC-␣ and PP1 expression increased during heart failure
(77, 207), and knock-down of PKC-␣ expression minimized contractile dysfunction in three separate models of
heart failure (81). These findings were used as a basis for
developing a gene therapy strategy (323). The delivery of
dominant negative PKC-␣ into rats 12 wk after cryoinfarction improved end-diastolic pressure and the positive
pressure derivative within 1 wk after gene transfer (81). It
should be noted that contractile function may be influenced via feedback of PKC-␣ on the PKA pathway. Constitutively active PKC-␣ decreased adenylyl cyclase activity in HEK cells after gene transfer, and this response was
enhanced by cotransfection with ␤-AR1 receptors. Based
on these findings, the PKC feedback response may be
mediated through direct actions on ␤-AR1 receptors in the
myocardium (302). Overall, the relationships between elevated levels of PKC agonists, increased classical PKC
isoform expression, downstream target protein phosphorylation, and contractile function under pathophysiological
states are only partially solved. Gene transfer may be a
key approach for obtaining substantial insight into the
changes in PKC signaling and their direct influence on
contractile function during heart failure, in addition to
further development of therapeutic targets aimed at this
signaling pathway.
II) PKC-␤. Expression of PKC-␤1 and -␤2 also increases significantly during heart failure (77). Transgenic
expression of wild-type PKC-␤2 or an inducible, constitutively active PKC-␤2 produced decreased and increased
contractile performance, respectively, in cardiac myo-
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enced by low or high levels of PKC-␧ expression. A negative inotropic response to phorbol ester was observed in
myocytes with 3- to 5-fold overexpression of PKC-␧, while
myocytes having 7- to 10-fold increases showed a brief
negative followed by a sustained positive inotropic response at room temperature. The differences in response
to PKC agonists were attributed to the relative amount of
sarcolemmal PKC-␧ localization (420), but these results
may also be agonist dependent. Additional considerations
that may explain the differences between studies in rabbit
versus rat myocytes include differences in Ca2⫹ cycling
between animal models, the recording temperature during cellular function studies, and the relatively small number of cells recorded in the later study.
The targets responsible for changes in shortening in
response to PKC-␧ have not been thoroughly investigated,
although work to date provides evidence that PKC-␧ may
influence contractile function more through influences on
protein expression than through phosphorylation of myofilament/Ca2⫹ cycling proteins. For example, viral-mediated gene transfer of dominant negative PKC-␧ or downstream activation of focal adhesion kinase (FAK) by Mansour et al. (523) demonstrated that PKC-␧ was necessary
for strain-induced recovery of sarcomere length in neonatal rat myocytes. In addition, gene transfer studies have
demonstrated that PKC-␧ downregulates SERCA2 expression in neonatal myocytes (702). Expression and phosphorylation of the gap junction protein connexin43 were
also influenced by PKC-␧ in myocytes (181).
V) PKC-␨. This atypical PKC is translocated from the
cytoplasm to the nucleus in cardiac myocytes during
ischemia (586). Future gene transfer studies are needed to
determine whether this isoform plays a direct role in
influencing cardiac myocyte physiology.
2. Downstream translocation and other
signaling pathways
The gene transfer approach remains to be utilized for
studying scaffolding proteins that bind PKC in cardiac
myocytes (Fig. 11). These proteins include receptors for
activated C kinases (RACKs) and substrates that interact
with C kinase (STICKs). To date, at least two RACKs have
been described, and these RACKs are responsible for
translocation of PKC isoforms (795). Gene transfer of
RACKs in other cell lines established the interrelationships between PKC and other signaling pathways, such as
the MAPKs (509, 740), PLC-␤2, and adenylyl cyclase (111).
Future studies utilizing RACKs or STICKs may be valuable
for determining their role in modulating contractile function and for understanding the role of RACKS in translocation with genetically coded fluorescent reporters within
the cardiac myocyte. An important caveat with these
proteins is that they may serve multiple functions within
the cell (795, 838), and their influence on PKC-mediated
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was translocated to caveolae, which are proposed to act
as a signaling platform (420, 769, 838). In addition, gene
transfer studies demonstrated that constitutively active
PKC-␦ activates MAPK, JNK and p38, and the Rac-1 signaling pathways in neonatal myocytes (348, 673). As with
other PKC isoforms, further cellular studies are needed to
gain a more specific understanding of the downstream
myofilament and/or Ca2⫹ cycling targets responsible for
any contractile response to PKC-␦. An additional consideration for future work is based on the evidence supporting differential activation by cellular localization of PKC-␦
(420) and stimulus-dependent differences in PKC-␦-mediated modulation due to tyrosine phosphorylation (838).
PKC-␦ also cross-regulates PKC-␧ (767), an isoform that
also influences contractile function (see below). These
alternative mechanisms modulating PKC-␦ activity may
play a role in influencing contractile function under physiological conditions and/or may alter the response during
pathophysiological stressors (838).
IV) PKC-␧. A substantial body of work has focused
on defining the role of PKC-␧ in modulating cardiac contractile function. Initial work demonstrated that activation and translocation of PKC-␧ in response to the PKC
agonist ET-1 produced a significant increase in the Ca2⫹
transient in AT-1-derived myocytes (411). These were followed by studies in transgenic mice, and a dose-dependent effect of PKC-␧ was observed on contractile function
(677). Low-level expression and expression of dominant
negative PKC-␧ had no significant influence on basal contractile function, but higher expression produced cardiac
hypertrophy, myocyte disarray, altered myofilament protein isoform expression, decreased systolic and diastolic
pressure, as well as reduced positive and negative pressure derivatives. The response to the PKC agonist ANG II
was also attenuated in this high expressing line. Due to
the presence of hypertrophy and myocyte disarray, it
became important to investigate the influence of PKC-␧
expression on myocyte function independent of these
adaptations.
To investigate the role of PKC-␧ on contractile function independent of changes in myofilament and Ca2⫹
cycling expression, Baudet et al. (40) used viral-mediated
gene transfer into adult rabbit myocytes. Two days after
gene transfer, a 28-fold increase in PKC-␧ activity was
observed without significant changes in the expression of
other PKC isoforms. However, PKC-␧ overexpression increased peak shortening and Ca2⫹ by 21% and prolonged
the twitch duration, which was attributed to the prolonged time to peak shortening. Relaxation was not significantly modified. In addition, the response to ET-1 was
attenuated in myocytes overexpressing PKC-␧. Later studies in rat myocytes proposed a biphasic influence of
PKC-␧ on contractile function in response to PKC activation (420). In contrast to the earlier gene transfer studies,
baseline contractile function was not significantly influ-
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peak shortening and the rate of contraction in myocytes
(271, 453). This alkalosis could also delay relaxation
(541). In the presence of the 5-N-ethyl-N-isopropylamiloride (EIPA), the relaxation response was still observed
while peak shortening was attenuated. These results suggested that Na⫹/H⫹ exchange contributes to the enhanced contractile shortening, but cTnI phosphorylation
plays a critical role in the relaxation response to ET.
The presence of three primary clusters (Ser-23/24,
Ser-43/45, and Thr-144) in purified cTnI that are phosphorylated by PKC (652) contributes to the challenge of understanding the role of PKC-mediated TnI modifications
on contractile function. These residues are Ser-23/24,
which also is phosphorylated by PKA, (Fig. 10; Refs. 454,
1004), Ser-43/45, and Thr-144 (Fig. 11). In myocytes expressing ssTnI or cTnI isoform chimeras, there is evidence that the three target clusters (Ser-23/24, Ser-43/45,
Thr-144) are phosphorylated in response to ET-1. ET-1
phosphorylated the N-card/slow-C TnI chimera (c/sTnI),
which contains the Ser-23/24 and Ser-43/45 sites, and
subsequently accelerated myocyte relaxation. These results provide evidence that at least one of the cTnI NH2terminal clusters (Ser-23/24 and Ser-43/45) contributes to
accelerated relaxation in response to ET-1. Only the Thr144 site is present in the TnI chimera N-slow/card-C.
While TnI phosphorylation in this chimera was not detected, relaxation time was intermediate between cTnI
and ssTnI, with no increase in peak shortening in response to ET-1. The lack of change in peak shortening and
in relaxation with N-card/slow-C TnI chimera may be
caused by conformational differences which influence the
ability of PKC to phosphorylate specific clusters within
the myofilament. Thus the role of Thr-144 in the ET-1
response was difficult to ascertain with these TnI chimeras.
Subsequent experiments focused on the direct contribution of cTnI Thr-144 phosphorylation to ET-induced
changes in myocyte contractile function (962). Thr-144 is
important because it is located within residues 138-149 (1,
869), the inhibitory peptide (IP) region of TnI (338, 652,
857). The IP region of TnI is the minimum sequence
needed to inhibit strong interactions between actin and
myosin in the absence of Ca2⫹ (857, 896), and it toggles
between actin (low [Ca2⫹]) and TnC during the Ca2⫹
transient. Thus cTnI Thr-144 phosphorylation may be an
important modulator of the TnI “switch” between actin
and TnC. Gene transfer and expression of the cTnI
Thr144Pro substitution delayed relaxation in response to
ET-1, providing evidence the Thr-144 residue significantly
contributed to the acute PKC-mediated acceleration of
relaxation in response to ET-1 (962).
The Ser-23/24 phosphorylation site is a well-documented phosphorylation site for PKA. There is now evidence that this cluster contributes to the accelerated
relaxation rate in response to agonist activation of PKC
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contractile function will require consideration of the scaffolding provided for other signaling complexes.
A) MYOFILAMENT TARGET PROTEIN, TROPONIN I. Activated
PKC directly phosphorylates the sarcomeric protein cTnI
(Fig. 11) and alters myofilament function. PKC-dependent
cTnI phosphorylation decreases myofilament Ca2⫹ sensitivity (92, 653) and correlates with peak PKC-dependent
contractile responses using multiple agonists (152, 426,
598, 688). However, the role cTnI phosphorylation plays
in the contractile response has been difficult to define, in
part because PKC phosphorylation of cTnI is linked to
both accelerated and decreased relaxation rates (689, 782,
958, 962). Gene transfer studies have established that
PKC-mediated cTnI phosphorylation accelerates relaxation rate in response to the neurohormone ET-1. The
addition of bis-1, a PKC antagonist, largely inhibited the
functional effects of ET-1, suggesting that PKC was a key
contributor to cTnI phosphorylation in response to ET-1.
MAPKs can act as downstream effectors of activated
PKC, and each pathway influences cardiac contractile
function (133, 411, 491, 684), but MAPK inhibitors did not
significantly influence acute ET-1-mediated cTnI phosphorylation. The PKA inhibitor H-89 did not significantly
influence cTnI phosphorylation in response to ET-1 (266).
In experiments with the neonatal cardiac isoform
slow skeletal TnI (ssTnI), ET-1 activation of PKC did not
significantly phosphorylate the ssTnI isoform (958). All
indices of relaxation were prolonged in response to ET-1
in myocytes expressing the nonphosphorylatable ssTnI.
The increased peak amplitude of shortening observed in
response to ET-1 was also significantly blunted in myocytes expressing ssTnI. These findings demonstrated that
PKC directly phosphorylates cTnI and cTnI phosphorylation during ET-1 activation of PKC accelerates relaxation
and contributes to the increased amplitude of contraction
(958).
The functional contribution of cTnI phosphorylation
relative to changes in Ca2⫹ cycling has also been investigated during ET-1 treatment. Enhanced peak shortening
and modest acceleration in relaxation rate were observed
in myocytes loaded with fura 2. However, the peak and
rate of Ca2⫹ transient decay were not altered in these
myocytes, which is consistent with the idea that ET-1induced PKC activation influences peak shortening and
relaxation through altered myofilament function rather
than the Ca2⫹ transient. The contribution of phosphorylated TnI to the ET-1-mediated change in contractile function was also investigated at different stimulation frequencies, and the frequency response (0.2 to 2 Hz) remained
unchanged with and without ET-1 (962).
Activation of the Na⫹/H⫹ exchange is also an important component of the contractile response to ET-1. Earlier, investigators showed ET-1 increased Na⫹/H⫹ exchange via PKC (271, 453). The ensuing alkalosis increased myofilament Ca2⫹ sensitivity (541) and enhanced
Physiol Rev • VOL
phosphorylation slows cross-bridge cycling and produces
a dominant decrease in relaxation rate. Another key remaining question is whether the three clusters independently and additively influence relaxation or, instead,
have a hierarchical influence on relaxation. These questions can be addressed using gene transfer into adult
myocytes.
Currently, it is controversial whether heart failure
causes significant changes in the PKC-dependent cTnI
phosphorylation state (66, 648). Decreases in overall PKCdependent phosphorylation have been reported in failing
human hearts (66), while increased Ser-43/45 phosphorylation has been correlated with decreased force in another set of failing human hearts (648). Thus, during heart
failure, the downstream effects of the temporal elevation
of ET-1, heightened PKC isoform expression, and altered
cTnI phosphorylation on contractile function are not well
understood and leave open an area of research primed for
gene transfer strategies.
3. Other targets
The strategy of studying the importance of multiple
phosphorylation sites and/or the role played by other
myofilament proteins and/or proteins involved in modulating Ca2⫹ cycling using gene transfer has not yet been
thoroughly applied to PKC signaling in myocytes. Instead,
biochemical techniques and transgenesis have been the
predominant approach. Proteins targeted by PKC that
may be of interest for future gene transfer studies include
TnT, MLC2, MyBP-C, connexin43, Na⫹/H⫹ exchanger
(NHE1), and DHPR.
A) PICOT. The protein interacting cousin of thioredoxin, PICOT, acts as an inducible inhibitor of hypertrophy. In addition, this protein has direct effects on contractile function (189, 408). PICOT was discovered in a
yeast-2 hybrid screen and showed 30% homology with
thioredoxin (973). After gene transfer into adult rat cardiac myocytes, PICOT increased peak shortening amplitude as well as the rate of contraction and relaxation.
Similar results were observed in myocytes isolated from
transgenic mice expressing PICOT, and comparable hemodynamic results were obtained in whole hearts. Analysis of the Ca2⫹ transient in myocytes indicated that more
efficient Ca2⫹ uptake by the SR played a key role in
accelerating relaxation rate after PICOT gene transfer.
This increase in relaxation rate was accompanied by increased phosphorylation of phospholamban and increased myofilament Ca2⫹ sensitivity in these myocytes.
Thus PICOT has a direct influence on contractile function,
and evidence to date suggests steps in Ca2⫹ cycling are an
important PICOT targets within myocytes.
Future studies may be expected to provide additional
insight into the role played by PICOT in cardiac myocytes.
For example, the peak Ca2⫹ amplitude and Ca2⫹ release
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(962). While a phospho-specific antibody aimed at this site
failed to show significant changes in cTnI Ser-23/24 phosphorylation in response to 10 min of ET-1, this cluster was
significantly phosphorylated with 60 min of ET-1. The
ET-1-induced cTnI Ser-23/24 phosphorylation was
blocked by the PKC inhibitor bis-1 but not by propranolol
or the PKA antagonist H-89. Treatment of myocytes with
other PKC agonists, including phenylephrine and phorbol
12-myristate 13-acetate, for 1 h also increased cTnI Ser23/24 phosphorylation. Collectively, these results suggested there are temporal changes in PKC-dependent
cTnI Ser-23/24 phosphorylation in response to multiple
PKC agonists. This hypothesis is supported by other earlier in vitro studies (650, 653). In functional studies, longer
term (60 min) activation with ET-1 continued to enhance
peak shortening and relaxation rate, which were both
attenuated in cTnI Ser-23/24 Ala-expressing myocytes.
The ET-1 response with cTnI Ser-23/24Ala was comparable to results observed with ssTnI. The combined results
from a phospho-specific antibody and cTnI Ser-23/24Ala
mutant gene transfer indicated that this cluster accelerates relaxation during more prolonged PKC activation by
ET (962).
The role of Ser-43/45 phosphorylation in intact myocytes is less clear. Substitution with negatively charged
residues at Ser-43/45 to mimic exhaustive phosphorylation produced a decrease in maximum force and Ca2⫹
sensitivity in reconstituted, permeabilized, fiber bundles
and slowed maximum in vitro sliding velocity (851). The
decreased Ca2⫹ sensitivity was expected to accelerate
relaxation. However, relaxation was substantially slower
in cTnIAsp5 transgenic mice (781) in which Asp was substituted for all five phosphorylation sites (e.g., Ser-23/24,
Ser-43/45, Thr-144). In agreement with this finding, the
converse knock-in with Ala-substituted cTnI to form
cTnIAla5 increased relaxation rates in response to PKC
activation by ET-1. A reasonable conclusion to draw from
these results is that PKC-mediated cTnI phosphorylation
decreases relaxation rate. The mechanism for this slowing of relaxation is more likely due to the decrease in
unloaded shortening velocity, rather than the decrease in
myofilament Ca2⫹ sensitivity (92). Decreased cross-bridge
detachment rate could account for the reduced velocity,
and it would slow relaxation in intact myocytes. However,
full phosphorylation of Ser-43/45 is not likely representative of physiological conditions. Further studies are
needed to determine the role Ser-43/45 plays in modulating PKC-mediated alterations in contractile function in
intact myocytes. A key question remaining from these
studies is whether the influence of cTnI Ser-43/45 phosphorylation on cross-bridge cycling dominates the influence on myofilament Ca2⫹ sensitivity and exclusively
slows relaxation. Alternatively, submaximal Ser-43/45
phosphorylation may decrease Ca2⫹ sensitivity and accelerate relaxation in intact myocytes, while more extensive
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D. Gene Transfer of Protein Phosphatases
1. PP1 and inhibitor 1 and 2
PP1 is modulated by two inhibitor proteins, inhibitor
1 (I-1) and inhibitor 2 (I-2). I-1 is a target for both PKA and
PKC phosphorylation (207, 775), with PKA phosphorylating Thr-35. PKA-mediated I-1 phosphorylation on Thr-35
enhances I-1 binding to and inhibition of PP1 activity
(210). The decrease in PP1 activity results in slower dephosphorylation of target proteins such as PLN and contributes to the positive inotropic and lusitropic actions of
␤-adrenergic agonists (207). The PKC-dependent phosphorylation site on I-1 was initially localized to Ser-67
(775), but more recently it was shown to involve Ser-65 or
Thr-75 (756). Phosphorylation of Ser-67 by cdk-5 is
needed in addition to Ser-65 to prevent dephosphorylation of I-1 by PP1. PKC-mediated modulation of I-1 and
PP1 was worked out with a combination of gene transfer
and transgenic studies (81). In these studies, overexpression of PKC-␣ decreased peak shortening amplitude in
isolated adult rat myocytes, while dominant negative
PKC-␣ (dnPKC-␣) or knock-down of PKC-␣ in mice increased the amplitude of shortening. In contrast to PKAmediated PP1 inhibition, increased PKC-␣ expression increased PP1 activity via phosphorylation of I-1, which
decreased I-1 binding to PP1. This increase in PP1 activity
significantly decreased PLN phosphorylation, increased
SR Ca2⫹ load, and slowed Ca2⫹ decay in myocytes. Conversely, gene transfer of dnPKC-␣ decreased I-1 phosphorylation at the PKC site and increased the binding of
Physiol Rev • VOL
I-1 to PP1, which decreased PP1 activity. The decreased
PP1 activity subsequently increased PLN phosphorylation
which, in turn, accelerated Ca2⫹ decay.
I-2 is another modulator of PP1. In vivo delivery of I-2
prevents heart failure in cardiomyopathic hamsters, as
indicated by the restoration of fractional shortening and
reductions in chamber size (985). Gene transfer of I-2
increased cytosolic PP1C␣ without a change in cytosolic
PP1 activity and decreased microsomal PP1C␣ and PP1
activity, which resulted in increased PLN phosphorylation
at Ser-16. Heightened PLN phosphorylation would be expected to accelerate relaxation (985). AAV delivery of I-2
to these hamsters prolonged survival time, suggesting a
new treatment that does not require activating PKA.
2. Calcineurin
The protein phosphatase calcineurin, or protein
phosphatase 2B (PP2B), plays a significant role in myocardial hypertrophy (596, 1005). Adenoviral-mediated
gene transfer of constitutively active calcineurin into neonatal rat cardiac myocytes significantly increased cell size
and protected against apoptosis (165). The influences of
calcineurin could be blocked by gene delivery of dominant negative nuclear factor of activated T cells (NFAT),
which is a downstream transcription factor in the calcineurin hypertrophy pathway (916). These effects were
mediated in part via NFAT3 and the Akt-PKB pathway.
Gene transfer of the Cain/Cabin-1 inhibitory domain for
calcineurin (⌬Cain) significantly reduced acute pressure
overload-induced hypertrophy in rats (7 days; Ref. 164)
without changing the pressure gradient. Cardiac specific
expression of ⌬Cain in transgenic mice also reduced hypertrophy. Other phosphatases, such as MAPK phosphatase (MKP-1), have been shown to inhibit ERK1/2, JNK,
and p38 activation using gene transfer techniques (88).
The direct and indirect influence of these phosphatases
on contractile function activity has not been investigated
using gene transfer.
E. Gene Transfer and MAPK Signaling
Cardiac gene transfer approaches have been used to
investigate each of the three main signaling cascades
within the MAPK signaling cascades. Mitogenic growth
factors activate Erk1/2, while cellular stressors activate
the stress-activated protein kinases, which include JNK
and p38 MAPK (350, 847). Signaling for all three MAPKs is
modulated by a set of two upstream kinases. Initially, a
MAPKKK or MEKK is activated, followed by MAPKK or
MEK activation, which modulates a specific MAPK. Several in-depth reviews have discussed the hypertrophic,
apoptotic, and other transcriptional control aspects of
these signaling cascades (159, 722, 848).
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rate did not contribute to the increased amplitude and
rate of contraction, respectively. Thus other potential
targets influenced by PICOT expression remain to be
determined. It is also unclear whether the PICOT-mediated influence on contractile function results from the
direct influence on one or more PKC isoforms and/or
downstream signaling proteins (e.g., MAPKs; see below).
Originally, PICOT was shown to associate with PKC-␪
rather than the endogenous cardiac PKC isoforms (973).
However, PKC-␪ protein is not detectably expressed in
adult rat myocytes (714, 768), and PICOT gene transfer
into neonatal cardiac myocytes did not significantly
change basal phosphorylation of PKC isoforms (408). Increased phosphorylation of the major PKC isoforms, including ␣, ␧, and ␰ in response to the PKC agonists ET-1
or phenylephrine, were observed in adult myocytes, but
the influence of PICOT on cellular targets involved in the
contractile response has not been thoroughly investigated
in adult myocytes. Expression of PICOT after gene transfer also inhibited agonist (e.g., ET-1, phenylephrine) activation of MAPKs, ERK1/2, and JNK, as well as downstream proteins such as activator protein 1 (AP-1) and
nuclear factor kB (NF-␬B) (305, 408, 775).
Physiol Rev • VOL
response (491). In contrast, the decay of the Ca2⫹ transient slowed, and SERCA2 expression decreased 3 days
after gene transfer of activated MKK6 (comparable to
MKK6bE) into neonatal rat cardiac myocytes (16), presumably due to activation of p38␤. Diastolic Ca2⫹ levels
also increased significantly after gene transfer of this
MKK6 in electrically paced cells, and diastolic Ca2⫹ levels
were restored after dual gene transfer of the activated
MKK6 and SERCA. On the basis of these results, the
combined activation of p38␣ and -␤ is expected to decrease myocyte contractile function via multiple targets.
However, it is presently unclear whether their combined
influence is additive, and whether their dose and temporal
activation influence other Ca2⫹ cycling proteins. In addition, transgenic studies indicate p38 MAPK serves as a
negative feedback regulator of PKA-mediated increases in
contractile function (1011), yet a gene transfer approach
has not been used to investigate this feedback loop in the
absence of potential compensatory adaptations that may
develop in transgenic animals. More detailed studies of
p38-mediated influences on contractile function are likely
to provide insight into these critical remaining questions.
F. Myocardial Nitric Oxide Synthase Signaling
and Contractile Function
Extensive studies have focused on nitric oxide (NO)
signaling in the myocardium. Nitric oxide synthase (NOS)
activity is particularly important during pathophysiological conditions including myocardial ischemia, preconditioning, and heart failure. Aside from the conventional NO
downstream signaling paradigm, NO regulates cardiac ion
channels (i.e., DHPR and RyR) and other subcellular components through a direct signaling mechanism in which
cysteine-thiol residues are posttranslationally modified in
a process known as S-nitrosylation (791). NOS isoforms
NOS-1 (neuronal or nNOS), NOS-2 (inducible or iNOS),
and NOS-3 (endothelial or eNOS) are each capable of
being expressed in cardiac myocytes. These experiments
are challengeing as protein expression and activity depend on the physiological or pathophysiological state,
species-dependent regional expression in hearts, and cellular localization of each NOS within myocytes. While the
paracrine and autocrine influences of NOS, the downstream and feedback signaling pathways, and the role of
NOS in heart failure have been extensively reviewed elsewhere (32, 70, 804), few of these investigations have
utilized gene transfer to determine the direct influence of
NOS or S-nitrosylation on contractile function. The lack
of gene transfer studies may be due to the appearance
that NO signaling plays its most important role during
pathophysiological conditions and NOS influences several
types of cells residing in the heart. The majority of gene
transfer studies to date have investigated the direct ef-
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Currently, there is little evidence to demonstrate a
direct influence of ERK1/2 on cardiac myocyte contractile
function. Gene transfer has been used to demonstrate that
this MAPK is a downstream target of PKC-␣ and -␦ in
cardiac myocytes (80, 673), which in turn activate small
GTPase MEKKs, including RhoA, Rac, Ras, and c-Raf-1
(722, 849). One study also has shown that excitation/
contraction influences ERK1/2 activity (475). Specifically,
gene transfer of Kv4.3 channels into neonatal myocytes
shortened action potential duration by decreasing Ca2⫹
influx, which reduced ERK1/2 activation and other indices
of hypertrophy (475).
More gene transfer studies have focused on JNKs,
which are also referred to as stress-activated protein kinases or SAPKs. Cellular stresses such as ischemia activate the upstream MEKK1/3, which in turn activates
MEK4/7 in the JNK pathway, and two isoforms of JNK are
expressed in cardiac tissue (722). As with ERK1/2, studies
focused on the JNK pathway indicated this cascade primarily influences cardiac function via transcriptional regulation. For example, gene transfer of a dominant negative MEKK4 (SEK-1 KR), which activates both JNK and
p38, inhibited pressure overload-induced hypertrophy
(118). However, it is interesting to note that gene transfer
into neonatal rat myocytes of constitutively active MEK7
(MEK7D), which specifically activates JNKs, acted as a
negative modulator of connexin43 expression (685). Connexin43 is a key subunit of gap junctions (917), and the
cellular uncoupling observed in this study indicated a loss
of gap junctions, and cellular uncoupling caused premature death in a transgenic mouse model expressing
MEK7D.
The p38 MAPK has been the target of most gene
transfer studies within the MAPK family, and MEK3/4/6
are the immediate upstream activators of this MAPK.
Activation of p38 MAPK is observed in response to pathophysiological conditions, including increases in hemodynamic load or myocardial ischemia (67, 234). In contrast
to ERK and JNK, a few studies have assessed the direct
influence of p38 on contractile function (491). In earlier
work using gene transfer of the constitutively active
MEKKs, MKK3bE and MKK6bE (325, 742), MKK3bE or
downstream p38␣, each increased apoptosis, and a dominant negative p38␣ suppressed this effect (943). In contrast, MKK6bE increased hypertrophic responses in myocytes, which was similar to the results obtained with
downstream activation of p38␤. The hypertrophic effects
of MKK6bE were suppressed by dominant negative p38␤.
Increased p38 MAPK activation achieved 24 h after gene
transfer of upstream MKK3bE also decreased cardiac
myocyte peak shortening and diminished the rates of
contraction and relaxation via a decrease in myofilament
Ca2⫹ sensitivity (491). There were no detected changes in
cellular Ca2⫹ cycling, and TnI phosphorylation and cellular pH did not appear to be involved in this contractile
1621
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DAVIS ET AL.
fects of NOS on contractile function using NOS-2 or
NOS-3 gene transfer (detailed below), but, in lieu of recent reports using mouse transgenesis to demonstrate a
role of NOS isoforms and S-nitrosylation in Ca2⫹ homeostasis (198, 281), there is an additional avenue in the
NOS field primed for using a gene transfer approach to
further understand the role of NO-mediated posttranslational modifications in cardiac function.
1. NOS1 or neuronal NOS
2. NOS2 or inducible NOS signaling
Inducible NOS (NOS2) activity increases in response
to stress or pathophysiological conditions, such as myocardial infarction in humans (reviewed in Ref. 804). During heart failure, NOS2 is expressed by cardiac myocytes
(250). Increased NOS2 expression in failing hearts had
little influence on basal contractile function but significantly reduced the contractile response to ␤-AR stimulation (1017). The mechanism responsible for this effect
was postulated to result from influences of NOS2 on Ca2⫹
handling proteins, including the L-type Ca2⫹ channel
(DHPR) and the RyR (1017). In contrast to this negative
influence on ␤-AR modulation of contractile function during heart failure, NOS2 has been shown to contribute
significantly to the late phase of ischemic preconditioning. Preconditioning was first described by Murry et al.
(620) as a brief ischemic event followed by reperfusion
that provides protection against the development of contractile dysfunction during a later, more prolonged bout
of ischemia (70). Short-term NOS2 expression after gene
transfer protected against ischemia via activation of cyclooxygenase-2 (487). More significantly, NOS2 levels also
increased and the myocardium was protected against
myocardial infarction initiated 1–2 mo after direct cardiac
adenoviral gene delivery of NOS2 to mouse hearts compared with LacZ-injected controls (486). Gene delivery of
NOS2 without infarction did not cause any significant
changes in baseline contractile function, although baseline heart rate was significantly depressed in this study.
3. NOS3 or endothelial NOS
There is currently disagreement about the role for
NOS3 in modulating cardiac contractile function. Moderately increased levels are observed after viral-mediated
gene transfer of NOS3 to isolated rat myocytes, without
changes in NOS2 expression (734). This increase in NOS3
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Adenoviral delivery of NOS1 has focused on delivery
to the neuronal compartment and/or ganglia involved in
autonomic regulation of the heart. Gene transfer of this
synthase to mice lacking NOS1 demonstrated the critical
role for nitric oxide production in the regulation of heart
rate (154, 594).
significantly increased contractility and relaxation under
basal conditions. Basal and peak Ca2⫹ transients were
also increased in fura 2-loaded myocytes. There was a
slight increase in Akt phosphorylation, and the enhanced
contractile function was blocked by the PI3K inhibitors
LY294002 and wortmannin. On the basis of these observations, the direct effect of NOS3 was concluded to be
mediated via PI3K activation of the PKB/Akt pathway,
although the contractile targets for this pathway were not
identified. Conflicting results were observed 3– 4 days
after gene delivery of myocyte-specific NOS3 into a
knockout mouse model (107). Viral delivery of NOS3 with
a cardiac-specific ␣-MyHC promoter restored the increased systolic pressure and decreased relaxation rate
observed in NOS3 knockout mice back toward wild type,
and the enhanced ␤-AR-mediated contractile function observed with this knockout model also returned to wildtype levels. Similar results were observed in a NOS3
overexpression transgenic model (87). The basis for these
divergent outcomes is not known, but it may result from
the level of NOS3 expression or the reduced transfection
efficiency in the in vivo versus in vitro models. Alternatively, these differences may result from compensatory
adaptations in the whole animal compared with the myocyte, or from acute versus long-term influences of this
synthase in wild-type versus knockout mice.
Under pathophysiological conditions, cardiac myocyte-specific NOS3 overexpression improved left ventricular function after infarction (64, 406). Gene delivery of
NOS3 4 days before infarction significantly increased
myocyte expression of NOS3 and reduced the infarct size
after acute ischemia and reperfusion, although the effects
on contractile function were not measured (5). One week
after infarction, gene delivery of NOS3 reduced the increase in end-diastolic pressure and the ischemia-associated increases in heart weight, myocyte size, fibrotic lesions, and apoptotic cells (829). There were also significant decreases in proteins involved in oxidative stress.
Some of this beneficial effect may be related to NOS3
effects on cardiac-associated neural and endothelial compartments (253, 415, 440, 441, 776). Liposome-mediated
gene delivery of NOS3 also improved donor heart survival
after transplantation in a rabbit model (395). In this study
and in earlier work on gene therapy into the vasculature
(175, 922), several beneficial effects were due to actions
on endothelial cells. However, there was also significantly
reduced NF-␬B and associated apoptosis in myocytes. In
comparison, direct myocardial injection of NOS3 reportedly increased myocyte apoptosis (430), which suggests
that cross-talk and dose may be important for the beneficial influences of endothelial-derived NOS3 on myocytes.
Studies have also demonstrated that heat shock protein 90 (Hsp90) acts as a scaffolding protein for NOS3,
and it modulates Akt-mediated phosphorylation of Ser-
G. Other Signaling Proteins of Interest
1. Superoxide dismutase
Oxidative stress resulting from the generation of reactive oxygen species superoxide (O⫺
2 ) followed by peroxide production decreases cardiac function during periods of stunning and ischemia-reperfusion (70; for pathway, see Ref. 89). Signaling via this pathway is closely
linked to NOS signaling (520). Three isoforms of superoxide dismutase (SOD) catalyze the breakdown of superoxide into peroxide, including copper/zinc SOD (Cu/ZnSOD) in the cytoplasm, extracellular SOD (EC-SOD)
present outside the cell, and manganese SOD (Mn-SOD)
in the mitochondrial matrix. Catalase detoxifies the peroxide (1013).
Woo et al. (978) initially demonstrated that delivery
of EC-SOD and catalase together improved cardiac function after ischemia-reperfusion in mice. Later studies by
this group demonstrated that catalase alone also restored
cardiac function in response to ischemia-reperfusion
(1013). Cu/Zn-SOD was used in an attempt to protect
against doxorubicin cardiotoxicity in neonatal rat myocytes, although there were no significant beneficial effects
(3). Gene delivery of EC-SOD alleviated cardiac stunning
and improved cardiac function, as well as decreased infarct size following ischemia-reperfusion (485). However,
the major mechanism of action appeared to be mediated
via improved endothelial cell function derived from the
increased availability of NO and H2O2, which act to normalize vasodilatation (470). These effects do not appear
to be mediated by direct influences on contractile function in cardiac myocytes (382).
Modulation of Mn-SOD is critical for tolerance to
oxidative stress and attenuates ischemia-reperfusion injury (4). To determine whether Mn-SOD would prove to
Physiol Rev • VOL
be beneficial during cryoprotection of hearts prior to
transplantation, gene delivery of Mn-SOD was followed by
orthotopic transplantation into donor rats. After 4 days,
hearts were removed and contractile function measured
before and after 6 h of global ischemia and 1 h of reperfusion. Developed pressure, and max and min dP/dt were
significantly higher after Mn-SOD delivery than with LacZ
delivery. Similar improvement was previously observed
using liposome gene delivery (5). Gene delivery of eNOS
also restored contractile function to a similar extent,
although Mn-SOD and eNOS together did not have an
additive effect (6). Further work is needed to determine
whether these beneficial influences are mediated directly
through the myocardium or via endothelial function.
2. Other signaling targets
The role of several signaling proteins has been investigated using gene transfer, although relatively few investigators have directly examined the influence on cardiac
and/or cardiac myocyte contractile function. For example, potentially important signaling via protein kinase D
(PKD) has been studied using gene transfer. The influence
of PKD on downstream phosphorylation of HDAC5 (380)
and on contractile proteins (345), such as TnI and
MyBP-C, has been studied using this approach. PKC activation of PKD phosphorylates and exports HDAC5 from
the nucleus, and it plays a significant role in hypertrophy
(923). Addition of PKD to skinned cardiac myocytes directly decreased myofilament Ca2⫹ sensitivity, which
would be expected to accelerate relaxation. Thus this
kinase pathway may play a significant role in physiological and/or pathophysiological cardiac function, as it is
also a downstream target for PKC.
Examining the contractile effects of other signaling
proteins like TIMP1, I␬B, G protein-coupled receptors,
small GTPases, and annexin VI by a gene transfer approach has also been initiated. For instance, gene transfer
of TIMP1 reduced matrix metalloproteinase activity 6 wk
after left anterior descending artery occlusion, preserved
both systolic and diastolic function, and reduced myocardial fibrosis (407). Overexpression of I␬B also improved
end-diastolic and systolic function after infarction (894).
Gene transfer of small GTPase proteins, including cdc42
(626), rhoA (906), rac1 (708), gem (616), and cyclinD1/
CDK4/ras (870) has shown the importance of these proteins in transcriptional regulation and/or hypertrophic signaling within the myocardium. In addition to these transcription-based effects, viral-gene delivery of gem into
guinea pig cardiac myocytes also acted as a Ca2⫹ channel
blocker, which significantly shortened the action potential and decreased the rates of contraction and relaxation
(616). Annexin VI also was shown to modulate Ca2⫹
cycling via modulatory influences on RyR and NCX in a
transgenic model (303). Further investigation of these
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1177 and calcineurin-mediated dephosphorylation of Thr495 within eNOS (86). Interestingly, gene transfer of
Hsp90 via liposomes reduced infarct size and improved
end-diastolic pressure and regional contractile function
during ischemia-reperfusion injury in a pig model (461).
These effects were primarily mediated via the effects of
Hsp90 scaffolding within the vascular compartment, and
it is unclear whether a similar strategy to increase Hsp90
expression within myocytes would also prove beneficial.
Gene transfer of NOS3 into NOS3 null cardiac myocytes
also restored lipopolysaccharide activation of p38 MAPK
and downstream activation of tumor necrosis factor-␣,
suggesting NOS3 is an upstream modulator of this signaling cascade (680). These types of studies demonstrate the
potential insights to be gained from using gene transfer
approaches to understand clinically significant and complicated physiological and pathophysiologic cardiac conditions.
1623
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DAVIS ET AL.
proteins may provide deeper insight into the direct and
indirect roles of these signaling pathways in modulating
contractile function.
VII. FUTURE DIRECTIONS
ACKNOWLEDGMENTS
We thank Dr. Wally Koch and Dr. Susan Hamilton for
helpful comments on various versions of the manuscript. We
also thank Dr. L. Craig Davis and Lorretta Davis for their extensive editorial comments.
Physiol Rev • VOL
GRANTS
This review was supported by grants from the National
Institutes of Health, American Heart Association, and the Muscular Dystrophy Association.
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The era of gene transfer-based therapeutics for enhanced myocardial performance, which was only a promissory note less than a decade ago, is now operational and
in practice today. Results primarily from small mammal
studies have provided the underpinnings for translational
studies spanning larger mammals and even humans. Presently, there are several human clinical trials featuring
gene therapies for acquired and inherited heart diseases.
Where does the field of gene-based cardiac therapeutics
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therapies in general. Large-scale efforts currently underway are devoted to enhancing genetic vectors and will
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Scalable production of vectors is also a major issue. This
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to this list in the future. As a final comment, it is important
to consider the prospect of titratable or inducible genebased systems for the heart. Ideally, the design of highly
effective inducible therapeutic cardiac gene expression
cassettes would include low read through in healthy myocardium and robust induction in diseased heart. Ongoing
efforts towards developing chemical-mediated induction
or, even better, transcriptional machinery sensitive to the
changing ionic/energetic environment of the diseased cardiac myocyte could offer the proverbial “guardian angel”
for heart-directed therapeutic gene expression in the face
of emergent cardiac disease.
Address for reprint requests and other correspondence:
J. M. Metzger, Dept. of Integrative Biology and Physiology, Univ.
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M. V. Westfall, M. Blankinship, T. J. Herron, G. Guerrero-Serna,
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Designing Heart Performance by Gene Transfer

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