Role of Cardiac Myocyte CXCR4 Expression in Development and

Role of Cardiac Myocyte CXCR4 Expression in
Development and Left Ventricular Remodeling After Acute
Myocardial Infarction
Udit Agarwal, Wael Ghalayini, Feng Dong, Kristal Weber, Yong-Rui Zou, Sina Y. Rabbany,
Shahin Rafii, Marc S. Penn
Rationale: Stromal cell– derived factor (SDF)-1/CXCR4 axis has an instrumental role during cardiac development
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and has been shown to be a potential therapeutic target for optimizing ventricular remodeling after acute
myocardial infarction (AMI) and in ischemic cardiomyopathy. Although a therapeutic target, the specific role of
cardiac myocyte CXCR4 (CM-CXCR4) expression following cardiogenesis and survival of cardiac myocyte and
left ventricular remodeling after AMI is unknown.
Objective: We hypothesized that cardiac myocyte derived CXCR4 is critical for cardiac development, but it may
have no role in adulthood secondary to the short transient expression of SDF-1 and the delayed expression of
CM-CXCR4 following AMI. To address this issue, we developed congenital and conditional CM-CXCR4ⴚ/ⴚ
mouse models.
Methods and Results: Two strains of CM-CXCR4flox/flox mice were generated by crossing CXCR4flox/flox mice
with MCM-Creⴙ/ⴚ mouse and MLC2v-Creⴙ/ⴚ mouse on the C57BL/6J background, yielding CXCR4flox/flox
MCM-Creⴙ/ⴚ and CXCR4flox/floxMLC2v-Creⴙ/ⴚ mice. Studies demonstrated recombination in both models
congenitally in the MLC2v-Creⴙ/ⴚ mice and following tamoxifen administration in the MCM-Creⴙ/ⴚ mice.
Surprisingly the CXCR4flox/floxMLC2v-Creⴙ/ⴚ are viable, had normal cardiac function, and had no evidence of
ventricular septal defect. CXCR4flox/floxMCMⴙ/ⴚ treated with tamoxifen 2 weeks before AMI demonstrated 90%
decrease in cardiac CXCR4 expression 48 hours after AMI. Twenty-one days post AMI, echocardiography
revealed no statistically significant difference in the wall thickness, left ventricular dimensions or ejection
fraction (40.9ⴞ7.5 versus 34.4ⴞ2.6%) in CXCR4flox/flox mice versus CM-CXCR4ⴚ/ⴚ mice regardless of strategy
of Cre expression. No differences in vascular density (2369ⴞ131 versus 2471ⴞ126 vessels/mm2; CXCR4flox/flox
versus CM-CXCR4ⴚ/ⴚ mouse), infarct size, collagen content, or noninfarct zone cardiac myocyte size were
observed 21 days after AMI.
Conclusions: We conclude that cardiac myocyte– derived CXCR4 is not essential for cardiac development and,
potentially because of the mismatch in timings of peaks of SDF-1 and CXCR4, has no major role in ventricular
remodeling after AMI. (Circ Res. 2010;107:00-00.)
Key Words: stem cells 䡲 myocardial infarction 䡲 cardiogenesis
S
be important by multiple laboratories is the stromal cell–
derived factor (SDF)-1/CXCR4 signaling pathway.4 – 8 This
pathway has been implicated in stem cell survival following
transplantation, homing of bone marrow– derived and cardiac
progenitor stem cells to the infarct zone heart, and cardiac
myocyte survival during AMI and chronic heart failure. The
SDF-1/CXCR4 axis has also been shown to be critical in
cardiac development.9 –11 Although SDF-1/CXCR4 axis is a
tem cell– and gene transfer– based strategies are being
pursued in an attempt to decrease infarct size, optimize
ventricular remodeling, and prevent the onset of chronic heart
failure in patients following acute myocardial infarction
(AMI). Although benefits have been demonstrated in several
clinical trials and recent metaanalyses,1–3 the mechanism
responsible for the benefits seen are under investigation. One
potentially important pathway that has been demonstrated to
Original received January 3, 2010; resubmission received May 3, 2010; revised resubmission received July 6, 2010; accepted July 7, 2010. In June
2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
From the Skirball Laboratory for Cardiovascular Cellular Therapeutics, Departments of Cardiovascular Medicine and Stem Cell Biology and
Regenerative Medicine (U.A., W.G., F.D., K.W., M.S.P.); and Center for Cardiovascular Cell Therapy, Heart and Vascular Institute (M.S.P.), Cleveland
Clinic, Ohio; Department of Genetics Medicine (S.Y.R., S.R.) and Howard Hughes Medical Institute (S.R.), Weill Medical College of Cornell University,
New York; Department of Microbiology (Y.R.Z.), Columbia University, College of Physicians and Surgeons, New York; and Bioengineering Program
(S.Y.R.), Hofstra University, Hempstead, NY.
Correspondence to Marc S. Penn, MD, Departments of Cardiovascular Medicine and Stem Cell Biology, Cleveland Clinic, NE3 9500 Euclid Ave,
Cleveland, OH 44195. E-mail [email protected]
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.110.223289
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September 3, 2010
Non-standard Abbreviations and Acronyms
AMI
CXCR4
CM-CXCR4
EGFP
GFP
LAD
LV
LVED
MCM
MI
MLC2v
MSC
pc
SDF
acute myocardial infarction
CXC chemokine receptor-4
cardiac myocyte–specific CXCR4
enhanced green fluorescent protein
green fluorescent protein
left anterior descending artery
left ventricular
left ventricular end dimension
MerCreMer, tamoxifen-induced ␣-myosin heavy chain
driven Cre
myocardial infarction
ventricular myosin regulatory light chain-2
mesenchymal stem cell
postconception
stromal cell– derived factor
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therapeutic target of interest, its natural role in myocardial
repair in adulthood is less clear because SDF-1 expression is
immediate and transient and cardiac myocyte CXCR4 expression is delayed and persistent following AMI.12,13 More
recently, the hypothesis has been forwarded that the basis of
benefit associated with stem cell therapy following acute
myocardial infarction is the restoration of the temporal
alignment of SDF-1 and CXCR4.14,15
CXCR4, a G protein– coupled 7-transmembrane receptor,
together with its ligand SDF-1, can play a crucial role during
embryonic development and in maintaining the stem cell
niche, homing of stem cells at the site of injury, and
preservation of the injured tissue. During embryogenesis,
CXCR4 expression starts as early as blastocyst formation and
is expressed in various different stages of development and a
variety of cell types.16 The importance of CXCR4 during
development is evident in the CXCR4-null mouse. In these
mice, the deficiency of CXCR4 is lethal as the developing
embryo acquires various developmental anomalies including
defective hematopoiesis (␤-lymphopoiesis and myelopoiesis), neurogenesis (abnormal cortex formation), and angiogenesis and cardiogenesis (ventricular septal defect).9 –11 Cardiac neural crest participate in the formation of ventricular
septum during cardiogenesis and express CXCR417; however,
the contribution of cardiac myocyte derived CXCR4 in
ventricular formation is not yet recognized.
Following AMI, SDF-1 expression is elevated immediately
and peaks within 24 hours. However, cardiac myocyte CXCR4
expression occurs between 48 and 72 hours after AMI.12 This
physiological mismatch in the peak of the ligand and its receptor
has led various researchers to overexpress SDF-1 in infarcted
regions. The prolonged expression of SDF-1 in infarcted tissue
leads to reestablishment of stem cell homing, neoangiogenesis,
myocardial preservation, and increased ventricular function.18 –21
Although SDF-1/CXCR4 axis has been shown to be critical in
myocardial reparative process after myocardial infarction (MI),
the exact role of CXCR4 derived from cardiac myocytes in
ventricular remodeling is not known.
To address this question directly, we have developed a
congenital (MLC-2Vcre) and conditional (MCM-cre) deletion of
cardiac myocyte CXCR4 using the CXCR4flox/flox mouse. We
have characterized these mice before and after AMI.
Methods
An expanded Methods section is available in the Online Data
Supplement at http://circres.ahajournals.org and includes detailed
information regarding immunostaining, western blotting, echocardiography, left anterior descending artery (LAD) ligation, and
cardiac remodeling.
Mice Generation and In Vivo Analysis of
CXCR4 Deletion
Previous studies have reported the generation of mice having
CXCR4flox allele, in which exon 2 (2.2 kbp) is flanked by 2 loxP
sequences.22 We crossed CXCR4flox/flox mice with the mice bearing
a transgene of ␣-MyHC-MerCreMer (MCM) and MLC2v-Cre. The
progeny was crossbred to yield MCM⫹/⫺CXCR4flox/flox and
MLC2vCRE⫹CXCR4flox/flox mice. Tamoxifen (40 mg/kg) in corn
oil was administered IP in 6-week-old MCM⫹CXCR4flox/flox male
mouse continuously for 5 days. The genomic DNA from the hearts
of 8-week-old mice from both types was isolated and subjected to
PCR to estimate CXCR4 deletion. The location of the primers used
to detect CXCR4 deletion is depicted in Figure 1. The primers used
were as follows: forward, 5⬘-CACTACGCATGACTCGAAATG-3⬘;
reverse, 5⬘-CCTCGGAATGAAGAGATTATG-3⬘. All animals were
housed in an animal facility of Cleveland Clinic approved by the
Association for Assessment and Accreditation of Laboratory Animal
Care International, and all animal protocols were approved by
Animal Research Committee. All animals had C57Bl6/J background.
SDF-1 mRNA Expression
Three after LAD ligation, the heart were perfused with 10 mL of saline
and infarcted left ventricles were cut at a level just below the ligation.
The tissue was snap-frozen and stored in ⫺80°C until used. The RNA
was harvested using TRIzol and cDNA was synthesized using SuperScript VILO cDNA synthesis kit from Invitrogen. The real-time PCR
reaction for SDF-1 and 18S was carried out using TaqMan primers on
Applied Biosystems 7500 Real-Time PCR System.
Stem Cell Homing Studies
Mesenchymal stem cells (MSCs) were isolated and prepared as we
have previously described.12 The cells were incubated at 37°C.
Confluent cells were passaged and plated out at 1:2 to 1:3 dilutions
until passage 6. Cells were assayed for their ability to differentiate
into the adipogenic, chondrogenic, and osteogenic lineages. Cells
were maintained in differentiation media for 2 to 3 weeks. Differentiation was validated by staining the cells with Oil Red (adipogenic
lineage), Alcian blue (chondrogenic lineage), or alkaline phosphatase
(osteogenic lineage). Cells we induced to express green fluorescent
protein (GFP) using a lentiviral construct encoding enhanced green
fluorescent protein (EGFP) under the CMV promoter. Seven days
after transfection, GFP⫹ cells were sorted by FACS. Animals were
infused via the tail vein with 250 000 GFP⫹ MSCs 24 hours after
LAD ligation. Hearts were harvested 72 hours after MSC infusion
and prepared as described above for immunostaining with a primary
antibody against GFP. The number of GFP⫹ cells per high-power
fields was quantified in the infarct border zone as described before,12
by blinded observers across a minimum of 4 sections and 12 fields
obtained from the mid–left ventricle per animal.
Statistical Analysis
Ventricular function was analyzed with one way ANOVA. All
values with P⬍0.05 were considered significant.
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Cardiac Myocyte CXCR4 Expression
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Figure 1. Characterization of MLC2vCREⴙ/ⴚCXCR4flox/flox mouse.
A, Exon 2 of CXCR4 gene is flanked by loxP sites. The primers bind
before and after the loxP sites to detect cleaved CXCR4. B, A PCR
analysis on genomic DNA from tail and heart was performed on
8-week-old CXCR4flox/flox, MLC2v-Cre⫹/⫺CXCR4flox/flox, and MLC2vCre⫹/⫺CXCR4flox/⫹ mice. i and ii show the presence of CXCR4 floxed
allele and Cre into the genome, and iii shows deletion of CXCR4 from
ventricular homogenates and not from the tail of the same animal. C,
CXCR4 staining (red) and phase contrast pictures of neonatal cardiac
myocytes from CXCR4flox/flox and MLC2v-Cre⫹/⫺CXCR4flox/flox mice
shows deletion of CXCR4 from neonatal cardiac myocytes of MLC2vCre⫹/⫺CXCR4flox/flox mice. White arrows point toward the cardiac
myocytes, and the green arrow points toward noncardiac myocyte
cells in culture. D, Representative Doppler echocardiography image showing normal outflow and inflow during systole and diastole in
MLC2v-Cre⫹/⫺CXCR4flox/flox mouse without any evidence of a ventricular septal defect. E, Representative image of view through right
ventricle to reveal normal IVS in MLC2v-Cre⫹/⫺CXCR4flox/flox mouse. F, Circumferential and radial strain in MLC2v-Cre⫹/⫺CXCR4flox/flox
and CXCR4flox/flox mice showing no significant difference (n⫽4 in each group).
Results
Generation of Congenital Cardiac
Myocyte–Specific CXCR4-Deficient Mice
CXCR4 deficiency is embryonic lethal and is known to
exhibit defective hematopoiesis, neural development, and
ventricular septal defects. The ventricular septum begins to
form in mouse at day 11 and is complete by day 12.5
postconception (pc).23 MLC2v expression begins at day 9 pc
in the primitive heart tube.24 Therefore, to assess the role of
cardiac myocyte–specific CXCR4 expression in cardiac development, we generated a mouse with congenital deletion of
CXCR4 specifically in cardiac myocytes using the MLC2vCre–mediated expression. We successfully crossed MLC2vCre⫹/⫺ (kind gift from Kenneth Chien) with CXCR4flox/flox
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Table. Baseline LV Function Obtained by Echocardiographic Parameters of Mice Used in Studies
Congenital Deletion
Tamoxifen
flox/flox
CXCR4
⫹/⫺
MLC2vCre
Conditional Deletion
flox/flox
CXCR4
⫹/⫺
MCM
CXCR4flox/flox
MCM⫹/⫺ CXCR4flox/flox
End diastole (mm)
IVS
1.15⫾0.09
1.14⫾0.06
1.23⫾0.04
1.27⫾0.07
LVPW
1.12⫾0.08
1.01⫾0.05
1.12⫾0.06
1.08⫾0.06
LVED
2.77⫾0.09
2.76⫾0.15
2.70⫾0.20
2.66⫾0.10
IVS
1.82⫾0.08
1.93⫾0.11
1.96⫾0.07
1.71⫾0.07*
LVPW
1.67⫾0.05
1.65⫾0.07
1.72⫾0.09
1.73⫾0.04
LVED
1.37⫾0.14
1.10⫾0.14
1.10⫾0.22
1.41⫾0.06*
87.4⫾2.5
92.1⫾1.9
91.3⫾2.5
83.1⫾1.5*
End systole (mm)
Ejection fraction (%)
Values are expressed as means⫾SEM (n⫽7 in each group). *P⬍0.05. IVS indicates interventricular septum; LVPW, left ventricular
posterior wall; LVED, left ventricular end dimension.
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(kind gift from Yong-Rui Zou) and CXCR4flox/⫺ to generate
the MLC2v-Cre⫹/⫺CXCR4flox/⫺ mouse and MLC2v-CRE⫹/⫺
CXCR4flox/flox mouse, respectively. All these mice were viable
with litter sizes similar to that observed with MLC2v-Cre⫹/⫺.
The genotype was determined by genomic PCR from the
homogenate obtained from tails (Figure 1A and 1B). The
loxP sites surrounds the exon 2 of CXCR4 gene (encodes
⬎90% of protein), and to determine the deletion of exon 2 of
CXCR4 gene, we used the primers that bind before and after
the loxP sites. Successful recombination was observed from
the ventricular homogenate and not from the tail. Immunofluorescence for CXCR4 in neonatal cardiac myocytes in
culture showed an absence of CXCR4 staining in MLC2vCre⫹/⫺CXCR4flox/flox mice. Importantly, noncardiac myocytes, as defined by phase-contrast microscopy from the
myocardium of MLC2v-Cre⫹/⫺CXCR4flox/flox, remained
positive for CXCR4 expression (Figure 1C).
Based on echocardiography and autopsy, no detectable ventricular septal defect or valvular defects were observed (Figure
1D and 1E, respectively). Echocardiography further revealed
normal cardiac function in the MLC-2vCre⫹/⫺CXCR4flox/flox
mice compared to littermates that were CXCR4flox/flox or
CXCR4flox/⫺ but lacked MLC2vCre allele. The baseline function was within normal limits and shown in the Table. We also
observed no difference in myocardial contractility as measured
by myocardial strain imaging (radial strain, 23.4⫾13.3 versus
21.9⫾9.8; circumferential strain, ⫺11.1⫾3.9 versus ⫺9.8⫾4.1
in the presence and absence of MLC2v-Cre in CXCR4flox/flox or
CXCR4flox/⫺ mice (Figure 1F). These data suggest that cardiac
myocyte CXCR4 expression is not required for normal heart
development.
Generation of Conditionally Regulated Cardiac
Myocyte–Specific CXCR4-Deficient Mice
To determine the role of cardiac myocyte– derived CXCR4 in
ventricular remodeling after MI, we also generated the
conditional cardiac-specific CXCR4-deficient mice. We
crossed CXCR4flox/flox and CXCR4flox/⫺ mice with the mercremer (MCM⫹/⫺) mice (from The Jackson Laboratories) to
generate MCM⫹/⫺CXCR4flox/⫺ and MCM⫹/⫺CXCR4flox/flox
mice, respectively (Figure 2A, i and ii). To verify recombi-
nation, we quantified CXCR4 deletion by performing quantitative PCR on the mice treated daily with 40 mg/kg IP of
tamoxifen dissolved in corn oil for 2, 4, 6, and 8 days. Using
the same PCR strategy described above, we observed no
further deletion of CXCR4 following tamoxifen administration beyond 4 days (Figure 2B; Figure 2A, iii).
We have previously demonstrated that cardiac myocyte
CXCR4 expression is upregulated beginning 48 hours after
acute myocardial infarction.12,25 Therefore, to quantify the
degree of cardiac myocyte CXCR4 recombination, MCM⫹/⫺
CXCR4flox/flox and MCM⫹/⫺CXCR4flox/⫺ mice were treated
with tamoxifen for 5 days, allowed to recover cardiac
function for 14 days, and then underwent induction of AMI
by LAD ligation. Two days following AMI, immunohistochemistry demonstrated downregulation of cardiac myocyte
but not endothelial CXCR4 expression 48 hours after LAD
ligation in the tamoxifen-treated group (Figure 2C). Based on
Western blots of infarcted heart homogenates, we achieved
⬇90% reduction in myocardial CXCR4 protein compared to
littermates that did not receive tamoxifen (Figure 2D). The
baseline ventricular function before and 14 days after tamoxifen was within normal limits, as shown in the Table. There
were modest statistically significant differences in left ventricular end diastolic dimension and ejection fraction in
MCM⫹/⫺CXCR4flox/flox that received tamoxifen 2 weeks
before echocardiography compared to those that did not.
Effect of CXCR4 on Ventricular Remodeling and
Function Before and After MI
MCM⫹/⫺CXCR4flox/flox mice were treated with tamoxifen
(40 mg/kg, IP) for 5 days at 6 weeks of age. As recently
reported in studies with the MCM mouse, following tamoxifen administration, we observed a transient decrease in
cardiac function that resolved 10 to 14 days later26; therefore,
we did not perform any procedures on the MCM mice until
14 days after tamoxifen administration. Mice that did
(MCM⫹/⫺CXCR4flox/flox) or did not receive tamoxifen and
(MLC2vCRE⫹/⫺CXCR4flox/flox and CXCR4flox/flox) underwent LAD ligation to induce AMI, and their cardiac function
was monitored by echocardiography. We hypothesized that
because SDF-1 expression is transiently expressed for a short
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Cardiac Myocyte CXCR4 Expression
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Figure 2. Characterization of MCMⴙ/ⴚCXCR4flox/flox mouse. A, PCR analysis on genomic DNA from tail and heart was performed on
8-week-old CXCR4flox/flox, MCM⫹/⫺CXCR4flox/flox, and MCM⫹/⫺CXCR4flox/⫹ mice. i depicts a wild-type and floxed CXCR4 band; ii depicts
CRE band in the genome; and iii depicts deleted band for CXCR4, which was only detected after tamoxifen administration in the ventricular
homogenates and not from the tail. B, A dose–response curve for tamoxifen-induced recombination of CXCR4flox/flox based on quantitative
PCR shows maximum deletion was achieved after 4 days of tamoxifen administration. C, Representative images of immunofluorescence for
CXCR4 48 hours after AMI shows knockdown of CXCR4 in cardiac myocytes of tamoxifen-treated MCM⫹/⫺CXCR4flox/flox and endothelial
cells positive for CXCR4 (green). Delineated area in CM-CXCR4⫺/⫺ image is shown below at higher magnificent to aid in identification of
nucleated cardiac myocytes devoid of CXCR4 expression. D, Western blot analysis of CXCR4 48 hours after AMI on infarct region shows
⬇90% deletion of CXCR4. E, Representative M-mode recording of CXCR4flox/flox and MCM⫹/⫺CXCR4flox/flox after tamoxifen administration.
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Figure 3. Ventricular function assessment of cardiac myocyte–specific
CXCR4-null hearts after AMI. Echocardiographic analysis of 8- to 10-week-old
CXCR4flox/flox and cardiac-specific
CXCR4⫺/⫺ mice as a function of time
after AMI. A, Anterior wall thickness
(AWT) (mm). B through D, Posterior wall
thickness (PWT) (mm) (B), LV end-diastolic dimension (LVEDd) (mm) (C), and
ejection fraction (%) (D) at 0, 3, and 21
days after AMI (n⫽7 for CXCR4flox/flox
and n⫽12 for CM-CXCR4⫺/⫺ at each
time point). There was no significant difference between the measured parameters at days 3 and 21 after AMI. Data
represent means⫾SEM. E, Representative M-mode recording of CXCR4flox/flox
and cardiac-specific CXCR4⫺/⫺ mice 21
days after AMI.
period of time immediately after AMI and begins to return to
baseline by the time cardiac myocyte CXCR4 expression is
significantly upregulated, the deletion of cardiac myocyte
CXCR4 expression would not alter cardiac function or cardiac
remodeling. Consistent with our hypothesis, 3 days after AMI,
the ejection fraction, anterior wall thickness, and posterior wall
thickness of the cardiac myocyte–specific CXCR4⫺/⫺ mice
(MCM⫹/⫺CXCR4flox/flox and MLC2vCRE⫹/⫺CXCR4flox/flox)
were: ejection fraction, 57.8⫾5.0%; anterior wall thickness,
0.71⫾0.07 mm; and PW thickness, 0.92⫾0.10 mm, respectively; there were not statistically different from mice that did
not receive tamoxifen (ejection fraction, 50.1⫾5.0%; anterior
wall thickness, 0.73⫾0.08 mm; and PW thickness,
1.04⫾0.11 mm, respectively) (Figure 3A through 3F). Twentyone days after AMI, the ventricular function and remodeling was
not statistically different between the absence and presence of
cardiac myocyte CXCR4 expression: ejection fraction,
34.4⫾2.6% versus 40.9⫾7.5%; anterior wall thickness,
0.36⫾0.12 versus 0.32⫾0.11 mm; and PWT, 0.81⫾0.21 versus
0.83⫾0.04 mm, respectively (Figure 3A through 3E).
We evaluated vascular density 21 days after AMI to determine whether cardiac myocyte CXCR4 may have had any effect
on vascular density. Using isolectin staining (Figure 4), we
measured vascular density in the infarct borderline zone and
found similar levels of vascular density in the presence and
absence of cardiac myocyte CXCR4 (CM-CXCR4) expression
(2471⫾126 versus 2369⫾131 vessels/mm2, respectively; Figure
4A). We further quantified vascular density as vessels per
cardiac myocyte in the noninfarct zone 21 days after AMI
(Figure 4B). We observed no difference in vessels per myocyte
between groups (vessels/cardiac myocyte: 1.15⫾0.09 versus
1.18⫾0.09, CXCR4f/f versus CM-CXCR4⫺/⫺, n⫽5 animals per
group, P⫽0.61)
To further characterize the cardiac remodeling 21 days
after MI (Figure 5A through 5D) , we measured infarct size
21 days after LAD ligation using Masson’s trichrome stain
(Figure 5B). We found no statistically significant difference
in the infarct size of CXCR4flox/flox animals (47.93⫾4.42% of
the left ventricular [LV] area, n⫽3) compared to CMCXCR4⫺/⫺ animals (45.89⫾7.61% of the LV area, n⫽3).
We further characterized the response to MI in the noninfarct zone to determine whether cardiac myocyte CXCR4 had
a role in remodeling in the noninfarct zone. The interstitial
fibrosis within the noninfarct zone of the myocardium 21
days after MI (Figure 5C) was 0.34⫾0.11% versus
0.38⫾0.16% of the LV area and cardiac myocyte diameter in
the posterior wall remote from the infarct zone (Figure 5D)
was 12.34⫾0.80 versus 12.89⫾0.46 ␮m in CXCR4flox/flox
(n⫽4) compared to CM-CXCR4⫺/⫺ animals (n⫽4).
SDF-1␣ mRNA levels were analyzed 3 days after LAD
ligation by real-time PCR. We observed by real-time PCR that
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Figure 4. A, Representative photomicrographs of vascular density from
CXCR4flox/flox (left) and CM-CXCR4⫺/⫺
(right) mice 21 days after MI (green,
isolectin; red, wheat germ agglutinin;
blue, DAPI). Vascular density calculated
as vessels/mm2 (B) and vessels/cardiac
myocyte (C) in untreated CM-specific
CXCR4flox/flox (n⫽4) and treated
CM-specific CXCR4⫺/⫺ (n⫽6) mice
showed no statistically significant difference among the groups; P⬎0.05.
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there was a 33% decrease (P⫽0.08, n⫽4 per group) in SDF-1
mRNA expression in animals that were CM-CXCR4⫺/⫺ compared to CM-CXCR4⫹/⫹(Figure 6). To determine whether this
decrease in SDF-1 expression significantly altered stem cell
homing, we infused 250 000 GFP⫹ MSCs via tail vein 1 day
after AMI and euthanized animals 4 days after AMI (3 days after
MSC infusion).12 Immunofluorescence was used to quantify the
number of MSCs per millimeter squared of tissue and revealed
no significant differences in MSCs present (CXCR4flox/flox:
17.4⫾7.33 cells/mm2 versus CM-CXCR4⫺/⫺: 17.2⫾3.1 cells/
mm2; P⫽0.99, n⫽5 per group), suggesting that the degree of
change in SDF-1 expression in the CM-CXCR4⫺/⫺ is not
sufficient to alter stem cell homing to the heart.
Discussion
Because significant body of literature has been developed that
has demonstrated a critical role for the SDF-1/CXCR4 axis in
cardiac development10,27 and myocardial response to cell
therapy12,14,28,29 the goal of this study was to analyze the
importance of cardiac myocyte derived CXCR4 expression
during development and repair following AMI. To address
this question we generated a congenital cardiac deletion of
CXCR4 mouse and a conditional tamoxifen inducible cardiac
myocytes specific CXCR4 deletion mouse. We characterized
the baseline function of these animals and then studied the
myocardial response to injury in these mice.
Figure 5. A, Masson’s trichrome staining
of CXCR4flox/flox and CM-CXCR4⫺/⫺ on
mice paraffin-embedded heart sections
21 days after MI. B, Infarct size assessment in mice 21 days after LAD ligation.
C, Interstitial fibrosis in viable myocardium 21 days after MI. D, Cardiac myocyte diameter (microns) in posterior wall.
Data represent means⫾SD (n⫽4 per
group).
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Figure 6. A, Representative SDF-1␣
mRNA expression by real-time PCR
reaction at 3 days after MI. B, Quantification of quantitative PCR of myocardial
SDF-1 expression 3 days after MI. Data
represent means⫾SD (n⫽3 to 4 animals
in each group).
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Cardiac Myocyte–Derived CXCR4 in
Cardiac Development
SDF-1 and CXCR4 are ubiquitously expressed during embryogenesis. However, the timing and the stage specific
expression of the cytokine and ligand orchestrate the migration of stem cells and development of various organs.
CXCR4- and SDF-1– deficient mice present with similar
phenotypes and exhibits ventricular septal defect in the
membranous part of the septum. Studies have demonstrated
that CXCR4 is expressed in the outflow tract and descending
part of bulbus cordis by cardiac myocytes, endothelial cells
and cardiac neural crest cells during septum formation.30,31
However, the contribution of specific cell type expression of
CXCR4 during septum formation is not known. MLC2v is
expressed in the outflow tract at day 9 pc, before formation of
the septum which starts at or near day 11 pc. We chose
MLC2v as a promoter to regulate cre expression to specifically remove cardiac myocyte CXCR4 expression before
formation of the septum to determine whether the lack of
cardiac myocyte CXCR4 expression would alter cell patterning leading to the development of ventricular septal defect
formation. Our results demonstrate that the CXCR4 derived
from cardiac myocytes does not play a role in the formation of
ventricular septal defects observed in CXCR4⫺/⫺ mice. This
finding suggests that CXCR4 expression is necessary for the
patterning of cells for the heart and that this patterning occurs
before expression of proteins in the endocardial cushions and
muscular septum32 that define cardiac myocytes.
Cardiac Myocyte–Derived CXCR4 in
Myocardial Repair
We generated the conditional deletion of CXCR4 mouse
because we initially hypothesized that the congenital deletion
of cardiac myocyte CXCR4 would potentially be lethal or
result in cardiac anomalies or dysfunction. Therefore, we
wanted to allow normal cardiac development in the presence
of cardiac myocyte CXCR4 and then conditionally delete
cardiac myocyte CXCR4 following the administration of
tamoxifen.
Of note, as has been recently reported,26 tamoxifen administration results in a transient decrease in cardiac function in
the MCM mice. Consistent with these observations, we
observed significant degrees of cardiac dysfunction following
the administration of tamoxifen to the mercremer mouse that
resolved 14 days after the final dose. Thus, all our studies
commenced 14 days after the final dose of tamoxifen and in
animals with recovered cardiac function.
Our initial hypotheses were that the lack of cardiac
myocyte CXCR4 would prove lethal secondary to abnormal
cardiogenesis and at the very least ventricular septal defect
formation. Our findings with the MLV2v-Cre mouse would
suggest this hypothesis was false. We further hypothesized
that the absence of cardiac myocyte CXCR4 would not
adversely affect left ventricular remodeling or function following AMI because SDF-1 is rapidly and transiently expressed following AMI before cardiac myocyte CXCR4 is
upregulated. This hypothesis proved to be true.
Recent studies have demonstrated that MSCs with or
without SDF-1 overexpression have beneficial effects on
ventricular function after MI.16 Various mechanisms including stem cell homing, neoangiogenesis, and decreased myocytes death have been suggested. Studies have also demonstrated that SDF-1 overexpression initiates CXCR4 signaling
in hypoxic cardiac myocytes and induce antiapoptotic pathway by Akt phosphorylation.12 CXCR4 following AMI is
also involved in the recruitment of endothelial progenitor
cells and MSCs to the site of injury.28,33–35 Furthermore, the
role of SDF-1/CXCR4 signaling is questionable in recruitment and differentiation of cardiac stem cells into cardiac
myocytes.29
It is interesting that we observed a decrease in myocardial
SDF-1 expression in the CM-CXCR4⫺/⫺ mice. A recent
study in which adenovirus encoding CXCR4 was used to
constitutively overexpress CXCR4 in the myocardium before
inducing myocardial infarction demonstrated an increase in
cardiac SDF-1 expression.36 Thus, it would appear, based on
this study and our findings, that cardiac CXCR4 expression is
correlated with SDF-1 expression in response to AMI. Importantly, with respect to our findings, we did not observe a
significant difference in stem cell homing between hearts that
had cardiac myocyte CXCR4 expression and those that did
not. However, it is yet undefined and awaits future studies to
determine whether the myocardial response to stem cell
Agarwal et al
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engraftment will be blunted in the absence of cardiac myocyte CXCR4 expression.
The MCM⫹/⫺CXCR4flox/flox mouse developed for these
studies will serve as a tool to dissect out the mechanisms
responsible for the beneficial effects associated with the
administration of SDF-1. In particular, because baseline and
post-AMI cardiac function is the same in the presence and
absence of cardiac myocyte CXCR4 expression, differences
in response to stem cell therapy will be able to be attributed
to the absence of cardiac myocyte CXCR4. We will further be
able to determine the importance and relative contribution of
CXCR4 on strategies associated with enhancing cardiac
myocyte survival such as ischemic preconditioning and
growth factor delivery.15,37 In conclusion, deletion of cardiac
myocyte CXCR4 does not alter cardiac development or
function. Similarly, deletion of cardiac myocyte CXCR4
before AMI does not alter myocardial response to injury.
These data, taken together, would suggest no role for cardiac
myocyte CXCR4 expression in development, normal physiology, or response to injury. Future studies will need to
determine whether there is a role for cardiac myocyte CXCR4
expression in the modulation of the myocardial response to
injury mediated by SDF-1 and/or stem cell therapy.
Sources of Funding
This work was funded by the Skirball Foundation. W.G. was the
recipient of funding from the Fuad Jubran Center for Middle East
Medical Education.
Disclosures
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
None.
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Novelty and Significance
What Is Known?
●
●
●
SDF-1/CXCR4 axis is important in stem cell– based myocardial repair.
Myocardial SDF-1 expression begins to decline before cardiac
myocyte CXCR4 expression is upregulated after acute myocardial
infarction (MI).
CXCR4-deficient mice die in utero with various birth defects including
ventricular septal defect.
What New Information Does This Contribute?
●
●
Cardiac myocyte CXCR4 expression is not essential for heart
development.
CXCR4 deletion from adult cardiac myocytes does not affect ventricular remodeling after MI in the absence of stem cell treatment.
The SDF-1␣/CXCR4 axis plays a major role in the recruitment of
bone marrow– derived and cardiac stem cells to the infarct zone
after acute myocardial infarction. Recent data suggest that
cardiac myocytes upregulate CXCR4 after MI and that extended
SDF-1 expression may increase cardiac myocyte survival.
Assessing these effects is complicated by the fact that CXCR4deficient mice die of various birth defects in utero. Thus, it
becomes critical to dissect out the role cardiac myocyte– derived
CXCR4 (CM-CXCR4) plays after MI and during heart development. To investigate these issues, we generated congenital and
conditional cardiac myocyte–specific CXCR4-deficient mouse
models. We found that congenital CM-CXCR4 deletion is not
essential for heart development and ventricular remodeling after
AMI. These are novel findings and have been reported for the
first time in literature. Moreover, the mouse models we have
generated in our laboratory will be useful for investigating the
contribution of CM-CXCR4 to improved ventricular remodeling
observed in response to stem cells delivery.
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Role of Cardiac Myocyte CXCR4 Expression in Development and Left Ventricular
Remodeling After Acute Myocardial Infarction
Udit Agarwal, Wael Ghalayini, Feng Dong, Kristal Weber, Yong-Rui Zou, Sina Y. Rabbany,
Shahin Rafii and Marc S. Penn
Circ Res. published online July 15, 2010;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Role of cardiac myocyte CXCR4 expression in development and left ventricular
remodeling after acute myocardial infarction
Udit Agarwal, MD1
Wael Ghalayini, MD1
Feng Dong, MD, PhD1
Kristal Weber, BS1
Yong-Rui Zou, PhD5
Sina Y. Rabbany, PhD3,4
Shahin Rafii, MD, PhD3,6
Marc S. Penn, MD,PhD1,2
1
Skirball Laboratory for Cardiovascular Cellular Therapeutics, Departments of
Cardiovascular Medicine and Stem Cell Biology and Regenerative Medicine, Cleveland
Clinic, Cleveland, OH
2
Center for Cardiovascular Cell Therapy, Heart and Vascular Institute, Cleveland Clinic,
Cleveland OH
3
Department of Genetics Medicine, Weill Medical College of Cornell University, New
York, NY
4
Bioengineering Program, Hofstra University, Hempstead, NY 11549
5
Department of Microbiology, Columbia University, College of Physicians and Surgeons
6
Howard Hughes Medical Institute, Weill Medical College of Cornell University, New
York, NY
Key words:
Stem Cells, Myocardial infarction, Cardiogenesis
Running Header: Cardiac myocyte CXCR4 expression
Word Count: 1018
Corresponding Author:
Marc S Penn MD, PhD
Departments of Cardiovascular Medicine and Stem Cell Biology
Cleveland Clinic, NE3
9500 Euclid Avenue
Cleveland,OH 44195, USA
Email: [email protected]
Ph:+1-216-445-1932
Fax: +1-866-299-7071
Materials and Methods :
Immunostaining: Mixed cellular population (containing cardiac myocytes, endothelial cells and
fibroblast cells) of Neonatal heart was isolated from the pups (1-3 days old) of MLC2vCRE+/CXCR4flox/flox parents with Neomyts Kit (Cellutron Life Technology Cat # nc-6031) and plated on
the 35mm plate. The cells were cultured for 24 hours and then fixed in 2% paraformaldehyde,
blocked with 1% BSA,1% donkey serum solution and then stained for CXCR4 (primary antibody
– 1:100 from abcam Catalog#7199 overnight at 4°C, secondary 1:1000- anti rabbit 594 from
abcam for 1 hour at room temperature).
Immunostaining in tissue was performed on the hearts harvested 48 hours post MI and 21 days
post MI from MCM+/-CXCR4flox/flox mice with and without tamoxifen treatment. The hearts were
fixed in formalin and embedded in paraffin blocks. 6μm sections were made and stained as
described1. Forty eight hours post AMI tissue were stained for CXCR4 (Primary - NB100-74396
(Novus Biologicals), secondary- donkey anti rabbit IgG Alexa Fluor 488 from molecular probes)
and 21 days post AMI tissue was stained to calculate vessel density with isolectin (Cat # FL1201 from Vectorlabs) and wheat germ agglutin to quantify cardiac myocyte density (Cat #RL1022 from Vectorlabs).
Western Blotting - Western blotting was done on the 48 hours post infarcted tissue
homogenate of CXCR4flox/flox and MCM+/-CXCR4flox/flox mice (with tamoxifen administration) as
described1. Briefly, the infarcted tissue was homogenized in PBS with 0.1% Triton X-100
supplemented with PMSF (100 mM), leupeptin (10 µg/ml) and aprotinin (10 µg/ml). Total protein
(50 μg) from each sample was prepared in 4x laemmli buffer (200 mM Tris HCl (pH 6.8), 8%
SDS, 0.1% bromophenol blue, 40% glycerol), subjected to 10% SDS PAGE and transferred on
PVDF membrane. The blot was finally probed with primary antibody (1:500 in 5% milk in
1xTBST) against CXCR4 (Abcam, cat# 2074) followed by incubation with peroxidaseconjugated anti-mouse secondary antibody (1:4000 in 5% milk in 1xTBST). Chemiluminescence
(Amersham Biosciences) was used to visualize the bands. Microtek scanner was used to scan
the blots and and the density of the bands was analysed using the NIH software, Image J.
Cardiac remodeling – Mice were anesthetized and their hearts were perfusion fixed with 10%
phosphate-buffered formalin under normal pressure 21 days after LAD ligation. The hearts were
embedded in paraffin and 5µm sections were cut from the apex to the level just below ligation
and alternating sections were stained with Masson trichrome as described2. 5X images were
taken to measure infarct size. Interstitial fibrosis was measured using the viable posterior wall of
non infracted ventricle. Cardiac myocyte diameter was measured along the shortest diameter
across the nucleus. 60 such readings were taken. All parameters were measured using ImagePro Plus (Media Cybernetics). Formula used for calculating infarct size - %infarct size =
(epicardial infarct length/ total epicardial circumference) X 100.
Echocardiography: Baseline 2D-echocardiography was performed on MLC2v-Cre+/CXCR4flox/flox , MCM+/-CXCR4flox/flox and CXCR4 flox/flox mice at 6 weeks of age using 15MHz linear
array transducer interfaced with a Sequoia C256 and GE vision 7 as described previously3.
Fourteen days post tamoxifen administration echocardiography was performed on MCM+/CXCR4flox/flox mice to determine the baseline function. Doppler and myocardial straining analysis
was done on MLC2v-Cre+/-CXCR4flox/flox mice at 6 weeks of age. Echocardiography was also
performed 3 and 21 days post AMI.
EF was calculated as the (LVEDareaLVESarea)/LVEDarea x 100 where the LVESarea and the LVEDarea are the the end systolic
and end diastolic areas of the left ventricle obtained in the parasternal long axis view.40, 41
Left Anterior Descending (LAD) artery ligation: LAD ligation was performed on 8-10 weeks
old MLC2vCRE+/-CXCR4flox/flox , MCM+/-CXCR4flox/flox (post tamoxifen injection) and CXCR4flox/flox
mice as previously described.4 Breifly, the animals were anesthetized with Xylazine/ketamine,
intubated and ventilated with room air at 105 breaths per minute using a rodent ventilator
(Harvard Apparatus). Sternotomy was performed and LAD was identified with the help of
surgical microscope (Leica M500). LAD was ligated by using 7-0 prolene. Immediate blanching
and anterior wall dysfunction revealed a successful ligation. The chest and skin were closed
using 6-0 prolene. The animals were removed from the ventilator and kept under oxygen until
they recover from anesthesia. Only the animals which survived first 24 hours of ligation were
considered for the study.
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