Thatte-Somah-CIRC

Development and Evaluation of a Novel Solution, Somah, for the Procurement
and Preservation of Beating and Nonbeating Donor Hearts for Transplantation
Hemant S. Thatte, Laki Rousou, Bader E. Hussaini, Xiu-Gui Lu, Patrick R. Treanor
and Shukri F. Khuri
Circulation published online Oct 12, 2009;
DOI: 10.1161/CIRCULATIONAHA.108.808907
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Transplantation
Development and Evaluation of a Novel Solution, Somah,
for the Procurement and Preservation of Beating and
Nonbeating Donor Hearts for Transplantation
Hemant S. Thatte, MSc, PhD; Laki Rousou, MS, MD; Bader E. Hussaini, MD, MPH;
Xiu-Gui Lu, MD; Patrick R. Treanor, CCP, LCP; Shukri F. Khuri, MD†
Background—Injury to myocytes, endocardium, and the coronary endothelium during harvesting and storage can
compromise outcomes after heart transplantation. Safeguarding of structure and function of cardiomyocytes and
endothelium in donor hearts may lead to improved patient survival after transplantation. Information gained from
porcine hearts stored in standard transplant solution was used to design a superior preservation solution that would
optimally protect and maintain organs from beating heart and/or nonbeating heart donors during long-term storage.
Methods and Results—Multiphoton microscopy was used to image deep within cardiac biopsies and coronary artery tissue
harvested from porcine hearts obtained from beating heart and nonbeating heart donors for analysis of myocyte and
endothelial cell structure and function. Cell structural integrity and viability, calcium mobilization, and nitric oxide
generation were determined with fluorescence viability markers, immunofluorescence, and Western blots. During
hypothermic storage in standard preservation solution, Celsior, myocyte, and endothelial viability was markedly
attenuated in hearts obtained from beating heart donors. In contrast, hearts from beating and nonbeating heart donors
stored in the newly formulated Somah solution demonstrated an increase in high-energy phosphate levels, protection of
cardiac myocyte viability, mitochondrial membrane polarization, and structural proteins. Similarly, coronary artery
endothelial organization and function, calcium mobilization, and nitric oxide generation were well maintained during
temporal storage in Somah.
Conclusions—The Celsior preservation solution in clinical use today has led to a profound decline in cardiomyocyte and
endothelial cell viability, whereas the newly designed Somah solution has safeguarded myocyte and endothelial integrity
and function during organ storage. Use of Somah as a storage medium may lead to optimized graft function and
long-term patient survival after transplantation. (Circulation. 2009;120:1704-1713.)
Key Words: endothelium 䡲 myocytes 䡲 nitric oxide synthase 䡲 transplantation
I
n the face of advances in transplantation, postoperative
outcomes have improved only moderately in the past
decade.1 This is due perhaps to increased acceptance of older
recipients with morbidities and, as a result of persistent organ
shortage, the inclusion of older, marginal, and nonbeating
heart organ sources in the donor pool.2 Use of organs from
marginal and older donors has resulted in decreased graft
function and survival compared with organs obtained and
used for transplantation from younger donors in previous
years.3 Preserving organ viability during storage is an important prerequisite for successful posttransplantation long-term
outcome. Most transplantation centers use cold static storage
in either extracellular- or intracellular-type storage solutions
to preserve organs, with variable outcomes.
Clinical Perspective on p 1713
The conventional preservation strategy for explanted hearts
involves storage of the organ in hyperkalemic, electrolyte
solutions that contain impermeants to maintain osmoregulation and control edema, reactive oxygen species scavengers to
prevent free radical damage, substrates to maintain energy,
and buffers to prevent acidosis. University of Wisconsin
Solution,4 Celsoir,5 Histidine-Tryptophan-Ketoglutarate Solution,6 and other storage solutions2 were designed to potentially counterbalance these adverse effects. However, damage
to cardiomyocytes and the endothelium by hypothermia,
hyperkalemia, edema, energy depletion, and reperfusion injury remains a problem.2,7 To offset the negative effects
associated with hyperkalemia-induced depolarized arrest and
Received July 22, 2008; accepted August 19, 2009.
From the Cardiothoracic Division, Department of Surgery, Harvard Medical School, VA Boston Healthcare System and Brigham and Women’s
Hospital, Boston, Mass.
†Deceased.
Guest Editor for this article was Robert A. Kloner, MD, PhD.
Correspondence to Hemant S. Thatte, MSc, PhD, Department of Surgery (112), VA Boston Healthcare System, 1400 VFW Pkwy, West Roxbury, MA
02132. E-mail [email protected]
© 2009 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.108.808907
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Thatte et al
New Solution to Preserve Hearts for Transplantation
storage, the concept of polarized or hyperpolarized arrest and
storage has been introduced and is being tested through the
design of new solutions that contain adenosine, ATPsensitive potassium channel agonists, and/or sodium channel
blockers to potentially prevent ionic imbalance and consequent energy depletion and edema during storage.7
We have previously demonstrated that our surgical conduit
storage solution, GALA, is able to preserve the endothelial and
smooth muscle function in ex vivo–stored vascular conduits for
an extended period.8 Therefore, using a similar strategy and
GALA as the foundation, we have formulated a novel organ
storage solution, Somah (ambrosia of rejuvenation in Sanskrit),
to counterbalance many of the impairing factors encountered by
the heart and other organs during storage. We hypothesized that
Somah solution will provide optimum cell protection and thus
preserve the structure and function of cardiac myocytes and the
coronary endothelium not only during short-term hypothermic
storage but also for a longer duration. Our objective was to
evaluate the comparative efficacy of Somah solution with the
commonly used organ preservation solution, Celsior, a solution
shown to be equivalent and/or superior2,5 to other marketed
solutions used in transplantation.
Methods
Materials
Celsior was purchased from Genzyme Corp (Cambridge, Mass). Creatine orotate was obtained from Man Sports Products Inc (San Diego,
Calif), and micronized creatine monohydrate and citrulline malate were
purchased from True Protein Inc (Oceanside, Calif). Diaminofluorescein AM ester (DAF-2/DA) was purchased from Calbiochem (La Jolla,
Calif). Calcein AM ester, ethidium homodimer (live-dead assay kit),
JC-1 mitochondrial polarization dye, and calcium orange were obtained
from Molecular Probes Inc (Eugene, Ore).
Animal Protocol
Heart Storage
The study was conducted in 9 female pigs, each weighing 40 to 50
kg, in accordance with a protocol approved by our Animal Studies
Subcommittee. The animals were divided into 3 groups of 3 animals.
Two different solutions were compared for their ex vivo preservation
of the heart: Celsior, the solution currently used in organ transplantation, and the newly formulated Somah solution. In groups 1 and 2,
hearts were harvested and immediately stored in Celsior (Celsior
beating heart [BH]) and Somah solution (Somah BH), respectively,
for 4 hours at 4°C. In group 3, hearts were harvested 1 hour after
death and then stored in Somah solution for 4 hours at 4°C (Somah
nonbeating heart [NBH]). Cardiomyocyte biopsies were obtained
from the anterior and posterior regions of the left ventricle at 0, 60,
120, 180, and 240 minutes. Left anterior descending coronary artery
(LAD) samples were collected from the hearts at the end of storage
time (240 minutes).
Surgical Procedure
General anesthesia was induced with intramuscular injections of
telazol 4 to 6 mg/kg and xylazine 2 mg/kg. After intubation, animals
were maintained with isoflurane (1% to 2%) inhalation anesthetic
with 100% oxygen and mechanically ventilated. The ear vein and
femoral artery were cannulated for intravenous access and blood
pressure monitoring, respectively. External ECG leads were placed,
and the chest was opened via a median sternotomy. The pericardium
was opened and elevated with 2-0 silk sutures to the skin. In
experimental groups 1 and 2, systemic heparinization with 300
mg/kg was achieved through the arterial line. Ten minutes after
heparinization, a 9F aortic vent cannula was placed in the aortic root
for infusion of cardioplegia. The aorta was cross-clamped, and 1 L
Table 1.
1705
Composition of Somah Organ Preservation Solution
Concentration
Components
mmol/L
Distilled water, L
g/L
1.00
Calcium chloride
1.30
0.191
Potassium chloride
7.00
0.522
Potassium phosphate (monobasic)
0.44
0.060
Magnesium chloride (hexahydrate)
0.50
0.101
0.50
0.123
Magnesium sulfate (heptahydrate)
Sodium chloride
125.00
7.31
Sodium bicarbonate
5.00
0.420
Sodium phosphate (dibasic; heptahydrate)
0.19
0.05
D-Glucose
11.00
1.982
Glutathione (reduced)
1.50
0.461
Ascorbic acid
1.00
0.176
L-Arginine
5.00
1.073
L-Citrulline
1.00
0.175
Adenosine
malate
2.00
0.534
Creatine orotate
0.50
0.274
Creatine monohydrate
2.00
0.298
L-Carnosine
10.00
2.26
L-Carnitine
10.00
2.00
0.50
0.075
Dichloroacetate
Insulin 10 mg/mL, mL/L
1.00
pH is adjusted to 7.5 with sodium bicarbonate or Tris-hydroxymethyl
aminomethane at desired temperature.
cardioplegia (4°C) was infused rapidly with a pressure bag at a
system pressure of 150 mm Hg (coronary pressure, 60 to 70 mm Hg)
in all 3 groups. The cardioplegia solutions were rapidly vented
through the inferior vena cava and left superior pulmonary vein. The
hearts of group 1 were arrested and flushed by rapid infusion of
Celsior until the hearts became uniformly pale and the effluent was
relatively clear. In contrast, hearts of pigs in group 2 were initially
perfused with 500 mL of 15 mmol KCl Somah cardioplegia solution,
followed by a washout with 500 mL of 7 mmol KCl Somah storage
solution. In group 3, the animals were prepared to the point of
application of the aortic cross-clamp as described above. The
animals were quickly exsanguinated through the superior vena cava
until cardiac arrest. One hour postmortem, the aorta was crossclamped and cannulated as above, and the hearts were rapidly
perfused with 7 mmol ice-cold KCl Somah storage solution (4°C).
The hearts from all 3 groups were quickly harvested, stored in
Celsior or Somah solution at 4°C, and transported to the laboratory
for evaluation.
Formulation of Somah Solution
Somah was formulated with the GALA solution8 as the base. GALA
is a physiological salt solution that has been shown to effectively
preserve the blood vessel endothelium8 but lacks key ingredients to
sustain complex organs (heart, liver, kidney, etc) over long-term ex
vivo storage. GALA was modified by changing the concentration of
some of its ingredients and by including additional key components
to formulate Somah solution (Table 1). The solution was freshly
made and chilled to 4°C, and the pH was adjusted to 7.5 with
Tris-hydroxymethyl aminomethane. The composition of Celsior
solution used in this study is given in Table 2.
Cell Viability Assay
Structural and functional viability of cardiomyocytes and endothelial
cells was assessed with a fluorescence-based live-dead assay and
multiphoton microscopy as described.8 –11
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Table 2.
Composition of Celsior
Concentration
Components
mmol/L
g/L
Mannitol
60.00
10.93
Lactobionic acid
80.00
28.66
Glutamic acid
20.00
2.94
Histidine
30.00
4.65
0.25
0.04
15.00
1.12
Calcium chloride
Potassium chloride
Magnesium chloride
Sodium hydroxide
Glutathione SH
13.00
2.64
100.00
4.00
3.00
0.92
o
pH 7.3 at 20 C.
mol/L ice-cold perchloric acid and homogenized for 30 seconds. The
homogenate was centrifuged at 1970g for 10 minutes at 0°C. An
aliquot of the supernatant was neutralized with an equal volume of
0.4 mol/L ice-cold KHCO3 solution and centrifuged as mentioned
above. The supernatant was stored at ⫺80°C for ATP and CP
measurements. The pellet was dissolved in an equal volume of 0.1
mol/L NaOH, centrifuged as above, and used for protein assay. ATP
and CP were measured with a bioluminescent assay kit and spectrofluorometer (PerkinElmer, Waltham, Mass) according to the
manufacturer’s protocol.
Calcium Mobilization and Generation of
Nitric Oxide
Bradykinin-induced calcium mobilization, eNOS activation, and
nitric oxide production in the endothelium of LAD were measured
with calcium orange dye and DAF-2/DA, respectively, with multiphoton microscopy, and the fluorescence data were normalized as
described.8 –11
Measurement of Esterase Activity
Multiphoton Microscopy
Conversion of Calcein AM ester (nonfluorescent) to fluorescent
Calcein by esterases in living cells was used as a marker of esterase
activity in the heart tissues. The enzyme activity was measured and
quantified as described.10,11
Imaging and semiquantitative fluorescence measurements were performed with a BioRad MRC 1024ES multiphoton imaging system
(BioRad, Hercules, Calif) coupled with a MaiTai laser (SpectraPhysics, Mountain View, Calif) tuned to 790 to 820 nm in the
transmission and epifluorescence mode as described.8 –11 The luminal endothelium and myocytes were identified by XYZ scanning and
imaged at depths of 50 ␮m in artery cross sections and 50 to 100 ␮m
in left ventricular biopsies from the site of excision.8 –11
JC-1 Assay: Evaluation of Mitochondrial
Membrane Potential
Cardiac myocytes and LAD segments were labeled with JC-1
membrane potential dye and imaged with multiphoton microscopy.
Fluorescence was excited at 790 nm and emission was measured at
520 and 585 nm with narrow bandpass filters. JC-1 forms
j-aggregates (red) in polarized mitochondria but remains dissociated
into monomers (green) in the cytoplasm in depolarized mitochondria.12 The ratio of red to green fluorescence (polarized to depolarized) was estimated with MetaMorph software. To reduce intersample and intrasample variability, the subsequent fluorescence
values were normalized to the values obtained at the start of the
experiments for each sample.
Immunofluorescence
Cardiomyocyte biopsy sections were labeled with primary anti–
myosin heavy chain, anti–myosin light chain, anti-actinin, anti-actin,
and anti–troponin C. LAD sections were labeled with caveolin 3,
endothelial nitric oxide synthase (eNOS), and von Willebrand factor
(vWF) antibodies as described.11
Western Blotting
Protein Extraction
Left ventricular tissue or LAD sample (20 mg) was suspended in
extraction buffer containing a protease inhibitor cocktail. Tissue was
homogenized for 30 seconds and centrifuged at 16 100g for 10
minutes, and the supernatant was collected. Equal amounts of total
protein (50 ␮g) from different samples were mixed with Laemmli
sample buffer containing 5% ␤-mercaptoethanol and heated at
100°C for 3 minutes.
Electrophoresis
Proteins were resolved on 7.5%, 10%, or 12% SDS-PAGE and
electroblotted onto the nitrocellulose membrane. Proteins were
identified through the use of antibodies (anti–myosin heavy and light
chain, anti-actinin, anti-actin, and anti–troponin C for cardiac tissue;
anti– caveolin 1, anti-eNOS, and anti-vWF for LAD samples) as
described.11
ATP and Creatine Phosphate Assay
ATP and creatine phosphate (CP) were measured in tissue extracts
from Somah BH and NBH as described.13 In short, 20 mg heart
tissue, cut into 300 small pieces, was suspended in 500 mL of 0.4
Statistical Analyses
The measurements and data extraction were performed in a blinded
fashion. The analyses were focused on comparing the Celsior BH
(n⫽3) with the Somah BH (n⫽3) and Somah NBH (n⫽3) groups to
evaluate the ability of preservation solutions to maintain viability of
the hearts. The esterase activity differences between groups were
evaluated by a linear mixed model to account for the potential
correlation between repeated measurements over time. Mitochondrial polarization was compared as a ratio change from baseline
between the 3 groups. Differences between groups were analyzed
with 1-way ANOVA, and the Tukey test was used to identify
specific differences between groups. A 2-sample t test was used to
evaluate the differences in normalized fluorescence intensity of
calcium mobilization and nitric oxide production at 10 minutes
between the Celsior BH and Somah BH and between the Celsior BH
and Somah NBH groups, respectively. A paired t test was used to
assess the difference in ATP and CP values between time 0 and 240
minutes in the Somah BH and Somah NBH groups, respectively.
Statistical significance was accepted at the 95% confidence level
(P⬍0.05). All values used were mean⫾SD unless otherwise indicated. All analyses were performed with SAS statistical software
version 9.1(SAS Institute, Cary, NC).
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Results
Cardiomyocyte Viability in Explanted Hearts
The effect of preservation solutions on myocyte structural
viability in donor hearts was investigated. As shown in Figure 1A,
myocytes obtained from all 3 groups showed robust green
fluorescence at the start of storage. However, within 60
minutes, Celsior BH myocytes began to exhibit a red fluorescence pattern and a simultaneous decrease in green fluorescence, indicating deterioration of myocyte structure. In
contrast, both Somah BH and Somah NBH demonstrated
minimal red fluorescence and a robust increase in green
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Thatte et al
New Solution to Preserve Hearts for Transplantation
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Figure 1. Viability assessment of stored hearts. A, Green fluorescence indicates cell viability; red fluorescence, compromised cells.
Extensive cell membrane damage was observed within 60 minutes of storage in Celsior. In contrast, myocytes remained progressively
viable in Somah BH and Somah NBH. Representative image of independent porcine experiments; magnification ⫻320. B, Viability of
cardiomyocytes was quantified by measuring green fluorescence. Celsior BH demonstrated temporally variable esterase activity. This
activity remained robust and increased significantly in a time-dependent manner in both Somah BH and Somah NBH. Each bar represents the mean⫾SEM of values for each group/time point.
fluorescence during 4 hours of storage, indicating viability
and recovery of the cardiomyocytes (Figure 1A).
Functional viability of cardiomyocytes was estimated by
measuring the quantum yield of Calcein green fluorescence
as a measure of esterase activity in living cells (Figure 1B).
The green fluorescence in the tissue decreased temporally in
Celsior BH but remained robust and increased with time in
both Somah BH and Somah NBH. Time-adjusted esterase
activity in Celsior BH, Somah BH, and Somah NBH was
99.03, 162.80, and 177.50 (arbitrary units), respectively. Both
Somah BH and Somah NBH groups had significantly higher
esterase activity compared with the Celsior BH group
(P⫽0.0012 and P⫽0.0004, respectively).
Polarization State of Mitochondria
in Cardiomyocytes
Functionality of the cardiomyocytes was further evaluated by
assessing the polarization state of the mitochondria. The
mitochondria in tissue samples from all 3 groups remained
initially polarized, as shown by the fluorescence pattern (red
fluorescence more than green fluorescence; Figure 2A).
However, in Celsior BH, the mitochondria depolarized within
60 minutes of storage. Mitochondrial function deteriorated
progressively with time, with a substantial attenuation of total
fluorescence (red and green) at the end of storage. In contrast, in
both Somah BH and Somah NBH, mitochondria remained
polarized during the time of storage (Figure 2B). These results
were confirmed by estimation of the mitochondrial membrane
polarization ratio (polarized to depolarized) in these samples. As
shown in Figure 2B, mitochondria in Celsior BH depolarized
progressively during the course of the experiment and remained
depolarized. In contrast, mitochondria in hearts stored in Somah
solution remained polarized with significant increases in polarization with time of storage (Figure 2B).
Structural and Contractile Components
of Cardiomyocytes
The immunofluorescence patterns of myosin, actin, actinin, and
troponin were not different between the 3 groups (Figure 3).
However, the immunofluorescence intensity of these la-
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Figure 2. Mitochondrial polarization in stored hearts. A, Mitochondria were polarized in all heart groups at the start of storage. Mitochondria progressively depolarized in Celsior BH (red fluorescence less than green fluorescence) but remained polarized in Somah BH
and Somah NBH during storage. Magnification ⫻320. B, Mitochondria in Celsior BH progressively depolarized. Mitochondria in Somah
and Somah NBH showed significant increases in polarization. Each bar represents the mean⫾SEM of measurement/time point;
*P⬍0.05, Celsior BH vs Somah BH; **P⬍0.05, Celsior vs Somah NBH.
beled proteins appeared to decrease in Celsior BH, indicating a potential loss or degradation during storage. In
contrast, Somah BH and Somah NBH showed robust
fluorescence labeling of these proteins, indicating preservation of the structural components.
We observed a sizable decrease in myosin, actinin, actin,
and troponin C and a substantial loss in myosin light chain
proteins in extracts from Celsior BH (Figure 4A). In contrast,
structural and contractile proteins were well preserved,
clearly resolved, and well labeled in cardiomyocyte extracts
from Somah BH (Figure 4A) and Somah NBH over the entire
course of storage (Figure 4B), confirming our observations
with immunofluorescence (Figure 3).
Viability of Coronary Arteries
The LAD obtained from the Celsior BH showed thinning of
the endothelial cells and substantial disruption of the smooth
muscle medial layer in the artery (Figure 5A). In contrast, the
endothelium and smooth muscle cells were well preserved,
intact, and morphologically typical in the LAD obtained from
Somah BH and Somah NBH (Figure 5A).
Viability of the coronary arteries was assessed in a manner
similar to that used for cardiomyocytes mentioned above.
Substantial membrane damage and extensive cell death were
observed in LAD from the Celsior BH, demonstrated by red
fluorescence and decreased green fluorescence. Additionally,
myocytes in the medial region also showed sizable damage,
characteristic of altered permeability of smooth muscle membranes (Figure 5B). In contrast, both endothelial and smooth
muscle cells exhibited robust green fluorescence, indicating
preservation of structural integrity, in Somah BH and Somah
NBH (Figure 5B). Similarly, esterase activity and mitochondrial polarization remained robust in LAD from Somah BH
and Somah NBH; in contrast, both these activities were
compromised in Celsior BH (not shown).
Caveolin, eNOS, vWF, and cadherin, components of endothelial cells, were identified with immunofluorescence in
the LADs (Figure 5C). In Celsior BH, LAD samples showed
noticeable disruption of caveolin and vWF and a decreased
labeling of eNOS and cadherin in the endothelium, with
potential attenuation of cell function, including nitric oxide
production, vasomotor regulation, and cell adhesion. In con-
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Thatte et al
New Solution to Preserve Hearts for Transplantation
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Figure 3. Immunofluorescence identification of structural and contractile proteins.
Myocyte proteins in left ventricular biopsies were labeled with fluorescent antibodies and imaged with multiphoton
microscopy. Myosin, actinin, actin, and
troponin C fluorescence labeling was
attenuated in cardiomyocytes from Celsior BH; Somah BH and Somah NBH
showed robust labeling of these proteins.
Magnification ⫻320.
trast, these proteins were robustly labeled and well preserved
in the LAD segments obtained from Somah BH and Somah
NBH (Figure 5C). Similarly, protein extracts of arteries from
Celsior BH showed complete absence of high-molecularweight vWF and a decrease in extractable eNOS and caveolin
proteins (Figure 6). However, these proteins are well preserved in LAD from Somah BH and Somah NBH, thus
substantiating our immunofluorescence results (Figure 5C).
Calcium Mobilization and Generation of Nitric
Oxide in Coronary Arteries
Bradykinin stimulation of LAD resulted in a minimal change
in calcium and nitric oxide fluorescence in Celsior BH
(Figure 7A and 7B), indicating attenuated eNOS activity and
potentially depressed vasomotor function. In contrast, a
significant, time-dependent 2.5- to 3-fold increase in endothelial calcium and nitric oxide fluorescence was observed in
LAD from both Somah BH and Somah NBH (Figure 7A and
7B). These results show that both the tonic- and agonistresponsive components of eNOS were fully functional in the
artery endothelium of these hearts, very similar to our
observation in freshly harvested porcine LAD.10 Normalized
fluorescence intensity for calcium mobilization and nitric
oxide generation at 10 minutes was significantly higher in the
Somah BH than in the Celsior BH group (2.51⫾0.64 versus
0.85⫾0.14, P⫽0.026; and 2.37⫾0.25 versus 1.04⫾0.19,
P⫽0.013, respectively). Similarly, the Somah NBH group
showed greater calcium mobilization and nitric oxide production compared with the Celsior BH group (2.63⫾1.70 versus
0.85⫾0.14, P⫽0.046; and 1.70⫾0.2 versus 1.04⫾0.1,
P⫽0.042, respectively).
Measurement of High-Energy Phosphates in
Somah Hearts
Figure 4. Contractile components in the cardiomyocytes. A,
Myofibrillar protein patterns from anterior (A) and posterior (P)
biopsies from the left ventricle demonstrated substantial difference between the heart groups. Celsior BH showed a sizable
decrease in myosin, actinin, actin, and troponin C and a substantial loss in myosin light chain (LC) proteins. These proteins
were clearly resolved and well preserved in extracts from
Somah BH and Somah NBH (B). Representative blot from n⫽3.
HC indicates heavy chain.
ATP and CP concentrations of Somah BH and Somah NBH
are given in Table 3. Interestingly, the low temperature of
storage notwithstanding, the hearts stored in Somah solution
were able to synthesize ATP and CP and thus increase their
concentrations (Table 3). ATP levels increased by 35% and
CP levels increased by 60% in the Somah BH. Similarly,
ATP levels increased by 75% and CP levels by 243% in the
Somah NBH during 4 hours of storage.
Discussion
The objective of this study was to find a solution for extended
temporal, extracorporeal preservation, and revitalization of
organs on implantation from beating and nonbeating heart
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Figure 5. Evaluation of structural integrity
of coronary arteries. A, Thinning of endothelium and disruption of smooth muscle
layers was observed in Celsior BH. Endothelium and smooth muscle layers were
well maintained in Somah BH and Somah
NBH. B, Live-dead assay. Extensive cell
membrane damage was observed in endothelial and smooth muscle cells in the
Celsior BH. Cell viability was well preserved in Somah BH and Somah NBH. C,
Celsior BH showed disruption in the
labeling pattern of caveolin and vWF and
decreased staining of eNOS and cadherin
in the endothelium. These proteins
showed robust, smooth, uniform labeling
in Somah BH and Somah NBH. Magnification ⫻320.
donors. We hypothesized that the loss of structural and
functional integrity in hearts stored in standard storage
solutions can be avoided by developing a synergistic physiological salt solution (Somah) that combines various energy
substrates, metabolic modulators, free radical scavengers and
antioxidants, ammonia chelators and nitric oxide synthase
substrates, intracellular and extracellular H⫹ chelators, and a
physiological concentration of calcium. We postulated that
optimizing these components would prevent edema, ionic
imbalance, and energy depletion and provide a favorable
environment and cellular support during ex vivo storage and
during reperfusion. The protection of structure and function
of cardiomyocytes and the endothelium that we observed in
BH and NBH donor hearts stored in Somah confirms this
hypothesis (Figures 1 through 7 and Table 3).
Formulation of Somah solution met the energy requirements of cardiomyocytes and coronary endothelium, accen-
Figure 6. Structural and functional components of the endothelium in stored hearts. There was no vWF protein immunoreactivity and a 20% and 35% decrease in eNOS and caveolin proteins in artery extracts from the Celsior BH. vWF, eNOS,
caveolin were well preserved in Somah BH and Somah NBH.
Representative blot from n⫽3.
tuating the cardioprotective mechanisms during hypothermic
and hypoxic storage. The components of Somah also prime
the organs with substrates and metabolites during storage to
facilitate resumption of biochemical, physiological, and mechanical work on reperfusion after transplantation. Preloading
the organs with selective synergistic constituents helps counterbalance the detrimental effects of initial hyperoxic state
and allows an uneventful transition to normoxic functional
state after transplantation.
The preservation of anaerobic metabolism in organs during
hypoxic storage is of critical importance. High-energy phosphates generated through the glycolytic pathway are crucial
in maintaining the functional state of cell membranes, during
periods of hypoxia and/or ischemia, and during normoxia in
the working heart.14 Maintenance of membrane polarity,
activity of electrogenic Na/K ATPase, calcium uptake by
storage organelles, ionic homeostasis, and membrane asymmetry is crucially dependent on ATP generated via the
glycolytic pathway.15,16 A relatively small amount of glycolytic ATP is essential to maintain homeostasis and to prevent
irreversible damage to the organ during hypoxic storage. Our
results show that Somah solution clearly provides these
metabolic requirements (Table 3).
Insulin translocates glucose transporters to the cell surface,
facilitating glucose uptake, and synthesis of glycolytic ATP.15–19
Insulin also stimulates intermediary pathways of metabolism
via phosphorylation, activating glucose influx, glycogen synthesis, and growth.15 These activities stimulated by insulin in
Somah solution were further accentuated by the addition of
dichloroacetate, which exerts pluripotent effects by stimulating glucose metabolism and reducing intracellular lactate
concentration by accelerating its conversion to pyruvate,
facilitating the production of alkali by the conversion of
carbon dioxide to bicarbonate.20 –22 Thus, dichloroacetate
simultaneously enhances both ATP generation and the buffering capacity of the tissue during storage.
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Thatte et al
New Solution to Preserve Hearts for Transplantation
1711
Figure 7. Calcium mobilization and nitric oxide generation in coronary arteries. A, Calcium (red) and nitric oxide (green) fluorescence
was attenuated in LAD in Celsior BH. Robust calcium and nitric oxide fluorescence was observed in Somah BH and Somah NBH. Magnification ⫻320. B, The integrated calcium and nitric oxide fluorescence intensity in the endothelium was quantified at 10 minutes after
bradykinin stimulation. Each time point was normalized to the fluorescence intensity measured at time 0 minutes (baseline⫽1). Each
bar represents the mean⫾SEM of 15 measurements for each group.
The normal oxygen tension of arterial blood is
⬇100 mm Hg (2⫻10⫺4 mol/L23,24). Therefore, although the
supply of oxygen is diminished in excised hearts, mitochondria need only minute amounts of oxygen (0.05 mm Hg; 10⫺7
mol/L) for an operational oxidative phosphorylation.14,23,25
However, once a critical anoxic state is reached, decreased
mitochondrial oxygen tension leads to membrane depolarization, release of cytochrome C, and induction of apoptosis.15
Interestingly, the concentration of dissolved oxygen in Somah
solution was 140 mm Hg (2.8⫻10⫺4 mol/L) at 4°C. We
speculate that this oxygen concentration was sufficient to
maintain the mitochondrial respiration and oxidative phosphorylation in the tissue during storage. Our data corroborate
Table 3.
this observation in that the mitochondria in the cardiomyocytes from both the Somah BH and Somah NBH remained
polarized compared with those stored in Celsior (Figure 2).
Furthermore, both the ATP and CP concentrations increased
significantly in hearts preserved in Somah (Table 3).
Creatine in Somah solution may play a multifactorial role
in the heart. It is involved in energy metabolism, protein
synthesis, membrane stabilization, and intracellular buffering; acts as an antioxidant; and prevents heart failure.26 The
orotate form of creatine was included to facilitate long-term
generation of ATP, with orotic acid forming an important
constituent of phospholipids and RNA, the building blocks of
tissue repair and synthesis. Orotic acid is ergogenic, provides
ATP and CP Concentrations in Somah-Stored Porcine Hearts
ATP, mmol/mg Protein
Treatment
CP, mmol/mg Protein
0 min
240 min
P*
0 min
240 min
P*
Somah BH (n⫽3)
0.20⫾0.10
0.27⫾010
0.011
0.25⫾0.10
0.40⫾0.11
0.034
Somah NBH (n⫽3)
0.40⫾0.05
0.70⫾0.20
0.147
0.70⫾0.20
2.40⫾0.97
0.159
*Paired t test.
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1712
Circulation
October 27, 2009
a pyrimidine intermediate in the generation of ATP via the
pentose-phosphate shunt, and enhances glucose uptake and
ATP synthesis.27,28
Carnosine, levocarnitine, and citrulline malate formed
important constituents of Somah solution. Carnosine, composed of ␤-alanine and L-histidine, provides cardioprotection
by intracellular buffering and calcium regulation and serves
as an antioxidant, antiglycating, aldehyde-quenching, metalchelating agent.29,30 Levocarnitine (L-carnitine) facilitates
fatty acid translocation across the mitochondria, enhances
glucose/carbohydrate metabolism during hypoxia/ischemia,31
stimulates anaerobic ATP generation required for maintenance of membrane function during organ storage,32 and acts
as an antioxidant during reperfusion. Citrulline mediates in
urea and nitric oxide cycle. Combined with malate, it has
been used in antiasthenic treatment33 and been shown to
reduce fatigue and improve muscle performance by increasing ATP production. It can also reduce lactic acid and
ammonia and increase recovery of ATP and CP.34,35 Additional organ-specific, synergistic effects of the components of
Somah include the breakdown and/or synthesis products of
carnosine, citrulline malate and creatine orotate, into
␤-alanine, histidine, malate, and fumarate, which enter the
Kreb cycle to generate additional ATP. Subsequently, histidine is also converted to glutamine, which then chelates
ammonia (from the transaminase reactions) to form carbamoyl phosphate, augmenting the nitric oxide cycle.
Adenosine in Somah solution is a precursor of ATP
synthesis and exerts cardioprotective effects via vasodilation,
catecholamine antagonism, and preconditioning by activating
protein kinase C and mitochondrial potassium channels.7,15,36
Adenosine also maintains the heart in a polarized and/or
hyperpolarized state, thereby contributing to the reduction in
ionic imbalance and associated degenerative changes in the
stored heart.7,36
Edema in the transplanted organ is a major clinical problem that impairs the proper functioning of the organs. Storage
solutions used in current practice have not addressed the role
of activation of hemichannels and/or aquaporins in the heart
in the development of edema during organ storage.37–39
Conditions during storage may lead to opening of hemichannels and aquaporins, thereby disrupting ionic homeostasis and
excessively increasing water uptake and cell volume.37 To
compensate for increased volume and to avoid rupture, the
cells rearrange the cytoskeleton, causing cell stiffness.37
Edema and stiffness can lead to organ failure after transplantation. Specific components of Somah solution, including a
physiological concentration of calcium (Table 1), address
these issues and thus prevent any of these detrimental
changes from taking place in the stored hearts.
Long-term outcomes of transplanted hearts are limited by
the development of cardiac allograft vasculopathy resulting
from immunologic and nonimmunologic risk factors precipitated by injury to and dysfunction of the endothelium.40
Therefore, the structural and functional viability of endothelium in coronary vasculature during storage is critical to the
long-term survival of a transplanted heart. Our results show
that in contrast to Celsior, the LAD endothelium is not injured
and remains functionally viable in both groups of Somah
hearts (Figures 4 through 6). Similarly, the physiological
processes of calcium mobilization, activation of eNOS, and
nitric oxide generation are also well preserved (Figure 7). We
have previously shown that these protective effects on endothelium and smooth muscle cells are a result of synergism of
properties of glutathione, ascorbic acid, and arginine in
vessels preserved in GALA,8 which also holds true for Somah
solution (Table 1).
Currently, numerous patients are awaiting cardiac (organ)
transplantation in the United States because of a lack of donor
organs or because of the age criterion. Of those patients who
receive heart transplants, survival at 5 years after transplantation is 68%.1–3 The majority of deaths are due to the
accelerated vasculopathy that occurs after cardiac transplantation. However, significantly increasing the storage time,
improving the condition of donor hearts/organs during storage, and allowing the use of NBH donors will have a
profound impact on the current landscape of transplant
surgery. We believe that replacing current preservation solutions with Somah would immediately affect the practice of
transplant surgery. Studies are ongoing to evaluate long-term,
in vitro preservation of organs in Somah to translate our
experimental observations into a clinical reality.
Acknowledgments
We thank Alona Birjinuik for data extraction, Dr Errol Baker for
statistical analysis, and Dr Maryrose Sullivan for editorial comments.
We also acknowledge Aditi Thatte for her encouragement and Mary
Beth Shertick, Marion Rapoza, and Nancy Healey for administrative
support.
Sources of Funding
This work was supported by grants from DoD/ONR (Dr Thatte)
and VA Medical Research Service (Drs Khuri and Thatte). Dr
Rousou was supported by the T32 National Institutes of Health
training grant.
Disclosures
None.
References
1. Ceka JM. The OPTN/UNOS Renal Transplant Registry 2003. Clin
Transpl. 2003;1–12.
2. Maathuis MJ, Leuvenink HGD, Ploeg RJ. Perspectives in organ preservation. Transplantation. 2004;83:1289 –1298.
3. Alexander JW, Zola JC. Expanding the donor pool: use of marginal
donors for solid organ transplantation. Clin Transplant. 1996;10:1–19.
4. Southard JH, Belzer FO. Organ preservation. Annu Rev Med. 1995;46:
235–247.
5. Menasché P, Termignon JL, Pradier F, Grousset C, Mouas C, Alberici G,
Weiss M, Piwnica A Experimental evaluation of Celsior, a new heart
preservation solution. Eur J Cardiothorac Surg 1994;8:207–213.
6. Bretschneider HJ. Myocardial protection. Thorac Cardiovasc Surg. 1980;
28:295–302.
7. Chambers DJ, Hearse DJ. Development in cardioprotection: polarized
arrest as an alternative to depolarized arrest. Ann Thorac Surg. 1999;68:
1960 –1966.
8. Thatte HS, Biswas KS, Najjar SF, Rhee JH, McGarry T, Birjiniuk V,
Crittenden MD, Michel T, Khuri SF. Multi-photon microscopic evaluation of saphenous vein endothelium and its preservation with a new
solution, GALA. Ann Thorac Surg. 2003;75:1145–1152.
9. Biswas K, Thatte H, Najjar S, Rhee JH, Birjiniuk V, Crittenden M,
Michel T, Khuri S. Multi-photon microscopy in the evaluation of human
saphenous vein. J Surg Res. 2001;95:37– 43.
Downloaded from circ.ahajournals.org by on October 13, 2009
Thatte et al
New Solution to Preserve Hearts for Transplantation
10. Dygert JH, Thatte HS, Kumbhani DJ, Najjar SF, Treanor PR, Khuri SF.
Intracoronary shunt induced endothelial cell damage in porcine heart.
J Surg Res. 2006;131:168 –174.
11. Rousou L, Taylor K, Lu XG, Crittenden M, Haime M, Khuri SF, Thatte
HS. Saphenous vein conduits harvested by endoscopic technique exhibit
structural and functional damage. Ann Thorac Surg. 2009;87:62–70.
12. Chen LB, Smiley ST. Probing mitochondrial membrane potential in
living cells by a J-aggregate forming dye. In: Mason WT, ed. Fluorescent
and Luminescent Probes for Biological Activity. New York, NY:
Academic Press Inc; 1993:124 –132.
13. Bessho M, Ohsuzu F, Yanagida S, Sakata N, Aosaki N, Tajima T,
Nakamura H. Different extractability of creatine phosphate and ATP from
cardiac muscle with ethanol and perchloric acid solution. Anal Biochem.
1991;192:117–124.
14. Weiss JN, Lamp ST. Glycolysis preferentially inhibits ATP-sensitive K⫹
channel in isolated guinea pig cardiac myocytes. Science. 1987;238:
67–70.
15. Opie LH, Lopaschuk GD. Fuels: aerobic and anaerobic metabolism. In:
Opie LH, ed. Heart Physiology: From Cell to Circulation. 4th ed. Philadelphia, Pa: Lippincott, Williams and Wilkins; 2004:306 –354.
16. Apstein CS, Gravino FN, Haudenschild CC. Determinants of a protective
effect of glucose and insulin on the ischemic myocardium: effect of
contractile function, diastolic compliance, metabolism, and ultra structure
during ischemia and reperfusion. Circ Res. 1983;52:515–526.
17. Lopaschuk GD, Stanley WC. Glucose metabolism in the ischemic heart.
Circulation. 1997;95:313–315.
18. Young LH, Renfu Y, Russell R, Hu X, Caplan M, Ren J, Shulman GI,
Sinusas AJ. Low flow ischemia leads to translocation of canine heart
GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo.
Circulation. 1997;95:414 – 422.
19. Russell RR III, Cline GW, Guthrie PH, Goodwin GW, Shulman GI,
Taegtmeyer H. Regulation of exogenous and endogenous glucose metabolism by insulin and acetoacetate in the isolated working rat heart: a
three-tracer study of glycolysis, glycogen metabolism, and glucose oxidation. J Clin Invest. 1997;100:2892–2899.
20. Stacpoole PW, Harman EM, Curry SH, Baumgartner TG, Misbin RI.
Treatment of lactic acidosis with dichloroacetate. N Engl J Med. 1983;
309:390 –396.
21. Smolenski RT, Amrani M, Jayakumar J, Jagodzinski P, Gray CC,
Goodwin AT, Sammut IA, Yacoub MH. Pyruvate/dichloroacetate supply
during reperfusion accelerates recovery of cardiac energetics and
improves mechanical function following cardioplegic arrest. Eur J Cardiothorac Surg. 2001;19:865– 872.
22. Stacpoole PW, Nagaraja NV, Hutson AD. Efficacy of dichloroacetate as
a lactate-lowering drug. J Clin Pharmacol. 2003;43:683– 691.
1713
23. Feigl EO. Coronary physiology. Physiol Rev. 1983;63:1–205.
24. Opie LH, Heusch G. Oxygen supply: coronary flow. In: Opie LH, ed.
Heart Physiology: From Cell to Circulation. 4th ed. Philadelphia, Pa:
Lippincott, Williams and Wilkins; 2004:279 –305.
25. Brown GC. Control of respiration and ATP synthesis in mammalian
mitochondria and cells. Biochem J. 1992;284:1–13.
26. Persky AM, Brazeau GA. Clinical pharmacology of the dietary supplement creatine monohydrate. Pharmacol Rev. 2001;53:161–176.
27. Ingwall JS. ATP and the Heart. Boston, Mass: Kluwer Academic Publishers; 2002.
28. Richards SM, Conyers RAJ, Fisher JL, Rosenfeldt FL. Cardioprotection
by orotic acid: metabolism and mechanism of action. J Mol Cell Cardiol.
1997;29:3239 –3250.
29. Stvolinsky SL, Dobrota D. Anti-ischemic activity of carnosine. Biochemistry (Mosc). 2000;65:849 – 855.
30. Zaloga GP, Roberts PR, Nelson TE. Carnosine: a novel peptide regulator
of intracellular calcium and contractility in cardiac muscle. New Horiz.
1996;4:26 –35.
31. Carter AL. Current Concepts in Carnitine Research. Atlanta, Ga: CRC
Press; 1992:1–5.
32. Ferrari R, Merli E, Cicchitelli D, Mele D, Fucili A, Ceconi C. Therapeutic
effect of L-carnitine and propionyl-L-carnitine on cardiovascular diseases: a review. Ann N Y Acad Sci. 2004;1033:79 –91.
33. Bendahan D, Mattei JP, Ghattas B, Confort-Gouny S, Le Guern ME,
Cozzone PJ. Citrulline malate promotes aerobic energy production in
human exercising muscle. Br J Sports Med. 2002;36:282–289.
34. Callis A, Magnan de Bornier B, Serrano JJ, Bellet H, Saumade R.
Activity of citrulline malate on acid-base balance and blood ammonia and
amino acid levels: study in animal and man. Arzneimittelforschung. 1991;
41:600 – 603.
35. Barbul A. Arginine: biochemistry, physiology, and therapeutic implications. JPEN J Parenter Enteral Nutr. 1986;10:227–238.
36. Opie LH, Heusch G. Myocardial reperfusion: stunning, hibernation, and
preconditioning. In: Opie LH, ed. Heart Physiology: From Cell to Circulation. 4th ed. Philadelphia, Pa: Lippincott, Williams and Wilkins;
2004:574 –598.
37. Quist AP, RShee SK, Lin H, Lal R. Physiological role of gap junctional
hemichannels: extracellular calcium dependent isosmotic regulation.
J Cell Biol. 2000;148:1063–1074.
38. Saez JC, Retamal MA, Basilio D, Bukauskas FF, Bennet MVL.
Connexin-based gap junction hemichannels: gating mechanisms. Biochim
Biophys Acta. 2005;1711:215–224.
39. John S, Cesario D, Weiss JN. Gap junctional hemichannels in the heart.
Acta Physiol Scand. 2003;179:23–31.
40. Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circ Res. 2006;99:801– 815.
CLINICAL PERSPECTIVE
Organ donation is limited by the number of organs available for transplantation, with organs being derived largely from
brain-dead, beating heart donors. In addition, the length of storage continues to be a limiting factor with currently available
solutions and devices, and the viability of stored organs and their cellular substructures, even within currently accepted
storage timeframes, may be compromised. In the case of heart transplantation, hearts must be transplanted within 4 to 6
hours of harvesting, thereby severely restricting the number of organs harvested for transplantation. A new capability to
harvest organs from nonbeating heart donors could open up a new paradigm in transplant medicine. Use of Somah solution
should enable the resuscitation of an arrested heart within 60 minutes of the arrest. Thus, it will make available a much
larger donor pool, considering that patients dying in the emergency room as a result of noncardiac trauma can become
potential heart donors. By increasing the number of available organs, one can increase the number of hearts for candidates
on the waiting list and offer hearts to patients who are not currently considered for transplantation. Besides the extension
of storage times beyond current conventions, hearts stored in Somah solution show complete preservation of coronary
endothelial cells. Considering that 40% to 50% of late rejection of the donor heart occurs in the presence of new and
accelerated arteriosclerosis, it is likely that Somah solution will have a significant impact on prolonging the life of patients
after cardiac transplantation.
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