Persistent Regional Downregulation in Mitochondrial Enzymes and Upregulation of Stress Proteins in Swine With Chronic Hibernating Myocardium
Hibernating myocardium is accompanied by a downregulation in energy utilization that prevents the immediate development of ischemia during stress at the expense of an attenuated level of regional contractile function. We used a discovery based proteomic approach to identify novel regional molecular adaptations responsible for this phenomenon in subendocardial samples from swine instrumented with a chronic LAD stenosis. After 3 months (n=8), hibernating myocardium was present as reflected by reduced resting LAD flow (0.75±0.14 versus 1.19±0.14 mL · min−1 · g−1 in remote) and wall thickening (1.93±0.46 mm versus 5.46±0.41 mm in remote, P<0.05). Regionally altered proteins were quantified with 2D Differential-in-Gel Electrophoresis (2D-DIGE) using normal myocardium as a reference with identification of candidates using MALDI-TOF mass spectrometry. Hibernating myocardium developed a significant downregulation of many mitochondrial proteins and an upregulation of stress proteins. Of particular note, the major entry points to oxidative metabolism (eg, pyruvate dehydrogenase complex and Acyl-CoA dehydrogenase) and enzymes involved in electron transport (eg, complexes I, III, and V) were reduced (P<0.05). Multiple subunits within an enzyme complex frequently showed a concordant downregulation in abundance leading to an amplification of their cumulative effects on activity (eg, “total” LAD PDC activity was 21.9±3.1 versus 42.8±1.9 mU, P<0.05). After 5-months (n=10), changes in mitochondrial and stress proteins persisted whereas cytoskeletal proteins (eg, desmin and vimentin) normalized. These data indicate that the proteomic phenotype of hibernating myocardium is dynamic and has similarities to global changes in energy substrate metabolism and function in the advanced failing heart. These proteomic changes may limit oxidative injury and apoptosis and impact functional recovery after revascularization.
Hibernating myocardium is characterized by viable, chronically dysfunctional myocardium that develops in response to repetitive myocardial ischemia.1,2 We have previously demonstrated that the relation between regional oxygen consumption, coronary flow, and function in response to stress is attenuated in hibernating myocardium and thus dissociated from the usual determinants of myocardial oxygen demand.3 By reducing regional energy utilization, hibernation prevents the development of ischemia after submaximal stress. This is supported by a lack of biochemical markers of ischemia and preservation of total ATP and creatine phosphate content in swine with hibernating myocardium3,4 as well as human biopsies from patients without significant fibrosis.5 Although there has been interest in identifying the role of increased glucose uptake in these responses, maximal insulin stimulated glucose uptake is unchanged in chronic hibernating myocardium, and alterations in other metabolic pathways responsible for the attenuated increase in oxygen consumption are unknown.2,4,6,7
We hypothesized that hibernating myocytes in viable dysfunctional myocardium can intrinsically downregulate their metabolic needs to achieve a balance between supply and demand at a reduced regional workload. As a first step to delineate the metabolic pathways involved, we used a discovery based proteomic approach to identify targets that are regionally modified in pigs with chronic hibernating myocardium. Enzymatic activity assays of the pyruvate dehydrogenase complex (PDC) were used to illustrate how changes in the expression of multiple protein components of an enzyme system can combine to produce a cumulative functional impact in vitro. Lastly, because depressed flow and function remain constant for at least 2 months in this model8 we assessed the stability of protein changes after the development of hibernating myocardium. The results demonstrate a chronic downregulation of mitochondrial enzymes that, while regional and associated with normal global function, is similar to that in the advanced failing heart.
Materials and Methods
Procedures and protocols conformed to institutional guidelines for the care and use of animals in research and are detailed in the online supplement. Briefly, farm-bred juvenile pigs were chronically instrumented with a 1.5- to 2.0-mm fixed diameter stenosis placed on the proximal left anterior descending (LAD) coronary artery. This model progresses from chronically stunned to hibernating myocardium after 3 months with physiological features persisting unchanged for up to 5 months after instrumentation.2,8,9 Sham animals were normal or underwent thoracotomy and dissection of the LAD without placement of an occlusive stenosis. Hibernating animals were studied 3 months (n=8) or 5 months (n=10) after instrumentation in the closed-chest sedated state (Telazol/xylazine IM and propofol 2 to 5 mg · kg−1 · min−1IV). Resting and vasodilated coronary flows were assessed using fluorescent microspheres as previously described, and LV function was assessed with M-mode echocardiography.10 To circumvent acute posttranslational modifications arising from pharmacological stimulation,11 animals were recovered and reanesthetized 72 hours later with hearts excised in the unstimulated state. Samples were flash frozen and total protein extracted from approximately 0.2g of myocardial tissue.
2D Differential-in-Gel Electrophoresis (2D-DIGE)
We used 2D-DIGE for analysis of changes in myocardial protein expression (Figure 1).12 Reagents, electrophoresis materials, and software were all obtained from GE Healthcare. Methodological details are provided in the online supplement (available online at http://circres.ahajournals.org).
Samples were labeled with CyDye DIGE Fluor (Cy3 or Cy5), and an internal standard (Cy2 labeled) contained an equal mix of protein from all animals or a pooled sham sample. Paired comparisons of LAD versus remote (3 months n=8; sham n=7) as well as unpaired LAD versus sham (3 months n=8; 5 months n=10) were performed on separate gel runs. For all analyses, Cy3 and Cy5 were randomly used to label LAD and control regions to prevent dye bias. Proteins were isoelectrically focused with immobilized pH gradient strips (24 cm, pH 3 to 10, nonlinear) using an IPGphor system, and subsequently electrophoresed on 12.5% polyacrylamide gels in an Ettan DALT SDS-PAGE system. Gels were scanned with a Typhoon 9410 imager, cropped (ImageQuant v5.2), and imported into DeCyder DIA software (v5.0) for spot identification and normalization. Spot analysis was performed using DeCyder BVA software.
MALDI-TOF and Protein Identification
Spots were excised from separate 2D-gels loaded with 500 to 1000 μg of total protein and stained with Sypro Ruby (Molecular Probes). In-gel digestion was performed using trypsin. Digests were concentrated and purified with ZipTips (Millipore), and eluted in a saturated solution of α-cyano-4-hydroxycinnamic acid. We used a Bruker Daltonics Biflex MALDI-TOF mass spectrometer (Bruker Daltonics) or a Bruker Daltonics Autoflex MALDI-TOF in TOF-TOF mode for peptide mass fingerprinting. Proteins were identified using Mascot. MOWSE scores greater than 61 were significant for the Swiss Protein database, whereas scores of greater than 67 were significant for the NCBI database (P≤0.05). The reported scores and accession numbers are from the Swiss Protein database unless otherwise specified. For MALDI-TOF-TOF data, the MS Ion search feature was used with the MSDB database. Scores of 39 or higher were significant.
Pyruvate Dehydrogenase Complex, Cytochrome C Oxidase, and Citrate Synthase Activity
Methodological details are provided in the online supplement. “Active” and “total” PDC activity were measured using 14CO2 collected during in vitro reaction with radioactivity measured with a scintillation counter. Dihydrolipoamide dehydrogenase (E3), cytochrome c oxidase and citrate synthase activity were measured spectrophotometrically.
Data are presented as the mean±SEM. Student’s paired t test was used to examine differences between LAD and remote regions of the same heart. For serial changes in hibernating myocardium evaluated 3 and 5 months after instrumentation, we used unpaired t tests. A probability value of <0.05 was significant.
In both 3- and 5-month swine with hibernating myocardium, TTC staining showed no evidence of infarction. Assessment of connective tissue demonstrated 4.7±0.6% in full-thickness samples taken from the hibernating LAD region compared with 3.0±0.3% for remote regions.
Flow and Function in 3- and 5-Month Swine With Hibernating Myocardium
Hemodynamics are summarized in the online supplement and were similar in each group. Transmural myocardial perfusion is summarized in Figure 2A. Resting subendocardial flow was reduced in both 3- and 5-month swine (LAD 0.75±0.14 versus 1.19±0.14 mL · min−1 · g−1 in 3-month swine; LAD 0.84±0.07 versus 1.65±0.15 mL · min−1 · g−1 in 5-month swine, both P<0.05). After adenosine vasodilation subendocardial flow was critically impaired and failed to increase above resting levels. Global function was normal and there was no clinical evidence of heart failure in either group. Regional function is summarized in Figure 2B. LAD wall thickening in hibernating LAD regions was reduced relative to remote myocardium in both groups (3-month LAD 1.93±0.46 mm versus 5.46±0.4 mm and 5-month LAD 2.73±0.35 versus 6.93±0.57; P<0.05 for both). With the exception of a small increase in resting LAD flow (P<0.05), hemodynamics, function, and perfusion were similar in swine studied at 3 or 5 months after instrumentation.
Regional Proteomic Changes in Hibernating Myocardium at 3-Months Paired Analysis With Remote Myocardium
To identify differentially expressed proteins, we initially used a paired analysis in the 3-month hibernating group using the remote normally perfused region from each heart as an internal control for potential posttranslational modifications related to tissue harvesting. Detailed quantitative analyses and protein identification data are provided in the online supplement. Whole tissue protein preparations revealed 1323 unique protein spots with 1019 detectable in at least half of the 2D gels. Of these, 191 were significantly altered (P<0.05) in hibernating LAD regions (70 increased and 121 decreased). We identified 52 differentially expressed protein spots representing 37 unique proteins (supplemental Table I). An additional 62 spots have been identified which were not significantly altered representing 35 unique control proteins. For all identified spots, the average Mascot Mowse score for each protein was 99 with 25% average peptide sequence coverage. Of the 72 unique proteins identified, 35 had known porcine sequences.
Abundance of hibernating LAD proteins relative to remote regions in hibernating (n=8) and sham animals (n=7) is tabulated (supplemental Table II) with selected examples summarized in Figure 3A and 3B. We observed a general downregulation of mitochondrial proteins including citric acid cycle enzymes (6 unique proteins) and electron transport chain and ATP-synthase subunits (8 unique proteins). There was also a downregulation of cytoplasmic CK, myoglobin, and long chain Acyl-CoA dehydrogenase. Glycolytic proteins were not altered with the exception of pyruvate kinase which was reduced. Contractile proteins such as myosin heavy chain, myosin light chain isoforms, troponin T, and tropomyosin exhibited multiple spots on the gels likely reflecting posttranslational modifications of which most were unchanged or reduced in hibernating myocardium. Regional differences in protein expression between LAD and remote regions of sham animals were infrequent and small (supplemental Table II).
In contrast to the regional reductions in mitochondrial proteins in hibernating myocardium, there were significant increases in stress proteins including αB-crystallin, HSP27, and HSP20-β6. Likewise, cytoskeletal proteins including desmin and vimentin, and antioxidant proteins such as superoxide dismutase and peroxiredoxin-2, were also increased early after the development of hibernating myocardium.
Amplification of Protein Subunit Changes for PDC
Many of the proteins identified were subunits of a given protein complex of which we chose PDC as an example to illustrate the cumulative impact on biological activity (Figure 4). We found modest reductions in individual PDC components by 2D-DIGE but their effects on PDC activity were more pronounced. “Total” PDC activity (LAD 21.9±3.1 versus 42.8±1.9 nmoles/mg/min, P<0.05) and E3 catalytic activity (1.04±0.11 versus 1.47±0.30 μmoles/mg/min, P<0.05) were significantly downregulated in samples taken from hibernating LAD regions. The greater reductions in PDC activity in hibernating myocardium reflected the net result of changes in the individual subunits as well as other unidentified regulatory components.
Temporal Evolution of Proteomic Changes in Chronic Hibernating Myocardium-Unpaired Analysis With Normal Controls
To determine the chronicity of the proteomic changes in hibernating myocardium we assessed protein expression ratios in LAD versus normal sham animals studied after 3 and 5 months. Data are summarized in supplemental Table II with selected proteins in Figure 5A and 5B. Of the 114 spots identified, 25 showed significant differences at 5 months in comparison with swine studied early after the development of hibernating myocardium (3 months). The trends were predominantly related to the initial differential protein expression normalizing. We found persistent reductions in mitochondrial protein expression in 5-month animals compared with 3-month animals with notable exceptions including long chain acyl-CoA dehydrogenase, ATP synthase, mitochondrial CK, and mitochondrial aspartate aminotransferase, all of which returned toward normal at 5 months. Pyruvate kinase became significantly downregulated at 5 months. Whereas increases in cytoskeletal proteins at 3 months normalized after 5 months, stress proteins such as αB-crystallin, HSP20-β6, and HSP-27 remained chronically elevated in hibernating myocardium. Figure 6 summarizes the regional and temporal alterations of mitochondrial proteins, and Figure 7 summarizes the results for cytoskeletal proteins early and late after the development of hibernating myocardium.
Cytochrome C Oxidase, Citrate Synthase, and Mitochondrial Protein Content in Hibernating Myocardium
To further support the functional importance of protein changes in hibernating myocardium identified from 2D-gels, we performed activity assays for cytochrome c oxidase and citrate synthase (Figure 8A). There was a reduction in activity for both enzymes in the LAD region compared with sham controls (P≤0.05). Subendocardial mitochondrial and total protein yield per gram of tissue was the same for all samples, suggesting that reductions in mitochondrial mass/volume were not the cause of these changes (Figure 8B).
The present study used a discovery based proteomic approach to demonstrate intrinsic downregulation of many of the mitochondrial enzymes responsible for oxidative metabolism and electron transport in response to a flow limiting coronary stenosis. These changes may contribute to the reductions in resting flow, function, and oxygen consumption that occur in the absence of ischemia or infarction in chronic hibernating myocardium. The chronic upregulation of stress proteins coupled with the transient increases in cytoskeletal proteins support the notion that the molecular mechanisms operative in hibernating myocardium are dynamic, protect myocytes from irreversible injury, and do not always lead to inexorable fibrosis.
Chronic Downregulation of Mitochondrial Function and Oxygen Consumption in Hibernating Myocardium
We found regional reductions in many of the enzymes involved in oxidative metabolism in hibernating myocardium. Interestingly, these changes involved entry points of carbohydrate (PDC) and fatty acid metabolism (acyl-CoA dehydrogenase) as well as many of the components of the electron transport chain. The majority of these changes persisted in swine where hibernating myocardium was present for at least 2 months (5-month animals). Changes in mitochondrial proteins paralleled chronic reductions in flow and function in the absence of progressive fibrosis as we have previously demonstrated in this model over the same time frame.8 The regional reductions in metabolic enzymes do not appear to be related to myocardial scar as there was no significant infarction by TTC and only trivial regional increases in myocardial connective tissue are found in this model. Furthermore, although not all protein spots could be identified using mass spectrometry, the majority of LAD protein spots were not significantly altered when compared with normal myocardium. Unchanged metabolic enzymes that were identified included GAPDH, triosephosphate isomerase, phosphoglycerate mutase, aminoacylase-1, and DRP-2. Thus, the regional reductions in mitochondrial enzyme expression appear to represent an intrinsic adaptation of the heart to hibernation.
There are in vivo as well as in vitro physiological correlates of the reduced mitochondrial enzyme levels. In previous work we have demonstrated that there is a reduction in myocardial oxygen consumption at rest and during submaximal stress that allows increases in external workload to occur without immediately precipitating subendocardial ischemia in hibernating myocardium.3 This “protection” against the development of an oxygen supply/demand mismatch is, however, accomplished at the expense of reduced regional LV function. Using isolated mitochondria from a similar model, McFalls demonstrated a reduction in the respiratory control index in hibernating myocardium which they postulated to reflect a basal uncoupling of mitochondrial respiration via increases in uncoupling protein-2.13 They also demonstrated that mitochondria from hibernating myocardium are better adapted to ischemia as evidenced by decreased superoxide formation and a preservation of state-3 mitochondrial oxygen consumption after a 20-minute period of anoxia-reoxygenation. Collectively, these observations support the central role of mitochondrial respiration in the adaptation of the heart to chronic repetitive ischemia.
The results of the present study may help to understand the temporal pattern of myocyte apoptosis in chronic hibernating myocardium. The progression from chronically stunned to hibernating myocardium is associated with an increase in myocyte apoptosis that leads to substantial regional myocyte loss and compensatory cellular hypertrophy in animals with hibernating myocardium that are studied at 3 months.14,15 Although the physiological findings of hibernating myocardium remain unchanged up to 5 months, data from our previous studies demonstrated that apoptosis returns to levels that are no different than normal myocardium (19±8 apoptotic myocytes/106myocytes versus 160±50 apoptotic myocytes/106 myocytes at 3 months, P<0.05).10,14 The reduction in the frequency of apoptosis to levels similar to normal animals supports the hypotheses that the downregulation in mitochondrial oxidative enzymes and upregulation of stress proteins serve to adapt the heart and prevent progressive myocyte loss.
Comparison With Stunned Myocardium and Pharmacological Preconditioning
The proteomic changes we found in chronic hibernating myocardium differ substantially from those found in infarcted hearts but have similarities as well as important differences with findings that have been reported in isolated hearts with acutely stunned myocardium and pharmacological preconditioning in isolated cardiac myocytes.11,16 White examined isolated rabbit hearts subjected to 15 minutes of ischemia and 60 minutes of reperfusion using a proteomic approach.16 Similar to hibernating myocardium, spots representing stress proteins such as HSP27 and αB-crystallin were increased after acute ischemia. In contrast to the concordant downregulation of mitochondrial proteins identified in hibernating myocardium, components of PDC and ATP synthase increased, whereas components of other enzymes such as electron transport complexes I and II and VDAC-1 showed bidirectional changes after acute ischemia. In isolated myocytes subjected to pharmacological preconditioning, Arrell found selected components of proteins involved in oxidative phosphorylation to increase.14 These included electron transport complex I, ATP synthase, isocitrate dehydrogenase, and the pyruvate dehydrogenase E3-binding protein, whereas other mitochondrial energetic proteins were unchanged. The protein changes observed after acute interventions were frequently in opposite directions from those in hibernating myocardium and most likely reflected posttranslational modifications because the time period from intervention to tissue harvesting was only 60 minutes in these studies. Nevertheless, the studies following acute ischemia and pharmacogical preconditioning support a fundamentally different response of the chronically adapted hibernating heart. Additional studies in swine subjected to reversible ischemia will be required to fully elucidate all of the differences and similarities in myocardial proteomics with hibernating myocardium but should provide insight into whether stunning or preconditioning are components of the chronic adaptive response to repetitive ischemia.
Regional Proteomic Changes in Hibernating Myocardium Versus Global Changes in Hypertrophied and Failing Myocardium
Interestingly, although this single vessel animal model of hibernating myocardium is associated with normal global LV function, it results in regional proteomic changes that are very similar to the global changes that have been reported in advanced congestive heart failure. Like hibernating myocardium, heart failure produces a reversion to a fetal phenotype of substrate utilization and suppression of adult protein isoforms with reductions in the activity of members of the citric acid cycle.17 Lei found PDC to be downregulated at the transcriptional and protein levels in pacing induced heart failure.18 Schott demonstrated chamber specific reductions in PDC and isocitrate dehydrogenase using a proteomic approach in pressure overloaded right ventricles without overt failure.19 Similarly, Jin reported a downregulation of PDC and isocitrate dehydrogenase in hypertrophied hearts from spontaneously hypertensive rats.20 Gallego-Delgado found NADH dehydrogenase components and cytochrome oxidase to be reduced in spontaneously hypertensive rats. ATP synthase β chain subunits were upregulated.21 Faber reported a downregulation in ATP synthase delta, electron transfer flavoprotein β subunit and the NADH dehydrogenase (ubiquinone) β subcomplex.22 Whereas these small animal models feature cardiac hypertrophy from pressure overload or failure of the left or right ventricle, similar regional alterations in the expression of regional oxidative enzymes accompany the development of hibernating myocardium.
Regional changes in the expression of cytosolic proteins such as myoglobin, stress proteins, and cytoskeletal structural proteins in hibernating myocardium were also similar to the global alterations reported in advanced heart failure. Myoglobin and creatine kinase M were reduced in hibernating myocardium as they are in heart failure.23,24 Upregulation in the expression of stress proteins and antioxidant enzymes were also similar to heart failure and probably reflect a general protective mechanism to minimize the production of reactive oxygen species that ultimately lead to cell death.25 Additional cardioprotective pathways previously identified using candidate proteins after more severe levels of short-term repetitive ischemia in swine include GSK-3β and H-11 kinase.26,27 The cessation of apoptosis despite persistent physiological changes of hibernating myocardium suggests that cardioprotective pathways and downregulation of regional energy utilization may attenuate myocyte loss from oxidative stress in the chronic state.
Although speculative, the common denominator potentially explaining regional and chamber specificity of the alteration in oxidative metabolism and stress proteins could be the development of myocyte cellular hypertrophy. Although there is no evidence of cardiac hypertrophy in terms of left ventricular mass in swine with hibernating myocardium, the regional myocyte loss from apoptosis leads to compensatory regional cellular hypertrophy that prevents wall thinning.14 This is further supported by the regional nature of these changes and the fact that they occur in the absence of pressure overload or global heart failure. Thus, rather than resulting from chronic ischemia, the proteomic alterations reflect the reversion of the hibernating region to regional cellular hypertrophy.
The upstream transcriptional signaling pathways responsible for the regional mitochondrial protein changes in hibernating myocardium are unknown. The synthesis of metabolic proteins is regulated by nuclear respiration factors (NRF-1 and NRF-2) and members of the peroxisome proliferator-activated receptor family (PPARα, PPARβ, and PPARγ).28–30 NRF-1 and NRF-2 are under the control of the PPARγ coactivator-1α (PGC-1α), which can downregulate metabolic enzymes in response to altered substrate availability. Similarly, because PGC-1α is required for activation of PPARα it could also coordinately downregulate enzymes of fatty acid oxidation in hibernating myocardium. Further studies will be required to elucidate the response of these pathways to ischemia and in hibernating myocardium.
The interpretation of our results needs to consider the cellular makeup of the whole myocardial tissue sample. Although myocytes only contribute approximately half of the cells, they still contribute the majority of protein in a whole tissue preparation. The total amounts will however be dominated by the most abundant proteins that include contractile and mitochondrial proteins. Although this does not diminish the important regional differences we have reported here, additional studies using subproteomic fractionation will be required to identify less abundant protein changes that may be “diluted” because of the dominance of contractile and mitochondrial proteins. In addition, differences in myocyte protein content per gram of tissue between hibernating and normal tissue have been recently reported in a different model by Bito.31 Although Bito showed that calculated mitochondrial volume fraction was increased in hibernating myocardium, we found that mitochondrial protein content per gram of tissue was similar in the 2 regions. Mitochondrial protein content was not diminished in hibernating myocardium in either study. Collectively, these findings support the notion that reductions in the specific mitochondrial proteins that we have observed are likely attributable to an adaptive response whereby the expression of specific genes are selectively downregulated, and that the reductions are not simply from a decrease in mitochondrial mass or numbers.
An additional limitation of our approach is the inability to identify specific posttranslational modifications (PTMs) that may account for some protein spots. Although mitochondrial proteins were generally reduced in hibernating myocardium, there were frequently directionally opposing changes in contractile proteins that had the same protein identity established by MALDI-TOF. More advanced approaches such as LC/MS-MS separation will be required to characterize important PTMs. Furthermore, the discovery based proteomic analysis we used identifies changes in protein expression but does not examine what effect chronic ischemia has on protein activity. Although we have demonstrated reductions in activity of specific mitochondrial enzymes, as is the case with PDC, the cascade of alterations found in hibernating myocardium would likely result in more profound changes in the in vitro activity for many other proteins, especially multi-subunit complexes. Nevertheless, the attenuated metabolic response and the cessation of apoptosis over time in hibernating myocardium provide integrated in vivo end points supporting the biological relevance of the protein changes identified.
Our study demonstrates regional reductions in the expression of mitochondrial oxidative enzymes and other proteins in hibernating myocardium that are similar to those that occur globally in hypertrophy, fetal heart, and the advanced failing heart. These may represent a common response of the myocyte to stress or cellular hypertrophy resulting in an attenuation of regional oxygen demand as external workload is increased at the expense of contractile function. We speculate that by limiting the development of a supply/demand imbalance, oxidative stress is minimized and further myocyte apoptosis and progressive degeneration is prevented. The reversibility of these proteomic alterations is currently unknown and has considerable implications for understanding the mechanisms responsible for viable dysfunctional myocardium in ischemic cardiomyopathy where function can be severely reduced in the absence of ischemia or fibrosis. If this reflects a phenotype secondary to compensatory cellular hypertrophy from apoptosis-induced myocyte loss, it could explain persistent contractile dysfunction after revascularization. Indeed, almost 1 in 4 dysfunctional segments without fibrosis by Gd MRI enhancement fail to improve after coronary revascularization.32 Experiments to evaluate the effects of revascularization and manipulate the proteomic changes independently of perfusion will be required to test this possibility.
We thank Anne Coe, Deanna Gretka, Elaine Granica, and Amy Johnson for technical assistance.
Sources of Funding
This work was supported by grants from the Department of Veterans Affairs, the American Heart Association, NHLBI (HL-55324, HL-61610), the Albert and Elizabeth Rekate Fund and the John R. Oishei Foundation.
Original received May 10, 2007; revision received October 3, 2007; accepted October 17, 2007.
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