Revascularization of chronic hibernating myocardium stimulates myocyte proliferation and partially reverses chronic adaptations to ischemia

Abstract

Background: The time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles.

Objectives: This study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction.

Methods: Swine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes.

Results: At 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post-percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit(+) cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted.

Conclusions: Delayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Keywords: coronary blood flow; myocyte regeneration; proteomics.

Figure 1. Selected Angiographic Images From an Animal With Hibernating Myocardium

The pre-PCI image demonstrates a severe proximal LAD stenosis. The 2-h post-PCI image demonstrates a widely patent LAD with no significant restenosis after 1 month. LAD = left anterior descending artery; PCI = percutaneous coronary intervention.

Figure 2. Coronary Flow Reserve and Regional Wall Thickening in Hibernating Myocardium Before and Immediately After PCI

LAD subendocardial flow reserve (A) and wall thickening (B) were reduced in hibernating myocardium (pre-PCI). Flow reserve normalized 2 h later (post-PCI), but LAD wall thickening remained depressed. Abbreviations as in Figure 1.

Figure 3. Delayed Time Course of Functional Improvement After PCI

Regional wall thickening was reduced at rest (pre-PCI). There was no immediate effect of revascularization, but a delayed improvement in function became evident after 1 week. No further functional improvement occurred at 1 month and LAD wall thickening remained depressed. Abbreviations as in Figure 1.

Figure 4. Heat Map Demonstrating Differential Expression of Proteins Correlating With Increased Flow and/or Function From Animals With Hibernating Myocardium

Following revascularization and the alleviation of ischemia, up-regulated (red) stress and cytoskeletal proteins and glycolytic enzymes generally normalized (yellow). In contrast, many mitochondrial and contractile proteins remained depressed (green) 4 weeks after revascularization.

Figure 5. Summary of Selected Proteins from 2D-DIGE

(A) Mitochondrial proteins were reduced in animals with hibernating myocardium, with little improvement after revascularization. (B) Revascularization had heterogeneous effects on cytosolic proteins. Increases in stress and structural proteins tended to normalize after alleviation of ischemia. In contrast, contractile proteins such as troponin T remained persistently down-regulated 4 weeks after revascularization. 2D-DIGE = 2-dimensional differential in-gel electrophoresis.

Figure 6. Variable Effects of Revascularization on Enzymatic Activity

Cytochrome c oxidase activity was reduced in hibernating myocardium (n = 9) and remained unchanged following revascularization (n = 12). In contrast, citrate synthase activity normalized following PCI. Abbreviations as in Figure 1.

Figure 7. Effect of PCI on Myocyte Diameter and Nuclear Density in Hibernating Myocardium

Untreated animals exhibited cardiomyocyte hypertrophy and a reduction in myocyte nuclear density. Four weeks after revascularization, myocyte nuclear density increased and myocyte diameter decreased, suggesting new myocyte formation. Abbreviations as in Figure 1.

Figure 8. Revascularization of Hibernating Myocardium Increases cKit⁺ Cells, Ki67⁺ Myocytes and pHH3⁺ Myocytes

(A) Confocal images of Ki67⁺ staining (green) in a myocyte (cardiac troponin I; red) and nonmyocyte after revascularization. Nuclei are stained with DAPI (blue). Revascularization significantly increased Ki67⁺ myocytes consistent with increased DNA synthesis. (B) Confocal images depicting a pHH3⁺ nucleus (green), indicating a myocyte undergoing mitosis. Revascularization increased pHH3⁺ myocytes as compared to nonrevascularized animals with hibernating myocardium and sham controls. (C) Confocal images of a cKit⁺ cell between cardiomyocytes. Myocardial cKit⁺ cells increased after revascularization. DAPI = 4′,6-diamidino-2-phenylindole; pHH3 = phosphorylated histone H3.

Figure 9. Central Illustration. Revascularization of Hibernating Myocardium

Delay in the functional recovery of hibernating myocardium is partly related to stimulating new myocyte formation with myocyte cellular hypertrophy replaced by small new myocytes (upper panels). Immunohistochemistry in the lower panel shows that revascularization increases proliferating myocytes (pHH3 and Ki67 stains), as well as cKit positive cardiac stem cells. pHH3= phosphorylated histone H3.