Apoptosis predominates in nonmyocytes in heart failure

Abstract

The goal of this investigation was to determine the distribution of myocardial apoptosis in myocytes and nonmyocytes in primates and patients with heart failure (HF). Almost all clinical cardiologists and cardiovascular investigators believe that myocyte apoptosis is considered to be a cardinal sign of HF and a major factor in its pathogenesis. However, with the knowledge that 75% of the number of cells in the heart are nonmyocytes, it is important to determine whether the apoptosis in HF is occurring in myocytes or in nonmyocytes. We studied both a nonhuman primate model of chronic HF, induced by rapid pacing 2-6 mo after myocardial infarction (MI), and biopsies from patients with ischemic cardiomyopathy. Dual labeling with a cardiac muscle marker was used to discriminate apoptosis in myocytes versus nonmyocytes. Left ventricular ejection fraction decreased following MI (from 78% to 60%) and further with HF (35%, P < 0.05). As expected, total apoptosis was increased in the myocardium following recovery from MI (0.62 cells/mm(2)) and increased further with the development of HF (1.91 cells/mm(2)). Surprisingly, the majority of apoptotic cells in MI and MI + HF, and in both the adjacent and remote areas, were nonmyocytes. This was also observed in myocardial biopsies from patients with ischemic cardiomyopathy. We found that macrophages contributed the largest fraction of apoptotic nonmyocytes (41% vs. 18% neutrophils, 16% fibroblast, and 25% endothelial and other cells). Although HF in the failing human and monkey heart is characterized by significant apoptosis, in contrast to current concepts, the apoptosis in nonmyocytes was eight- to ninefold greater than in myocytes.

Fig. 1.

The increase of apoptosis after chronic myocardial infarction (MI) and after MI + heart failure (HF). A: representative examples of the staining used to discriminate apoptosis in myocytes from nonmyocytes (left). Myocytes were stained with cardiac troponin I (TnI) (green; A, right, top) and counterstained with rhodamine wheat germ agglutinin (WGA; red; A, right, bottom). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). A, middle: merged image. B: quantification of apoptosis in myocytes and nonmyocytes in adjacent and remote regions. In all conditions, the increase in apoptosis was greater in nonmyocytes than myocytes. Sham-operated (n = 6), MI (n = 5), MI + HF (n = 9) monkeys are represented. TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick end labeling. *P < 0.05, nonmyocytes vs. myocytes; †P < 0.05, compared with MI vs. MI + HF. These sections were obtained from myocardium sufficiently distant from the chronic infarct, so that myocyte structure remained intact and cardiac troponin I could be used as a marker for the myocytes.

Fig. 2.

Immunohistochemical staining of cleaved caspase-3 in remote and adjacent myocardium. A, left: representative photograph of double staining with cleaved caspase-3 and WGA. Arrows signify a cleaved caspase-3-positive nonmyocyte and myocyte in the myocardium from a monkey with HF. The staining for cleaved caspase-3 is green. The outlines of myocytes were shown by WGA staining (red), and nuclei were stained with DAPI (blue). A, right: quantification of cleaved caspase-3-positive cells. The number of cleaved caspase-3-positive nonmyocytes is significantly greater (*P < 0.05) than that of myocytes, both in the adjacent and remote areas of the myocardium. B, left: correlation of TUNEL and cleaved caspase-3 in chronic HF. In the representative photographs (B, top), TUNEL-positive myocytes and nonmyocytes (green) are shown. B, bottom: shown in green are a cleaved caspase-3-positive myocyte (left) and a nonmyocyte (right).

Fig. 3.

Dual immunostaining for macrophages and cleaved caspase-3 and cell types of nonmyocytes. A: representative photographs of apoptotic macrophages in the myocardium of a monkey with HF. The macrophages were stained with HAM56 (red) and cleaved caspase-3 (green). B: quantification of apoptotic macrophages among apoptotic nonmyocytes. C: different cell types of nonmyocytes (red); macrophages, neutrophils, fibroblasts, and endothelial cells. D: a quantitative distribution of cell types of apoptotic nonmyocytes. Values for each cell type were rounded to 2 integers, and, therefore, the sum on the chart is greater than 100%. If we had used 3 integers, this would most likely not have occurred.

Fig. 4.

Apoptosis of myocardium in patients with HF. A: representative photograph of dual staining with TUNEL and WGA. Arrows signify TUNEL-positive nonmyocytes (positive nuclei are green). B: quantification of apoptotic rate (in %). The percentage of TUNEL-positive nonmyocytes was greater than that of myocytes (*P < 0.05) in the human cardiomyopathy tissue, similar to that observed in the monkey HF model (Fig. 1).