Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways

Abstract

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

Fig 1.

Relation of gp120, Nef, and TNF-α to HIV-1 cardiomyopathy and cardiomyocyte apoptosis. The hormone BNP was determined in the plasma of 12 patients by using a competitive enzyme immunoassay (A). The left ventricular sections from 18 AIDS hearts were stained by the trichrome technique to detect fibrosis (B), immunocytochemistry with anti-gp120, -Nef (C), and -TNF-α (E) , and the TUNEL and DAPI techniques (D) (note bright contracted nuclei corresponding to TUNEL-positive apoptotic nuclei). Maximum staining was identified at low power, and then three high-power fields were scanned in a predetermined order by using IMAGE PRO software. Zero value indicates that no positive staining was found anywhere. The mean ± SD for each group (HIVCM-positive n = 5 and HIVCM-negative, n = 13) was plotted on the y axis and the data were analyzed by the t test (P values: BNP = 0.004, fibrosis = 0.091, gp120 = 0.008, Nef = 0.315, apoptosis = 0.004, TNF-α = 0.051), and the Mann–Whitney nonparametric test (P values: BNP = 0.001, fibrosis = 0.04, gp120 = 0.08, Nef = 0.3315, apoptosis = 0.004, TNF-α = 0.003).

Fig 2.

Cleaved caspase-9 and Fas ligand are overexpressed in HIVCM compared with non-HIVCM. Heart tissues from a control patient, three AIDS patients without cardiomyopathy, and four patients with non-HIV-1 cardiomyopathy were immunostained by using antiactive caspase-9 and anti-Fas ligand antibodies [×40 (a, b, e, and f); ×100 (c, d, g, and h)].

Fig 3.

Apoptosis of cultured rat myocytes is induced by HIV-1 or gp120 through the mitochondrial pathway. (A) NRVMs were sham-infected (a), infected with HIV-1_MN (b), or treated with gp120 (1 μg/ml) (c) and were stained with DAPI (Left), TUNEL (Center), antisarcomeric antibody MF-20 (Right, b), or anti-gp120 (Right, a and c). (B) NRVMs were treated as indicated with HIV-1 (active HIV_MN or vaccine HIV_MN) or gp120, intact, boiled or neutralized by antibody. gp120-treated cells were pretreated with the caspase-8 inhibitor Z-IETD-FMK (10 μM), caspase-9 inhibitor Z-LEHD-FMK (10 μM), the CCR5 antagonist AOP-RANTES (100 nM), pertussis toxin (Ptx) (1 μg/ml), or heparin (20 μg/ml), and stained by the TUNEL technique. The results were scanned and mean OD plotted. (C) NRVMs were either sham-infected or infected with HIV-1_MN and were stained with Hoechst 33342 (a), JC-1 (b), and anticytochrome C (c). JC-1 staining (red and green) results were scanned. A high red/green ratio indicates a high mitochondrial membrane potential.