Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x

Cytokine-induced NO production depresses myocardial contractility and has b een shown to be cytotoxic to cardiac myocytes. However, the mechanisms of c ytokine-induced cardiac myocyte cell death are unclear. To analyze these me chanisms in detail, we treated neonatal cardiac myocytes in serum-free cult ure with a combination of the macrophage-derived cytokines interleukin-1 be ta, tumor necrosis factor-alpha, and interferon-gamma. These cytokines caus ed a time-dependent induction of cardiac myocyte apoptosis, but not necrosi s, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase , reflecting caspase activation. Apoptosis was preceded by a >50-fold induc tion of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/mu g protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxida nts (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokine s also mediated an NO-dependent, sustained increase in myocyte expression o f the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione al so induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effec ts of cytokines, including poly(ADP-ribose) polymerase cleavage, could be a ttributed to interleukin-1 beta; interferon-gamma and tumor necrosis factor -alpha had no independent effects on apoptosis or on NOx production. We con clude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part th rough the generation of oxygen free radicals. The rate and extent of this a poptosis is modulated by alterations in the cellular balance of Bak and Bcl -x(L), which respond differentially to cytokine-induced and exogenous NO an d by the availability of oxidant species.