Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia

Objective: Nitric oxide (NO) has been shown to induce apoptosis in cardiomy ocytes under normoxic conditions. The ability of NO to induce apoptosis aft er ischemia-reperfusion, a situation of increased NO release in vivo, has n ot been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on th is pathway in cardiomyocytes. Methods: The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a gluc ose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexci nV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclus ion of propidium iodide and (2) by laddering of genomic DNA. Results: Incub ation of cardiomyocytes with the NO-donor (+/-)-S-nitroso-N-acetylpenicilla mine (SNAP, 100 mu M) induced apoptosis in 14.1+/-1.9% of the cells and nec rosis in 24.4+/-4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. A poptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT -cGMP (100 mu M, 9.6+/-0.6% apoptotic cells) or YC-1 (75 mu M, 14.6+/-2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis ind uced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9+/-0.7% or 13.4+/-2.4% apopt otic cells). A correlation of the apoptotic response to SNAP or YC-1 with a n increased activity of soluble guanylyl cyclase, determined by measurement s of intracellular cGMP contents, was found. Conclusions: NO induces apopto sis in a cGMP dependent manner in isolated adult cardiomyocytes whereas ind uction of necrosis seems cGMP-independent. After simulated in vitro ischemi a the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection. (C) 2000 Elsevier Scie nce B.V. All rights reserved.