Abstract

Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca²⁺-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca²⁺ concentration was adjusted with Ca²⁺-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca²⁺ (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N^G-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3–3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca²⁺ (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca²⁺ actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca²⁺, occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.