Cardiac mitochondrial cGMP stimulates cytochrome c release

Abstract

Although the existence of cardiac mitochondrial cGMP has been reported previously [Kimura and Murad (1974) J. Biol. Chem. 249, 6910-6916], the physiological and pathophysiological properties of cGMP in cardiac mitochondria have remained unknown. The aim of the present study was to clarify whether cardiac mitochondrial cGMP regulates the apoptosis of cardiomyocytes. In the presence of GTP, the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine; 1 mmol/l) and SNP (sodium nitroprusside; 1 mmol/l) each markedly increased the cGMP level in a highly purified mitochondrial protein fraction prepared from left ventricular myocytes of male Wistar rats, and these increases were inhibited by 1 micromol/l ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), an inhibitor of NO-sensitive guanylate cyclase. In purified mitochondria, both SNAP (1 mmol/l) and the membrane-permeant cGMP analogue 8-Br-cGMP (8-bromo-cGMP; 1 mmol/l), but not cGMP (1 mmol/l), increased cytochrome c release from succinate-energized mitochondria without inducing mitochondrial swelling and depolarization of the mitochondrial membrane as factors of activation of MPT (mitochondrial permeability transition). The cytochrome c release mediated by SNAP was inhibited in the presence of 1 micromol/l ODQ. On the other hand, 1 mmol/l SNAP induced apoptosis in primary cultured adult rat cardiomyocytes in a time-dependent manner, and this induction was significantly inhibited in the presence of ODQ. Furthermore, apoptosis induced in primary cultured cardiomyocytes by hypoxia/re-oxygenation was also inhibited by ODQ. These results suggest that the acceleration of cGMP production in cardiac mitochondria stimulates cytochrome c release from mitochondria in an MPT-independent manner, resulting in apoptosis.

Nitric oxide modulates mitochondrial activity and apoptosis through protein S-nitrosylation for preimplantation embryo development

“…The NO/GC/cGMP pathway is also involved in cell survival [32,35]. Previous studies have associated NO in apoptosis with the formation of highly reactive oxidants, such as peroxynitrite (ONOO-) or hydroxyl peroxide [26,30].…”

Cytosolic Ca2+–Induced Apoptosis in Rat Cardiomyocytes via Mitochondrial NO-cGMP-Protein Kinase G Pathway

“…We have shown that NO donors, to increase mitochondrial cGMP concentration, induce cytochrome c release from mitochondria, followed by cardiomyocyte apoptosis; we also showed that 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), an NO-sensitive guanylyl cyclase inhibitor, inhibits cardiomyocyte apoptosis under ischemia-reperfusion conditions (Seya et al, 2007). However, NO and cGMP do not induce mitochondrial swelling or membrane depolarization.…”

“…The adequacy of anesthesia was monitored from the disappearance of righting reflex or pedal withdrawal reflex. Each heart was isolated, the aorta was cannulated under artificial respiration, and both were quickly removed as a unit and hung on a Langendorff-type apparatus to start the coronary perfusion with a Ca Rat cardiac mitochondria were prepared as described previously (Seya et al, 2007). Briefly, heparinized adult Wistar rats (n 5 23, 350-400 g) were anesthetized with pentobarbital sodium (50 mg/kg i.p.).…”

“…The animals were maintained for 1 week at a temperature of 22 6 2°C and a relative humidity of 57% on a 12-h light/12-h dark schedule (lights on 08:00-20:00) and had free access to water and food. Ventricular myocytes were isolated from adult Wistar rats (n 5 18, 350-400 g) by collagenase perfusion (Ono et al, 2000;Seya et al, 2007). Briefly, each rat was anesthetized with pentobarbital sodium (50 mg/kg i.p.).…”

“…To assess mitochondrial membrane function, mitochondrial swelling was determined by adding mitochondrial suspension (0.05 mg of protein per milliliter) to Ca 21 -EGTA buffered medium and measuring decreased absorbance at 540 nm (Seya et al, 2007;Seya et al, 2009). Mitochondrial membrane potential was assessed by adding mitochondrial suspension (0.1-0.2 mg of protein per milliliter) to Ca 21 -EGTA buffered medium containing 0.5 mg/ml diS-C3(5) and recording fluorescence intensity at 670 nm in a fluorescence spectrophotometer (FP-750, JASCO Co. Japan) following excitation at 620 nm.…”