Masaki Shibuya

Pleiotropic Effects of the β-Adrenoceptor Blocker Carvedilol on Calcium Regulation during Oxidative Stress-Induced Apoptosis in Cardiomyocytes

Carvedilol is a nonselective β-adrenoceptor blocker with multiple pleiotropic actions. A recent clinical study suggested that carvedilol may be superior to other β-adrenoceptor blockers in the treatment of heart failure. Despite numerous investigations, the underlying mechanisms of carvedilol on improving heart failure are yet to be fully established. The purpose of this study is to clarify the pleiotropic effect of carvedilol on cytosolic and mitochondrial calcium regulation during oxidative stress-induced apoptosis in cardiomyocytes. Carvedilol (10 μM), but not metoprolol (10 μM), reduced H2O2 (100 μM)-induced apoptosis in neonatal rat cardiomyocytes. During the process, changes in cytosolic calcium concentration ([Ca2+]i) and mitochondrial calcium concentration ([Ca2+]m) and mitochondrial membrane potential (ΔΨm) were measured by fluorescent probes [Fluo-3/acetoxymethyl ester (AM), Rhod-2/AM, and tetramethylrhodamine ethyl ester, respectively] and imaged by laser confocal microscopy. The results showed that H2O2 caused [Ca2]m overload first, followed by [Ca2+]i overload, leading to ΔΨm dissipation and the induction of apoptosis. Carvedilol (10 μM) significantly delayed these processes and reduced apoptosis. These effects were not observed with other β-adrenoceptor blockers (metoprolol, atenolol, and propranolol) or with a combination of the α (phentolamine)- and the β-adrenoceptor blocker. The antioxidant N-acetyl-l-cysteine (NAC, 5 mM) and the combination of NAC and propranolol (10 μM) showed an effect similar to that of carvedilol. Therefore, the effect of carvedilol on H2O2-induced changes in [Ca2+]m, [Ca2+]i, and ΔΨm is independent of α- and β-adrenoceptors but is probably dependent on the antioxidant effect.

NO donor-activated PKC-delta plays a pivotal role in ischemic myocardial protection through accelerated opening of mitochondrial K-ATP channels.

Nitric oxide (NO) can activate protein kinase C (PKC) and the activation of mitochondrial ATP-sensitive potassium (K-ATP) channels is cardioprotective. However, interactions among NO, PKC, and mitochondrial K-ATP channels remain vague. To clarify the cardioprotective mechanism induced by nicorandil, we compared its ability to activate PKC isoforms with that of the mitochondrial K-ATP channel opener, diazoxide. We induced myocardial infarction in rats by 30 minutes of ischemia followed by reperfusion, then assessed the infarct size 3 weeks later. We also examined the translocation of PKC isoforms in the isolated perfused rat heart. Nicorandil and diazoxide reduced infarct size, and the effect of nicorandil, but not of diazoxide attenuated by the PKC inhibitor, chelerythrine, or by the NO quencher, carboxy PTIO. Immunoblotting revealed that nicorandil translocated PKC-δ to the mitochondria, and that this was inhibited by carboxy PTIO. The protective effect of nicorandil against myocardial infarction partly depended on the translocation of PKC-δ to the mitochondria, which we attributed to the NO donor effect of nicorandil. The PKC-δ- dependent activation of mitochondrial K-ATP channel opening might be synergistic with its direct effect, making nicorandil an efficient opener of such channels.