Mitochondrial Ca -Activated K Channels in Cardiac Myocytes A Mechanism of the Cardioprotective Effect and Modulation by Protein Kinase A

Background—The large-conductance Ca -activated K (BKCa) channel in the cardiac inner mitochondrial membrane (mitoKCa channel) has been shown to protect the heart against ischemic injury. However, questions about the cardioprotective mechanism and the kinase-mediated regulation of mitoKCa channels remain to be answered. Methods and Results—Flavoprotein fluorescence in guinea pig ventricular myocytes was measured to assay mitoKCa channel activity. The mitochondrial Ca concentration ([Ca ]m) and membrane potential ( m) were measured by loading cells with rhod-2 and JC-1, respectively. Cell death was assessed by trypan blue permeability. The BKCa channel opener NS1619 reversibly increased the flavoprotein oxidation in a concentration-dependent manner. NS1619 (30 mol/L) attenuated the ouabain (1 mmol/L)-induced elevation of [Ca ]m with accompanying depolarization of m. These effects of NS1619 were completely antagonized by the BKCa channel blocker paxilline (2 mol/L) but not by the mitochondrial ATP-sensitive K (mitoKATP) channel blocker 5-hydroxydecanoate (500 mol/L). Paxilline, however, failed to block the oxidative effect of diazoxide (100 mol/L), a mitoKATP channel opener. The combined application of submaximally effective concentrations of NS1619 (10 mol/L) and diazoxide (30 mol/L) produced additive effects. NS1619 (30 mol/L) blunted the rate of cell death during exposure to ouabain; this cardioprotective effect was prevented by paxilline. Activation of cAMP-dependent protein kinase by 8-bromoadenosine 3 5 -cyclic monophosphate (0.5 mmol/L) and forskolin (10 mol/L) potentiated the NS1619-induced flavoprotein oxidation. Conclusions—Opening of mitoKCa channels, which is modulated by cAMP-dependent protein kinase, depolarizes the m and attenuates the mitochondrial Ca overload. Our study further indicates that mitoKCa channel activation confers cardioprotection in a manner similar to but independent of mitoKATP channel activation. (Circulation. 2005;111:198-203.)