Activation of Mitochondrial SK Channels in Cardiomyocytes Derived from Hypertrophic Hearts Attenuates Ca2+-Dependent Arrhythmia by Reducing Mitochondrial ROS Production Thereby Stabilizing RyRs

Abstract

Small conductance Ca2+-activated K+ (SK) channels were recently discovered in mitochondria inner membrane. Activation of these voltage-independent channels was found to be protective against ischemia-reperfusion injury by reducing intracellular levels of reactive oxygen species (ROS). Considering submicromolar [Ca2+] sensitivity of SKs we hypothesized that these channels may play an important role in cardiac hypertrophy. To investigate the role of mSK channels in cardiac hypertrophy we used a rat model of pressure-overload induced by ligation of ascending aorta (TAB). Using confocal Ca2+ imaging and patch-clamp we found that inhibition of SK channels with cell-permeable inhibitor UCL-1468 (1 uM) increased frequency of spontaneous Ca2+ waves (SCWs) and delayed afterdepolarizations (DADs) in TAB ventricular myocytes (TCMs). Conversely, membrane-permeable SK enhancer CyPPA (10 uM) attenuated pro-arrhythmic SCWs and DADs in TCMs. Furthermore, inhibition of mSKs enhanced; and activation reduced ROS production by mitochondria in TCMs measured with MitoSox. Monobromobimane assay demonstrated that increased oxidation of ryanodine receptors (RyRs) in TCMs was reversed by CyPPA. Experiments in permeabilized myocytes showed that CyPPA was unable to completely reverse increase in spark frequency in TCMs. However, incubation of TCMs with CyPPA restored SR Ca2+ content, suggestive of substantial improvement in RyR function. Optical mapping experiments of TAB hearts using dual Ca2+ and membrane potential imaging revealed that incubation with membrane-permeable SK activator NS309 (2 uM) improved aberrant Ca2+ homeostasis and abolished DADs and VT/VF induced by beta-adrenergic stimulation. These data suggest that pharmacological activation of mSK channels in hypertrophy protects from Ca2+-dependent arrhythmia via reduction of mitochondrial ROS, and thereby reduction of oxidized RyR leading to its stabilization.