Expression and Activation of BKCa Channels in Mice Protects Against Ischemia-Reperfusion Injury of Isolated Hearts by Modulating Mitochondrial Function

Abstract

<b>Aims:</b> Activation and expression of large conductance calcium and voltage-activated potassium channel (BK_Ca) by pharmacological agents have been implicated in cardioprotection from ischemia-reperfusion (IR) injury possibly by regulating mitochondrial function. Given the non-specific effects of pharmacological agents, it is not clear whether activation of BK_Ca is critical to cardioprotection. In this study, we aimed to decipher the mechanistic role of BK_Ca in cardioprotection from IR injury by genetically activating BK_Ca channels. <b>Methods and Results:</b> Hearts from adult (3 months old) wild-type mice (C57/BL6) and mice expressing genetically activated BK_Ca (Tg-BK_Ca ^R207Q, referred as Tg-BK_Ca) along with wild-type BK_Ca were subjected to 20 min of ischemia and 30 min of reperfusion with or without ischemic preconditioning (IPC, 2 times for 2.5 min interval each). Left ventricular developed pressure (LVDP) was recorded using Millar's Mikrotip® catheter connected to ADInstrument data acquisition system. Myocardial infarction was quantified by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Our results demonstrated that Tg-BK_Ca mice are protected from IR injury, and BK_Ca also contributes to IPC-mediated cardioprotection. Cardiac function parameters were also measured by echocardiography and no differences were observed in left ventricular ejection fraction, fractional shortening and aortic velocities. Amplex Red® was used to assess reactive oxygen species (ROS) production in isolated mitochondria by spectrofluorometry. We found that genetic activation of BK_Ca reduces ROS after IR stress. Adult cardiomyocytes and mitochondria from Tg-BK_Ca mice were isolated and labeled with Anti-BK_Ca antibodies. Images acquired via confocal microscopy revealed localization of cardiac BK_Ca in the mitochondria. <b>Conclusions:</b> Activation of BK_Ca is essential for recovery of cardiac function after IR injury and is likely a factor in IPC mediated cardioprotection. Genetic activation of BK_Ca reduces ROS produced by complex I and complex II/III in Tg-BK_Ca mice after IR, and IPC further decreases it. These results implicate BK_Ca-mediated cardioprotection, in part, by reducing mitochondrial ROS production. Localization of Tg-BK_Ca in adult cardiomyocytes of transgenic mice was similar to BK_Ca in wild-type mice.