Expression and Activation of BK(Ca) Channels in Mice Protects Against Ischemia-Reperfusion Injury of Isolated Hearts by Modulating Mitochondrial Function

Aims: 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. Methods and Results: 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. Conclusions: 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.