Abstract
Aims: Activation and expression of large conductance calcium and voltage-activated potassium channel (BKCa) 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 BKCa is critical to cardioprotection. In this study, we aimed to decipher the mechanistic role of BKCa in cardioprotection from IR injury by genetically activating BKCa channels.Methods and Results: Hearts from adult (3 months old) wild-type mice (C57/BL6) and mice expressing genetically activated BKCa (Tg-BKCaR207Q, referred as Tg-BKCa) along with wild-type BKCa 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-BKCa mice are protected from IR injury, and BKCa 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 BKCa reduces ROS after IR stress. Adult cardiomyocytes and mitochondria from Tg-BKCa mice were isolated and labeled with Anti-BKCa antibodies. Images acquired via confocal microscopy revealed localization of cardiac BKCa in the mitochondria.Conclusions: Activation of BKCa is essential for recovery of cardiac function after IR injury and is likely a factor in IPC mediated cardioprotection. Genetic activation of BKCa reduces ROS produced by complex I and complex II/III in Tg-BKCa mice after IR, and IPC further decreases it. These results implicate BKCa-mediated cardioprotection, in part, by reducing mitochondrial ROS production. Localization of Tg-BKCa in adult cardiomyocytes of transgenic mice was similar to BKCa in wild-type mice.
Highlights
The large conductance calcium and voltage-activated potassium channels (MaxiK, BKCa, KCa1.1) encoded by Kcnma1 gene are ubiquitously expressed in excitable and non-excitable cells [1, 2]
Our results demonstrate that expression and activation of BKCa are vital for cardioprotection from IR injury as well as IPCmediated cardioprotection
Cardioprotection mediated by BKCa is possibly modulated by mitochondrial reactive oxygen species (ROS) production
Summary
The large conductance calcium and voltage-activated potassium channels (MaxiK, BKCa, KCa1.1) encoded by Kcnma gene are ubiquitously expressed in excitable and non-excitable cells [1, 2]. Studies involving activation [10,11,12,13,14,15] and inactivation [11, 16] with pharmacological and genetic tools, including global [10], and tissue-specific knockouts [17], have implicated BKCa channels in cardiac function, neuroprotection [18], and cardioprotection from ischemia-reperfusion (IR) injury, in addition to IR-induced inflammation and mucosal barrier disruption in the small intestine [19]. Tissue-specific knockouts in which BKCa was ablated in adult cardiomyocytes showed that expression of mitochondrial BKCa is responsible for its cardioprotective effect [17]. Modifying BKCa in mice by introducing a mutation responsible for its constitutive activation [8], independent of pharmacological agents, can further support the role of BKCa in cardioprotection from IR injury
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