Abstract 238: Molecular Identity of Cardiac Mitochondrial Chloride Intracellular Channel Proteins and Their Role in Mitochondrial Function

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Introduction: Mitochondria are primary target organelles for cardioprotection. Rabbit heart and cardiomyocytes showed increased infarction upon treatment with IAA-94, a known chloride intracellular channel (CLIC) inhibitor implicating role of CLICs in cardioprotection. There are six CLIC homologs (CLIC1-6), but the molecular identity of cardiac mitochondrial CLICs and their functional roles are not deciphered. We aim to determine the identity of cardiac mitochondrial CLICs and establish their role in mitochondrial function to understand the mechanism of CLIC-mediated cardioprotection. Methods: Hearts were isolated from 2-3 months old SD rats for all the experiments. qPCRs were performed to determine the abundance of CLIC1-6 and corroborated by Western blot using specific antibodies. Immunofluorescence was performed on neonatal and adult cardiomyocytes. Percoll-purified mitochondria were also probed with Western blot, immuno organelle chemistry and Mass spectrometry to establish the presence of CLICs. In order to determine their role in mitochondrial function, spectrofluorimetric analysis were performed to measure the reactive oxygen species (ROS) production using amplex red and calcium retention capacity (CRC) using calcium green. Results: We have discovered that CLIC4, CLIC5 and CLIC1 as the most abundant paralogs in rat heart using qRT PCR and corroborated their presence by Western blot. CLIC4 and CLIC5, not CLIC1 were found to be present in mitochondria of neonatal and adult cardiomyocytes. Percoll-purified mitochondria further showed presence of CLIC4 and CLIC5 where they colocalizes with mitochondria (45+/-2 and 74+/-3, n=3, respectively). Over-expressing CLICs in H9C2 cells showed mitochondrial localization of CLIC4 and CLIC5 and not CLIC1 establishing them as mitochondrial anion channels. Blocking of CLICs with IAA-94 modulated the ROS production at both complex I and II/III of electron transport chain of mitochondria. Further IAA-94 reduced CRC of mitochondria by 83+/-6%, n=4. Conclusions: We have established that CLIC4 and CLIC5 are the cardiac mitochondrial anion channels and their activity regulates the function of mitochondria hence playing a direct role in cardioprotection from ischemia-reperfusion injury.