Cardioprotective role of inner membrane chloride channel, CLIC5 from ischemia-reperfusion injury.

Abstract

Mitochondrial chloride channels by regulating mitochondrial volume, pH and membrane potential are important in determining the cell fate. However, very few of them are identified in mitochondria. Previously, we demonstrated the localization of a member of chloride intracellular channel (CLIC) family, CLIC5 in the inner mitochondrial membrane and its importance in regulating reactive oxygen species (ROS) generation. Herein, we show the novel role of an alternative N-terminal splice variant clic5b in cardiac mitochondrial physiology. Mass spectrometry and qPCR analysis indicated the presence of the splice variant, clic5b in cardiac tissue in addition to the prominent transcript variant, clic5a. Interestingly, the presence of splice variant clic5b was correlated with its mitochondrial localization in myoblast cells, H9C2 (55.12 ± 7.1%) but not in mouse lung epithelial cells (11 ± 2.1%). Further, using transfection studies we showed that the N terminal region of clic5b is integral for its mitochondrial targeting in neonatal cardiomyocytes and in HEK cells as transfection of clic5a does not exhibit mitochondrial localization. We also demonstrate increased mitochondrial ROS generation in clic5-/- mouse heart (N=4, p≤0.05) in comparison to wild type (wt) mice. On the other hand, lungs, that only contain splice variant clic5a, neither demonstrate mitochondrial localization of CLIC5 nor any change in ROS production. The absence of CLIC5 exacerbated myocardial infarction (52 ± 9.5% versus 38.6 ± 4.5%, n≥3, p≤ 0.05) and reduced left ventricular function upon ischemia-reperfusion (IR) injury in clic5-/- mice in comparison to wt mice suggesting a cardioprotective role of CLIC5. Furthermore, in human heart failure samples, we observed a differential expression of the splice variants clic5a and clic5b in comparison to healthy hearts. Thus, our study highlights the role of splice variant clic5b in regulating cardiac mitochondrial physiology and cardioprotection from IR injury.