Identification of Cardiac Mitochondrial Chloride Intracellular Channel (CLIC) Proteins and their Physiological Function

Abstract

Emerging evidences show involvement of chloride channels present in cardiac cells, in the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Previously it was demonstrated that blocking cardiac chloride channels with IAA-94, an inhibitor for chloride intracellular channel proteins (CLICs), prevented the protective effects of ischemic preconditioning and cyclosporine A-mediated cardioprotection but the molecular identity and specific role of CLICs in cardiac tissue is not known. Therefore, we attempted to establish molecular identity of CLICs and understand mechanism of IAA-94-mediated cardio deleterious effect. We demonstrate that IAA-94 increases myocardial infarction after ischemic/reperfusion injury in in vivo rat model also. Amongst the six paralogs of CLICs present in mammals, qPCR showed that CLIC1 (63.2+3.8%), CLIC4 (89.6+10.4%), and CLIC5 (66.7+14.0%) are the most abundant CLICs in the heart. We recorded IAA-94 sensitive channel conductance of 110+10 pS in isolated mitoplasts as reported for CLICs. Further, organelle-immunochemistry showed that CLIC5 (69.1±12.2%), CLIC4 (48.9±6.9%), but not CLIC1 (29.5±11.2%) localized to the ultrapure mitochondria. DmCLIC, an ortholog of mammalian CLIC present in D. melanogaster also localizes to cardiac mitochondria. We further demonstrate that rate of reactive oxygen species (ROS) production is reduced upon IAA-94 (100 µM) addition in both cardiac mitochondria (82.5±3.0%) and H9C2 cells (20.1±4.6%). ROS modulation was specific to IAA-94 and was not observed with other chloride channel inhibitors. IAA-94 significantly reduced the calcium retention capacity of the mitochondria (90.0±6.0%), hence reducing a threshold for opening mitochondrial permeability transition pore, but did not affect its membrane potential. Our study highlights the presence of CLIC4 and CLIC5 in cardiac mitochondria, where they modulate mitochondrial function, therefore playing a potential role in cardioprotection.