Identification of Role of Mitochondrial Chloride Intracellular Channel (CLIC) Protein, CLIC4 and CLIC5 in Cardioprotection from IR Injury via Probably Modulating the Opening of MPTP Pore

Abstract

Chloride intracellular channels (CLICs) are widely distributed across species and play a pivotal role in many physiological and pathophysiological process such as cell cycle regulation, tumor progression, metastasis and pulmonary hypertension. They are unique class of dimorphic ion channel proteins existing in soluble and integral membrane form. Recently, we demonstrated that indanyloxyacetic acid 94 (IAA-94), a known CLIC blocker, increased myocardial infarction (MI) by 25±8% (p=0.01, n=5) upon in vivo ischemia-reperfusion (IR) injury. Further, the calcium retention capacity (CRC) of mitochondria upon IR injury in IAA-94 treated groups were reduced by 21±1.5% (p<0.05, n>3) in comparison to controls. Thus, suggesting the targets of IAA-94 such as CLICs are important mediators of cardioprotection from IR injury. Interestingly, we observed that amongst the six mammalian paralogs of CLICs, CLIC4 (49±7%) and CLIC5 (69±12%) localize to Percoll-purified ultra-pure cardiac mitochondria. We also detected IAA-94 sensitive CLIC like channel activity in cardiac mitoplast further indicating their functional activity. Cardiac mitochondria from clic5-/- mice exhibited increased ROS (p< 0.05, n=3) production. Moreover, we demonstrate that lack of CLIC4 and CLIC5 reduce the mitochondrial CRC by ∼17% and ∼20% (p=0.01, n≥7 for each) respectively, signifying their role in mitochondrial permeability transition pore (mPTP) opening. In support, pull down assays and mass spectrometry analysis reveal the interaction of CLIC4 and CLIC5 to each other as well as Cyclophilin D (CypD) and ATP synthase, the probable component of mPTP pore. To conclude, our results highlight the significance of mitochondrial chloride channels CLIC4 and CLIC5 in cardioprotection from IR injury probably by regulating the opening of mPTP via interacting with ATP synthase and Cyclophilin D.