We demonstrate that CLIC CLIC and CLIC are

We demonstrate that CLIC1, CLIC4 and CLIC5 are the most abundant CLIC transcripts present in the heart. CLIC4 and CLIC5 but not CLIC1 localize to adult cardiac mitochondria. CLIC localization in the GDC-0994 sale was also observed in cardiac tubes of Drosophila melanogaster. Further, we show that CLIC4 is enriched in the outer mitochondrial membrane (OMM) whereas CLIC5 localizes to inner mitochondrial membrane (IMM). Also, cardiac mitochondria from CLIC5 knockout (KO) mice showed increased reactive oxygen species (ROS) generation, thus implicating a direct role of CLIC5 in modulating mitochondrial ROS generation.
Methods
Results IAA-94 abrogated the protective effects of IPC as it increased myocardial infarction (MI) due to IR injury in vitro (Diaz et al., 1999), and also prevented cyclosporine A mediated cardioprotection (Diaz et al., 2013). Moreover, IAA-94-sensitive currents were observed in cardiac mitoplast (Misak et al., 2013) but the molecular identity of cardiac mitochondrial CLICs is not yet elucidated. Therefore, we first set onto examine the identity of CLICs in cardiac mitochondria. As there are six paralogs of CLIC in humans and rats, we tested the abundance of each CLICs in cardiac tissue. Real time qPCR analysis revealed the relative abundance of CLIC4 (89.6±10.4%), CLIC5 (66.7±14.0%), CLIC1 (63.2±3.8%), CLIC2 (35.6±10.6%), CLIC3 (4.5±0.7%) and CLIC6 (0.4±0.2%) (Fig. 1A, B) in the rat heart. Relative abundance was normalized to GAPDH (Fig. 1A) transcript levels. Negative controls without reverse transcriptase (−RT) showed no detectable signals (Fig. 1B). Expression of CLIC1, CLIC4 and CLIC5 proteins was corroborated using CLIC-specific antibodies (Supplementary Fig. 1) on Western blot of whole rat heart lysates (Fig. 1C). Calculated molecular weights of CLIC1, CLIC4 and CLIC5 are ~26.98, 28.63, and 28.23kDa, respectively. However, on 4–20% SDS gel, CLIC1 migrated at ~30kDa, CLIC4 at ~28kDa and CLIC5 at ~30 and ~50kDa. Two distinct bands were observed for CLIC5 in the heart lysate which could either be attributed to a dimer or presence of another isoform (NP_001107558.1). Additional lower molecular weight bands were also observed for CLIC4 in kidney lysates which could be attributed to protein degradation/proteolysis as reported for other ion channels (Knaus et al., 1995). After establishing the presence of CLIC1, CLIC4 and CLIC5 in the heart, we probed their cellular localization in neonatal and adult cardiomyocytes isolated from rat. We quantified their subcellular localization in isolated neonatal cardiomyocytes. CLIC5 showed higher degree of localization to mitotracker-labeled mitochondria of postnatal day 3 cardiomyocytes (73.2±11.5%, n≥5) in comparison with CLIC1 and CLIC4 [(41.7±7.3%; 41.2±7.3%); n≥5 respectively] (Fig. 2). We also investigated the presence of CLIC1, CLIC4 and CLIC5 in the endoplasmic reticulum (ER) of neonatal cardiomyocytes. We found that CLIC4 (35±1.6%, n=3) and CLIC1 (37±1.0%, n=3) localizes to the ER labeled with ER-Tracker™ red dye, but localization of CLIC5 to ER marker was evidently lower (15±1.1%, n=3) (Supplementary Fig. 2).