br Methods br Results IAA abrogated the protective
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 CDK4 inhibitor (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). Furthermore, quantitative analysis of localization of CLIC1, CLIC4, and CLIC5 to the mitochondria in isolated adult cardiomyocytes (Fig. 3) revealed similar distribution of CLICs as observed in neonatal cardiomyocytes (Fig. 2). However, CLIC4 and CLIC5 showed a higher degree of colocalization to the mitochondria of adult cardiomyocytes in comparison to CLIC1 [Fig. 3 (I, III) (46.3±2.5%); 57.1±2.2%); vs. (35±4.1%), n=5, respectively]. There was no localization of CLIC1, CLIC4 or CLIC5 observed in the nucleus of adult cardiomyocytes (Fig 3D′, H′ and L′). D. melanogaster possesses a single CLIC ortholog, DmCLIC, and its localization in cardiac tubes was also tested. Cardiac tubes of flies showed localization of DmCLIC in mitochondria colabeled with anti-ATP synthase antibodies [Fig. 3(II, III)]. Clic, a protein-null mutant showed no significant labeling with anti-DmCLIC antibodies (Supplementary Fig. 3). Taken together, we have shown that mammalian cardiac CLIC4 and CLIC5 as well as DmCLIC localize to the mitochondria. Majority of mitochondrial proteins are known to possess canonical mitochondrial targeting sequence (MTS) (Omura, 1998), hence we investigated the presence of potential MTS in CLICs. Surprisingly, we did not detect any canonical MTS, and further in silico analysis (Ferre et al., 2013) revealed a very low probability (<0.005) for each CLIC protein being in mitochondria (Table 1). VDAC2 was taken as a control to validate the in silico analysis as it is a well-established OMM protein. All of our analysis algorithms predicted VDAC2 to be a mitochondrial protein.