Role of ROS signaling in regulating cell death pathways
Role of ROS signaling in regulating cell death pathways has been well established. Involvement of CLIC1 in ROS production has been studied earlier. It was observed that macrophages from clic1 mice showed ~30% decrease in cellular ROS generation which effects macrophage function (Jiang et al., 2012). Pharmacological inhibition of CLIC1 by IAA-94 reduced colon cancer cell migration and invasion via modulation of cellular ROS (Wang et al., 2014). Additionally, it was observed that β-amyloid-induced Cl conductance and ROS generation were prevented by the inhibition of CLIC1 by IAA-94, thus reducing the β-amyloid-mediated neurotoxicity (Milton et al., 2008). In addition increased ROS levels are also known to upregulate CLIC4 BTL-104 in ovarian cancers (Yao et al., 2009a, Yao et al., 2009b), thus indicating strong inter-regulation of CLICs and ROS generation. As CLIC5 preferentially localizes to the IMM it is expected to play an active role in mitochondrial function such as ROS generation. In our study we did observe an increase in the rate of ROS production upon activation by complex II/III substrate (succinate) in cardiac mitochondria of clic5 mice but no significant change in clic1 or clic4 mice. An absence in the modulation of ROS by CLIC1 is not surprising since CLIC1 does not localize to cardiac mitochondria.
ROS generation by mitochondria plays crucial role in damaging cellular components and initiating cell death pathways (Garlid et al., 2013, Kalogeris et al., 2014). Chloride channel blockers like 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) protected cardiomyocytes from ROS induced apoptosis during IR injury (Wang et al., 2015, Wang et al., 2005), whereas IAA-94 surprisingly ablated the cardioprotection due to IPC. These pharmacological data using IAA-94 implicate cardiac CLICs in cardioprotection, however it is not clear whether CLICs can modulate ROS generation in the heart. During IR, it is observed that pH recovery, calcium overload, and increase in ROS can cause rapid opening of mPTP thereby contributing to cardiomyocyte death (Perrelli et al., 2011). Our genetic approach using clic5 mice shows increase in mitochondrial ROS production. These results support the finding that blocking CLICs with IAA-94 increases myocardial infarction due to IR injury (Diaz et al., 2010, 1999) possibly due to an increase in ROS levels. Therefore, the specific role of CLIC5 in modulating ROS production will be vital to elucidate and study its contribution to cardioprotection from IR injury due to IPC.
Acknowledgments We would like to thank Dr. Olimpia Meucci (DUCoM) for providing reagents. HS is supported by CTRI, CURE, and AHA SDG (11SDG7230059) and startup funds from DUCoM.