The Contribution of Mitochondrial Calcium Flux to Permeability Transition Pore Opening

Abstract

Mitochondria are important dynamic organelles which play a vital role in cellular bioenergetics, Ca2+ homeostasis and cell death pathways. One of the causative events of mitochondrial dysfunction is mitochondrial permeability transition pore (mPTP) opening. mPTP is a well-known phenomenon in which permeability of inner mitochondrial membrane increase abruptly. The transient opening of mPTP has important physiological implications including reactive oxygen species (ROS) homeostasis and Ca2+ mobilization. Whereas long-term mPTP opening causes pathological consequences in ischemia reperfusion injury, diabetic kidney disease and hepatic stenosis among others. Several factors including Ca2+ are known to induce mPTP opening. Despite extensive research, the injuries caused by mPTP are still challenging. Therefore, this study was designed to modulate Ca2+ homeostasis using a blocker of Sodium-Calcium-Lithium exchanger (NCLX), CGP37157, to investigate mPTP as a therapeutic target. mPTP opening was evaluated in isolated mitochondria both from kidney and liver on a microplate-based spectrophotometer using different concentrations of calcium chloride (CaCl2). Furthermore, the concentration dependent effect of CGP37157 on mPTP opening in the presence and absence of Na+ (regulator of NCLX) was also studied. The differential mPTP opening in both liver and kidney was also evaluated. Our data showed a concentration-dependent effect of CaCl2 on mPTP opening. However, no observable effect of CGP37157 was found on CaCl2 induced-mPTP opening at any used concentration both in the presence and absence of Na+ in mitochondria isolated from both kidney and liver. For finding the role of MCU in mPTP opening, we blocked MCU with Mitoxantrone, but no inhibitory effect was shown on mPTP. The differential comparison data showed that there was elevated but non-significant higher mPTP opening in liver as compared to kidney. On this basis resting MCU protein level was examined in liver and kidney with no difference observed at protein level. In brief, this study found that MCU and NCLX-mediated Ca2+ flux is dispensable for mPTP formation in mitochondria isolated from both kidney and liver. Moreover, the findings demonstrated that liver mitochondria are more prone to mPTP opening in comparison to kidney mitochondria. Key words: Mitochondrial permeability transition pore (mPTP), Calcium homeostasis, Ischemia reperfusion injury (I/RI), Sodium-Calcium-Lithium exchanger (NCLX), CGP3715