The “M” Conformation of Adenine Nucleotide Translocase Enhances Cyclosporine A-Induced Delay of Mitochondrial Ca2+ Uptake after Cardiac Ischemia/Reperfusion Injury

Abstract

Mitochondria can take up large amounts of Ca2+ during IR injury. How excess mitochondrial Ca2+ leads to formation/opening of the mitochondrial permeability transition pore (mPTP) is poorly understood. Adenine nucleotide translocase (ANT), while not a mPTP component, may regulate mPTP opening. Our aim was to assess if ANT has a role in regulating mPTP after IR injury of isolated guinea pig hearts. We measured Ca2+ retention capacity (CRC), a marker of bioenergetic activity, in mitochondria isolated after no IR (time control, TC) or after IR injury. Cyclophilin D (CypD) interacts with mitochondrial proteins, including ANT, to initiate mPTP opening. Cyclosporine A (CSA) blocks CypD, thus delaying mPTP opening induced by Ca2+ overload if CSA is given during initiation of Ca2+ efflux. Therefore, we examined if ANT inhibitors carboxyatractyloside (cATR) or bonkrekic acid (BKA) alter CSA-induced delay of mPTP opening. We found that CSA-induced delay of mPTP opening (assessed by Ca2+ release) was greater for IR+BKA vs. IR only; all data vs. TC (100%): For substrates NaPyr and NaSucc: IR+BKA, 88±10%, 97±10% (p<.05) vs. IR, 61±7%, 64±7%, respectively. In contrast IR+cATR exhibited no difference from IR (p>0.5) with NaPyr or NaSucc, 55±7%, 71±6%. BKA induces the “m” conformation of ANT (only ADP/ATP transport), while cATR induces the “c” conformation of ANT (ADP/ATP+nonspecific transport), so the differential effect of BKA on CRC suggests at least a partial role for modulation by ANT in the “m” conformation when CSA was given to delay mPTP opening. These results indicate enhanced efficiency of CSA to stall mPTP opening in IR injured mitochondria based on the contention that BKA promotes ANT to “m” being favorable over the “c” conformation induced by cATR or by IR injury.