Slow Ca2+ Efflux by Ca2+/H+ Exchange in Cardiac Mitochondria Is Modulated by Ca2+ Re-uptake via MCU, Extra-Mitochondrial pH, and H+ Pumping by FOF1-ATPase

Mitochondrial (m) Ca2+ influx is largely dependent on membrane potential (Delta Psi(m)), whereas mCa(2+) efflux occurs primarily via Ca2+ ion exchangers. We probed the kinetics of Ca2+/H+ exchange (CHEm) in guinea pig cardiac muscle mitochondria. We tested if net mCa(2+) flux is altered during a matrix inward H+ leak that is dependent on matrix H+ pumping by ATP(m) hydrolysis at complex V (FOF1-ATPase). We measured [Ca2+](m), extra-mitochondrial (e) [Ca2+](e), Delta Psi(m), pH(m), pH(e), NADH, respiration, ADP/ATP ratios, and total [ATP](m) in the presence or absence of protonophore dinitrophenol (DNP), mitochondrial uniporter (MCU) blocker Ru360, and complex V blocker oligomycin (OMN). We proposed that net slow influx/efflux of Ca2+ after adding DNP and CaCl2 is dependent on whether the Delta pH(m) gradient is/is not maintained by reciprocal outward H+ pumping by complex V. We found that adding CaCl2 enhanced DNP-induced increases in respiration and decreases in Delta Psi(m) while [ATP](m) decreased, Delta pH(m) gradient was maintained, and [Ca2+](m) continued to increase slowly, indicating net mCa(2+) influx via MCU. In contrast, with complex V blocked by OMN, adding DNP and CaCl2 caused larger declines in Delta Psi(m) as well as a slow fall in pH(m) to near pH(e) while [Ca2+](m) continued to decrease slowly, indicating net mCa(2+) efflux in exchange for H+ influx (CHEm) until the Delta pH(m) gradient was abolished. The kinetics of slow mCa(2+) efflux with slow H+ influx via CHE m was also observed at pH(e) 6.9 vs. 7.6 by the slow fall in pH(m) until Delta pH(m) was abolished; if Ca2+ reuptake via the MCU was also blocked, mCa(2+) efflux via CHE m became more evident. Of the two components of the proton electrochemical gradient, our results indicate that CHEm activity is driven largely by the Delta pH(m) chemical gradient with H+ leak, while mCa(2+) entry via MCU depends largely on the charge gradient Delta Psi(m). A fall in Delta Psi(m) with excess mCa(2+) loading can occur during cardiac cell stress. Cardiac cell injury due to mCa(2+) overload may be reduced by temporarily inhibiting FOF1-ATPase from pumping H+ due to Delta Psi(m) depolarization. This action would prevent additional slow mCa(2+) loading via MCU and permit activation of CHEm to mediate efflux of mCa(2+).