Total Matrix Ca(2+) Modulates Ca(2+) Efflux via the Ca(2+)/H(+) Exchanger in Cardiac Mitochondria

Mitochondrial Ca(2+) handling is accomplished by balancing Ca(2+) uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca(2+) buffering in the matrix and Ca(2+) efflux mainly via Ca(2+) ion exchangers, such as the Na(+)/Ca(2+) exchanger (NCLX) and the Ca(2+)/H(+) exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca(2+) regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca(2+) efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca(2+) load via repetitive application of a finite CaCl(2) bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca(2+) efflux at different matrix Ca(2+) loads was revealed by inhibiting Ca(2+) uptake or reuptake with Ru360 after increasing number of CaCl(2) boluses. In Na(+)-free experimental buffer and with Ca(2+) uptake inhibited, the rate of Ca(2+) efflux and steady-state free matrix Ca(2+) [mCa(2+)](ss) increased as the number of administered CaCl(2) boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca(2+) buffering while maintaining a constant [mCa(2+)](ss), decreased the rate of Ca(2+) efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca(2+) buffering rate and decreased [mCa(2+)](ss.) A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca(2+) efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca(2+) efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca(2+) efflux. The results indicate that Ca(2+) efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca(2+). A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca(2+) buffering system and the matrix free Ca(2+) concentration.