Total Matrix Ca Modulates Ca Efflux Via the Ca/H Exchanger in Cardiac Mitochondria

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