Total Matrix Ca2+ Modulates Ca2+ Efflux via the Ca2+/H+ Exchanger in Cardiac Mitochondria

Mitochondrial Ca²⁺ handling is accomplished by balancing Ca²⁺ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca²⁺ buffering in the matrix and Ca²⁺ efflux mainly via Ca²⁺ ion exchangers, such as the Na⁺/Ca²⁺ exchanger (NCLX) and the Ca²⁺/H⁺ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca²⁺ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca²⁺ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca²⁺ load via repetitive application of a finite CaCl₂ bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca²⁺ efflux at different matrix Ca²⁺ loads was revealed by inhibiting Ca²⁺ uptake or reuptake with Ru360 after increasing number of CaCl₂ boluses. In Na⁺-free experimental buffer and with Ca²⁺ uptake inhibited, the rate of Ca²⁺ efflux and steady-state free matrix Ca²⁺ [mCa²⁺]_ss increased as the number of administered CaCl₂ boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca²⁺ buffering while maintaining a constant [mCa²⁺]_ss, decreased the rate of Ca²⁺ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca²⁺ buffering rate and decreased [mCa²⁺]_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 Ca²⁺ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca²⁺ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca²⁺ efflux. The results indicate that Ca²⁺ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca²⁺. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca²⁺ buffering system and the matrix free Ca²⁺ concentration.