Altering buffer Ca 2+ and pH stimulates activation of mitochondrial Ca 2+ /H + exchanger

Calcium overload in mitochondria (m) is well known to contribute to myocyte damage in cardiac ischemia and reperfusion injury. Integral proteins across the inner mitochondrial membrane facilitate transmembrane influx and efflux of Ca 2+ . Influx of Ca 2+ occurs primarily by the mitochondrial Ca 2+ uniporter (mCU) and efflux by the Na + /Ca 2+ exchanger (NCE). Evidence for activation of Ca 2+ /H + exchange (CHE), an additional mechanism for Ca 2+ influx or efflux tied to trans‐matrix pH, has been investigated recently but requires further mechanistic understanding. We hypothesized that altered buffer (extra‐matrix) pH and [Ca 2+ ] can induce CHE activity as demonstrated by reciprocal changes in matrix pH m and [Ca 2+ ] m when Ca 2+ influx through mCu is in equilibrium and Ca 2+ efflux through NCE is prevented by using Na + free buffer and an NCE blocker (CGP 37157). Our aim was to determine if changing extra‐matrix buffer pH alters [Ca 2+ ] m, inferring CHE activity, when Na + and ADP are absent to prevent Ca 2+ efflux through NCE and hydrogen (H + ) uptake through complex V, respectively. To do so, mitochondria were isolated from guinea pig hearts using differential centrifugation, suspended in isolation buffer, and loaded with fura‐4‐AM, a fluorescent probe used to measure [Ca 2+ ] m . Isolated mitochondria were placed in one of 3 experimental buffers (acidic, pH 6.9; physiologic, pH 7.15; or alkaline, pH 7.6). Mitochondria were charged with the Na + ‐free substrate pyruvic acid (0.5 mM). Matrix levels of Ca 2+ were monitored spectrofluorometrically before and after adding a bolus of 40 μM CaCl 2 (EGTA ~38–40 μM) to the mitochondrial buffer. Matrix Ca 2+ levels were monitored for 30 min to permit primary uptake through the mCU and to attain equilibrium. Matrix pH and mitochondrial membrane potential (Δψ m ) were also measured using the fluorescent probes BCECF‐AM and rhodamine 123, respectively. We found that mitochondria suspended in pH 6.9 buffer exhibited a delayed, secondary increase in [Ca 2+ ] m precluded by a reciprocal decrease in matrix pH. These changes were absent in mitochondria suspended in pH 7.15 and 7.16 buffers. Δψ m was unaffected by any protocol suggesting that changes in pH and Ca 2+ were not due to a change in Δψ m . Although changing buffer pH and [Ca 2+ ] caused changes to pH m and [Ca 2+ ] m , there may be other factors in addition to CHE that explain these observations. Assessment of mitochondrial respiration and proton pumping by blocking complexes I, III, IV, V, and mCU may be necessary to fully evaluate CHE independent of other causes of H + and Ca 2+ flux. A better understanding of mitochondrial Ca 2+ uptake, including the role of CHE, may be helpful in treating and reducing cardiac ischemia and reperfusion damage in patients. Support or Funding Information NHLBI Training Grant T35 HL072483Isolated mitochondria suspended in experimental buffer of varying pH's: 6.9, 7.15, and 7.6. Mitochondria were given a 40 mM CaCl 2 bolus at 120 seconds and internal mitochondrial matrix Ca 2+ levels were monitored using fura‐4‐AM. Mitochondria suspended in acidic buffer (pH 6.9) experienced a secondary uptake of Ca 2+ at approx. 800 seconds. Secondary uptake of Ca 2+ was not observed in mitochondria suspended in pH buffers of 7.15 and 7.6.BCECF‐AM was used to measure mitochondrial matrix pH over 30 minutes. A 40 mM bolus of CaCl 2 was added at 120 seconds. Mitochondria suspended in acidic buffer (pH 6.9) experienced a decline in mitochondrial matrix pH to approx. 7. Data collection was ended at 30 minutes.Mitochondrial membrane potential was measured using rhodamine‐123 at three different buffer pH's. Mitochondria were added to the experimental buffer at 30 sec. A 40 mM bolus of CaCl 2 was added at 210 sec. and 36 μM FCCP, a mitochondrial membrane uncoupler, was added to the suspended mitochondria at 1740 sec. to stop data collection.