Acidification reduces mitochondrial calcium uptake in rat cardiac mitochondria

Cardiac ischemia-reperfusion (I/R) injury is accompanied by intracellular acidification that can lead to cytosolic and mitochondrial calcium overload. However, the effect of cytosolic acidification on mitochondrial pH (pH(m)) and mitochondrial Ca2+ (Ca-m(2+)) handling is not well understood. In the present study, we tested the hypothesis that changes in pH(m) during cytosolic acidification can modulate Ca-m(2+) handling in cardiac mitochondria. pHm was measured in permeabilized rat ventricular myocytes with the use of confocal microscopy and the pH-sensitive fluorescent probe carboxyseminaphthorhodafluor-1. The contributions of the mitochondrial Na+/H+ exchanger (NHEm) and the K+/H+ exchanger (KHEm) to pH(m) regulation were evaluated using acidification and recovery protocols to mimic the changes in pH observed during I/R. Ca-m(2+) transport in isolated mitochondria was measured using spectrophotometry and fluorimetry, and the mitochondrial membrane potential was measured using a tetraphenylphosphonium electrode. Cytosolic acidification (pH 6.8) resulted in acidification of mitochondria. The degree of mitochondrial acidification and recovery was found to be largely dependent on the activity of the KHEm. However, the NHEm was observed to contribute to the recovery of pH(m) following acidification in K+-free solutions as well as the maintenance of pH(m) during respiratory inhibition. Acidification resulted in mitochondrial depolarization and a decrease in the rate of net Ca-m(2+) uptake, whereas restoration of pH following acidification increased Ca-m(2+) uptake. These findings are consistent with an important role for cytosolic acidification in determining pHm and Ca-m(2+) handling in cardiac mitochondria under conditions of Ca2+ overload. Consequently, interventions that alter pHm can limit Ca-m(2+) overload and injury during I/R.