Ca2+ Dysregulation Induces Mitochondrial Depolarization and Apoptosis: ROLE OF Na+/Ca2+ EXCHANGER AND AKT

Abstract

We previously reported that constitutively activated Galpha(q) (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca(2+) handling linked Galpha(q) activity to apoptosis because resting Ca(2+) levels were significantly increased prior to development of apoptosis. Treating cells with EGTA lowered Ca(2+) and blocked both loss of mitochondrial membrane potential (an indicator of permeability transition pore opening) and apoptosis (assessed by DNA fragmentation). When cytosolic Ca(2+) and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca(2+) oscillations (time-to-peak approximately 4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca(2+), directly paralleled by loss of mitochondrial membrane potential. Ca(2+) transients generated by caffeine-induced release of SR Ca(2+) were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca(2+). Indeed, the Na(+)/Ca(2+) exchanger (NCX), which removes Ca(2+) from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca(2+) levels and prevented DNA fragmentation in cells expressing Q209L. Interestingly, constitutively activated Akt, which rescues cells from Q209L-induced apoptosis, prevented the decrease in NCX expression, normalized cytosolic Ca(2+) levels and spontaneous Ca(2+) oscillations, shortened caffeine-induced Ca(2+) transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca(2+) can lead to Ca(2+) overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca(2+) dysregulation is a target of Akt-mediated protection.