Ca²⁺ dysregulation induces mitochondrial depolarization and apoptosis: Role of Na⁺/Ca²⁺ exchanger and Akt

We previously reported that constitutively activated Gα_q (Q209L) expression in cardiomyocytes induces apoptosis through opening of the mitochondrial permeability transition pore. We assessed the hypothesis that disturbances in Ca²⁺ handling linked Gα_q activity to apoptosis because resting Ca²⁺ 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 Ca²⁺ and mitochondrial membrane potential were simultaneously measured by confocal microscopy, sarcoplasmic reticulum (SR)-driven slow Ca²⁺ oscillations (time-to-peak ∼4 s) were observed in Q209L-expressing cells. These oscillations were seen to transition into sustained increases in cytosolic Ca²⁺, directly paralleled by loss of mitochondrial membrane potential. Ca²⁺ transients generated by caffeine-induced release of SR Ca²⁺ were greatly prolonged in Q209L-expressing cells, suggesting a decreased ability to extrude Ca²⁺. Indeed, the Na⁺/Ca²⁺ exchanger (NCX), which removes Ca²⁺ from the cell, was markedly down-regulated at the mRNA and protein levels. Adenoviral NCX expression normalized cytosolic Ca²⁺ 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 Ca²⁺ levels and spontaneous Ca²⁺ oscillations, shortened caffeine-induced Ca²⁺ transients, and prevented loss of the mitochondrial membrane potential. Our findings demonstrate that NCX down-regulation and consequent increases in cytosolic and SR Ca²⁺ can lead to Ca²⁺ overloading-induced loss of mitochondrial membrane potential and suggest that recovery of Ca²⁺ dysregulation is a target of Akt-mediated protection.