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

Shigeki Miyamoto,Amy L Howes,John W Adams,Gerald W Dorn,Joan Heller Brown

doi:10.1074/jbc.m505223200

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.

Highlights

Cardiomyocyte apoptosis has been suggested to be a major factor contributing to the transition from compensated hypertrophy to dilated cardiomyopathy and heart failure [1,2,3,4,5,6], as well as to ischemia/reperfusion injury [7]
When cells expressing Q209L were cultured in normal medium to which EGTA was added, cytosolic Ca2ϩ was maintained at control levels (Fig. 1A) and apoptosis was markedly attenuated (Fig. 1C)
We previously reported that Q209L expression resulted in mitochondrial membrane depolarization and that this effect was prevented by bongkrekic acid, a permeability transition pore (PT-pore) inhibitor [14]

Summary

Introduction

Cardiomyocyte apoptosis has been suggested to be a major factor contributing to the transition from compensated hypertrophy to dilated cardiomyopathy and heart failure [1,2,3,4,5,6], as well as to ischemia/reperfusion injury [7]. Transgenic (TG) mice expressing the heterotrimeric G protein ␣ subunit (G␣q) show increased susceptibility to development of cardiomyopathy, heart failure, and apoptosis in response to aortic banding [8] and in the peripartal period [2, 5]. Moderate activation of G␣q is a well established mediator of cardiac hypertrophy, excessive G␣q activation can lead to apoptosis that contributes to the transition from hypertrophy to heart failure [2, 10]. Previous studies examining cardiomyocytes from G␣q transgenic mice revealed prolongation of Ca2ϩ transients and decreases in cardiomyocyte Naϩ/Ca2ϩ exchanger (NCX) [16]. These findings suggested that the ability to decrease cytosolic Ca2ϩ was compromised. We demonstrated that protective effects of NOVEMBER 18, 2005 VOLUME 280 NUMBER 46

Methods

Results

Conclusion