title: 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.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Miyamoto
authors.forename: Amy L
authors.initials: AL
authors.lastname: Howes
authors.forename: John W
authors.initials: JW
authors.lastname: Adams
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Dorn
authors.forename: Joan Heller
authors.initials: JH
authors.lastname: Brown
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D015536:Down-Regulation; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005434:Flow Cytometry; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005786:Gene Expression Regulation; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology*; D009991:Oscillometry; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D012333:RNA, Messenger; D019831:Sodium-Calcium Exchanger; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D002110:Caffeine; D004533:Egtazic Acid; D004247:DNA; D051057:Proto-Oncogene Proteins c-akt; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium