Ca2+ Dysregulation Induces Mitochondrial Depolarization and Apoptosis

Abstract

We previously reported that constitutively activated G␣ 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 G␣ 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 ϳ4 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 ؉

Akt mediates mitochondrial protection in cardiomyocytes through phosphorylation of mitochondrial hexokinase-II

“…Mitochondrial membrane potential can be assessed in cardiomyocytes loaded with TMRE, using confocal microscopy and fluorescence imaging, as validated in our previous work. 6 As shown in Figure 1a treatment with 100 mM H 2 O 2 lead to a dramatic decrease in TMRE fluorescence within 60 min, indicative of mitochondrial depolarization. This response was inhibited by pretreatment with EGTA or cyclosporine A (Figure 1b), consistent with involvement of elevated Ca 2 þ and PT-pore opening in H 2 O 2 -induced mitochondrial depolarization.…”

“…Cardiomyocytes were first loaded with TMRE then treated with LIF or vehicle for 15 min, after which time the plasma membrane was permeabilized by addition of saponin (50 mg/ml) for 60 s. As we have previously reported, mitochondrial membrane potential is not perturbed by this saponin permeabilization protocol. 6 The extramitochondrial Ca 2 þ concentration was then raised to induce mitochondrial membrane depolarization (Figure 2). In control cells, raising Ca 2 þ to 2 mM (from the physiological resting level of 100 nM) decreased TMRE fluorescence and further elevating Ca 2 þ to 10 mM fully depolarized the mitochondria (Figure 2a).…”

“…Confocal imaging was carried out using a Bio-Rad MRC1024ES imaging system as described and validated previously. 6 TMRE was excited at 568 nm and fluorescence was recorded using a photomultiplier and emission filter of HQ598/40. Fluorescence values were background-subtracted and expressed as a relative value (100F/F 0 , where F is the measured fluorescence and F 0 is that at the beginning of the experiment).…”

“…Cells were loaded with TMRE and permeabilized with saponin (50 mg/ml, 60 s) as described previously. 6 An intracellular buffer containing magnesium methansulphonate (5.25 mM), potassium methansulphonate (40 mM), potassium chloride (75 mM), ATP (5.3 mM), PIPES (20 mM), E-64 (1 mg/ml), and EGTA (10 mM). Thapsigargin (1 mM) was also added to block sarcoplasmic reticulum function and succinate (5 mM) was added to energize mitochondria.…”

“…1,2 The protein kinase Akt and its downstream signaling cascade, support cell survival in many systems including the heart. [3][4][5][6] Receptor stimuli that have been reported to elicit Akt-mediated cell protection in the heart include ligands for receptor tyrosine kinases (insulin-like growth factor-1:IGF-1), 3,4 glycoprotein 130 (cardiotrophin-1; CT-1, leukemia inhibitory factor; LIF) 7,8 and G-protein-coupled receptors (sphingosine 1-phosphate; S1P). 9 Sustained expression of constitutively active Akt (myr-Akt) prevents cardiomyocyte death induced by ischemia/reperfusion both in vitro 3,4 and in vivo, 4,5 as does expression of nuclear-targeted Akt, suggesting that transcriptional events mediate the cardioprotective effects of Akt.…”