Abstract 17807: Cyclophilin D Deficiency Attenuates Ca2+ Waves During Mitochondrial Depolarization in Mouse Cardiomyocytes
Recent studies have indicated that mitochondrial Ca flux plays a significant role in modulating micro-domain Ca2+ levels, near the ryanodine receptors, and spontaneous Ca wave (CaW) behaviors in isolated cardiomyocytes. In the present study, we have used a genetic mouse model which lacks cyclophilin D (CypD KO), a necessary component for the opening of the mitochondrial permeability transition pore (mPTP), to further assess the hypothesis that mPTP plays a central role in the regulation of CaWs during mitochondrial depolarization. Ventricular myocytes were isolated from wild type (WT) and CypD KO mice. Mitochondrial calcein release, as estimated from the fluorescence decrease, was used as the index of mPTP opening. Cytosolic Ca2+ was imaged using Fluo-4-AM. Spontaneous CaWs were induced in the presence of high external Ca2+ (4 mM). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) was used to depolarize mitochondrial membrane potential (ΔΨm). WT myocytes showed a significant decrease (61%) in calcein fluorescence in the presence of FCCP (10 μM), compared to CypD KO myocytes (28%; p < 0.05, unpaired t-test). These results indicate less opening of mPTP in response to ΔΨm depolarization in CypD KO myocytes. Consistent with these results, FCCP (50-500 nM) significantly increased the frequency of CaWs in WT myocytes, however no significant increase was observed in CypD KO myocytes. For example, 50 nM FCCP significantly increased the frequency of CaWs from 12.9 ± 2.2 to 32.2 ± 5.3 min-1 in WT myocytes ( p < 0.05, paired t-test), while no significant increase was seen in CypD KO myocytes (26.6 ± 4.3 to 24.4 ± 4.4 min-1). The effects of FCCP on Ca2+ waves in WT mice were attenuated by the mPTP blocker cyclosporin A. Finally, FCCP increased CaW frequency when cellular ATP consumption was inhibited by oligomycin A (1 μM), an ATP synthase inhibitor, supporting the notion that mitochondrial Ca fluxes play a vital role in the regulation of CaWs. In conclusion, cyclophilin D deficiency attenuates Ca2+ waves during mitochondrial depolarization induced by FCCP, suggesting that mitochondrial Ca release via mPTP opening plays an important role in the regulation of intracellular CaW and arrhythmogenesis.
- © 2013 by American Heart Association, Inc.