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"Mitochondrial Ca2+ Flux as a Modulator of Arrhythmogenesis"

Rick Gordan
Cell Biology and Molecular Medicine Program
M.S. 2009, University of Medicine and Dentistry of New Jersey
B.A. 2007, Franklin & Marshall College, PA

Thesis Advisor: Lai-Hua Xie, PhD, FAHA
Associate Professor
Department of Cell Biology and Molecular Medicine

Thursday, May 12, 2016
1:00 P.M., MSB G609


Background: Recent reports have shown that mitochondria play a significant role in Ca2+ homeostasis of cardiomyoyctes. In the present study, we aimed to investigate how mitochondrial membrane potential (ΔΨm) depolarization influences the local Ca2+ concentrations near the sarcoplasmic reticulum (SR) micro-domain, promoting proarrhythmic Ca2+ waves (CaWs) and Ca2+ alternans. Arrhythmias may also be caused by iron overload as a result of genetic disorders or frequent blood transfusions, however the underlying mechanism is not well understood. Thus, we have also assessed the role that iron overload plays in promoting arrhythmias via a similar mechanism: ROS production-mediated mPTP opening.
Methods and Results: To assess the role of the mitochondrial permeability transition pore (mPTP) in this regulation, we employed both the mPTP inhibitor cyclosporine A (CsA) and a genetic mouse model which lacks cyclophilin D (CypD KO), a necessary component for mPTP opening. Ventricular myocytes isolated from WT mice showed a significant depolarization in ΔΨm and decrease in mitochondrial calcein fluorescence (indicating mPTP opening) in response to the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) treatment. FCCP also raised basal Ca2+ levels and increased the frequency and amplitude of spontaneous CaWs. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effects of FCCP on CaWs were counteracted by mPTP inhibition by CsA, suggesting the involvement of the mPTP. Consistent with this notion, in response to FCCP, CypD KO myocytes showed similar ΔΨm depolarization, however less mPTP opening, significantly lower release of mitochondrial Ca2+, and a smaller increase in the frequency of CaWs than WT myocytes. The proarrhythmic effect of FCCP (30 nM) and the involvement of mPTP were compared in ex-vivo, Langendorff-perfused WT and CypD KO hearts. The incidences of ST-T-wave alternans and arrhythmias induced by programmed S1-S2 stimulation were evaluated separately. Alternans were observed in a significantly higher number of WT hearts perfused with FCCP, than in WT hearts pre-treated with CsA, and did not occur in CypD KO hearts. Arrhythmia scores for WT hearts perfused with FCCP were significantly higher than WT hearts pretreated with CsA, or CypD KO hearts. Perfusion with Fe3+/8-HQ significantly increased relative cellular ROS levels in myocytes and significantly depolarized ΔΨm. Additionally, Fe3+/8-HQ significantly increased the CaW rate, however the CaWs were significantly attenuated in CypD KO myocytes, or WT cells treated with CsA, or the antioxidants MitoTEMPO or EUK-8. ECG recording and arrhythmia induction testing were conducted as with FCCP. Hearts treated with Fe3+/8-HQ had increased arrhythmia scores compared to hearts pretreated with CsA or CypD KO hearts, indicating that mPTP inhibition can ameliorate the proarrhythmic effects of iron overload.
Conclusions: Our results suggest that mitochondrial Ca2+ release via the mPTP influences local Ca2+ levels in the micro-domain near the SR and plays an important role in regulating intracellular CaWs, alternans, and arrhythmogenesis. Additionally, our results suggest that iron overload increases ROS production, depolarizes the ∆Ψm, and promotes CaWs and arrhythmogenesis. Understanding the role of mitochondrial Ca2+ regulation in arrhythmogenesis may be helpful in developing new approaches for the treatment of cardiac arrhythmias.

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