| Summary: | Iron (Fe) plays an essential role in many physiological processes. Hereditary hemochromatosis or frequent blood transfusions often cause iron overload (IO), which can lead to cardiomyopathy and arrhythmias; however, the underlying mechanism is not well defined. In the present study, we assess the hypothesis that IO promotes arrhythmias via reactive oxygen species (ROS) production, mitochondrial membrane potential (∆<i>Ψ</i><sub>m</sub>) depolarization, and disruption of cytosolic Ca dynamics. In ventricular myocytes isolated from wild type (WT) mice, both cytosolic and mitochondrial Fe levels were elevated following perfusion with the Fe<sup>3+</sup>/8-hydroxyquinoline (8-HQ) complex. IO promoted mitochondrial superoxide generation (measured using MitoSOX Red) and induced the depolarization of the Δ<i>Ψ</i><sub>m</sub> (measured using tetramethylrhodamine methyl ester, TMRM) in a dose-dependent manner. IO significantly increased the rate of Ca wave (CaW) formation measured in isolated ventricular myocytes using Fluo-4. Furthermore, in ex-vivo Langendorff-perfused hearts, IO increased arrhythmia scores as evaluated by ECG recordings under programmed S1-S2 stimulation protocols. We also carried out similar experiments in cyclophilin D knockout (CypD KO) mice in which the mitochondrial permeability transition pore (mPTP) opening is impaired. While comparable cytosolic and mitochondrial Fe load, mitochondrial ROS production, and depolarization of the ∆<i>Ψ</i><sub>m</sub> were observed in ventricular myocytes isolated from both WT and CypD KO mice, the rate of CaW formation in isolated cells and the arrhythmia scores in ex-vivo hearts were significantly lower in CypD KO mice compared to those observed in WT mice under conditions of IO. The mPTP inhibitor cyclosporine A (CsA, 1 µM) also exhibited a protective effect. In conclusion, our results suggest that IO induces mitochondrial ROS generation and ∆<i>Ψ</i><sub>m</sub> depolarization, thus opening the mPTP, thereby promoting CaWs and cardiac arrhythmias. Conversely, the inhibition of mPTP ameliorates the proarrhythmic effects of IO.
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