Iron Overload Promotes Arrhythmias via ROS Production and Mitochondrial Membrane Potential Depolarization

Abstract

Genetic disorders or frequent blood transfusions often cause iron overload which can lead to arrhythmias. However, the underlying mechanism is not well known. In the present study, we assess the hypothesis that iron-overload promotes arrhythmias via reactive oxygen species (ROS) production and mitochondrial membrane potential (ΔΨm) depolarization, opening the mitochondrial permeability transition pore (mPTP) and promoting Ca waves. ROS levels were measured by DCF fluorescence. Perfusion with Fe2+ (1 – 10 mM) significantly increased relative ROS levels. For example, 1 mM Fe2+ raised ROS levels 465 ± 14% compared to the control (p <0.01). TMRM fluorescence decrease was used as an index of ΔΨm depolarization. Fe2+ significantly decreased TMRM fluorescence (1 mM: 8.1 ± 2.3%; 10 mM: 94.8 ± 2.9 %), compared to the baseline in myocytes, indicating ΔΨm depolarization by Fe2+. Intracellular Ca2+ was imaged with Fluo-4-AM. Spontaneous Ca waves were induced in high external Ca2+ (4 mM). Furthermore, Fe2+ significantly increased the rate of Ca waves: 1 mM Fe2+ significantly increased the rate from 23.9 ± 1.7 to 39.8 ± 7.6 min−1 (p < 0.05). Fe2+-promoted Ca waves were significantly reduced by the mPTP inhibitor cyclosporine A (CsA; 1 µM). ECGs were recorded from Langedorff-perfused hearts. Arrhythmia induction testing was conducted through programmed S1-S2 stimulation. Hearts treated with Fe2+ (1 mM) alone significantly increased arrhythmia scores compared to hearts pretreated with CsA prior to Fe2+ (p < 0.05), indicating mPTP inhibition can ameliorate the proarrhythmic effects of iron-overload. These observation were confirmed in the cyclophilin D knockout (CypD KO) mice, in which mPTP opening is impaired. In conclusion, our results suggest that exogenous Fe2+ treatment increases ROS production, depolarizes the ΔΨm, opens the mPTP, and promotes Ca waves and arrhythmogenesis.