Methylene Blue Counteracts H2S-Induced Cardiac Ion Channel Dysfunction and ATP Reduction

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H₂S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca²⁺ homeostasis disrupted by H₂S poisoning. In vivo, MB restores cardiac contractility severely depressed by sulfide and protects against arrhythmias, ranging from bundle branch block to ventricular tachycardia or fibrillation. To dissect the cellular mechanisms by which MB reduces arrhythmogenesis and improves bioenergetics in myocytes intoxicated with H₂S, we evaluated the effects of H₂S on resting membrane potential (E_m), action potential (AP), Na⁺/Ca²⁺ exchange current (I_NaCa), depolarization-activated K⁺ currents and ATP levels in adult mouse cardiac myocytes and determined whether MB could counteract the toxic effects of H₂S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H₂S (100 µM) significantly depolarized E_m, reduced AP amplitude, prolonged AP duration at 90% repolarization (APD₉₀), suppressed I_NaCa and depolarization-activated K⁺ currents, and reduced ATP levels in adult mouse cardiac myocytes. Treating cardiomyocytes with MB (20 µg/ml) 3 min after H₂S exposure restored E_m, APD₉₀, I_NaCa, depolarization-activated K⁺ currents, and ATP levels toward normal. MB improved mitochondrial membrane potential (?ψ_m) and oxygen consumption rate in myocytes in which Complex I was blocked by rotenone. We conclude that MB ameliorated H₂S-induced cardiomyocyte toxicity at multiple levels: (1) reversing excitation–contraction coupling defects (Ca²⁺ homeostasis and L-type Ca²⁺ channels); (2) reducing risks of arrhythmias (E_m, APD, I_NaCa and depolarization-activated K⁺ currents); and (3) improving cellular bioenergetics (ATP, ?ψ_m).