Methylene Blue Counteracts H ₂ S-Induced Cardiac Ion Channel Dysfunction and ATP Reduction

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H ₂ S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca ²⁺ homeostasis disrupted by H ₂ S 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 H ₂ S, we evaluated the effects of H ₂ S on resting membrane potential (E _m ), action potential (AP), Na ⁺ /Ca ²⁺ 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 H ₂ S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H ₂ S (100 µM) significantly depolarized E _m , reduced AP amplitude, prolonged AP duration at 90% repolarization (APD ₉₀ ), 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 H ₂ S exposure restored E _m , APD ₉₀ , 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 H ₂ S-induced cardiomyocyte toxicity at multiple levels: (1) reversing excitation–contraction coupling defects (Ca ²⁺ homeostasis and L-type Ca ²⁺ channels); (2) reducing risks of arrhythmias (E _m , APD, I _NaCa and depolarization-activated K ⁺ currents); and (3) improving cellular bioenergetics (ATP, ∆ψ _m ).