Preconditioning with hydrogen gas produces cardioprotective effects through autophagy activation in rat cardiomyocytes

It is controversial whether hydrogen (H2) gas can activate cellular survival cascades and autophagy in the myocardium. This translational study aimed to investigate the hypothesis that preconditioning with inhaled H2 gas had a cardio-protective effect mediated through the activation of cellular signaling cascades and autophagy. Rat cardiomyocytes were isolated following 3% H2 gas inhalation for 60 min and were then incubated in a solution with or without 10 μM of 3-methyladenine (MA), an autophagy inhibitor. Intracellular Ca2+ concentration after 10-min perfusion of hydrogen peroxide, Ca2+ mobilization from the endoplasmic reticulum (ER), and opening of the mitochondrial permeability transition pore (MPTP) were estimated using fluorescence imaging. The expressions of extracellular signal-regulated kinase (ERK) and Akt and the levels of mitochondrial membrane potentials (ψm) were determined using flow cytometry. Furthermore, autophagosomes and microtubule-associated light chain (LC) 3-II were estimated using for autophagy activation flow cytometry. H2 gas inhalation effectively inhibited the increase in the intracellular Ca2+ concentration and Ca2+ mobilization from the ER. The inhibitory effects of H2 gas were completely abolished by pretreatment with 3-MA. H2 gas inhalation also prolonged the time to MPTP opening, which was considerably shortened by the 3-MA pretreatment. The expression of signaling molecules and the levels of ψm decreased after 1.8 mM Ca2+ stimulation. Regardless of the stimulation, H2 gas inhalation preserved the ERK expression and ψm levels and increased autophagosomes and the LC3-II expression, but it did not affect the Akt expression. The 3-MA pretreatment completely abolished the cardio-protective effects of H2 gas as well as the autophagy activation. Preconditioning with H2 gas confers the cardio-protective effect in association with the activation of the mitogen-activated protein kinase/ERK pathway and autophagy in the myocardium. Thus, it may be feasible and effective as preventive intervention in patients undergoing cardiac surgery.