Hydrogen inhibits isoproterenol‑induced autophagy in cardiomyocytes in vitro and in vivo

Abstract:

A previous study from our group has demonstrated that hydrogen administration can attenuate cardiovascular hypertrophy in vivo by targeting reactive oxygen species‑dependent mitogen‑activated protein kinase signaling. The aim of the present study is to determine the effect of hydrogen on cardiomyocyte autophagy during β‑adrenoceptor activation in vivo and in vitro. We prepared hydrogen‑rich medium, and the concentration of hydrogen was measured by using the MB‑Pt reagent method. For the in vitro study, H9c2 cardiomyocytes were stimulated with isoproterenol (ISO; 10 µM) for 5, 15 and 30 min, and then the protein expression levels of the autophagy marker microtubule‑associated protein 1 light chain 3β II (LC3B II) were examined by western blotting. The effect of hydrogen‑rich medium was then tested by pretreating the H9c2 cardiomyocytes with hydrogen‑rich medium for 30 min, then stimulating with ISO, and examining the protein expression levels of the autophagy marker LC3B II. For the in vivo study, mice received hydrogen (1 ml/100 g/day, by intraperitoneal injection) for 7 days prior to ISO administration (0.5 mg/100 g/day, by subcutaneous injection), and subsequently received hydrogen with or without ISO for another 7 days. Hypertrophic responses were examined by heart weight (HW) and heart weight/body weight (HW/BW) measurements. The protein expression of autophagy markers Beclin1, autophagy‑related protein 7 (Atg7) and LC3B II were examined. The results demonstrated that excessive autophagy occurred following 5 min of ISO stimulation in vitro. This enhanced autophagy was blocked by pretreatment with hydrogen‑rich medium. Furthermore, hydrogen improved the deteriorated hypertrophic responses and inhibited the enhanced autophagic activity mediated by ISO administration in vivo, as indicated by decreasing HW and HW/BW, and suppressing the protein expression levels of Beclin1, Atg7 and LC3B II. Therefore, the results of the present study demonstrated that hydrogen inhibited ISO‑induced excessive autophagy in cardiomyocyte hypertrophy models in vitro and in vivo.