Effect Of Hydrogen Sulfide On Reactive Oxygen Species Level In Rat Cardiomyocyte Subjected To Hypoxia/Reoxygenation

Ischemia/reperfusion (I/R) injury is important in human pathophysiology. It is a clinical problem, and occurs in a wide variety of important clinical conditions. Myocardial I/R injury is a common phenomenon associated with thrombolysis, angioplasty and coronary bypass surgery, which effectively restores blood flow to ischemic myocardium. Although the mechanism of myocardial damage induced by I/R is not completely elucidated, one of the major mechanisms is provided by oxidative stress and the burst of reactive oxygen species (ROS) production. Accumulated evidences have shown that ROS cause the oxidation of proteins, lipids and nucleic acids, resulting in the structural and functional changes of proteins, leading to severe functional and metabolic disorders. Recently, hydrogen sulfide (H2S) has been proposed as a new gaseous messenger molecule. It is produced endogenously in mammalian tissues, and exerts many important biological functions. Convincing evidence has shown that H2S participate in myocardial I/R injury. No matter in global or cellular models of I/R, it was reported that H2S is protective against I/R injury of the heart. The molecular mechanisms of H2S induced protection are still unclear, and the underlying mechanism(s) may involve NO release, PKC activation, and KATP channel opening and so on. Moreover, it is reported that H2S could exert myocardial protection at least in part through direct scavenging of oxygen-free radicals and hydrogen peroxide. Enlightened by this opinion, we intended to figure out whether H2S could decrease intracellular ROS level by the modulation of generation and/or clearance of ROS in rat cardiomyocytes subjected to I/R.In the present study, we investigated the regulatory effect of H2S on the cellular ROS levels in neonatal rat cardiomyocytes following hypoxia/reoxygenation (H/R) that simulated I/R condition. Isolated and cultured neonatal rat cardiomyocytes in control group were maintained in normal conditions. Both H/R group and H/R+NaHS group were incubated in non-serum media with hypoxia condition for4hours followed by1h of reoxygenation. Different concentrations of exogenous H2S donor, NaHS, were applied in H/R+NaHS group30min prior to the onset of hypoxia and maintained until60min reoxygenation. During reoxygenation, intracellular ROS levels at different time points were assessed using the cell-permeable probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). After60min reoxygenation, the mitochondria were harvested from cardiomyocytes for the measurements of the activities of mitochondrial respiratory chain complex I, II, III, IV, and the activities of endogenous antioxidases incluing superoxide dismutase (SOD), catalase(CAT), and glutathione peroxidase(GPx) were determined by corresponding enzyme activity assays, respectively. In addition, Western blot was used to detect the expressions of manganese superoxide dismutase (Mn-SOD), copper/zinc superoxide dismutase (CuZn-SOD) and peroxisome proliferator-activated receptors gamma (PPARy) in cardiomyocytes. The results showed that exogenous NaHS (H2S donor) effectively inhibited the increase in cellular ROS levels over the period from the5min to60min after the initiation of reoxygenation. Among the different concentrations of NaHS used, the corresponding optimal dose was50μmol/L. The activities of cardiomyocyte mitochondrial respiratory chain complex Ⅰ, Ⅲ, Ⅳ decreased significantly in H/R and H/R+NaHS group, compared with those in control group. It is noteworthy that NaHS could greatly reduce the activity of complex IV injuried by H/R. Intracelluar CAT and GPx activities were not significant affected by the treatment with H/R and/or NaHS, while SOD activity of the cardiomyocytes were significantly decreased in H/R group compared with control group, and recovered with NaHS treatment. We found that NaHS could directly interact with SOD protein and enhance the superoxide anions scavenging activity of SOD, which may involve in the mechanism for NaHS to increase intracellular SOD activity. Moreover, NaHS increased protein levels of MnSOD, with no contribution to expression of Cu/Zn SOD. Furthermore, the expression of PPARy, which was recently identified as an important transcriptional determinant of Mn-SOD expression in cardiomyocytes, was not found to be affected by NaHS. In summary, these results suggest that H2S can reduce ROS levels of cardiomyocytes subjected to H/R, which was associated with the modulation of mitochondrial electron transport and the upregulation of the activity and expression of Mn-SOD.