| Hydrogen sulfide （H2S） is a kind of toxic gas with the characteristic smell of rotten eggs. In a range of mammalian cells and tissues, endogenous H2S is synthesized from L-cysteine and homocysteine mainly by two enzymes, cystathionine (3-synthase （CBS） and cystathionine y-lyase （CSE）. CBS is mainly found in the central nervous system （CNS） whereas CSE is the main H2S-forming enzyme in the cardiovascular system. 3-mercaptopyruvate sulfurtransferase （3-MST）, the third enzyme in H2S production, has been found in brain and vascular endothelium.3-MST could produce H2S along with cysteine aminotransferase （CAT） using both L-cysteine and alpha-ketoglutarate as substrates. In recent years, there is growing evidence that H2S plays vital roles in many physiological and pathological processes such as myocardial protection, angiogenesis, anti-apoptosis, vasorelaxation, anti-inflammatory, and so on. It’s now clear that H2S is considered to be the third gasotransmitter together with nitric oxide （NO） and carbon monoxide （CO）. However, the molecular mechanisms underlying the H2S actions, in particular the potential targeting molecular for H2S, remains unknown.Superoxide dismutases （SODs） are critical enzymes that could effectively scavenge reactive oxygen species （ROS） in biological systems by catalyzing the dismutation of superoxide radical （O2-） into oxygen and hydrogen peroxide. The hydrogen peroxide will be further scavenged by catalase to water and oxygen. Cu/Zn-SOD and Mn-SOD are two main types of SOD in mammalian cells. Mn-SOD is found in mitochondrial matrix while Cu/Zn-SOD is located in cytosol, intermembrane space of mitochondria, and nucleus. There are two different types of metal ions in Cu/Zn-SOD, including copper ion and zinc ion. The copper ion is localized in the active site and plays a vital role in maintaining the enzyme activity whereas the zinc ion has a very important structural role. In addition, the active center of Mn-SOD contains one manganese ion which carries out catalysis by experiencing a series of redox reactions. Recent studies have found that H2S could effectly enhance the activities of both Cu/Zn-SOD and Mn-SOD, but the underlying mechanisms remain unknown. Therefore, we mainly investigated the effects of H2S on both Cu/Zn-SOD and Mn-SOD and further provided new knowledge on the possible mechanisms.First, the hypoxia/reoxygenation model in cultured neonatal Sprague-Dawley （SD） rat ventricular myocytes was established. We then studied the effects of exogenous H2S on cell apoptosis, ROS generation, the activities and expression levels of Cu/Zn-SOD and Mn-SOD. The results showed that NaHS （50 μmol/L） could dramatically reduce the level of apoptosis and ROS generation. Moreover,50 μmol/L NaHS could enhance the activities of Cu/Zn-SOD and Mn-SOD without any effect on protein expression, which indicated that H2S may enhance the activities of Cu/Zn-SOD and Mn-SOD by changing their structures. In addition, we found that when the concentration of NaHS was higher than 50 μmol/L, both the cell apoptosis and ROS generation gradually increased with the activities of Cu/Zn-SOD and Mn-SOD decreased significantly. Therefore, according to the above research, we can make a conclusion that exogenous H2S in certain range of concentrations could protect cardiomyocytes against the ROS damage by increasing the activities of Cu/Zn-SOD and Mn-SOD. However, when the concentration of NaHS was higher than a threshold level, the cytotoxic effect of H2S will become evident and do harm to the growth of cardiomyocytes.We further investigated the effects of exogenous H2S on cell apoptosis, ROS generation, activities and expression levels of Cu/Zn-SOD and Mn-SOD after cardiomyocytes transfected with Cu/Zn-SOD si RNA or Mn-SOD si RNA followed by H/R procedure. Our results showed that the capability of exogenous H2S to increase the activities of both Cu/Zn-SOD and Mn-SOD has weakened. In addition, we found that there existed several compensatory pathways which may play important roles in maintaining the state of homeostasis in cardiomyocytes.We have also studied the influence of endogenous H2S on cell apoptosis, ROS generation, activities and expression levels of Cu/Zn-SOD and Mn-SOD after cardiomyocytes transfected with CSE si RNA, CBS si RNA, or 3-MST si RNA for 48h followed by H/R procedure. Compared with H/R group, the level of apoptosis and ROS generation increased dramatically with the activities of Cu/Zn-SOD and Mn-SOD decreased in cardiomyocytes transfected with CSE siRNA or CBS si RNA, but there were no significant changes in protein expression of Cu/Zn-SOD and Mn-SOD. However, there were no obvious changes in the level of apoptosis, ROS generation, and protein expression of Cu/Zn-SOD and Mn-SOD in cardiomyocytes transfected with 3-MST siRNA compared with H/R group. These phenomena indicated that three H2S-forming enzymes including CSE, CBS, and 3-MST could be simultaneously detected and they played different roles in maintaining the activities of Cu/Zn-SOD and Mn-SOD in cardiomyocytes. We also found that the expression levels of CBS and 3-MST increased to compensate for the loss of H2S induced by transfection with CSE siRNA in cardiomyocytes. After transfected with CBS siRNA, the expression level of 3-MST also increased. But transfected with 3-MST siRNA, there were no significant changes in protein expression of CSE and CBS. Therefore, we can draw a conclusion that CSE, CBS and 3-MST could play different roles in synthetizing H2S and follow the order of CSE> CBS> 3-MST in cardiomyocytes. In addition, we also found that exogenous H2S could protect cardiomyocytes from injury induced by transfection with CSE siRNA or CBS siRNA. Thus we can conclude that both exogenous and endogenous H2S could play important roles in protecting cardiomyocytes.We then investigated the mechanism of H2S in improving the activity of Cu/Zn-SOD through a series of experiments including radioactive ligand-receptor binding test, UV-Vis absorption analysis, quantum chemical calculation, and so on. Our results showed that H2S could enhance the activities of Cu/Zn-SOD and Mn-SOD probably through replacing the water that weakly coordinated to the copper ion or manganese ion at the active center of Cu/Zn-SOD or Mn-SOD.In addition, we detected the binding affinity between epidermal growth factor receptor （EGFR） and H2S according to the radioactive ligand-receptor binding test.We also cloned the a-MHC/CMV promoter and CSE gene and then constructed the adenoviral expression vectors which can specifically express the CSE protein in rat cardiomyocytes for further investigation.In conclusion, our results showed that H2S could enhance the activities of Cu/Zn-SOD and Mn-SOD probably through replacing the water that weakly coordinated to the copper ion or manganese ion at the active center of Cu/Zn-SOD or Mn-SOD.We found that three H2S-forming enzymes including CSE, CBS, and 3-MST could be simultaneously detected in rat cardiomyocytes and they could play different roles in synthetizing H2S with the order of CSE> CBS> 3-MST. In addition, we also found several compensatory pathways which may play important roles in maintaining the state of homeostasis in cardiomyocytes.
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