Hydrogen Sulfide Attenuates Cardiomyocyte Ischemia-Reperfusion Injury By Inhibiting Autophagy

BackgroundClinical treatment of MI by thrombolytic therapy and revascularization by percutaneous coronary intervention or coronary artery bypass graft surgery are effective. However ischemia-reperfusion injury (IR) following the revascularization may contribute to subsequent myocardial dysfunction which will increase the time and cost of in-patient department. So it is important to find the endogenous protective mechanism against IR. It has been widely accepted that necrosis and apoptosis were the main injury during IR, while recent papers suggests that another form of programmed cell death, autophagy, may play a significant role in IR injury. It has been suggested to be an essential function for cell homeostasis and cell defense and adaptation to an adverse environment. While autophagy was associated with a variety of cardiac pathologies,such as ischemia, heart failure, and aging when it is excessive. Furthermore the early stages of autophagy are required for apoptosis, and the apoptosis ratio, which was induced by IR, was attenuated when the autophagy was inhibited. Autophagy plays a different role in the heart during ischemia and reperfusion. It was protective during ischemia, but switched to a detrimental role during reperfusion. So it may be beneficial for the heart against IR injury to find a way of anti-autophagy.Studies on mechanisms of ischemia-reperfusion injury and myocardial protection are the spotlight in present day. In order to fight for cardiomyocytes ischemic necrosis and protect myocardium, lots of medicine was studied for myocardial protection (for example,β-antagonist, free-radical scavenger, calcium antagonist). Recently hydrogen sulfide (H2S) was accepted to have cardium protectiveeffect against IR. Hydrogen sulfide is known for decades only as a pungent toxic gas. Now H2S is increasingly recognized as a member of a growing family of"gasotransmitters"; together with nitric oxide (NO) and carbon monoxide (CO). Endogenous H2S is generated in mammalian tissues by two pyridoxal-5'-phosphate-dependent enzymes, including cystathionineβ-synthase (CBS) and cystathionineγ-lyase (CSE). In the heart, there is little cystathionine-synthase, whereas CSE is plentiful, and it has been demonstrated that H2S could exert a cardioprotective effect in response to IR, for example enhancing cell survival rate and reducing the infarced area. But it is still unknown whether H2S protect myocardium via inhibiting autophagy of cardiomyocytes.ObjectivesIn our experiments, cultured neonatal rat cardiomyocytes and adult SD rats were performed to prepared ischemia-reperfusion injury in vitro and in vivo, and were interfered with NaHS. The cardiomyocyte vitality was methered with MTT method, the heart injury induced by IR was evaluated by the infarcted area and LDH detection, the cells were stained with MDC, and flow cytometry was performed to identify the autophagy of cardiaomyocytes induced by ischemia-reperfusion injury, and RT-PCR and western-blot were performed to detect the mRNA and protein level of autophagy-related genes (Atg). Furthermore, it was observed whether PI3K/Akt was involved in the signal transduction of H2S'anti-autophagy effect. These will help us to demonstrate the mechanism of anti-autophagy effect of H2S during IR, and provide new theory and method of protecting the heart against IR during peroperative period.Methods and results1. IR model was performed and verified in vitro and in vitro1.1 Neonatal rat cardiomyocytes culture and ischemia-reperfusion model preparation in vitro1.1.1 ischemia-reperfusion model preparation in vitroCardiomyocytes subjected to hypoxic substrate-free solution for 24h containing elevated potassium, acidosis, lactate accumulation and reinstated with normal culture conditions for 3h to mimic Ischemia-Reperfusion injury.1.1.2 To identify the efficacy of the ischemia-reperfusion modelThe cells were randomlized into 2 groups, control and IR group. The MTT method was used to compare the cell vitality, and LDH was used to determine the cell injury induce by IR. It was found that the LDH of IR group was higher than that of the control group (P<0.05). At the same time, the cell vitality of IR group was decreased to 61.45±3.85% compared the con group (P<0.05) .1.2 Ischemia-reperfusion model preparation in vivo1.2.1 The method of establishing IR model in vivo IR was induced by ligating the left anterior descending artery (LAD) for 30min, followed by loosening the ligature for 120min. Successful ligation of LAD was evidenced by immediate regional cyanosis in the anterior ventricular wall and the apex of the heart with color change greater than 40% of the left ventricle (LV) and confirmed by electrocardiography (ECG).1.2.2 To identify the efficacy of the ischemia-reperfusion model in vivo The SD rats were randomlized into 2 groups, control and IR group. The myocardial infarct size, which was measured by Evans blue and TTC staining, was used to determine the heart injury induced by IR, together with LDH. It was found that the infarct size of IR group was larger than the control group (P<0.05). The LDH of IR group was higher than the con group (P<0.05). The±dp/dtmax of IR group was lower than that of control group (P<0.05).2. Hydrogen sulfide attenuates cardiomyocyte IR injury by inhibiting autophagy2.1 In vitro study2.1.1 Groups: the cells were randomlized into 6 groups A: control group (Con), where the cells were cultured with 5%CO2 and 95% air for 26.5h;B: HR group (HR), where the cells were treated with 24 h hypoxia (3%O2, 5%CO2, 92%N2) and 2 h reoxygenation (5%CO2, 95%air);C: 10uM H2S pre-treatment+HR groups (S10+ HR), where the cells were treated with NaHS of 10uM 30min before HR.D: 30uM H2S pre-treatment+ HR groups (S30+ HR), where the cells were treated with NaHS of 30uM 30min before HR.E: 50uM H2S pre-treatment+ HR groups (S50+ HR), where the cells were treated with NaHS of 50uM 30min before HR.F: 100uM H2S pre-treatment+ HR groups (S100+ HR), where the cells were treated with NaHS of 100uM 30min before HR.2.1.2 The ratio of autophagy cells was measured with flow cytometryCardiomyocytes were incubated at 37°C in 50uM monodansylcadaverin (MDC), and was then determined with the indo (blue) flow cytometry system. It was found that the autophagy ratio of group B was the highest in all groups( P<0.05). That of C, D, E and F groups were all lower than group B (P<0.05). Among these groups, the autophagy ratio of group D and E was lower than group C and F (P<0.05), and has no significant difference with group A (P<0.05). These suggested that autophagy could be induced by IR, and could be attenuated by H2S, which was concentration-dependent.2.1.3 The measurement of cardiomyocytes injury2.1.3.1 Cardiomyocytes vitality measurement with MTTThe cell vitality was measured with MTT method.It was found that the vitality of group B was decreased to 61.45±3.85% compared with group A (P<0.05), while that in group C, D, E and F was 73.6±3.66%,87±5.49%,81.7±5.82% and 72.6±5.24% compared with group A, respectively, which was higher than group B (P<0.05). Among these groups, group D was higher than group C, E, and F (P<0.05). These suggested that the cardiomyocyte was injuried by IR, and was protected by H2S.2.1.3.2 LDH detectionLDH may identify the cardiomyocytes injury. It was found that LDH in group B, C, D, E and F were higher than group A (P<0.05). LDH in group D and E were lower than group B (P<0.05).2.2 In vitro study2.2.1 Groups: the cells were randomlized into 5 groupsA: Control group, where the rats underwent thoracotomy without ligation;B: IR group, where the LAD was ligated to imitate ischemia for 30 min, and the ligation was loosed to imitate reperfusion for 120 min;C: 10uM/kg H2S pre-treatment+IR groups (S10+IR), where the rats were treated with NaHS of 10uM/ Kg 30min before IR.D: 30uM/kg H2S pre-treatment+IR groups (S30+IR), where the cells were treated with NaHS of 30uM/ Kg 30min before IR.E: 100uM/kg H2S pre-treatment+IR groups (S100+IR), where the cells were treated with NaHS of 100uM/kg 30min before IR.2.2.2 The measurement of cardiomyocytes injury2.2.2.1 The infarct size measyred with evans blue and TTCThe entire ventricular tissue was dissected, cut into six horizontal slices and sections, and was incubated in 1% (w/v) 2, 3, 5-triphenyl tetrazolium chloride (TTC) solution. Percentage infarct size (INF) and the area at risk (AAR) was calculated as INF/AAR. It was found that INF/AAR of group B was higher than group A (P<0.05). That of group D was lower than group B (P<0.05). These suggested that the myocardium was injuried by IR, and was protected by H2S.2.2.2.2 LDH detectionLDH may identify the myocardium injury. It was found that LDH in group B, C and E were higher than group A (P<0.05). LDH in group D was lower than group B (P<0.05).2.2.2.3 Mechanics of myocardiumThe±dp/dtmax of group B was lower than that of group A (P<0.05). The±dp/dt of group D was significantly lower than that of group B(P<0.05). 3. The mechanism of anti-autophagy effect of H2S3.1 Groups: same to the part 23.2 H2S attenuated the mRNA expression of Beclin1 induced by IR3.2.1 In vitroThe Beclin1 mRNA was determined by RT-PCR. It was found that the Beclin1 mRNA of group B was 2.41±0.86 times than group A (P<0.05), and that of group D and E were 1.07±0.16 and 1.5±0.76 times than group A, which were significantly lower than group B (P<0.05). These suggested that H2S could attenuate Beclin1 mRNA expression induced by IR.3.2.2 In vivoIt was found that the Beclin1 mRNA of group B was 1.83±0.61 times than group A (P<0.05), and that of group D was 1.05±0.31 times than group A, which was significantly lower than group B (P<0.05). While that of group C and E were 1.43±0.44 and 1.66±1.21 times than group A, which were higher than group D (P<0.05).These suggested that H2S could attenuate Beclin1 mRNA expression induced by IR, and it.was concentration-dependented.3.3 H2S attenuated the mRNA expression of Atg5 induced by IR3.3.1 In vitroThe Atg5 mRNA was determined by RT-PCR. It was found that the Atg5 mRNA of group B was 1.85±0.41 times than group A (P<0.05), and that of group C, D, E and F was 1.47±0.64, 1.09±0.73, 1.03±0.46, and 1.25±0.66 times than group A, among which Atg5 mRNA of group D and E were significantly lower than group B (P<0.05). These suggested that H2S could attenuate Atg5 mRNA expression induced by IR.3.3.2 In vivoIt was found that the Atg5 mRNA of group B was 2.63±0.29 times than group A (P<0.05), and that of group D was 1.18±0.34 times than group A, which were significantly lower than group B (P<0.05). While that of group C and E were 2.33±1.72 and 2.18±0.99 times than group A. These suggested that H2S could attenuate Beclin1 mRNA expression induced by IR,and it was concentration-dependented3.4 H2S regulated the protein expression of LC3-Ⅱinduced by IR3.4.1 In vitroThe LC3-Ⅱprotein was determined by western-blot, and the ratio of LC3-Ⅱ/ LC3-Ⅰwas use to stand for the effect of H2S on LC3 protein. It was found that the ratio of LC3-Ⅱ/ LC3-Ⅰof group B,C,D,E and F was 2.21±0.55, 1.68±0.30, 1.05±0.26, 1.19±0.28 and 1.74±0.26 which was higher than group A (0.44±0.23, P<0.05). Among these groups, that of group C, D and E were significantly lower than group B (P<0.05), and that of group D has no significant difference with E. These suggested that H2S could attenuate LC3-Ⅱprotein expression induced by IR , and it was concentration-dependented.3.4.2 In vivoIt was found that the ratio of LC3-Ⅱ/ LC3-Ⅰof group B was 2.76±0.25 which was higher than group A (1.43±0.3, P<0.05), and that of group D was 1.54±0.20, which was significantly lower than group B (P<0.05). These suggested that H2S could attenuate LC3-Ⅱprotein expression induced by IR,and it was concentration-dependented.4. The role of PI3K/Akt in the mechanism of anti-autophagy effect of H2S4.1 Groups: the cells were randomlized into 4 groupsA: HR group (HR), where the cells were treated with 24 h hypoxia (3%O2, 5%CO2, 92%N2) and 2 h reoxygenation (5%CO2, 95%air).B: H2S pre-treatment+IR groups (S+IR), where the cells were treated with NaHS of 30uM 30min before IR. C: Tricribine pre-treatment+IR groups (Tri+S+IR), where the cells were treated with tricribine of 1uM 30min before H2S pre-treatment, then same to group B.D: Tricribine+H2S pre-treatment+IR groups (Tri+S+IR), where the cells were treated with tricine of 1uM 30min before H2S pre-treatment, then same to group B.4.2 Tricribine attenuated the down-regulation of Beclin1 effect induced by H2SThe Beclin1 mRNA was determined by RT-PCR. It was found that the Beclin1 mRNA of group B was 0.43±0.13 times than group A (P<0.05), and that of group C and D were 0.92±0.09 and 0.69±0.07 times than group A, which were significantly higher than group B (P<0.05). These suggested that PI3K/Akt was involved in anti-autophagy effect induced by H2S.4.3 Tricribine attenuated the down-regulation of Atg5 effect induced by H2SThe Atg5 mRNA was determined by RT-PCR. It was found that the Atg5 mRNA of group B was 0.5±0.12 times than group A (P<0.05), and that of group C and D were 1.08±0.16 and 0.77±0.13 times than group A, which were significantly higher than group B (P<0.05). These also suggested that PI3K/Akt was involved in anti-autophagy effect induced by H2S. 4.3 Tricribine attenuated the down-regulation of LC3-Ⅱeffect induced by H2S The LC3-Ⅱprotein was determined by western-blot. It was found that the ratio of LC3-Ⅱ/ LC3-Ⅰof group A,C and D were 2.1±0.32, 2.22±0.64, and 1.5±0.38 which were higher than that of group B (0.72±0.4, P<0.05). These suggested that PI3K/Akt was involved in anti-autophagy effect induced by H2S.ConclusionIn our experiments, cultured neonatal rat cardiomyocytes and adult SD rats were useded to prepared ischemia-reperfusion injury in vitro and in vivo, and were interfered with H2S. The cardiomyocyte autophagy and the degree of cell injury were detected with flow cytometry and MTT method respectively. The mRNA and protein expression of Atg family were detected with RT-PCR and western-blot method. These stiudy will help us to demonstrate that H2S attenuates cardiomyocytes autophagy induced by IR, and its effect on the Atg family.The main conclusions were as follows: 1. The method of hypoxia-reoxygenation culture and ligation-loosing the LAD were effective for IR model.2. H2S attenuated cardiomyocytes autophagy induced by IR, and was concentration-dependented.3. H2S regulated Atg family (Atg5, Beclin1 and LC3-Ⅱ). mRNA and protein expression.4. PI3K/Akt was involved in the anti-autophagy effect of H2S.In summary, H2S attenuated the cardiomyocyte autophagy induced by IR by regulating the mRNA and protein expression of the Atg family.