The Role Of Autophagy In The Protective Effect Of Hydrogen Sulfide On Diabetic Myocardial Injury

BackgroundDiabetic cardiomyopathy, characterized by a set of structural and functional abnormalities in the heart, is acknowledged as one of the major cardiac complications in diabetic patients. Diverse pathogenic mechanisms contribute to diabetic cardiomyopathy, including left ventricular dysfunction, cardiomyocyte apoptosis and myocardial fibrosis. Diabetic cardiomyopathy manifests initially as asymptomatic diastolic dysfunction that eventually progresses to symptomatic heart failure. It is well established as one of the major causes of heart failure in diabetic patients. Despite lots of researches on the mechanisms of DCM have been reported, there is currently no special clinical interventions for DCM.The pathogenesis of diabetic cardiomyopathy is not completely understood, and it appears to be multifactorial involving hyperglycemia, hyperinsulinemia, abnormal fatty acid metabolism and oxidative stress, cardiac autonomic neuropathy and the local renin-angiotensin-aldosterone system overactivation. Autophagy is a physiological self-degradation and recycling process that proceeds within double-membrane organelles via the lysosomal digestive pathway and functions to maintain the intracellular environment. Autophagy occurs not only in healthy hearts under basal conditions, but is also activated under pathological conditions, including heart failure, cardiac hypertrophy, ischemic cardiomyopathy, acute myocardial infarction, post infarctionremodeling and cardiac senescence. This suggests that autophagy plays a key role in maintaining cardiac function but may also contribute to the pathogenesis of heart disease. Furthermore, one would expect autophagy to be centrally involved in diabetic cardiomyopathy, where apparent metabolic disorder and obvious impairment of contractile function coexist. Recent studies have demonstrated that autophagy is also an important contributor to the development and the progression of DCM.Hydrogen sulfide (H2S) has been qualified as the third kind of gasotransmitter following with carbon monoxide (CO) and nitric oxide (NO). Studies have shown that endogenous hydrogen sulfide is involved in many physiological and pathological processes of cardiovascular system. In diabetic cardiomyopathy model, administration of hydrogen sulfide can reverse myocardial hypertrophy, alleviate fibrosis, and improve insulin resistance. In vitro, hydrogen sulfide can reduce the high glucose induced cardiac myocytes damage. The mechanisms included:anti-inflammation, anti-oxidation and anti-apoptosis.According to the previous results, we hypothesized that early administration of H2S has a protective effect on DCM by regulating myocardial autophagy. Thus, we established the type 2 DCM model to explore the effect of H2S on DCM and the mechanism of this action.Objective1. To establish the type 2 DCM rat model2. To observe the expression of autophagy-related factor in the myocardium of type 2 DCM rat model.3. To verify the cardioprotective effects and mechanisms of H2S in the pathogenesis DCM.MethodsThirty-four male Sprague-Dawley rats were purchased from the experimental animal center of Shandong University of Traditional Chinese Medicine. After 1 week of acclimatization, all rats were randomly assigned to a control group (n= 10) or diabetic model group (n= 24). The control group received normal chow and the diabetic model group were fed a high fat diet. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed after 4 weeks. The rats with insulin resistance in the diabetic group were given a single intraperitoneal injection of streptozotocin (Sigma, St. Louis, MO; 40mg/kg i.p. in 0.1 mol/L citrate buffer, pH 4.5). One week after streptozotocin administration, rats with plasma glucose levels>11.1 mmol/L were considered a diabetic rat model. The diabetic model group (n= 24) was redivided into two groups:a diabetes group (n=12) and a diabetes + NaHS group (n=12). The diabetes+NaHS group was given a dose (14umol kg-1day-1) of NaHS by intraperitoneal injection for 6 weeks. Then rats were sacrificed.At the end of the experiment, the following parameters were measured:(1) Blood analyses:fasting blood glucose, total cholesterol, triglycerides and fasting insulin. Insulin sensitivity index (ISI) was calculated using the following formula:ISI= In [([fasting blood glucose] × [fasting insulin])-1]. (2) Measurement of blood pressure and heart rate:heart rate, systolic blood pressure, diastolic blood pressure were measured with a noninvasive tail-cuf system (Softron, Tokyo, Japan). (3) Echocardiographic evaluation:Transthoracic echocardiography was performed using the Vevo 770 imaging system. Assessment of left ventricular diastolic and systolic function in rats by echocardiography. (4) Histology and morphometric analysis:The sections were stained with HE and MassonTrichrome. Collagen volume fraction (CVF) were obtained by quantitative morphometry with automated image analysis. (5) TUNEL assay was used to detect the apoptosis of the myocardial cells of rats in each group. (6) Histopathology staining:Immunohistochemistry were performed for detecting the expressions of Atg 5. (7) Western blot analysis for the expression of Bcl-2?Bax?LC3?Atg5. Values are presented as mean ± SEM. SPSS 17.0 was used for statistical analysis. Diferences between experimental groups were determined by one-way ANOVA. p<0.05 was considered statistically significant.Results1. General characteristics of diabetic ratsAt the end of the experiment, our results found that water intake, food intake, urine volume were significantly higher in the diabetic group than the control group. There were three rats died in the experimental process. Two of them were from diabetes group, and one from the NaHS intervention group.2. Blood pressure and blood analysesAt the end of the experiment, DM group showed significantly increased FBG? TG?TC and FINS levels compared with the control group (p<0.05), and rats in DM group also showed impaired insulin sensitivity (p<0.05). There were no significant differences in SBP and DBP between diabetic and control rats. Compared to NC group, DM group were a significant increase in heart rate. NaHS improved these metabolic normities and impaired insulin sensitivity (p<0.05).3. Changes of left ventricular structure and function indexThe heart weight/body weight ratio (HW/BW) of DM group was significantly higher than that of NC group (p<0.05). HE staining showed that the left ventricular wall thickening, myocardial hypertrophy, distortion, arrangement disorder, and the gap increased in DM group, and the size of the nucleus was not very regular. Masson's trichrome staining of heart sections revealed cardiac fibrosis, with destroyed and irregular collagen network structure in the interstitial area. Compared with the DM group, the HW/BW, collagen volume fraction (CVF) were significantly decreased after NaHS intervention, and the difference was significant (p<0.05).Both systolic and diastolic indexes were evaluated by echocardiography in our study. According to the results, E/A were significantly decreased in the diabetic group compared with the control group, while EDT and IVRT were significantly increased (all p<0.05). There were no significant differences in LVEF and FS between diabetic and control rats (p>0.05). The above results suggest that diabetic rats had been with left ventricular diastolic dysfunction. After the intervention of NaHS, left ventricular diastolic dysfunction can be significantly improved in diabetic rats (p<0.05).4. The change of apoptosisTUNEL staining demonstrated that TUNEL-positive cells were seldom identified in the control group, but numerous TUNEL-positive cells were observed in the diabetic group. These TUNEL-positive cells in DM rats were reduced by NaHS treatment(p<0.05). Consistently, western analysis showed a significant increase in Bax and decrease in Bcl-2 in the DM group compared with the control group (p<0.05), which were significantly improved in DM+NaHS group (p<0.05).5. The changes of autophagyThe diabetic group revealed lower ATG5 and LC3?/? protein levels compared with the control (p<0.05), indicating a reduction in cardiac autophagy. Meanwhile ATG5 and LC3 ?/? protein levels were significantly higher in the DM+NaHS group than in the DM group(p<0.05).Conclusions1. By high fat diet and intraperitoneal injection of STZ, the DCM rat model was successfully established, which provided a reliable platform for the study of the pathogenesis of DCM;2. At the early stage of type 2 DCM, autophagy in the myocardium was significantly inhibited, along with the left ventricular remodeling and diastolic function decreased;3. Exogenous H2S could reverse myocardial remodeling and improve left ventricular dysfunction in DCM rats, and the protective effects of H2S on DCM was related to enhancement of autophagy.BackgroundDiabetic cardiomyopathy (DCM) is characterized by myocardial dysfunction occurring independently of coronary artery disease (CAD), valvular heart disease, or hypertension in diabetes mellitus (DM) patients. Although the pathological mechanism of DCM still remains multifactorial, hyperglycaemia is considered as the main underlying pathogenic factor for myocardial damage in this condition. Indeed, the cardiotoxic roles of hyperglycemia have been demonstrated in numerous cells and animal studies. Hyperglycaemia causes cardiomyocyte death, which is contributed to the production of oxidative stress, accelerated apoptosis, mitochondrial damage, hypertrophy, impaired calcium homeostasis, and fibrosis. Vitro experiments also showe that High glucose (HG) directly induces the increases in reactive oxygen species (ROS) levels and apoptosis, while the autophagy is suppressed in glucose-induced cardiomyocyte injuries. However, the underlying mechanisms of DCM are still incompletely and at present, the clinical treatment cannot effectively attenuate DCM and heart failure in human treatment. Therefore, it is reasonable to assume the molecules mechanisms and develop a potential therapeutic strategies for DCM. Recently, the effects of dysregulated autophagy and H2S on hyperglycaemia-induced cardiotoxicity in DCM have attracted considerable attention.Autophagy is an essential intracellular catabolic pathway that the long-lived proteins and damaged organelles are transfered to and degraded in the lysosomes, resulting in maintaining cellular homeostasis undergoing starvation or various other stresses. General macroautophagy is referred as autophagy and tightly controlled by a variety of positive and negative regulators. As is known to all, under normal circumstances, the low level of autophagy is a protective mechanism of cellular stress. However, excessive autophagy can lead to cell damage. Recent researches have proved that high autophagy levels are appeared in circumstances of pressure overload, ischemia/reperfusion, heart failure, myocardial infarction, and cardiac hypertrophy, suggesting that autophagy plays a significant role in the pathogenesis of heart diseases. Indeed, there are evidences pointing out that the change of autophagic response promotes the maintenance of heart functions and morphology. However, the role of autophagy in DCM is more complex. For example, inhibited autophagy appears adaptive feature in STZ diabetic mice (type 1 diabetic model) but maladaptive feature in HFD-induced diabetes (type 2 diabetic model). Although many researches have observed the altered autophagy in HG-induced cardiocytotoxicity, the pathophysiologic roles of autophagy in HG-induced cardiomyocytes injures remain completely understood.Hydrogen sulfide (H2S) has been qualified as a new gasotransmitter along with carbon monoxide (CO) and nitric oxide (NO) with multiple physiological functions including anti-oxidant, anti-apoptosis, preservation of mitochondrial function, anti-inflammatory in physiology and pathophysiology conditions. In recent years, accumulating evidences suggest that H2S has cardioprotective roles. Exogenous H2S has been proved to attenuate myocardial necrosis via reactive oxygen species signal pathways in streptozotocin (STZ)-treated rats and rescue contractile activity by preventing cardiomyocyte apoptosis in isoproterenol-induced rat. H2S also promotes ostischemic left ventricular function and mitochondrial respiration during myocardial ischemia-reperfusion (MI/R) damage. These findings gave us rationale that H2S might be a therapeutic strategy for diabetic-associated diseases. Recently, researches interest in the protection effects of H2S in DCM has drawn much attention. H2S alleviates the development of DCM through attenuation of oxidative stress, apoptosis and inflammation. H2S also attenuates HG-induced cardiotoxicity in H9c2 cells. Notably, H2S restores MI/R-impaired autophagic flux and reverses high-fat-inhibited autophagy activity. Hence, we speculate that the promotion of autophagy may be beneficial to the protective effect of H2S against HG-induced cardiotoxicity in human AC 16 cardiac cells.In the first part of the experiment, we have confirmed the protective effect of H2S on diabetic myocardial cell damage in diabetic rats, and observe the corresponding change of autophagy, suggesting a protective effect of H2S on diabetic myocardial cell damage by regulating cardiomyocyte autophagy. The second part of the experiment, we used different concentrations of HG to explore and establish hyperglycemia-induced cardiotoxicity model, then investigated the effects of bafilomycin A1 (Baf, an autophagy inhibitor) on exogenous H2S-induced protective functions in HG-treated human AC 16 cardiac cells, so as to provide a theoretical basis and a new therapeutic target for the treatment of DCM.Objectives1. To verify the cardiotoxicity of human AC 16 cardiac cells induced by high glucose;2. To clarify the protective effect of H2S on the cytotoxicity of human AC 16 induced by high glucose;3. To explored the role and underlying mechanism of autophy in H2S-induced protective functions in HG-treated human AC 16 cardiac cells.Methods1. Cell cultureThe human AC 16 cardiac cell lines were cultured with DMEM containing normal D-glucous (5.5 mmol/L) or various concentrations of high D-glucose (HG, 11mmol/L?22mmol/L?33mmol/L?44mmol/L).2. Cells were divided into several groups:AC16 cardiomyocytes were treated with normal D-glucous (5.5 mmol/L) or various concentrations of HG in the presence or absence of NaHS (30 min) or Bafilomycin A1 (Baf,30 min) or both for 24 h, which were divided into the following groups according to different interventions:Control group, HG group, HG+NaHS ?, Control+ NaHS?? HG+ NaHS+Baf??control+Baf??3.Cell viability and Lactate dehydrogenase (LDH) release assay(1) The viability of AC16 cells was accessed with CCK-8 kit according to the manufacture's instruments. The cell survival rate (%) was calculated as the percentage of viable cells in comparison with the control group.(2) The levels of LDH in the culture supernatant were determined by LDH release assay kit according to the manufacturer's instructions to asssess the degree of cellular injure and cytotoxicity.4. Cardiomyocyte apoptosis assay(1) Cells stained with Hoechst 33258, the morphology of apoptotic cells was observed by fluorescence microscopy. (Apoptotic cells were unevenly stained, with strong blue fluorescence, while the morphology of the normal nuclei was complete and showed uniform blue fluorescence)(2) The activity of caspase-3 was determined by caspase-3 enzyme-linked immunosorbent assay (ELISA) Kits according to the manufacture's instruments.5.Western blot analysisProtein were extracted from cardiomyocytes underwent different stimulation. The protein expression of Bax?Bcl-2?LC3?Beclin-1, p62and ?-actin was analyzed in our experiment.6.Statistical analysisAll data were presented as the means ± standard error of the mean (SEM). The difference between groups was determined by one-way analysis of variance (ANOVA) using SPSS 17.0 software (Chicago, IL, USA), and followed by the LSD test. P< 0.05 was considered significant.Results1. High glucose reduces the viability of human AC16 cardiac cellsCompared with the control, the CCK-8 result shown that treatment of AC 16 cells with HG at 33 and 44 mM concentrations for 24 h significantly decreases the cell viability (p<0.05), while low concentrations of glucose (11 and 22 mM) have no effect on the cell viability (p>0.05). Mesnwhile, HG(22mM,33mM and 44mM) concentration-dependently increased the levels of LDH in culture supernatant (p<0.05)2. High glucose induces apoptosis in human AC 16 cardiac cellHoechst 33258 staining result shown that 33 and 44 mM of HG obviously exhibit the phenomenon of nuclear cracking and condensation in human AC 16 cells comparing with control group (p<0.05). We found that HG at concentrations 22,33 and 44 mM markably increase the activity of caspase-3 (p<0.05). In addition, western blot results shown that treatment with HG significantly causes the up-regulation of Bax protein (p<0.01) and down-regulation of Bcl-2 protein in human AC 16 cardiac cells (p<0.01).3. High glucose inhibits autophagy in human AC16 cardiac cellsCompared with normal control, treatment with HG (22mM,33mM and 44mM) for 24 h concentration-dependent decreased the expression levels of beclin-1 and ratio of LC3-?/? (p<0.05) in human AC16 cardiac cells, while the level of p62 protein was obviously up-regulated by HG treatment (p<0.05).4. NaHS mitigates high glucose-caused cardiomyocytes damages in humanNaHS, a donor of H2S. we pre-treated of human AC 16 cells with NaHS (200?M and 400?M) for 30 min prior to HG (33 mM) for 24 h and measured the cell viability and apoptosis. CCK-8 assay result shown that pre-treatment with NaHS (200 and 400 ?M) for 30 min weakened the HG-induced the down-regulation of cell viability (p<0.05). In addtion, NaHS improved the phenomenon of nuclear condensation and cracking induced by HG (33 mM) in human AC 16 cardiac cells. NaHS (400 ?M) also reversed HG-induced the up-regulation of caspase-3 activity and Bax protein expression as well as the down-regualtion of Bcl-2 protein expression in a concentration-dependent manner(p<0.05).5. NaHS reverses high glucose-exhibited the inhibition of autophagy in human AC16 cardiac cellsAs shown by western bolt assay, the decreases in the levels of beclin-1 protein and ratio of LC3-?/? induced by HG (33 mM) were significantly reversed by pre-treatment with NaHS (200 and 400 ?M) for 30 min, respectively(p<0.05). NaHS also reduced the increase in the expression of p62 protein induced by HG (33 mM) in a dose-independence manner in human AC 16 cells (p<0.05).6. Inhibition of autophagy attenuates the protective effects of NaHS on HG-induced cardiocytotoxicity in human AC16 cardiac cellsTo further assess autophagy to the protective effects of H2S, AC 16 cells were pre-treated with Bafilomycin A1 (Baf, an inhibitor of autophagy,50 nM) for 30 min. NaHS-caused recovery of AC 16 cells activity was obviously reversed by pre-treated with Baf (p<0.05). Additionally, Hoechst 33258 staining result shown that NaHS-induced improvement of cell morphology throughout HG treatment was also abrogated by pre-treatment with Baf. Simultaneously, the mitigation effect of NaSH on the increased in the activity of caspase-3 (p<0.01) and expression of Bax protein (p<0.05), and the decreased in the expression of Bcl-2 protein (p<0.01) were also abolished by Baf.Conclusions1. High glucose can induce the injury of AC 16 myocardial cells;2. The decrease of autophagy and the increase of apoptosis were involved in the myocardial injury induced by high glucose;3. Exogenous H2S could alleviate the myocardial injury induced by high glucose, and reverse the inhibition of autophagy;4. Inhibition of autophagy offset the protective effect of exogenous H2S on attenuating HG-induced cardiotoxicity.