| BackgroundHeart failure (HF) is a chronic, progressive illness that is highly prevalent in the worldwide. Despite its place among the leading causes of morbidity, pharmacological and mechanic remedies have only been able to slow the progression of the disease. There is a critical need to further understand its underlying mechanisms. Autophagy is a major catabolic pathway by which mammalian cells degrade and recycle macromolecules and organelles. It plays a critical role in removing protein aggregates, as well as damaged or excess organelles, to maintain intracellular homeostasis and to keep the cell healthy. Recnetly, Autophagic vacuoles are found in cardiomyocytes in ischemic hearts, and in human and hamster cardiomyopathic failing hearts. However, the precise role of autophagy in the HF is still unclear and remains to be elucidated.ObjectivesEstablish the transverse aortic arch constriction (TAC) model, investigate the autophagic flux and autophagic gene expression in the progression of HF, and thus discuss the underlying mechanisms. Experiment IMethods1. Animals170male C57BL/6mice were divided into three groups:the normal group (n=50), the sham group (n=50) and the TAC group (n=70).8-11mice were sacrificed at0,3,7,14or28days after surgery in each group.2. Pressure-overload HFThe use of a horizontal incision at the level of the suprasternal notch allows direct visualization of the transverse aorta without entering the pleural space and thus obviates the need for mechanical ventilation. The transverse aorta was banded between the right innominate and left carotid arteries to the diameter of a27-gauge needle using a7-0silk suture. Sham operations were served as a control for the TAC group.3. Echocardiography AnalysisLeft ventricular mass weight (LVMW) and Ejection fraction (EF%) were detected to evaluate cardiac function.4. Histological and Immunochemical AnalysisHaematoxylin eosin (HE) and Masson staining were preformed to investigate cardiac hypertrophy myocardial fibrosis. Specific immunohistochemical staining was also performed with antibodies against microtubule-associated protein light chain3(LC3), Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon), monocyte chemotactic protein-1(MCP-1), intercellular adhesion molecule (ICAM-1), toll-like receptor4(TLR4), myeloid differentiation factor88(Myd88), matrix metallo proteinase-9(MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1),4-hydroxy-2-Nonenal (4-HNE),8-hydroxydeoxyguanosine (8OHdG).5. Real-time quantitative polymerase chain reaction (Real-time PCR)Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b, Rubicon, Beclin-1, Autophagy5(Atg5) and Atg7.a-Actin was a normalization control.6. Statistical analysis Statistical analysis involved use of SPSS11.5for Windows. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for two groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.Results1. Survival rateSurvival probability in the TAC group is lower than the normal group and the sham group (P<0.01).2. Echocardiography analysisCompared to the sham group, the TAC group showed enhanced cardiac hypertrophy, EF%was significantly decreased from73.4±3.5%to41±10.4%on the3rd day (P<0.01) and further decreased to36.2±5.7%on the28th day (P<0.01); LVMW was significantly increased, it increased from42.2±10.2to52.5±12.7on the3rd day after surgery (P<0.05) and further increased to58.2±11.0on the28th day (P<0.05).3. Histological and Immunochemical AnalysisHistology staining:HE and Masson staining revealed increased myocardium cross-sectional area and more cardiac fibrosis in hearts of the TAC group than the normal group and the sham group especially on the28th day (P<0.05or P<0.01).Autophagosomal marker assay:We analyzed the expression of autophagosomal markers such as LC3, P62, Beclin-1, Rubicon, Compared with the normal group and the sham group, the protein expression levels of LC3, P62significantly increased and peaked on the7th day and the28th day (P<0.05or P<0.01); The expression level of Beclin-1was increased on the3rd day and the7th day (P<0.01) and Rubicon expression level markedly increase on the3rd day, the7th day, the14th day and the28th day(P<0.05or P<0.01).Inflammation assay:The protein expression level of inflammation markers such as MCP-1, ICAM-1, TLR4, Myd88, MMP-9, TIMP-1were significantly increased in the TAC group at different time point after TAC surgery (P<0.05or P<0.01). Oxidative stress assayBoth4-HNE and8OHdG protein levels were increased in TAC mice on the3rd day, the14th day and the28th day (P<0.05or P<0.01).4. Real-time PCRCompared to the normal group and the sham group, the mRNA level of LC3a was markedly increased in TAC mice on the7th day (P<0.01), it showed no significant difference at other time points. The mRNA level of LC3b was increased on the3rd day (P<0.01) and the7th day (P<0.05) compared to the normal group and the sham group. Compared to the normal group and sham group, the mRNA level of Atg5were significantly increased on the3rd day, the7th day and the14th day while the expression of Atg7and Beclin-1increased on the3rd day (P<0.01). The mRNA level of Rubicon was higher at the7rd,14th and28th day than the normal group and the sham group (P<0.05or P<0.01). There's no significant different between the normal group and the sham group (P>0.05). Experiment â…¡Methods1. Animal180male C57BL/6mice were divided into three groups:the normal group (n=50), the sham group (n=50) and the TAC group (n=80). To investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline or chloroquine or rapamycin by intraperitoneal injection4h before being killed.8-10mice were killed at the7th day and the28th day after surgery in each subgroup.2. Pressure-overload HFTAC model was produced by the method in experiment â… .3. Autophagic flux assayTo detect autophagic flux in the heart, we measured expression of LC3by immunohistochemistry and Real-time PCR.Results1. Histological and Immunochemical AnalysisThe protein level of LC3was markedly increased in TAC mice on the7th day (21.43±2.9%, P<0.01) and the28th day (28.2±03.24%, P<0.01) compared with the normal group and the sham group. Rapamycin significantly increased the expression of LC3in all conditions (P<0.05), and combination with rapamycin and chloroquine further increased the expression LC3except the TAC group on the7th day (P<0.01). Rapamycin significantly increased the expression of LC3(P<0.05or P<0.01), and combination with rapamycin and chloroquine further increased the expression LC3in the normal group and the sham group on the28th day (P<0.05or P<0.01). Rapamycin slightly increased the expression of LC3(P>0.05) and combination with rapamycin and chloroquine couldn't further increase the expression LC3in TAC mice on the28th day (P>0.05).2. Real-time PCRThe mRNA expression of LC3a and LC3b was significant increased at the end of the7th day compared with the normal group and the sham group (P<0.05or P<0.01).On the7th day, rapamycin significantly increased the mRNA expression of LC3a except the TAC group (P<0.05), and increased the mRNA expression of LC3b in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3a and LC3b in every group (P<0.05or P<0.01). On the28th day, rapamycin significantly increased the mRNA expression of LC3a and LC3b in every group (P<0.05or P<0.01). Combination with rapamycin and chloroquine further increased the expression of LC3a and LC3b in every group except the TAC group.3. Autophagic flux assayAutophagic flux induced by rapamycin made no significant difference in every groups on the7th day. But it significantly decreased on the28th day in the TAC group compared with the normal group and the sham group.Conclusions1. Autophagy was compensatory enhanced at early stage of pathological pressure overload and showd normal autophagic flux induced by rapamycin in the TAC group. At the late stage, it showed autophagy dysfunciton and lower autophagic flux induced by rapamycin.2. Autophagy genes change dynamically and play an important role in the progression of cardiac remodeling in TAC mice.3. Autophagy and inflammation/oxidative stress are closely related in the progression of cardiac remodeling in TAC mice, and TLR4-Myd88pathway may be involved in the underlying mechanisms. BackgroundHeart failure (HF) is the leading cause of morbidity and mortality worldwide. There is a critical need to explore new therapeutic approaches in heart failure. Autophagy is a cellular response to starvation as well as a quality-control system that can deliver damaged organelles and long-lived proteins from the cytoplasm to lysosomes for clearance. Autophagy helps clear intracellular protozoa, bacteria and viruses and functions in antigen presentation. Growing evidence suggested that autophagy took part in the development and progression of heart diseases. Traditional Chinese drug has been used in the treatment of HF. Basic and clinical research showed that Qiliqiangxin could up-regulation the contents of ATP, improve the hemodynamics, inhibit neurohormonal activation, thus effectively improve cardiac function and the prognosis in patients. But the role of Qiliqiangxin in the autophagic mechanisms is still unclear in the progression of HF.Objectives1. To test the hypotheses that the traditional Chinese medicine Qiliqiangxin can improve cardiac function in the compensated stage and the decompensated stage of HF in transverse aortic arch constriction (TAC) mice.2. To elucidate the autophagic mechanism of the traditional Chinese medicine Qiliqiangxin in the compensated stage and the decompensated stage of HF in TAC mice.Methods1. Animal C57BL/6mice underwent TAC surgery (n=300), a model of pressure-overload heart failure, or sham surgery (n=60). We randomly divided the TAC mice into3groups for treatment (n=80each):the perindopril group, the Qiliqiangxin group and the TAC group. Mice were killed on the14th and28th day, to investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline (0.2ml), chloroquine (30mg/kg) or rapamycin (2mg/kg) by intraperitoneal injection4h before being killed.2. Pressure-overload HF:TAC model was produced by the method in Part I3. Echocardiography AnalysisLeft ventricular mass weight (LVMW) and LV ejection fraction (EF%) were detected to evaluate cardiac function.4.Histological and Immunochemical AnalysisHaematoxylin eosin (HE) and Masson were preformed to investigate cardiac hypertrophy myocardial fibrosis. Specific immunohistochemical staining was also performed to detect the expression of microtubule-associated protein light chain3(LC3) in every subgroups, and detect the expression of Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon), matrix metallo proteinase-9(MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1),4-hydroxy-2-Nonenal (4-HNE),8-hydroxy-2'-deoxyguanosine (8OHdG) in mice that were injection with physiological saline.5. Real-time quantitative polymerase chain reaction (Real-time PCR)Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b in every subgroups, and the levels of Rubicon, Beclin-1, Autophagy5(Atg5), and Atg7in in mice that were injection with physiological saline. a-Actin was a normalization control.6. Statistical analysisStatistical analysis involved use of SPSS11.5for Windows. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for2groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.Results1. Echocardiography AnalysisOn the14th day after surgery, acute pressure overload significantly decreased EF%and increased LVMW compared to the sham group (P<0.01). Both the perindopril group and the Qiliqiangxin group showed higher EF%and lower LVMW compared to the TAC group (P<0.05or P<0.01).On the28th day after surgery, chronic pressure overload further decreased EF%and increased LVMW in the TAC group compared to the sham group (P<0.01). Either perindopril or Qiliqiangxin could improve EF%(P<0.01) and LVMW (P<0.05) in the TAC mice. There is no significant difference between the perindopril group and the Qiliqiangxin group.2. Histological and Immunochemical Analysis(1) Histology stainingHE and Masson staining revealed increased myocyte cross-sectional area and more cardiac fibrosis in hearts of TAC mice than sham mice especially on the28th day (P<0.01). Either perindopril or Qiliqiangxin could help relieve such pathological change both on the14th day and the28th day (P<0.05).(2) Autophagic functionOn the14th and the28th day, the expression of LC3was significantly higher in the TAC group, the perindopril group and the Qiliqiangxin group in comparasion with the sham group (P<0.01). Compared with the TAC group, the perindopril group and the Qiliqiangxin group showed lower LC3positive rate (P<0.05or P<0.01).On the14th day, rapamycin significantly increased the expression of LC3in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3except the TAC group on the14th day (P<0.05or P<0.01).On the28th day, rapamycin significantly increased the expression of LC3(P<0.05or P<0.01) and combination with rapamycin and chloroquine further increased the expression LC3in every group (P<0.05or P<0.01)except the TAC group on the28th day.(3) Autophagic geneOn the28th day, the p62protein expression level in the TAC group was significantly increased compared with the perindopril group, the Qiliqiangxin group and the sham group (P<0.01).On the14th day and the28th day, the expression of Beclin-1in the sham group was significantly lower than the other groups (P<0.01).It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the14th day (P<0.05or P<0.01).On the28th day, it was higher in the TAC group than the perindopril group and the Qiliqiangxin group (P<0.05).On the14th and the28th day, the expression of Rubicon in the sham group was significantly lower than the other groups (P<0.01), and it was much lower in the perindopril group and the Qiliqiangxin group than the TAC group (P<0.05or P<0.01).(4) Oxidative stress markersOn the14th day and the28th day, the expression of4-HNE and8OHdG were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). The4-HNE and8OHdG protein expression levels were much higher in the TAC group than the perindopril group and Qiliqiangxin group (P<0.05or P<0.01) on the28th day.(5) MMPs/TIMPsOn the14th day, the expression of MMP-9were increased in the TAC group and the Qiliqiangxin group compared with the sham group and the perindopril group (P<0.01). On the28th day, the expression level of MMP-9was significantly increased in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01), and it was lower in the perindopril group and Qiliqiangxin group than the TAC group (P<0.05).On the14th day, there is no significant difference in every groups,on the28th day, the expression of TIMP-1were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the28th day (P<0.01).3. Real-time PCR(1) LC3expressionOn the14th day, the mRNA expression of LC3a in the TAC group, the perindopril group and the Qiliqiangxin group were significantly increased in comparison with the sham group (P<0.05or P<0.01). Compared with the TAC group, the mRNA expression of LC3a was significantly higher (P<0.01). On the28th day, the mRNA expression of LC3a was significantly higher in the perindopril group and the Qiliqiangxin group compared with the sham group and the TAC group (P<0.01).The mRNA expression of LC3b was significantly increased in the perindopril group and the Qiliqiangxin group compared with the TAC group and the sham group on the14th day (P<0.01). The mRNA expression of LC3b in the TAC group, the perindopril group and the Qiliqiangxin group were significantly increased in comparison with the sham group on the28th day (P<0.05or P<0.01). Furthermore, the mRNA expression of LC3b in the perindopril group were significantly higher in comparison with the TAC group on the28th day (P<0.05)..(2) Autophagic fluxOn the14th day, rapamycin significantly increased the mRNA expression of LC3a in every group (P<0.01), combination with rapamycin and chloroquine further increased the expression LC3a in every group (P<0.01).On the28th day, rapamycin significantly increased the mRNA expression of LC3a in every group (P<0.01); combination with rapamycin and chloroquine further increased the expression LC3a in every groups except the TAC group (P<0.01).On the14th day, rapamycin significantly increased the mRNA expression of LC3b in every group, combination with rapamycin and chloroquine further increased the expression LC3b in every group, P<0.01).On the28th day, rapamycin significantly increased the mRNA expression of LC3b in every group (P<0.05or P<0.01); combination with rapamycin and chloroquine further increased the expression LC3b in every groups except the TAC group (P<0.01).Autophagic flux was much lower in the TAC group but made no significant differedce on the14th day. On the28th day, the autophagic flux was further decreased in the TAC group compared to the other groups (P<0.01). And it was lower in the perindopril group and the Qiliqiangxin group compared to the sham group (P<0.05).(3) Autophagic geneOn the14th day, the expression of Atg5were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much lower in the perindopril group than the TAC group (P<0.05). On the28th day, compared to the sham group and the TAC group, the mRNA level of Atg5was significantly increased in the perindopril group and the Qiliqiangxin group (P<0.01).On the14th day and the28th day, the expression of Atg7were higher in the perindopril group and the Qiliqiangxin group than the TAC group and the sham group (P<0.05or P<0.01).The mRNA level of Beclin-1in the perindopril group and the Qiliqiangxin group were significantly higher than the other groups on the14th day (P<0.05or P<0.01), and it was higher in the TAC group than the other groups on the28th day (P<0.01).On the14th and the28th day, the mRNA expression of Rubicon were much higher in the TAC group, the perindopril group and the Qiliqiangxin group than the sham group (P<0.01). It was much higher in the perindopril group and the Qiliqiangxin group than the TAC group on the14th day (P<0.01), and lower in the perindopril group and the Qiliqiangxin group than the TAC group on the28th day (P<0.01).Conclusions1. A reversing effect of Qiliqiangxin on the cardic remodeling in TAC mice was observed in the present study.2. Qiliqiangxin could anti-oxidative stress and improve autophagic function, and has the same effect as perindopril. BackgroundHeart failure (HF) is a major public health concern associated with significant mortality and morbidity. Symptoms of psychological stress, such as anxiety and depression, are common among patients with HF. Furthermore, the presence of these disorders significantly leads to increased symptoms of HF, poor quality of life and an opposite effect on prognosis. Thus, psychological stress may have an important role in the development of HF. However, previous studies were prospective observational studies, so mechanisms relating psychological stress to HF are still not totally understood.Depression and anxiety disorders were linked to dysfunction of the renin-angiotensin system (RAS) and generation of Angiotensin â…¡(Angâ…¡). Angâ…¡ contributes to the progression of HF, and recent study showed that Angâ…¡ takes part in cardiomyocyte autophagy.Autophagy is a highly conserved cellular mechanism that plays a key role in the turnover of long-lived proteins, RNA, and other macromolecules. In the heart, autophagy occurs at low levels under normal conditions, then defects in the process cause cardiac dysfunction and HF. Autophagy may be an important mechanism underlying the progression of HF. Whether psychological stress induces autophagic dysfunction in HF is not totally understood. Psychological stress may affect cardiac autophagy through Angâ…¡, thus adversely affecting cardiac remodeling.The angiotensin-converting enzyme inhibitor (ACEI) captopril can reduce the activity of the renin-angiotensin system (RAS), blocking the conversion of AngI to Ang â…¡.We observed autophagy in mice with HF induced by transverse aortic constriction (TAC) and investigated whether psychological stress influences autophagy in HF and the role of captopril in this progression, thus discuss the underlying mechanism.Objectives1. To observe autophagy in early stage of HF in TAC mice, investigated whether psychological stress influence the autophagy in HF.2. To discusse the role of Ang â…¡ in this progression, thus studied the underlying mechanisms. Experiment IMethods1. Experimental Protocol150male C57BL/6mice (7-8weeks old) were underwent TAC surgery (n=120), a model of pressure-overload HF, or sham surgery (n=30). On the3rd day after surgery, we randomly divided the TAC mice into3groups for treatment (n=40):the combined acoustic and restraint stress group (stress), the stress plus captopril group (captopril) and the TAC control group (TAC). To investigate autophagic flux, mice in each group were randomly assigned to treatment with physiological saline(0.2ml), chloroquine (30mg/kg) or rapamycin (2mg/kg) by intraperitoneal injection4h before being killed.2. Combined acoustic and restraint stressStressed mice were exposed to combined acoustic and restraint stress for7days. They were placed in50-ml conical centrifuge tubes with multiple ventilation holes without pinning the tail for2h. Meanwhile, acoustic stress was produced by an alarm fixed on the top of the stimulator and introduced every3min at110dB lasting for5s.3. Splash testThe splash test was performed at the beginning and end of the stress procedure. An amount of10%sucrose solution was squirted on the dorsal coat of mice that were in their home cage. Grooming behaviour was recorded as total frequency.4. Echocardiography and hemodynamic analysisEchocardiography and hemodynamic analysis was performed at the beginning and end of1-week stress treatment. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) were measured by the tail-cuff method. Echocardiography was performed and left ventricular mass weight (LVMW) and ejection fraction (EF%) were detected to evaluate cardiac function.5. Survival analysisSurvival rate of every group was record.6. Blood collection and biochemical measurementsAfter1-week stress, all animals were killed, and blood was collected. Serum samples were prepared. Enzyme-linked immunosorbent assay (ELISA) kits were used to assay serum concentration of corticosterone and Ang â…¡.7. Histology and immunohistochemical stainingTissues were embedded in paraffin, and coronal sections (5μm) underwent general histological staining with haematoxylin&eosin (HE) and Masson. Specific immunohistochemical staining was also performed with antibody against microtubule-associated protein light chain3b (LC3b) in every group and performed with antibodies against Bcl-2interacting coiled-coil protein-1(Beclin-1), RUN domain protein as Beclin1-interacting and cysteine-rich containing (Rubicon),4-hydroxy-2-Nonenal (4-HNE) and8-hydroxydeoxyguanosine (8OHdG,) in groups injected with physiological saline.8. Real-time quantitative polymerase chain reaction (Real-time PCR)Real-time PCR was used to analyze the mRNA levels of LC3a, LC3b in every group and Beclin-1, Rubicon, Autophagy5(Atg5), and Atg7in groups injected with physiological saline.9. Statistical analysisStatistical analysis involved use of SPSS11.5for Windows. Survival was assessed by the Kaplan-Meier method with chi-square analysis. Normally distributed data were analyzed by one-way ANOVA and non-normally distributed data by Kruskal-Wallis one-way ANOVA. Student t test was used to compare data for2groups. Data are expressed as mean±SD. P<0.05was considered statistically significant.Results1. Grooming frequencyStressed mice groomed significantly less than did unstressed mice (P<0.01).2. Survival rateKaplan-Meier survival analysis showed cumulative survival probability significantly shorter for stressed mice (75%,27/36) than TAC mice (91%,33/36, P<0.05).3. Bichemical studies Seven-day stress increased serum corticosterone levels in the stress group and the captopril group (6-7fold, P<0.01) and compared with the sham group and the TAC group.The serum level of Ang II was significantly higher in the stressed than the TAC and captopril mice (P<0.01), with no difference in Ang â…¡ level between the captopril and the TAC mice (P>0.05). Moreover, the serum level of Ang â…¡ was higher but not significantly in the TAC than the sham mice (P<0.05).4. Hemodynamic analysis and echocardiographyStress significantly elevated SBP, DBP and HR in the stress and the captopril groups (P<0.01). The levels were lower with captopril than with stress alone. Compared to the TAC group, the stressed group showed enhanced cardiac hypertrophy, including increased LVMW (P<0.01) and ratio of heart weight to body weight (HW/BW, P<0.01). Most importantly, EF%was significantly lower in stressed than TAC mice after1week of stress (P<0.01). The captopril group showed higher EF%, lower LVMW and HW/BW compared to the stress group (P<0.01).5. Histology staining for cardiac fibrosisMasson staining revealed more cardiac fibrosis in hearts of stressed than TAC mice (P<0.05). Masson staining revealed less cardiac fibrosis exists in hearts of captopril mice, with no significant difference (P>0.05).6. Autophagic flux assayTo determine whether stress induces autophagic dysfunction in the heart, we measured expression of LC3b by immunohistochemistry and Real-time PCR. The level of LC3b was markedly increased in TAC mice (P<0.01). Furthermore, stress further upregulated the expression of LC3b (P<0.01). Administration of captopril decreased expression of LC3b compared to stress alone (P<0.01).Autophagic flux was markedly increased in TAC mice as compared with the sham group but significantly decreased in the stressed group as compared with the TAC group. The captopril group showed upregulated autophagic flux as compared with stress alone. Rapamycin significantly increased the expression of LC3b in all conditions (P<0.05or P<0.01), which indicates no autophagosome formation dysfunction under stress. Therefore, decreased clearance occurred after stress, and such autophagic responses could be reduced by captopril.7. Autophagosomal marker assayStress significantly increased the mRNA levels of Beclin-1, Rubicon, Atg5and Atg7(P<0.01). The expression of Rubicon and Beclin-1were lower with captopril than stress alone (P<0.01). Immunohistochemical staining results were consistent with Real-time PCR results.8. Cardiac oxidative stress assayTo investigate oxidative stress in the heart, we examined the expression of4-HNE and8OHdG. Both4-HNE and8OHdG levels were increased in TAC mice (P<0.01) and further exaggerated in stress mice (P<0.01). Moreover, levels were lower with captopril than stress alone (P<0.01). Experiment IIMethod1. Cell StimulationIn some experiments, cells were pretreated with Ang II (0.1μM), N-acetylcysteine (NAC,50μM), rapamycin (1μM) or bafilomycin A1(Baf-A1,50nM).2. ImmunofluorescenceThe protein levels of LC3b and4-HNE were measured by immunofluorescence.3. Western blotThe protein levels of LC3b, Rubicon, Beclin-1, β-action were measured by Western blot.4. Real-time PCRLC3a, LC3b, Atg5, Atg7, Beclin-1, Rubicon mRNA levels were measured by Real-time PCR after RNA extraction and reverse transcription.Result1. Ang â…¡ promotes autophagyTo further confirm the role of Ang â…¡ in autophagy, H9c2cells were incubated with vehicle (control) or Angâ…¡ (0.1μM) for22h, then rapamycin or bafilomycin Al were added to the medium and co-incubated for2h., finally underwent immunofluorescence. Ang â…¡ promoted the expression of LC3b (P<0.01). Ang â…¡ enhanced autophagic flux. Rapamycin or Baf-A1enhanced Angâ…¡-induced increased ofLC3b.2. Role of oxidative stress in induction of autophagyTo evaluate the role of oxidative stress in Ang â…¡-induced autophagy induction, cells were treated with vehicle (control) or Ang â…¡ with or without NAC (50μM), then underwent immunofluorescence and western blot analysis. Ang â…¡ enhanced LC3b and4-HNE expression (P<0.01), which was partly inhibited by NAC (P<0.01). Furthermore, Rapamycin or Baf-Al enhanced Ang â…¡-induced increased of LC3b which was partly inhibited by NAC (P<0.01).3. Expression of Autophagy gene Angâ…¡ enhanced Atg5, Atg7, Beclin-1, Rubicon mRNA expression and Beclin-1(P<0.05or P<0.01)., Rubicon protein expression, which was partly inhibited by NAC (P<0.05or P<0.01).Conclusions1. Stress may induce autophagic flux dysfunction in clearing autophagosomes and adversely affects cardiac hypertrophy and HF in mice with TAC.2. Endogenous Ang â…¡ plays a key role in autophagic dysfunction via the oxidative stress response to stress.
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