Autophagy In Right Ventricular Myocardium In Monocrotaline Induced Rats With Pulmonary Hypertension And Potential Mechanisms

Pulmonary arterial hypertension(PAH)is a progressive disorder that affects both the pulmonary vasculature and the heart.Although the initial insult in PAH involves the pulmonary vasculature,right ventricular(RV)function is a major determinant of prognosis of patients with PAH.Proliferative vascular remodeling and not vasoconstriction underlies PAH pathology in most patients,however,it became apparent that these therapies do limit symptoms and improve the patients' quality of life,but they do not prolong survival nor reverse the disease.It is very concerning that no RV-specific therapy for myocardial failure has been described.Presently,most knowledge of mechanisms of RV remodeling and treatment are learn from experiences of left ventricle(LV).Regretfully,most the beneficial medicines of LV showed little or even no effect on RV.Intensive studies of the cellular and molecular mechanisms of RV remodeling in PAH is of an important scientific significance and clinical value for the development of RV-specific therapy,reversal of right ventricularremodeling,and to improve the prognosis of patients.Autophagy is an evolutionarily conserved process from yeast to mammals for the bulk degradation and recycling of long-lived proteins and organelles.Intracellular components are surrounded by double membrane-bound autophagic vesicles which then fuse with lysosomes to form autolysosomes for degradation.Autophagy was proved to play important roles in regulation of cell survival and death.There are evidences showing that autophagy is involved in heart remodeling in different heart diseases.It has been proved myocardial ischemia/hypoxia are structural derangements contributing to the progression of RV failure in PAH.There are two crucial pathways involved in the regulation of autophagy during ischemia/hypoxia.One is the adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)and the other is hypoxia inducible factor 1?(HIF-1?)/adenovirus E1 B 19 kDa protein-interacting protein 3(BNIP3)/Beclin-1 pathway.However,the dynamic changes of autophagy of RV in PAH,and whether the two pathway are underlying mechanisms have not been clarified.Additionally,the potential role of autophagy in different stages of RV remodel is not well understood.This study may provide an initial step into understanding the PAH-related RV myocardial autophagy,their potential mechanisms and roles in monocrotaline(MCT)induced PAH rats.These findings may have broad implications in better designing pharmacological strategies to protect against RV remodeling and dysfunction in PAH in a stage-specific manner.Part I: Establishment of monocrotaline-induced PAH rat model and alternation of oxygen supply related markers of RV.Objective: To establish monocrotaline-induced PAH rat model,and tostudy dynamic change of RV structure and function,as well as alternation of oxygen supply related markers of RV in the model.Methods: The SD rats(n=60)were randomly assigned to either the MCT(n=36)or control(n=24)group.The MCT group received a single 60 mg/kg subcutaneous injection of MCT,while the control group received an equivalent volume of saline.Within each group,rats were further subdivided according to injection durations(2 weeks,4 weeks,6 weeks).Echocardiography was performed and RV structure and functional parameters were obtained in the living rats at end of each time points.Invasive right heart hemodynamic study was performed and pulmonary systolic and mean artery pressures were obtained.Hematoxylin-eosin staining(H&E)was performed to study the structure of lung and RV tissues.Masson's trichrome stain was used to study collagen volume fraction.Wheat germ agglutinin(WGA)stain and CD31 immunofluorescence were performed to assess RV cardiomyocyte cross-sectional area and capillary density,respectively.Moreover,Real-time quantitative polymerase chain reaction(RT-qPCR),immunohistochemical stain and Western blot were used to study the mRNA and protein expression of RV myocardial myoglobin.Results: Two weeks after MCT injection,pulmonary systolic and mean pressures increase,and mild RV hypertrophy and larger cardiomyocytes diameter without RV dilation was observed.Further enlargement of cardiomyocytes diameter and accumulation of fibrosis resulting in RV enlargement with moderately decreased function were observed at 4 week,whereas typical characteristics of RV decompensation and failure occurred at 6weeks,thus demonstrating the progression of RV remodeling in the MCT model.Compared to the control,slightly increase of RV cardiomyocyte cross-sectionalarea without significant alternation of capillary density and myoglobin at 2weeks after MCT injection.Whereas,RV cardiomyocyte cross-sectional area was strongly increase,and capillary density,myoglobin decreased at 4th and 6th week.These changes were more obviously in 6th week compared with 4th week.Conclusion: PAH rat model has been established and progression of RV remodeling has been duplicated by MCT injection.At the 2 week,an enlargement of RV cardiomyocytes have presented;while at the 4 and 6 week,further enlargement of cardiomyocytes and RV capillary rarefaction resulted in decrease coronary artery perfusion,as well as reduction in RV myoglobin protein leading to RV myocardial ischemia/hypoxia are structural derangements contributing to the progression of RV failure in PAH.Part II: Changes of cardiomyocyte autophagy during RV remodeling in PAH rats and potential mechanisms.Objective: To investigate the time course changing of autophagy in RV remodeling in MCT induced PAH rat model,and to clarify whether the hypoxia-related pro-autophagy pathways are involved in RV autophagic regulation.Methods: Using RT-qPCR and immunochemistry,the expression of the autophagosomal marker Microtubule associated light chain protein 3(LC3)was assessed.Moreover,western blot was used to study LC3-II/LC3-I ratio and P62 protein.In addition,transmission electron microscopy(TEM)was utilized to verify autophagy structure.Using Western blot analysis,the protein expression of each signaling components was assessed,including mTOR(Ser 2481),AMPK?(Thr172),p70S6K(Ser424),Beclin-1 HIF-1?,Bcl2,and BNIP3,in MCT-treated rats and controls at each time point.Results: LC3 mRNA and LC3 protein(as measured by densitometry)as well as LC3-II/LC3-I ratio became markedly and gradually upregulated in the RV myocardium during the progression of PAH in MCT rats,and reached its peak at sixth week,while P62 decreased after MCT treatment.Similarly,the number of autophagosomes increased in the MCT rats was observed by TEM.In contrast,both LC3 mRNA,LC3,LC3-II/LC3-I ratio together with P62 remained low and constant in the controls at every time point assessed after MCT treatment.Autophagosomes were rarely observed in controls.Additionally,no significant differences in these parameters could be observed in the LV of MCT rats at these different time points.Interestingly,LC3 levels in the MCT-treated groups strongly correlated to PASP(r2=0.626,P<0.01).Compared to controls,downregulation of phosphorylated mTOR and phosphorylated p70S6K(normalized to total levels)was observed in RV of the 2 and 4 week subgroup MCT rats;phosphorylated mTOR became upregulated in the RV of the6 week subgroup MCT rats.Inversely,as phosphorylated AMPK?(normalized to total levels)trended towards increased activation in the RV of 2 and 4 week MCT rats compared to controls;however,it appears to decline in the RV of 6th week MCT rats compared with the 2 and 4 week MCT rats.Moreover,expression of BNIP3 and Beclin-1 was relatively low in the RV of the 2 and 4week MCT rats,and then became significantly upregulated in the 6 week MCT rats compared with age-matched controls.However,it appears that the BNIP3 activation was not totally dependent on Hif-1?.HIF-1? levels were slightly increased in the RV of the 2 week MCT group and became significantly increased in the RV of the 4 week MCT group,but then slightly decreased in 6week.Additionally,Bcl2 peaked in expression in the RV of the 2 week MCT group,and then went on a gradual decline in the 4 and 6 week.No alternation of these proteins in the control groups in different time point.Conclusion: Pro-autophagy signaling pathways were activated in a RV remodeling stage-dependent manner as phospho-AMPK were primarily upregulated and phospho-mTOR suppressed in the RV at 2 and 4 weeks post-MCT injection.In contrast,BNIP3 and beclin-1 expression were relatively low at these stages,but were significantly upregulated after 6 weeks in this model,BNIP3 activation are not totally Hif-1?independent.Part III: The roles of AMPK/mTOR and BNIP3/Beclin-1 pathways mediated autophagy activation in cardiomyocyte with hypoxia.Objective: To investigate roles of AMPK/mTOR and BNIP3/Beclin-1pathways mediated autophagy activation in cardiomyocyte with hypoxia.Combined with the second part of the study,to further reveal the mechanisms of autophagy in PAH induced RV remodeling.Methods:(1)One group of H9C2 rat cardiomyocytes were randomly divided into following four subgroups,including normorxia,hypoxia(for 1 days),hypoxia+Compound C and hypoxia+AICAR.Western blot was performed to assess phospho-AMPK,AMPK,phospho-mTOR,mTOR,and LC3 protein expression.Monodansylcadaverin(MDC)stain was used to imaging autophagosomes.WGA stain was performed to image size of cardiomyocytes.RT-qPCR was used to assess atrial natriuretic peptide(ANP),Type B natriuretic peptide(BNP)mRNA levels.CCK8 was used to detect relative vialibility of cardiomyocytes.Flow cytometry was utilized to assess apoptosis rate of cardiomyocytes.Hypoxia and hypoxia+AICAR group were treated withbafilomycin A1 to verify activation of autophagy.Another group of rat cardiomyocytes were randomly divided into following three subgroups,including hypoxia,hypoxia+AICAR,hypoxia+AICAR+3-MA.MDC stain was used to imaging autophagosomes.Western blot was performed to assess LC3 protein expression.CCK8 was used to detect relative vialibility of cardiomyocytes.Flow cytometry was utilized to assess apoptosis rate of cardiomyocytes.(2)One group of cardiomyocytes were divided into following five subgroups,including normorxia,hypoxia(for 3 days),negative,hypoxia+lentiviral vector with siBNIP3,and hypoxia+ lentiviral vector with BNIP3.Western blot was performed to assess BNIP3,Beclin-1,and LC3 protein expression.MDC stain was used to imaging autophagosomes.Flow cytometry was utilized to assess apoptosis rate of cardiomyocytes.Hypoxia and hypoxia+lentiviral vector with BNIP3 group were treated with bafilomycin A1 to verify activation of autophagy.Another group of rat cardiomyocytes were randomly divided into following three subgroups,including hypoxia,negative,hypoxia+lentiviral vector with BNIP3,hypoxia+ lentiviral vector with BNIP3+3-MA.MDC stain was used to imaging autophagosomes.Western blot was performed to assess LC3 protein expression.Western blot was performed to assess LC3 protein expression.CCK8 was used to detect relative vialibility of cardiomyocytes.Flow cytometry was utilized to assess apoptosis rate of cardiomyocytes.Results:(1)Compared with normoxia cardiomyocytes,the hypoxia group presented higher ratio of phospho-AMPK/AMPK and lower phospho-mTOR/mTOR;higher fluorescence intensity of MDC and LC3-II/LC3-I ratio;lower relative vibility and higher percentage of apoptosis.Compared to the hypoxia group,Compound C treated lead lower ratio ofphospho-AMPK/AMPK and higher ratio of phospho-mTOR/mTOR,lower fluorescence intensity of MDC,LC3-II/LC3-I ratio as well as relative viability of cardiomyicytes,while evern higher percentage of apoptosis.However,AICAR treated lead opposite results.There are no differences of size,ANP and BNP mRNA expression in different group of cardiomyocytes.Bafilomycin A1 increased hypoxia and hypoxia+AICAR group fluorescence intensity of MDC stain and LC3-II/LC3-I ratio.Compared with hypoxia+AICAR group,hypoxia treated with AICAR plus 3-MA showed lower fluorescence intensity of MDC and LC3-II/LC3-I ratio,lower viability and higher number of apoptosis.(2)Compared with normoxia cardiomyocytes,the hypoxia group presented higher BNIP3 and Beclin-1 expression,fluorescence intensity of MDC and LC3-II/LC3-I ratio;while lower relative viability and higher percentage of apoptosis.Compared with hypoxia cardiomyocytes,hypoxia treated with BNIP3-RNAi lentiviral transfection cardiomyocytes resulted in lower BNIP3 and Beclin-1 expression,fluorescence intensity of MDC and LC3-II/LC3-I ratio,as well as lower number of apoptosis;whereas higher relative vibility.Compared with hypoxia group,the BNIP3 overexpress cardiomyocytes showed higher BNIP3 and Beclin-1 expression,fluorescence intensity of MDC and LC3-II/LC3-I ratio,and higher number of apoptosis;while lower relative viability.No differences of these markers of empty lentiviral vector treated group compared with hypxia group.Bafilomycin A1 increased hypoxia cardiomyocytes or hypoxia with BNIP3 overexpress cardiomyocytes fluorescence intensity of MDC stain and LC3-II/LC3-I ratio.Compared with BNIP3 overexpress cardiomyocytes,hypoxia treated with BNIP3 plus 3-MA showed lower fluorescence intensity of MDC and LC3-II/LC3-I ratio,higher relative viability,but no differences of percentage of apoptosis.Conclusion: AMPK/mTOR pathway activated autophagy serves as a negative regulator against cardiomyocytes apoptosis and death;whereas,BNIP3/Beclin-1 pathway mediated autophagy acts as regulator of pro-death mechanisim of cardiomyocytes independent of apoptosis regulation in hypoxia.