The Effects Of Rapamycin On Cardiomyocyte Apoptosis And Autophagy In Rats With Postinfarction Heart Failure

Background and purpose:Heart failure（HF）is the ultimate outcome of various heart diseases,and myocardial infarction（MI）is the most common cause of HF.After MI,the neuro-endocrine system is activated,and neuroendocrine factors such as angiotensin Ⅱ（Ang Ⅱ）,epinephrine,and endothelin are released,and progressive ventricular remodeling is initiated.Following ventricular remodeling,cardiomyocyte hypertrophy and cardiomyocyte apoptosis occurs.Cardiomyocyte apoptosis leads to the lost of cardiac contractile protein and cardiac diastolic dysfunction.Low level but persistent cardiomyocyte apoptosis can lead to fatal HF.On the other hand,upregulated cardiomyocyte autophagy is often found in failing myocardium.Autophagy is another form of cell death,but it is currently believed that autophagy was originally a mechanism that can protect cardiomyocytes from apoptosis by decomposing and recovering useful components inside the cell and supplementing metabolic substrates for the cell during HF.Therefore,anti-apoptosis and pro-autophagy of cardiomyocytes may be effective strategies for the treatment of postinfarction HF.Our previous studies have shown that multiple genes associated with the mechanistic target of rapamycin（mTOR）pathway in the myocardium of post-MI rats are differentially expressed compared with control rats.mTOR is an intracellular atypical serine/threonine kinase.The mTOR kinase is present in two distinct multiprotein complexes,mTOR complex 1（mTORC1）and mTORC2 which are involved in the regulation of cell growth,survival and autophagy.The functions of mTORC1 and mTORC2 are distinct and interrelated.mTORC1 mainly promotes cell growth through physiological processes such as protein synthesis.Furthermore,mTORC1 is a key protein complex that inhibits catabolism,such as cell autophagy.However,the function of mTORC2 has not been fully elucidated yet.Previous studies indicates that mTORC2 may play an important role in the regulation of cell polarity and cell survival.Rapamycin is an inhibitor of mTOR,and it is also a commonly used antitumor drug and coronary stent coating drug in clinical practice.Rapamycin has a rapid and direct inhibitory effect on mTORC1.Long-term treatment of rapamycin can also inhibit the function of mTORC2 because it"occupies"mTOR and affects the assembly of mTORC2.In pressure overload-induced myocardial hypertrophy and acute myocardial ischemia animal models,it has been found that rapamycin can reduce myocardial hypertrophy,activate cardiomyocyte autophagy,and reduce the area of MI by inhibiting mTORC1.However,postinfarction HF often develops from old MI,and often manifests as a chronic course,which is not exactly the same as the pathogenesis of pressure overloaded-induced myocardial hypertrophy and acute myocardial ischemia.In postinfarction HF,can rapamycin exert its anti-cardiomyocyte apoptosis and improve cardiac function by inhibiting mTORC1pathway and activating cardiomyocyte autophagy;whether the anti-apoptosis effect of rapamycin will be affected when the function of mTORC2 is inhibited long-term rapamycin treatment;and the mechanism involved in the anti-apoptosis effect of rapamycin have not been reported.In the present study,we investigated the effects of rapamycin on cardiomyocyte apoptosis,autophagy,and cardiac function and the mechanisms involved in the anti-cardiomyocyte apoptosis of rapamycin by constructing post-MI rat and Ang Ⅱ-induced H9c2 cell apoptosis models.Methods:Post-MI rat model was constructed by ligating the anterior descending coronary artery branch of Wistar rats.Cardiac function was assessed by echocardiography at 4weeks after MI.Then,rapamycin（1.4 mg/kg/day）or DMSO（vehicle）was injected for 4 weeks and rats were sacrificed after cardiac function assessment at 8 and 12weeks after MI.The heart was taken to observe its general morphology,and the left ventricle was separated for HE,Masson,and caspase-3 immunohistochemical staining.Myocardial tissue of an area far from the infarction site was separated for Western blot detection of apoptosis-,autophagy-,mTORC1 pathway-,mTORC2 pathway-and endoplasmic reticulum stress pathway-related proteins.Ang Ⅱ was used to create a pathological humoral environment for H9c2 cells.To determine the optimal concentration of Ang Ⅱ for inducing apoptosis,an in vitro cell viability assay was performed.Following short-term and long-term pretreatment with rapamycin,H9c2cells were evaluated by flow cytometry,caspase-3 enzyme activity detection,immunofluorescence and Western blot detection of apoptosis-and autophagy-related proteins.Western blot analysis of mTORC1-,mTORC2-and endoplasmic reticulum stress pathway-related proteins was performed to analyze the regulatory mechanism of rapamycin on H9c2 cell apoptosis and autophagy.Results:1.Transcriptome expression profile of myocardium in rats with postinfarction HF.The transcriptome expression profile of myocardium in rats with postinfarction HF（8 weeks after MI）was significantly different from that in sham rats.Among these differentially expressed genes,10 genes were upregulated and 26 genes were downregulated.KEGG pathway annotation of differentially expressed genes were mainly focused on pathways associated with apoptosis,protein synthesis,gene transcription and translation,inflammation,autophagy,amino acid and lipid metabolism.The functions of multiple differentially expressed genes are closely associated with the mTOR pathway.2.Rapamycin attenuatea ventricular remodeling and improve cardiac function in rats with postinfarction HF.At 8 weeks after MI,compared with the DMSO group,the left ventricular ejection fraction（57±2（4）vs.47±3（4）,P（27）0.05）and left ventricular shortening（28±1（4）vs.20±1（4）,P（27）0.05）of the rapamycin group were significantly increased,the left ventricular end diastolic diameter（600±45 mm vs.833±33 mm,P（27）0.05）and the left ventricular end systolic diameter（400±45 mm vs.650±34 mm,P（27）0.05）were significantly reduced,and the area of cardiomyocyte were smaller.At 12 weeks after MI,rapamycin still improved the left ventricular ejection fraction（55±2（4）vs.40±3（4）,P（27）0.05）and left ventricular shortening（25±1（4）vs.17±1（4）,P（27）0.05）in rats with HF.3.Rapamycin inhibited the cleavage of caspase-3 and promote autophagy in cardiomyocytes.At 8 weeks after MI,compared with DMSO group,the level of cleaved caspase-3 in myocardium of the rapamycin group was significantly reduced,the level of autophagy marker,LC3B Ⅱ in myocardium of the rapamycin group was increased,and the level of autophagy substrate p62 in myocardium of the rapamycin group was significantly decreased.Similar results were also observed at 12 weeks after MI.4.Rapamycin inhibited the expression of key molecules in the mTOR pathway.At 8 weeks after MI,the level of RibS6^Ser235/236 downstream of mTORC1 in the myocardium of the DMSO group was significantly increased,suggesting that mTORC1 activity was increased and protein synthesis was activated after MI.Compared to the DMSO group,rapamycin significantly inhibited the expression of RibS6^Ser235/236 and Akt^Ser473 downstream of mTORC2.At 12 weeks after MI（4 weeks after rapamycin was discontinued）,compared with the DMSO group,the expression of RibS6^Ser235/236 in the rapamycin group was still slightly suppressed,and the expression level of Akt^Ser473 returned to the baseline.5.Rapamycin inhibited apoptosis and activated autophagy in H9c2 cells.Ang Ⅱ（100 nM,24 hours）induced an apoptosis model of H9c2 cells in vitro,which is similar to the cardiomyocyte apoptosis pattern in failing myocardium in vivo.The results of flow cytometry,cell caspase-3 enzyme activity and Western blot detection of cleaved caspase-3 showed that both short-term（30 minutes）and long-term（24 hours）pretreatment with rapamycin significantly reduced the rate of Ang Ⅱ-induced H9c2 cell apoptosis.The results of Western blot and cellular immunofluorescence detection of autophagic marker protein expression showed that both short-term and long-term rapamycin pretreatment significantly upregulated basal and Ang Ⅱ-induced autophagy levels in H9c2 cells.6.Rapamycin significantly inhibited the expression of key molecules of the mTORC1 pathway.The expression of RibS6^Ser235/236 in Ang Ⅱ-induced H9c2 cells was upregulated.Short-term and long-term rapamycin pretreatment significantly inhibited the expression of RibS6^Ser235/236,but had no significant effect on the expression of AktSer473.7.Rapamycin inhibited the mTORC1-endoplasmic reticulum stress pathway and reduced Ang Ⅱ-induced H9c2 cell apoptosis and cardiomyocyte apoptosis in rats with postinfarction HF.In rats with postinfarction HF and Ang Ⅱ-induced H9c2 cells,rapamycin significantly inhibited the expression of phosphorylated JNK and DDIT3,which were key molecules that induced apoptosis in the endoplasmic reticulum stress pathway.Conclusion:In postinfarction HF,rapamycin may mainly reduced neonatal protein synthesis and increased autophagic protein degradation,thereby inhibiting downstream endoplasmic reticulum stress pathways,reducing cardiomyocyte apoptosis,delaying ventricular remodeling and improving cardiac function by inhibiting the mTORC1pathway.