Melatonin Ameliorates Pressure Overload-induced Cardiac Hypertrophy By Attenuating Atg5-dependent Autophagy And Activating Akt/mTOR Pathway

Heart failure(HF),a complex clinical syndrome,is the terminal state of all heart diseases.HF is the leading cause of mortality and threatens health of human beings in our country and worldwide.Pathological cardiac hypertrophy,which is a pathological characteristic common to numerous forms of heart disease,such as hypertension and valvular dysfunction,is generally considered to be an independent risk factor of heart failure.Despite the cardiac structure and function in patients with early stage changes for an increase in cardiac output,the heart will gradually decompensate,leading to heart failure.Therefore,cardiac hypertrophy has become an important therapeutic target for heart failure.With its unique advantages,3D printing technology can present the structure of the lesion in a more intuitive and complete form,which greatly improves the success rate of surgery and has been widely used in clinical treatment.The synthesis and degradation of protein and intracellular organelles in cardiomyocytes is a dynamic process.Autophagy is a dynamic process that turns over damaged proteins and organelles through a lysosome-associated degradation system and physiologically maintains cellular homeostasis at basal level.In the past decade,autophagy has been widely studied in regulation of cardiac structure and function.Autophagy related protein 5(Atg5),an E3 ubiquitin ligase,is an essential protein that plays a crucial role in the formation and autophagy process of autophagosomes.Previous studies have shown that atg5-deficient mice lead to left ventricular dilation and systolic dysfunction.The relationship between Atg5 and stress-induced hypertrophy is not clear.A growing number of studies have shown that autophagy plays an important role in the development of myocardial hypertrophy,and may aggravate myocardial injury by causing cell death.Impaired autophagy in the heart is associated with almost all forms of cardiovascular disease.New research evidence suggests that regulating autophagy may be a potential therapeutic strategy and drug target for heart disease.It has been demonstrated that certain signaling pathways,such as the Akt/mTOR pathway,are involved in the regulation of autophagy in response to basal state and stress.Melatonin(N-acetyl-5-methoxytryptamine),the main secreted product of the pineal gland,is considered to be a significant protective endocrine hormone,which can prevent pathological changes in myocardial remodeling caused by heart damage and cardiac hypertrophy.A large number of studies have confirmed that melatonin and its metabolites play an important role in reducing oxygen free radicals,cell apoptosis and oxidative stress.However,the effect of melatonin on apoptosis and autophagy in stress-induced myocardial hypertrophy and its specific mechanism have not been confirmed.Therefore,in this study,we evaluated the feasibility and effectiveness of aortic constriction model(TAC)using 3D printing technology,and explored the role of melatonin in improving autophagy by activating the Akt/mTOR signaling pathway and regulating Atg5 to reduce TAC induced cardiac hypertrophy.Objectives:(1)3D printing technology in evaluation of mice TAC model;(2)To investigate the role of Akt/mTOR pathway and autophagy in melatonin-alleviated TAC-induced cardiac hypertrophy;(3)To elucidate the role of Akt/mTOR pathway and Atg5 in improving autophagy and reducing TAC-induced cardiac hypertrophy.Methods:Part I 3D printing technology in evaluation of mice TAC modelThirty healthy 10-week-old C57BL/6 adult male mice were randomly divided into the Sham group and the aortic constriction model group(TAC group),with 15 mice in each group.C57BL/6 mice in TAC group were anesthetized with isoflurane,fixed to a thermostatic operating table,intubated,connected to a small animal ventilator,and kept anesthetized during the operation.On the sternal notch cut in the skin,a longitudinal incision for 1 cm,cut open the sternum,thoracic dilator open incision,separation and thymus tissue,clearly showing the aortic arch,near Aortic site between innominate and left common carotid artery wear 7-0 silk,with silk aorta with preset 25 G needle ligation,elastic,right cut ligature and pull out the needle,closed chest one by one.The Sham group adopted the same surgical method.After tissue was separated layer by layer to the aortic arch,the chest was closed layer by layer without treatment after 7-0 silk thread was worn.The hemodynamic changes of constriction site were observed by ultrasound.CT scan reconstruction image and 3D printing technology were used to print the blood vessel in the constricted part into a physical mold.The formation of myocardial hypertrophy was detected by traditional methods such as heart weight/weight ratio(HW/BW),heart weight/tibial length ratio(HW/TL),HE staining,Masson staining and western blot.Part II The role of Akt/mTOR pathway and autophagy in melatonin-alleviated TAC-induced cardiac hypertrophySixty healthy C57BL/6 adult male mice aged 8-10 weeks were randomly divided into the Sham group,melatonin drug control group(Mel group),aortic constriction model group(TAC group)and melatonin intervention aortic constriction group(Mel+TAC group),with 15 mice in each group.The surgical method was the same as before,while melatonin 10 mg/kg/d was intraperitoneal injected into the Mel group and Mel+TAC group for 8 weeks.HE and Masson staining,HW/BW and ANP,BNP and β-MHC m RNA levels were used to evaluate myocardial hypertrophy.Ultrasonic examination of cardiac function;Western blot was used to detect the protein expressions of apoptosis-related proteins Bcl-2,Bax,cleaved caspase-3 and cytochrome c,as well as the phosphorylation levels of Akt/mTOR signaling molecules,mTOR and its substrates S6 K and 4E-BP1.Apoptosis rate was observed by TUNEL staining.Protein expressions of autophagy related proteins Atg5,p62 and LC3I/II were detected by western blot.The number of autophagosomes was observed by LC3 B immunofluorescence staining.Transmission electron microscopy was used to observe the formation of autophagosomes.Part III The role of Atg5-dependent autophagy and Akt/mTOR pathway in melatonin-alleviated TAC-induced cardiac hypertrophyEighteen healthy 8-10 week old C57BL/6 adult male mice were randomly divided into the Sham control group,the no-load adenovirus control group(Ad-C),and the cardiac-specific high-expression Atg5 adenovirus group(Ad-Atg5).Another sixty healthy C57BL/6 adult male mice aged 8-10 weeks were randomly divided into model aortic constriction group(TAC),melatonin intervention aortic constriction group(Mel+TAC),LY294002 combined with melatonin intervention aortic constriction group(Mel+LY294002+TAC)and cardiac-specific high expression Atg5 combined with melatonin intervention aortic constriction group(Mel+Ad-Atg5+TAC),with 15 mice in each group.The surgical method was the same as before,while melatonin 10 mg/kg/d was intraperitoneally injected into the Mel group and(Mel+TAC)group for 8 weeks,and the adenovirus was injected into the myocardium 3 days before the modeling.HE and Masson staining,HW/BW and ANP,BNP and β-MHC m RNA levels were used to evaluate myocardial hypertrophy.Ultrasonic examination of cardiac function;Western blot was used to detect the protein expressions of apoptosis-related proteins Bcl-2,Bax,cleaved caspase-3 and cytochrome c,as well as the phosphorylation levels ofAkt/mTOR signaling molecules,mTOR and its substrates S6 K and 4E-BP1.Apoptosis rate was observed by TUNEL staining.Protein expressions of autophagy related proteins Atg5,p62 and LC3I/II were detected by western blot.The formation of autophagosomes was observed by LC3 B immunofluorescence staining.Transmission electron microscopy was used to observe the formation of autophagosomes.ResultsPart I 3D printing technology in evaluation of mice TAC model1.Vascular ultrasound results indicated significant constriction of the aorta.CT scan reconstruction images were combined with 3D printing technology to print the aorta of the TAC group into a physical mold,from which the constriction of aortic vessels could be more visually seen.2.Compared with Sham group,HE staining,Masson staining,HW/BW and HW/TL were significantly increased in TAC group.Meanwhile,western blot showed that protein expression levels of hypertrophy related indicators ANP,BNP and β-MHC were significantly increased.Part II The role of Akt/mTOR pathway and autophagy in melatonin-alleviated TAC-induced cardiac hypertrophy1.Compared with Sham group,HW/BW increased in TAC group,HE and Masson staining showed increases in myocardial cell cross-sectional area and degree of fibrosis in TAC group,significantly increased m RNA levels of hypertrophy markers ANP,BNP and β-MHC,and decreased LVEF and LVFS.After Mel treatment with TAC,HW/BW,cell cross-sectional area,degree of fibrosis,and m RNA levels of ANP,BNP,and β-MHC were significantly reduced,while LVEF and LVFS were increased.2.Compared with Sham group,the expression of apoptosis-related proteins Bax,cleaved caspase-3 and cytochrome c increased in TAC group,while the expression of Bcl-2 decreased.However,after treatment with Mel,the expression of Bax,cleaved caspase-3 and cytochrome c protein decreased,while the expression of Bcl-2 increased.TUNEL staining showed that the apoptosis rate of TAC group significantly decreased.3.Compared with Sham group,the protein levels of p-mTOR,p-Akt,p-S6 K and p-4E-BP1 were significantly decreased in TAC group,while levels of autophagy related proteins Atg5 and p62 and LC3II/LC3 I ratio were increased,and LC3 B fluorescence staining showed increased autophagosomes.After TAC treatment with Mel,the expression of p-mTOR,p-Akt,p-S6 K and p-4E-BP1 increased,while the expression of Atg5 and p62 decreased,the LC3II/LC3 I ratio decreased,and autophagosomes decreased.Part III The role of Atg5-dependent autophagy and Akt/mTOR pathway in melatonin-alleviated TAC-induced cardiac hypertrophy1.Compared with Sham group,Green flluorescent protein(GFP,negative control)and cardiac-specific c TNT promoter(Ad-c TNT-Atg5)adenovirus expressing Atg5 had no effect on normal heart structure and function,while the expression of Atg5 protein in the Ad-Atg5 group was significantly increased.2.Compared with TAC group,TAC treatment with Mel increased the expression of p-mTOR,p-Akt,p-S6 K and p-4E-BP1,and decreased the expression of autophagy related proteins Atg5 and p62,the LC3II/LC3 I ratio,and LC3 B fluorescence staining showed that the autophagosomes decreased.Both LY294002 and Ad-Atg5 inhibited the regulation of Akt/mTOR signaling pathway by Mel and the improvement of autophagy.3.Compared with TAC group,TAC treatment with Mel decreased HW/BW,myocardial cell cross-sectional area and degree of fibrosis,and increased LVEF and LVFS.Both LY294002 and Ad-Atg5 inhibited the effects of Mel on TAC-induced cardiac hypertrophy.Conclusion1.3D printing technology can be applied to evaluate the sucess of mice aortic coarctation model.2.Melatonin alleviates TAC-induced cardiac hypertrophy by activating Akt/mTOR pathway.3.Melatonin alleviates TAC-induced cardiac hypertrophy by activating Akt/mTOR pathway and Atg5-dependent autophagy.