Aerobic Exercise Training Alleviates Ischemia/Reperfusion Injury By Reducing METTL3-mediated M~6A RNA Methylation In Myocardial Cells Of Mice

Background:Ischemic cardiomyopathy represents one of the most life-threatening diseases in our country.Swift restoration of perfusion is a crucial step to rescue the ischemic heart.However,this process is accompanied by severe myocardial ischemia/reperfusion injury(MIRI),and effective clinical intervention methods are still lacking.Among the various mechanisms of ischemia/reperfusion(I/R)injury,oxidative stress stands out as one of the most important pathophysiological processes and is currently a target of widespread attention for prevention and treatment.Aerobic exercise training(AET),as a means to enhance physical fitness,has been shown to improve myocardial tissue structure,affect platelet activation,regulate vascular endothelial function,promote angiogenesis,and protect the myocardium against I/R injury.However,the protective mechanism of AET on MIRI remains unknown.N6-methyladenosine(m~6A)is the most common modification in RNA methylation,mainly occurring on adenine in the RRACH sequence.It can regulate m RNA splicing,transport,localization,translation and degradation by influencing RNA metabolism.m~6A methylation modification is dynamically regulated by m~6A methyltransferases(such as METTL3,METTL4,METTL14,WTAP,etc.)and m~6A demethylases(such as FTO,ALKBH5,etc.),also it is recognized by reader proteins(including YTHDF1,YTHDF2,YTHDF3,YTHDC1,etc.).m~6A methylation is involved in various cardiovascular diseases processes.For instance,methyltransferase METTL3 can regulate m RNA stability,thereby affecting subsequent metabolic levels and playing a significant role in controlling cardiac homeostasis and cardiac hypertrophy.While m~6A RNA methylation has been closely linked to myocardial I/R injury,whether it mediates the protective effects of exercise on MIRI remains poorly understood.Therefore,investigating whether AET enhances myocardial resistance to I/R injury in correlation with the m~6A RNA methylation level of myocardial tissue and further exploring the role of m~6A RNA methylation in myocardial oxidative stress is warranted.Objective:This study aims to investigate the role of AET in MIRI by establishing an AET model and a MIRI model in C57BL/6 mice,and to elucidate the impact of AET on the m6A RNA methylation level of myocardium in vivo.Furthermore,we aim to examine the expression of m~6A methylation modification mediated by methyltransferase METTL3 in MIRI and its effect on the myocardial oxidative stress pathway(Nrf2/HO-1),along with its possible mechanism through in vivo and in vitro experiments.We aim to provide a theoretical basis for the prevention and treatment of MIRI through this study.Methods:1.Male C57BL/6 mice aged 8～10 weeks were selected as experimental subjects and randomly divided into Sedentary+Sham group(SE+Sham),Swim training+Sham group(SW+Sham),Sedentary+ischemia/reperfusion injury group(SE+MIRI)and Swim training+ischemia/reperfusion injury group(SW+MIRI)(n=5～8 mice/group).Mice in the SW+Sham and SW+MIRI groups underwent 4 weeks swimming training before myocardial I/R surgery,while mice in the remaining groups did not.On the day of surgery,the SE+MIRI and SW+MIRI groups underwent myocardial I/R surgery,with ischemia lasting for 30 minutes followed by 24 hours of reperfusion.Basic indicators such as body weight(BW),heart weight(HW)and tibia length(TL)were measured after swimming training.Cardiac ultrasound was performed to evaluate the structure and function of the heart after myocardial I/R surgery.Myocardial tissues were collected for analysis.Evans blue-TTC staining was employed to detect the area of myocardial infarction.Electrochemiluminescence immunoassay was used to measure the expression of myocardial injury markers such as c Tn I,CK-MB and LDH.Pathological sections of myocardial tissue were prepared and wheat germ agglutinin(WGA)staining was utilized to observe myocardial size,Masson staining was employed to assess collagen fiber content in myocardial tissue,and the TUNEL method was used to determine the level of myocardial cell apoptosis.2.The Dot Blot method was used to detect m~6A RNA methylation levels in mice myocardial tissues after swim training and MIRI,the q RT-PCR method was used to detect the expression of m6A RNA methylation-related proteins,Western blot method was used for further verification.to clarify the effects and targets of swim training and MIRI on m6A RNA methylation in myocardial tissue.Those above experimental procedures were used to clarify the effects and targets of swim training and MIRI on m~6A RNA methylation in myocardial tissues.1.Primary cardiomyocytes were isolated and cultured from mice,whether they underwent swimming exercise or not(SE vitro and SW vitro).Hypoxia/reoxygenation(H/R)injury was induced in cardiomyocyte during logarithmic growth phase to simulate MIRI.Cardiomyocyte viability was measured using the CCK8 method,cardiomyocyte mitochondrial membrane potential was assessed using the JC-1 method,cardiomyocyte apoptosis was detected using flow cytometry,and apoptosis-related protein expression was analyzed using the Western blot method.These in vitro experiments were conducted to ascertain the effects of swim training and H/R on m~6A methylation in primary cardiomyocytes of mice,and to explore the role of METTL3.2.The myocardial tissues of mice after MIRI were collected,and m~6A modified RNA(Me RIP)was immunoprecipitated and sequenced.The sequencing data were further used to conduct KEGG signaling pathway analysis to explore the m~6A RNA methylation-related regulatory targets and signaling pathways during mouse MIRI.Western blot was used to detect the expression of nuclear Nrf2(Nu-Nrf2),and q RT-PCR was used to detect the levels of Nrf2 downstream antioxidant molecules HO-1,NQO1 and GCLM.In in vitro experiments,immunofluorescence staining was used to verify the intracellular localization of Nrf2,Western blot was used to detect the effects of transfection of METTL3 and Nrf2 si-RNAs on the expression of Nu-Nrf2 and HO-1in primary cardiomyocytes of mice,and flow cytometry was used to detect apoptosis of cardiomyocytes.Results:1.AET significantly reduces myocardial damage after I/R.(1)AET improves cardiac function following I/R injury.Four weeks of swim training markedly increased the heart weight of C57BL/6 mice,leading to physiological hypertrophy of the myocardium.Echocardiographic findings revealed impaired cardiac function in C57BL/6 mice following MIRI,as evidenced by increased left ventricular end-systolic diameter(LVESD)and decreased cardiac ejection fraction(EF)and left ventricular short-axis shortening rate(FS).However,AET significantly increased the aforementioned cardiac dysfunction associated with ischemic myocardium.(2)AET reduces myocardial tissue damage caused by I/R injury.Histological assessments including H&E staining,Masson staining,TUNEL staining and biochemical detection revealed that MIRI resulted in myocardial cell necrosis,apoptosis,as well as myocardial fibrosis and collagen deposition.Following swim training,myocardial cells exhibited physiological hypertrophy and showed decreased myocardial cell necrosis,apoptosis,myocardial fibrosis,and collagen deposition caused by I/R injury.2.m~6A RNA methylation mediates swim training to resist MIRI.(1)AET down-regulates the increase of m~6A RNA methylation level in myocardial tissues of C57BL/6 mice caused by I/R injury.(2)qRT-PCR and Western blot methods were used to detect the expression of m6A RNA methylation modification molecules,and the results suggested that methyltransferase METTL3 may be the main regulatory molecule for MIRI protection during swim training.3.Response of primary cardiomyocytes to H/R in mice that underwent swim training.(1)Knocking down METTL3 expression in SE vitro cardiomyocytes can reduce cardiomyocyte damage and LDH release caused by H/R,downregulate cleaved Caspase-3/total Caspase-3 and Bax/Bcl2 ratios,increase cardiomyocyte mitochondrial membrane potential,and reduce cardiomyocyte apoptosis.(2)The m6A RNA methylation level and the expression of methyltransferase METTL3 were reduced in primary cardiomyocytes of swimming-trained mice,and the results were consistent with the in vivo experimental results.Swim training can resist H/R-induced SW vitro cardiomyocyte damage,LDH release and cell apoptosis.4.Sequencing results showed that m~6A RNA methylation levels in myocardial tissues increased after MIRI.Moreover,oxidative stress-related protein signaling pathways in myocardial tissue were widely activated,with the activation of Nrf2-related signaling pathways potentially regulated by m6A RNA methylation.5.AET mediates the Nrf2/HO-1 signaling pathway to resist MIRI by regulating METTL3-related m~6A methylation.(1)AET can reduce the level of myocardial oxidative stress,and swim training down-regulates MDA expression and increases SOD activity in myocardial tissue after I/R in C57BL/6 mice.Me RIP results suggest that swim training and MIRI increase the m~6A RNA methylation modification level of Nrf2 in the myocardial tissues of C57BL/6mice.Western blot and q RT-PCR results showed the expression of myocardial cell nucleus Nrf2 and downstream antioxidant molecules HO-1,NQO1 and GCLM were significantly increased in mice attacked by MIRI after swim training.(2)Immunofluorescence staining results confirmed that the nuclear expression of Nrf2 increased significantly after H/R in SW vitro cardiomyocytes.(3)Knockdown of METTL3 expression in SE vitro cardiomyocytes increases the nuclear transfer of Nrf2 protein,increases the expression of downstream antioxidant factor HO-1,and enhances the antioxidant capacity of cardiomyocytes.(4)Nrf2 si RNA transfection led to a decrease in the expression of Nu-Nrf2 protein and downstream antioxidant enzyme HO-1 in swimming-trained cardiomyocytes and a significant increase in cardiomyocyte apoptosis.Conclusion:1.AET alleviates MIRI in C57BL/6 mice by improving myocardial antioxidant capacity.2.The m~6A RNA methylation level of cardiomyocytes injured by I/R in C57BL/6mice increased,accompanied by a pronounced oxidative stress response and cardiomyocytes apoptosis.m6A sequencing and signaling pathway enrichment analysis showed that cardiomyocytes METTL3-mediated m~6A RNA methylation level is closely related to the Nrf2/HO-1 oxidative stress pathway in cardiomyocytes.3.AET reduces the m~6A RNA methylation level of cardiomyocytes by inhibiting METTL3 expression and mediates myocardial resistance to I/R injury.4.AET reduces oxidative stress and apoptosis of cardiomyocytes after I/R by reducing METTL3-related m~6A RNA methylation levels in cardiomyocytes and activating the Nrf2/HO-1 antioxidant signaling pathway.