Exercise-induced Myocardial Hypertrophic Preconditioning Attenuates Myocardial Ischemia/Reperfusion Injury Mediated By Upregulation Of LncRNA Mhrt779

Background and Objective:We have first reported the myocardial hypertrophic preconditioning and exercise hypertrophic preconditioning(EHP).The latter one refers to the preservation of molecular memory against pathological myocardial hypertrophy even after the regression of exercise induced physiological myocardial hypertrophy subsides.The life span of former athletes was increased about 5 years longer,and they have a lower risk of suffering from cardiovascular disease,suggesting that the cardioprotection mediated by exercise still exist even after exercise termination for long time.Although exercise has been recommended as an important approach for the prevention and treatment of cardiovascular diseases,it is unclear whether EHP also exerts a protective effect on myocardial ischemia reperfusion injury.Based on the above evidence and our bioinformatics clues,we hypothesized that the EHP-induced molecular memory of LncRNA Mhrt779 is capable of resisting myocardial ischemia reperfusion injury.Methods1.The protocol of swimmingC57BL/6 male mice,8 weeks old,weighing about 25g,were purchased from Southern Medical University Animal Center.The mice were randomly divided into exercise or sedentary group.Mice were swimming at 15 minutes at the beginning for adaption.And then the exercise time was increased 15 minutes every two days until 90 minutes and maintain this intensity.The mice were trained twice a day in 5 days each week for total 21 days.The interval should be 7 hours at least between each exercise session.The mice should be monitored during swimming for avoiding choking or hypoxia.The sedentary group were moved freely in cages.One week after exercise training,the mice were underwent ischemia reperfusion(I/R)or sham surgery.2.Preparation of myocardial I/R modelMice were anesthetized by intraperitoneal injection of 0.5%isopentobarbital.After the mice respiration support by a ventilator,opened the chest and exposed the heart.Used 8-0 nylon suture to ligate the left anterior descending branch.The marked elevation of the ST segment and the whitening of the myocardium below the ligation were the signs of success.After 45 minutes of myocardial ischemia,loosen the ligature suture.Closed the chest cavity,and then placed the mice in a cage for waking up naturally.The sham operation group performed same operation without ligate the vessel.24 hours after the operation,the infarct area of the mice was detected,and the echocardiogram was performed seven days after the operation.3.Measurement of myocardial infarction areaAnesthetized the mice(as described above),connected a ventilator to support breathing.Cut and fixed the mice thorax with hemostats.Located the position during I/R surgery and ligated the same place with 8-0 nylon thread.Separated the thymus and then clamped the aorta a vascular clamp.0.5%Evans blue dye was injected into the aorta.After 5 to 10 seconds,removed the heart and freeze it.Cut it into 5 pieces below the ligature line and put them into 1%TTC dye solution for 30 minutes.Fixed the slices with 4%paraformaldehyde and photographed.Image J was used to detect the infarct area(IA),left ventricular area(LV),and area at risk(AAR)of the heart.4.Echocardiography assessment the cardiac function after surgeryVevo2100 system was used to evaluate the cardiac function.Adjust the concentration of isoflurane to maintain heart rate at 450-550 beats/min.Obtain the clear M-mode image at the level of the short axis papillary muscle of the left ventricle(LV),and measured the left ventricular end-diastolic and end-systolic diameters(LVEDd,LVESd).Calculated the left ventricle Ejection fraction and fractional shortening(LVEF,LVFS).Putted the mice into the cages for natural awake.5.Isolation,culturation and adenovirus transfection in cardiomyocytesThe primary cardiomyocytes and fibroblasts were separated from neonatal mice.After cultured 36 hours,added Sh-Mhrt779(Ad-Hu6-MCS-CMV-EGFP-shRNAMhrt779)or corresponding empty virus(Scramble)for 24 hours.Then changed the medium to serum-free and low sugar DMEM.Then put the peri dish into hypoxic or normoxic environment for 12 hours.After 24 hours of reoxygenation,Tunel staining was used to detect apoptosis of cardiomyocytes.6.Statistical analysisThe measurement data in this article were expressed as mean±standard error(mean±SEM).statistically significant difference analysis uses two independent sample t-test or one-way or two-way ANOVA,and multiple comparisons are corrected by Bonferroni’S.In all the statistical analyses in this study,P<0.05(two tailed)represents the test standard.Results1.3 weeks of swimming can induce physiological myocardial hypertrophy,and it subsides after 1 week of restMice in 3 weeks of swimming(exercise),1 week after exercise(exercise preconditioning)and sedentary group were sacrificed by cervical vertebrae.The heart weight to body weight ratio(HW/BW mg/g)and heart weight to tibia length ratio(HW/TL mg/mm)in exercise group were increased about 7.6%than sedentary group(P<0.05).1 week after the termination of exercise,there was no significant difference in HW/BW and HW/TL between exercise preconditioning group and sedentary group.2.EHP improves cardiac function dysfunction caused by ischemia reperfusion(I/R)surgeryThere was no significant difference in AAR between the exercise preconditioning and sedentary group.While exercise preconditioning group was significantly smaller than sedentary group in infarcted area,about 10%(P<0.05).The levels of Bax、Caspse3 and TUNEL positive cells of cardiomyocytes in exercise preconditioning group was significantly lower than sedentary group.7 days after surgery,we used echocardiography to evaluate the cardiac function in mice.The LVEF in exercise preconditioning group was higher than the sedentary group,about 11%(P<0.05),while the LVESd was 5%smaller than sedentary group(P<0.05).there was no significant difference in LVEDd between the above groups.3.EHP up regulated the expression of Mhrt779We performed transcriptomic sequencing analysis on mouse hearts of exercise preconditioning and sedentary group.By analysis the sequence results,we found that Mhrt779 in exercise preconditioning group was significantly up regulated twice than the sedentary group(P<0.05).We further verify the consistent results in sequence data by using qPCR.4.Knockdown of Mhrt779 increases ischemia-reperfusion injury.Knockdown Mhrt779 in myocardium can significantly reduce the expression level of Mhrt779.Five days after adenovirus injection,I/R or sham operation was performed.24 hours after surgery,Evans blue/TTC staining showed that knocking down Mhrt779 would increase the area of myocardial infarction.Western blot experiments showed that knocking down Mhrt779 increased the content of caspase3 caused by I/R and decreased the ratio of Bcl2/Bax.Seven days after surgery,echocardiography showed that knocking down Mhrt779 would further reduce the decrease in LVEF caused by I/R,and increase LVEDd and LVESd.5.Knockdown of Mhrt779 can promote apoptosis induced by hypoxia and reoxygenationWe cultured neonatal mouse cardiomyocytes transfected with Sh-Mhrt779.The TUNEL positive cardiomyocytes in hypoxia reoxygenation group was higher than normoxic group,about 5%(P<0.01).Knockdown of Mhrt779 further increased the TUNEL positive cells about 10%which induced by hypoxia reoxygenation(P<0.01).6.Exercise preconditioning up regulates methylation at the promoter of Mhrt779After termination exercise,the histone 3 lysine 4 trimethylation(H3K4me3)in exercise group was significantly higher than sedentary group,about 20%(P<0.05),while the methylation level of H3K4me3 further increased in exercise preconditioning group,about 60%(P<0.05).The level of methylation histone 3 lysine 36 trimethylation(H3K36me3)in exercise group were increased by 64%(P<0.05)than sedentary group,while the methylation level of H3K36me3 in exercise preconditioning group were further increased twice than sedentary group(P<0.05).ConclusionsExercise-induced myocardial hypertrophic preconditioning attenuates myocardial ischemia/reperfusion injury mediated by increasing the histone methylation of LncRNA Mhrt779 promoter.