Antihypertrophic Memory After Regression Of Exercise-induced Physiological Myocardial Hypertrophy Mediated By LncRNA Mhrt779

Background and ObjectiveHeart failure(HF)is the end stage of progression of various cardiovascular diseases,and imposes an enormous clinical and economic burden worldwide.In spite of the current pharmacological therapies,including inhibition of the renin-angiotensin-aldosterone system and sympathetic nervous system,and angiotensin receptor-neprilysin inhibitors,HF has high morbidity and mortality.New therapeutic strategies have become an impelling priority.Exercise is a well-known nonpharmacological intervention capable of improving cardiovascular fitness,and exercise training has been recommended as an important component of therapy for HF patients.However,not all patients are adequate for exercise therapy.Clarifying the underlying molecular mechanisms responsible for the benefit of exercise would provide new therapeutic targets for HF.Pathological cardiac hypertrophy is a major independent risk factor for the development of HF;in contrast,exercise-induced physiological cardiac hypertrophy is beneficial.Former athletes had significantly lower systolic blood pressure than their age-matched controls in later life,and the former elite athletes survived 5-6 years longer than controls,implying that an elite athlete career during young adulthood brings cardioprotection in later life even the physiological hypertrophy has regressed.We previously reported a phenomenon termed hypertrophic myocardial preconditioning,in which short-term imposition of pathological hypertrophic stress on the heart has a protective effect against subsequent hypertrophic stress and slows progression to HF.However,after the regression of physiological cardiac hypertrophy,such as that induced by physical exercise,its effect on the resistance to subsequent pathological hypertrophic stress was hardly studied.Therefore,we hypothesized that an exercise-induced antihypertrophic memory exists even after regression of physiological hypertrophy due to exercise termination.In this study,we used swimming-trained mice to test this hypothesis and confirmed an underlying mechanism related to a heart-enriched long noncoding(Inc)RNA termed myosin heavy chain associated RNA transcript(Mhrt).Methods1.Endurance exercise modelC57BL/6 male mice(8-10 weeks old and weighing 22-25 g)were provided by the Animal Center of Southern Medical University.Swimming exercise started in a ramp-up protocol starting at two sessions of 15 min,with a 15-min increase each 2-day until two sessions of 90 min were reached(exercise with 5 days/week,2 days rest).Ninety minutes daily of swimming was continued for the next 7 days.The protocol was ended after totally 21 days.The two swimming sessions were separated from each other by at least 6 hours.The mice were closely observed at all times to avoid relative hypoxia.Mice in the sedentary group were free to move in the cage.1 week after the end of exercise training,all mice underwent transverse aortic constriction(TAC)or sham surgery.2.Thoracic aortic constrictionMale C57/BL6 mice were anaesthetized by a mixture of ketamine(100 mg/kg,IP)and xylazine(5 mg/kg,IP).The mice were then ventilated and the chest cavity was opened to expose the aortic arch.A 7-0 silk suture were put between the innominate and left carotid arteries.A 26-gauge needle was put around the suture and the suture was tied.The needle was then removed and a stenosis of the aorta was formed.The chest of mice was then closed and the mice were put into cages for recovery.The sham-operated mice were underwent the same procedure leaving the suture untied.Four weeks after the surgery,mice were subjected to echocardiographic measurements and hypertrophic analysis.3.Echocardiography and Measurement of Cardiac HemodynamicsAt 4 weeks after surgery,echocardiography was performed with a Vevo 2100 system(Fujifilm Visual Sonics,Ontario,Canada).The inhalational flow of isoflurane was adjusted to anaesthetize the mice while maintaining their heart rate at 450-550 beats/min.M-mode images were recorded in the short axis view of the LV at the papillary muscle level,and were used to measure the LV end-diastolic and systolic diameter(LVEDd,LVESd),LV diastolic and systolic posterior wall thickness(LVPWd,LVPWs),LV ejection fraction and fractional shortening(LVEF,LVFS).LV hemodynamics was evaluated before the animals were sacrificed.Mice were anesthetized,intubated,and ventilated as described above.A 1.0 F catheter(Millar Instruments,Inc.,Houston,TX)was inserted into the right carotid artery and advanced to the LV.Data on the LV systolic and end-diastolic pressure(LVSP,LVEDP),and the maximum and minimum rates of change of the LV pressure(max dp/dt and min dp/dt,respectively)were digitized and recorded.LV contractility and the exponential time constant of LV relaxation(?)were calculated using Power Lab software(Blood pressure module;AD Instruments,Australia).4.Overexpression or Silencing lncRNA Mhrt779 in vivoRecombinants with adeno-associated virus 9(AAV9)or adenovirus(Ad)expressing or silencing mouse Mhrt779 were constructed and produced by Shanghai Genechem Biotechnologies Co.For in vivo infection,pAAV9-CMV-ZsGreen-Mhrt779(AAV-Mhrt779),pAAV9-CMV-ZsGreen-shMhrt779(AAV-shMhrt779)or control(Scramble)virus particles(1×1011 viral genomes/ml)were administered by direct injection in the LV free wall,apex and posterior wall(three sites,10 ?l/site)in mice at age of 8 weeks using a syringe with a 30-gauge needle.Four weeks later,mice were subjected to endurance exercise training,then sham or TAC surgery was performed.5.Histological ExaminationEach mouse was weighed at 4 weeks after TAC.Following euthanasia,the heart,lungs and a tibia were harvested for analysis.After the heart was fixed,dehydrated,embedded in paraffin,and sectioned,hematoxylin and eosin staining(HE),wheat germ agglutinin staining(WGA),and Masson's trichrome staining(masson)were performed for myocardial hypertrophy and myocardial fibrosis.6.Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry(ChIRP-MS)The heart was reversibly cross-linked with formaldehyde and then hybridized with biotin-labeled RNA anti-sense.After washing off the non-specific binding protein under strong denaturing conditions,the resulting RNA-binding protein(RBP)was identified and quantified by LC-MS/MS.The proteins that specifically interact with the target RNA were significantly different between the two groups.7.Isolation and culture of cardiomyocytes,and adenovirus transfectionIsolation and culture of primary neonatal rat or mouse cardiomyocytes and fibroblasts were carried out.After culture for 48 hours,cardiomyocytes were co-cultured with ad-Mhrt779(Ad-hU6-MCS-CMV-EGFP-Mhrt779)or ad-shMhrt779(Ad-hU6-MCS-CMV-EGFP-shRNA-Mhrt779)for 24 hours,and then the culture medium was replaced with serum-free DMEM/F12 for 12 h before stimulation with angiotensin ?(Ang ?)(1?M).Cells were cultured in serum-free DMEM/F12 for 24 hours under different treatment to test the effect of silencing(Ad-shMhrt)or overexpressing(Ad-Mhrt)Mhrt779 on cardiomyocyte hypertrophy.Finally,the cultured cardiomyocytes were harvested for analysis of cell surface area,western blotting and PCR.8.Statistical AnalysisQuantitative data are reported as the mean ±standard error of the mean.Statistical analysis for comparison between two groups was performed using two-tailed unpaired t-test.Comparisons among multiple groups were performed using either one-way or two-way(if there are two factor levels)variation analysis followed by Bonferroni's correction for post hoc multiple comparisons.and P<0.05 was considered statistical significance.Results1.Exercise Preconditioning Attenuates Pathological Myocardial HypertrophyAfter swimming for 21 days,an about 10%increase of heart weight/body weight(HW/BW),HW to tibia length(HW/TL)and cardiomyocyte cross-sectional area was noted,and no changes were found on myocardial fibrosis and hypertrophic markers of Nppa and Myh7 genes.At 7 days after TAC,the swimming mice(exercise hypertrophic preconditioning group,EHP)showed less increase in LV wall thickness(LVPWd and LVPWs)than sedentary group.Compared with sedentary group,preconditioning treatment significantly reduced the increase of HW/BW and HW/TL ratios,myocardial cell cross-sectional area,myocardial fibrosis and expression of embryonic genes Nppa and Myh7 in TAC mice.2.Exercise Preconditioning Delays HF ProgressionWe extended the post-TAC observation period to 4 weeks to test the effect of EHP on HF.Four weeks after TAC,compared with the Sed+TAC group,echocardiography showed the LVPWd,LVPWs,LVEDd,and LVESd were smaller in the EHP+TAC group,while LVEF and LVFS were higher.In contrast to sedentary TAC group,EHP TAC mice had significantly larger LV dp/dt max,LV dp/dt min,and LV contractility,as well as significantly smaller LVEDP and ?.Four weeks after TAC,EHP can significantly reduce the increase of indicators of myocardial hypertrophy(HW/BW and HW/TL)and indicators of TAC-induced congestive heart failure(LW/BW and LW/TL).Masson staining confirmed that myocardial fibrosis was lighter in the EHP+TAC group than in the Sed+TAC group.The above results indicate that EHP can inhibit pathological myocardial hypertrophy and delay the progression of HF.3.EHP up-regulates expression of lncRNAMh779 in myocardiumWe screened by qPCR and found that lncRNA mhrt779(Mhrt779),which is rich in the nucleus of cardiomyocytes,was significantly upregulated in cardiomyocytes of EHP mice.After pressure overload,Mhrt779 in the EHP+TAC group was significantly higher than that in the Sed+TAC group at 1 week and 4 weeks of TAC.4.Overexpression or knockdown of Mhrt779 can enhance or weaken the anti-hypertrophic effect of exercise hypertrophic preconditioningMyocardial injection of AAV-Mhrt779 or AAV-shMhrt779 significantly increased or decreased myocardial expression level of Mhrt779 at 4weeks later.All mice were subjected to EHP after AAV injection 4 weeks,and sham operation or TAC was performed later.Echocardiography showed that the overexpression of Mhrt779 significantly reduced the increase in LVPWd and LVPWs caused by TAC at 4 weeks Compared with the Scramble+TAC group,HW/BW,HW/TL,myocardial cell cross-sectional area,and myocardial fibrosis were significantly reduced in the Mhrt779+TAC group.When Mhrt779 was knocked down in the myocardium,compared with the Scramble+TAC group,LVPWd,LVPWs,HW/BW,HW/TL,myocardial cell cross-sectional area,and myocardial fibrosis increased significantly in the Sh-Mhrt779+TAC group.5.Mhrt779 binds to Brg1/Hdac2 complex and inhibits Hdac2/Akt/GSK3? signaling pathwayWe confirmed that Brgl is a specific binding protein of mhrt779 by CHIRP-MS,and found that Brgl and Hdac2 bind to each other to form a complex by co-immunoprecipitation in the hearts of 4 week TAC mice.The results of Western blotting confirmed that overexpression of Mhrt779 inhibited the activation of Hdac2/Akt/GSK3? signaling pathway under stress,while knocking down Mhrt779 enhanced the activation of Hdac2/Akt/GSK3? signaling pathway under stress.6.Mhrt779 Attenuates Ang ?-induced Cardiomyocyte HypertrophyMhrt779 overexpression significantly suppressed myocyte hypertrophy and hypertrophic markers(Nappa,Myh7)caused by Ang II in NRCMs.Western blot results show that Ang ?-activated Hdac2/Akt/GSK3p pathway was blocked by Mhrt779 overexpression.In Ang ?-stimulated NMCMs,treatment with Ad-shMhrt779 significantly increased myocyte cross section area,gene expression of Nappa and Myh7,protein expression of Hdac2,p-Akt and p-GSK3?.Conclusions1.Exercise can induce physiological myocardial hypertrophy.After the myocardial hypertrophy subsides,it still has the ability to resist pathological myocardial hypertrophy,thereby delaying the progress of heart failure,that is,the phenomenon of "pre-adaptation to exercise hypertrophy".2.LncRNA mhrt779 is significantly up-regulated in pre-adapted cardiomyocytes of exercise hypertrophy,and has the effect of resisting pathological myocardial hypertrophy.3.LncRNA mhrt779 inhibits the activation of Hdac2/Akt/GSK3? signaling pathway caused by pathological stress by binding to Brgl/Hdac2 complex.