MiR-214-5p Regulates Heat Shock Transcription Factor1in Pathological Cardiac Hypertrophy Attenuated By Exercise Preconditioning

BackgroundIn the past few decades, heart failure is the fastest growing sub-category of theincidence of cardiovascular diseases. Currently cardiovascular disease has become ahazard to the health of our people, the number of deaths due to cardio-cerebralvascular diseases was rapidly rising, and more and more tend to be younger.Pathological cardiac hypertrophy is accompanied by hypertension, aortic stenosis,myocardial ischemia and valve defects, increased myocardial cell volume, theformation of new sarcomere proteins, increased cardiac decompensation in the earlystages and increased cardiac fibrosis, myocardial necrosis or apoptosis in the latestages, all of which result in decompensated cardiac function and lead to heartfailure. Recent studies have recognized that pathological cardiac hypertrophy is thepreliminary lesion in heart failure in addition to the independent risk factors forheart failure, including stroke, coronary heart disease and sudden death.It is the mostcommon to make a model of pathological cardiac hypertrophy by pressure over-loadand in which transverse aortic constriction (TAC) is the most widely used.Exercise training attenuates cardiovascular circulation and decreasescardiovascular disease risk factors including coronary blood flow,endothelium-dependent vasodilation and cardiac oxygen and substrate supply.Furthermore, exercise training increases sensitivity to adenosine vascular resistance.Due to these effects, exercise training can reduce the harmful burden of myocardialneuroendocrine proinflammatory cytokines associated with heart failure, reducingmyocardial oxidative stress and thereby improving energy metabolism. Exercisepreconditioning (EP), formally proposed by Yamashita in1999, can enhancetolerance of cardiac ischemia-reperfusion injury (IRI) through endogenousprotection generated by exercise training and can also improve heart function.The microRNAs refer to a subfamily of small non-coding RNA species that aredesigned to influence gene expression in nearly all cell types. Gain-andloss-of-function studies using in vitro and in vivo models have revealed distinct rolesfor specific microRNAs in cardiovascular development, physiological functions, andcardiac pathological conditions. The microRNAs not only target single genes butoften functionally related gene networks, yielding complex gene regulatory networks.The recognition of microRNAs as essential regulators of cardiac morphology and function has fundamentally changed our view of the pathological aspects of thecardiovascular system.Next-generation sequencing technology is a new, unique research platform,which can be applied to the quantitative analysis of small RNA and depth of eachidentified. High-throughput sequencing of small RNA technology can sequence andexpress quantification to all the small RNA family in samples,and can resolve tinyRNA, small interfering RNA, Piwi-interacting RNA(piRNA) and other non-codingRNA and the sequence of corresponding target gene. The technology collect samplesof18-30nt RNA fragments using plastic separation technology, to access to millionsof single-base resolution of RNA sequence information in small-time. the use ofbiological information analysis platform can identify known small RNA, and predictnew small RNA and its target genes.The heat shock transcription factor1has been identified as an endogenouscardioprotective factor, which can regulate the nuclear transcription factor κB (NF-κB)subunit: RelA/P65to achieve the level of cardiomyocyte apoptosis regulation anddecrease its translocation from the cytoplasm to the nucleus to play a protective rolein inflammation. These facts indicated that HSF1may negatively regulate RelAthrough up-regulating HSP70and HSP90. NFκB was well known to act, in general,worsening cardiac remodeling or dysfunction mainly through activation of aproinflammatory pathway enhancing Heart Failure.Part Iexercise preconditioning attenuates pathological cardiachypertrophy induced by pressure-overload in ratsObjective: Exercise preconditioning(EP)， which has been universallyacknowledged to enhance tolerance of cardiac ischemia-reperfusion injury，is aphysiologic phenomenon of tissue protection observed in many organs, including theheart, in which brief periods of repetitive ischemia can protect cells from thecorresponding area during a subsequent ischemic insult.In this research we willinvestigate the effect of exercise preconditioning to pathological cardiac hypertrophyinduced by pressure-overload and heart failure,and to explore new animal models todelay and treat heart failure． Methods: sixty6-wks wild-type male Sprague-Dawley rats were randomlydivided into four groups(20rats in each group)： Sham-operation group(Shamgroup)， transverse aortic constriction control group(TAC group) and exercisepreconditioning+transverse aortic constriction group(EP+TAC group)．At4wks and8wks after TAC， the heart structure and function were evaluated byechocardiography and the molecular heart failure markers were measured by westernblotting and quantitative RT-PCR analysis.Results: On the basis of comparative analysis of echocardiographic andpathological results, compared with the Sham group, the indicators measured in twosurgical groups were significantly changed at the two point-in-time.Furthermore, theindicators of EP+TAC group was better than TAC group. Compared with sham group,the protein and mRNA expression of two heart failure markers: A-type natriureticpeptide (ANP) and B-type natriuretic peptide (BNP) in the left ventricle of the TACgroup was significantly increased. Though the levels of the EP+TAC group were notchanged at4wks after TAC, but were increased significantly at8wks after TAC.Moreover, compared with the TAC group, the mRNA expression of the two indicatorswere decreased47%and62%at4wks after TAC and were decreased44%and28.1%at8wks after TAC; the protein expression were decreased22.3%and48%at4wksafter TAC and were decreased21.5%and38.3%at8wks after TAC,and thedifference of expression at4wks after TACmore significant than8wks after TAC.Conclusion: Exercise preconditioning can attenuate cardiac pathologicalhypertrophy induced by pressure-overload delay the process of heart failure. Base onthis study,we can further explore the mechanism of EP protectiion againstpathological cardiac hypertrophy. Part IIExpression and mechanisms of heat shock transcriptionfactor1in attenuatement of EP to pathological cardiachypertrophy induced by pressure-overloadObjective: To investigate the Expression and mechanisms of heat shock transcription factor1in attenuatement of EP to pathological cardiac hypertrophyinduced by pressure-overload.Methods: myocardial factor related signaling pathway were detected in eachgroup of samples stored in liquid nitrogen.HSF1, NF-κB and HSP70mRNAexpression were detected by fluorescence quantitative reverse transcriptionpolymerase chain reaction (RT-PCR).detection of HSF1, NF-κB p65and HSP70,was used SDS-PAGE gel Western blotting.Results: According to mRNA and protein of HSF1and HSP70at4weeks and8weeks after TAC, the expression of EP+TAC group on two time points was higherthan the TAC control group (P <0.05), the difference statistically significant, but thedifferences between the two groups decreased over time. However,The expressionof NF-κB p65in cytoplasm and nucleus were just the opposite, the expression of EP+TAC group was lower than the TAC group.Conclusion: During the process of EP attenuated pathological cardiachypertrophy,the expression of HSF1and HSP70were increased, and expression ofNF-κB p65in cytoplasm and nucleus were decreased,furthermore, EP promoted theexpression of HSF1and HSP70, thereby inhibited NF-κB p65expression andactivation, to attenuate pathological cardiac hypertrophy. Part IIImicroRNA high-throughput sequencing in left ventricularObjective: screen microRNA related EP using Illumina HiSeqTM2000high-throughput sequencing technology.Methods: Nine samples were sequenced through standard processes usingIllumina HiSeqTM2000high-throughput sequencing technology., According to theamount of differential expression, microRNAs associated with EP were identified ineach group.Results: The sequencing was successfully completed, and microRNAs associatedwith EP were found.in which some microRNAs have negative regulation,such as:rno-miR-741-3p, rno-miR-155-3p, rno-miR-3593-3p, rno-miR-196c-3p,rno-miR-873-5p, rno-miR-879-3p, rno-miR-214-5p, rno-miR-1188-3p, rno-miR-471-5p, rno-miR-489-3p, rno-miR-212-5p, rno-miR-23a-5p,rno-miR-331-5p, rno-miR-218a-2-3p, rno-miR-3075. The other microRNAs havepositive regulation,such as: rno-miR-299a-5p, rno-miR-3586-5p, rno-miR-3547,rno-miR-328a-5p, rno-miR-215, rno-miR-34a-3p, rno-miR-293-5p, rno-miR-1224,rno-miR-292-3p, rno-miR-592, rno-miR-509-5p, rno-miR-6331, rno-miR-211-5p.Conclusion: MicroRNAs associated with EP were found and provided a basis forfurther research. Part IVmiR-214-5p targeted regulation of heat shock transcriptionfactor1in vitro validationObjective:To confirm that miR-214-5p targeted regulation of heat shocktranscription factor1and determine a target of miR-214-5p.Methods: Neonatal rat cardiomyocytes cultured in vitro went to six-well plates.After cell fusion area of30-50%, miR-214-5p mimics (M-rno-miR-214-5p), miR-214-5p inhibitors (I-rno-miR-214-5p) and miR-control (rno-miR-NC) weretranfected in cardiomyocytes. miR-214-5p potential target genes was found atOnline database query. HSF1gene expression was detected by quantitativeRT-PCR. HSF1protein level was further validated by Western blot. Finally, HSF1is confirmed miR-214-5p target gene by dual-luciferase reporter.Results: In this study, we demonstrate that miR-214-5p negatively regulatesHSF1. Compared with the other groups, heat shock transcription factor1mRNA andprotein level in I-miR-214-5p group were significantly improved (p <0.01). Andcomparison with blank and negative control groups， expression level of HSF1inthe miR-214-5p over-expression was not statistically significant. Dual-luciferasereporter assay confirmed that HSF1is direct targets of miR-214-5p.Conclusions: We report a completely novel role of miRNA-214-5p as a regulatorof heart failure by its direct targeting of HSF1after EP. Targeting of miRNA-214-5pcould serve as a novel therapeutic strategy to delay progressive heart failure duringEP attenuates pathological cardiac hypertrophy.