Genetic (gene Mutation) And Epigenetic (microRNA) Regulation Of Myocardial Remodeling

Objective:Cardiovascular diseases,such as coronary heart disease,stroke,peripheral vascular disease,hypertension and congenital heart disease,pose an important threat to human health and is also the leading cause of mortality in China.With the development of social economy,changes have taken place in our lifestyles,especially with the aging of the population and the acceleration of the urbanization process,the prevalence of cardiovascular risk factors in China is constantly rising.As a result,a continuous increase exists in the number of individuals with cardiovascular disease.Although visible efforts have been made in the study of cardiovascular development and pathogenesis,and great progress have been achieved in the understanding of clinical cell and molecular processes,there is still no significant improvement in prevention,early diagnosis or treatment of cardiovascular disease.miRNAs are a small non-coding RNA with a length of 22 nucleotides,covering different biological processes,including developmental time,differentiation,proliferation,cell death and metabolism.In recent years,many studies have proved that small non-coding RNAs(miRNAs)are involved in a variety of physiological and pathophysiological processes(including the physiological processes of the cardiovascular system).miRNAs researches have added a new dimension to a variety of disease treatments and interventions.The level of miR-451 in the left ventricle is significantly lower in patients with hypertrophic cardiomyopathy compared with healthy controls.Our previous studies have shown that overexpression of miR-451 in neonatal rat cardiomyocytes decreases surface area of cell,and inhibition of miR-451 expression results in a significant increase surface area of cell,through a possible target TSC1.However,whether miR-451 regulates cardiac remodeling in vivo,and the mechanism by which miR-451 regulates cardiac remodeling remains unclear.Therefore,we constructed miR-451 knockout rats to explore the role and mechanism of miR-451 in cardiac remodeling,expected to provide experimental evidence for elucidating the mechanism of heart failure and providing new therapeutic targets.Methods and Results:To investigate whether miR-451 was involved in the regulation of cardiac function and the involvement of cardiac remodeling and remodeling in the in vivo condition,we successfully constructed the miR-451 knockout rat model using CRISPR/Cas9 technique.There was no significant difference in the ratio of heart weight to body weight,left ventricular weight to body weight of 4-month-old miR-451 knockout rats compared with wild-type rats.The results of real-time quantitative PCR showed that there were no significant increase in the level of ANP and BNP.Masson trichrome staining showed that there was no significant increase in the area of cardiac fibrosis in miR-451 knockout rats.TUNEL staining showed that there was no signi:licant difference in the percentage of cardiomyocyte apoptosis between miR-451 knockout rats and wild-type rats.M-mode echocardiography showed that there was no significant change in left ventricular internal diameter at the end of diastole(LVIDd),left ventricular internal diameter at the end of systole(LVIDs),the left ventricular posterior wall thickness at the end of diastole(LVPWd),the left ventricular posterior wall thickness at the end of systole(LVPWs),the left ventricular anterior wall thickness at the end of diastole(LVAWd),the left ventricular anterior wall thickness at the end of systole(LVAWs),Left ventricular ejection fraction(EF%),left ventricular short axis shortening(FS%)between miR-451 knockout rats and wild-type rats.In summary,knockout of miR-451 did not cause a significant change in the morphology,function and function of the rats.In order to explore the role of miR-451 in myocardial tissue further,we established an adriamycin-induced heart failure rat model to investigate whether miR-451 knockout rats were able to attenuate adriamycin-induced heart failure.Before intraperitoneal injection of doxorubicin,there was no significant difference in body weight between miR-451 knockout rats and their wild-type littermates.After the injection,body weight were significantly decreased in both groups,but there was no significant difference in body weight between the two groups.There was no significant difference in ratio of heart weight to body weight,heart weight and tibial length,ratio of lung wet weight to lung dry weight between the miR-451 knockout rats group and the wild-type group.However,compared with the wild-type group,the ratio of myocardial fibrosis was significantly lower and the survival rate was significantly higher in the knockout group.Cardiac function shown by M-mode echocardiography in the miR-451(-/-)group was significantly better than that in the miR-451(+/+)group.LVIDs and left ventricular end systolic volum(LV vol s)in the miR-451(-/-)group were significantly lower than that in the miR-451(+/+)group,while LVPWs,EF and FS were significantly higher than those of miR-451(+/+)rats.The results suggest that knockout miR-451 can attenuate doxorubicin-induced heart failure.Conclusions:In summary,knockout of miR-451 did not affect the cardiac structure and function,but was able to attenuate adriamycin=induced heart failure in rats.Objective:Heart failure(HF)is caused by a variety of heart structure and/or functional diseases lead to ventricular filling and/or ejection function damage.Its 5-year mortality rate was 50%.The process of heart failure is often accompanied by myocardial remodeling.MircroRNAs(miRNAs)are a class of RNA molecules with a length of about 21?25 nt.They are widely found in various tissues of various species,promoting mRNA degradation or inhibits mRNA translation by 3'UTR binding,and is involved in regulating various biological processes.More and more experiments showed that autophagy was involved in the process of myocardial remodeling..Our previous work showed that miR-221 inhibits myocardial autophagy by modulating p27/CDK2/mTOR pathway,promotes myocardial remodeling and heart failure.Our results showed that at 4 weeks of age,Tg-miR-222 group significantly increased cardiac volume,ratios of heart weight to body weight increased significantly.Left ventricular wall was significantly thickened in the Tg-miR-222 mice at 4 and 16 weeks of age,and the left ventricular end diastolic diameter increased significantly in the left ventricle Short-axis shortening rate was significantly reduced.Real-time quantitative PCR results showed that expression of ANP and BNP were significantly increased in Tg-miR-222 mice.Masson trichrome staining showed a significant increase in myocardial fibrosis in the Tg-miR-222 group.TUNEL staining showed that the number of cardiomyocyte apoptosis in Tg-miR-222 mice was significantly increased.Previously,we found that overexpression of miR-221 induces heart failure in mice.The miR-222 and miR-221 share the same gene cluster,however,the role of miR-222 in the regulation of cardiac function remained ill-defned.Methods and Results:Western blot was used to evaluate the expression level of mTOR,p27,p-S6,p-mTOR,LC3-?,LC-?,p62.Western blot results showed that the expression level of p27 in the myocardium of Tg-miR-222 mice was significantly decreased.The expression of p27 in the myocardium of neonatal rats was miR-222 analogues,the same occurrence of p27 expression was significantly down-regulated.Previous studies have shown that autophagy may play an important regulatory role in myocardial remodeling.The results of Western blot analysis showed that in the 4-month-old Tg-miR-222 mouse myocardium,autophagic marker protein LC3 lipidated LC3-? level was significantly down-regulated;p62(with the autophagic marker,its expression is just opposite to the autophagy)expression was significantly up-regulated.We found that the mTOR pathway was activated,p-mTOR substrate p-S6 also significantly increased,in 4-week-old Tg-miR-222 mouse myocardium.Conclusions:The downregulation of p27 and the activation of mTOR pathway may be involved in miR-222-induced heart failure and autophagy inhibition.Thus,targeting miR-222 expression may be a therapeutic strategy against pathological cardiac remodeling.Object:It has been reported that mutations in MYH6 gene,which encodes human a-myosin heavy chain(a-MHC),may be associated with the pathogenesis of hypertrophic cardiomyopathy and dilated cardiomyopathy.While there is still no animal model wtih mutations in specific sites of MYH6.In order to study whether the mutations in human ?-myosin heavy chain were pathogenic,we used the CRISPR/Cas9 technique to generate ?-MHC R453C knock-in rat to confirm the pathogenicity of the mutation.Methods and Result:p-MHC R453C heterozygous type(?.MHCR453C/+)male rats and ?-MHC R453C heterozygous type(?-MHCR453C/+)female rats from different nest were mixed and bred.These newborn rats were separated by different sex and were labeled,and their genomic DNA was extracted.Primers were designed and mapped downstream of the mutant sites,and different genotypes were identified by sequencing.We recorded 21 neonatal nests of a total 191 neonatal rats.There were 62?-MHC wild type(WT)and 129 ?-MHC R453C/+newborn rats,while p-MHC R453C Homozygous type(?-MHCR453C/R453C)rat was not detected,suggesting that rats with homozygous ?-MHC R453C mutation were not viable.Next,we compared the echocardiography findings between the 4-month-old?-MHC R453C/+rats and their WT littermates.Compared with WT rats,left ventricular end diastolic diameter(LVIDs),left ventricular end-systolic volume were significantly dicreased(P<0.05),and the shortening of left ventricular short axis(FS%),ejection fraction(EF%)were significantly increased(P<0.05)in ?-MHC R453C/+rats Therefore,we hypothesized that ?-MHC R453C mutations might result in increased myocardial contractility in rats.However,there was no significant difference in left ventricular end diastolic diameter(LVIDd),Ieft ventricular end diastolic posterior wall thickness(LVPWd),left ventricular end systolic posterior wall thickness(LVPWs),left ventricular end diastolic wall thickness(LVAWd),left ventricular end systolic anterior wall thickness(LVAWs)between ?-MHCR453C/+and WT rats.There was no significant difference in the gross morphology of the heart of the naked eye,and in the cross-sectional area and ratios of heart weight to body weight in HE staining between ?-MHC R453C/+and WT rats.Masson trichrome staining and Sirius red staining showed a significant increase in myocardial fibrosis ratio in ?-MHCR453C/+rats compared with WT rats.There was no significant difference in left ventricular end systolic pressure(Max Pressure),left ventricular end diastolic pressure(Min Pressure),heart rate(Max dP/dt),left ventricular pressure maximum descent rate(Min dP/dt)by hemodynamic measurements between ?-MHC R453C/+and WT rats.Conclusions:In conclusion,rats with homozygous ?-MHC R453C mutation were not viable.The morphological and M-type echocardiography findings of the rats with?-MHC R453C heterozygous mutation did not change.