| Aims:Chronic Heart Failure(CHF)is the final stage of various cardiovascular diseases and is the most common cause of death.Despite significant advances in the treatment of cardiovascular disease,the prevalence of chronic heart failure is still increasing rapidly.Pathological myocardial remodeling is an independent risk factor for the morbidity and mortality of cardiovascular diseases,and is the major pathophysiological basis for the development of various cardiovascular diseases into CHF.Hence to determine the mechanism of myocardial remodeling is particularly urgent for the prevention and treatment of CHF.Long noncoding RNAs are a novel class of gene regulators and may contribute to disease cause,and growing research has shown that it plays an important role in cardiovascular disease.The microarray technology was used to investigate the expression profiles of long noncoding RNAs and their potential functional roles in chronic heart failure,of which LncRNA H19 changed the most.LncRNA H19 is one of the first-known imprinted genes.It is expressed only in myocardium and skeletal muscle after birth.It takes part in the process of growing development as well as tumorigenesis and development.Recent studies indicated that H19 was involved in the regulation of cardiomyocyte development,apoptosis,death,etc.Our earlier research also demonstrated that its SNPs was closely linked with the incidence and severity of Coronary Heart Disease(CHD),and its expression increased in CHD patients' plasma.This study aims to explore the function and possible mechanisms of LncRNA H19 in the pathological remodeling of CHF,and to provide a new perspective for its prevention and treatment.Methods:1.We applied a well-established chronic heart failure rat model and performed long noncoding RNA microarray experiments on the left ventricular tissue of rats with chronic heart failure and under sham control.Differentially expressed long noncoding RNAs and mRNAs were identified through fold-change filtering.Bioinformatic analyses were performed to predict the potential biological roles of key long noncoding RNAs.According to the results of microarray,we used qRT-PCR to verified the expression of differentially expressed LncRNAs.2.(1)In vivo study,C57BL/6 mice were injected into tail vein with AAV9 to interfere or over-expression the level of H19 in myocardium.Then the heart failure model was contructed.The cardiac function was examined by echocardiogram.The following parameters were measured: left ventricular ejection fraction(EF),left ventricular fractional shortening(FS),left ventricular end systolic diameter and left ventricular posterior wall end.HE and Masson staining were used to observe the extent of myocardial hypertrophy and fibrosis.The ANP?BNP and myh7 mRNA levels in myocardial were detected by qRT-PCR to assess pathological myocardial hypertrophy.(2)In vitro study,Adv-H19 and Adv-si H19 were contructed and transfected to primary myocardial cells.Then pathological hypertrophy model was contructed by isoproterenol.Immunofluorescence and qRT-PCR were performed to test the volume of myocardial and pathological myocardial hypertrophy.3.(1)The IGF2 and IGF2 R expression levels in myocardial of heart failure model or pathological myocardial hypertrophy model by western blotting and qRT-PCR.(2)The AAV was contructed to over-express or interfere the expression level of H19 in myocardial and the expression level of IGF2 was detected by western blotting and qRT-PCR.(3)The expression level of IGF2 and H19 were over-expressed or interfered by transfecting AAV,and the volume of myocardial and pathological myocardial hypertrophy by immunofluorescence and qRT-PCR.The IGF2?IGF2R?Gaq and phosphorylation of PKC-? protein levels in myocardial by western blotting.(4)Leu27IGF2,one of the IGF2 analogue,which could activate IGF2 R signaling pathway on the basis of over-expression of H19 in pathological hypertrophy model induced by ISO.The volume of myocardial and pathological myocardial hypertrophy by immunofluorescence and qRT-PCR.Western blotting was used to detected the protein levels of IGF2?IGF2R?Gaq and phosphorylation of PKC-? in myocardial.Results: 1.We found that 1197 LncRNAs and 2066 mRNAs were upregulated,whereas 1403 LncRNAs and 2871 mRNAs were downregulated in failing hearts(fold-change > 2.0).We also identified 331 pairs of differentially expressed LncRNAs and nearby coding genes,and the most significant process putatively affected by these differential expression of LncRNAs was the MAPK signaling pathway.Expression levels of six LncRNA–mRNA pairs,which might be involved in the pathogenesis of CHF,were confirmed by quantitative real-time PCR.LncRNA H19 had the most significant difference in the left ventricular tissue of CHF rats.2.Our study first indicated that LncRNA H19 could significantly reduce HW/BW ratio,HW/TL ratio,LVsd and LVPWd,alleviate pathological cardiac remolding and notably improve cardiac function.In addition,we intervened the expression of LncRNA H19 in primary cardiomyocytes,and found that over-expression of H19 could inhibit ISO-induced pathological cardiac hypertrophy while inhibition of H19 could promote pathological cardiac hypertrophy.3.LncRNA H19 can reversely regulate the expression of IGF2 in cardiomyocytes.Interference of IGF2/IGF2 R signaling pathway can depress pathological cardiac hypertrophy caused by the silence of LncRNA H19,while activation of this pathway can weaken the depression of pathological cardiac hypertrophy.Conclusion: Our study identified a set of long noncoding RNAs that were aberrantly expressed in rats with chronic heart failure and might be involved in the pathogenesis of chronic heart failure.LncRNA H19 reduced the phosphorylation of Gaq / PKC-?via regulating IGF2 / IGF2 R signaling pathway to inhibit pathological myocardial remodeling and improve cardiac function.The results of our study may provide a novel perspective for pathological myocardial remodeling and chronic heart failure. |
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