Study On The Function Of H19 In Cardiac Hypertrophy

The heart is the first organ to form during embryogenesis, and also the most important functional organ in human body. Heart consists of cardiomyocytes and other cells(fibroblasts, lymphatic vessels, vessels, etc.). 25% of the number of cells in heart are cardiomyocytes, which accounts for 75% of the volume of the heart. Heart failure is one of the leading causes for the deaths of human beings, and it is often caused by cardiac hypertrophy, so it is of great realistic significance to detect the molecular mechanism of cardiac hypertrophy. Cardiac hypertrophy is an adaptive reaction of the heart against various stresses to maintain cardiac function at the early stage. However, sustained cardiac hypertrophy with maladaptive cardiac remodeling often leads to increased risk for heart failure and cardiac death. Although a variety of specific peptide hormones, growth factors, and mi RNAs have been identified as the regulators of cardiac hypertrophy, the underlying molecular mechanisms of cardiac hypertrophy are still not fully understood.Long non-coding RNAs(lnc RNAs) are transcribed RNA molecules greater than 200 nucleotides in length, but have no potential of protein-coding. Lnc RNAs have been shown to play important roles in various physiological processes, such as RNA processing, modulation of apoptosis and invasion, chromatin modification, and as a competing endogenous RNA(ce RNA). Recent studies have indicated that lnc RNAs also function in heart development and diseases. Braveheart can stimulate stem cells to differentiate into heart cells during mouse embryonic period. SNP(single nucleotide polymorphism) mutation of exon 5 of MIAT increases the transcription of MIAT, which may be associated with the development of myocardial infarction. Disorder of ANRIL, a lnc RNA located in 9p21, results in the anomaly proliferation of cardiomyocytes and coronary disease. RNA-seq data showed that the expressions of many lnc RNAs were changed in the hearts of mice after TAC, suggesting that lnc RNAs might play important roles in the process of cardiac hypertrophy and heart failure. Therefore, the study on lnc RNAs is becoming a new hotspot and keystone of basic and clinical cardiovascular research.As one of the first identified imprinted genes, the lnc RNA H19 gene has been verified as an important regulator in mammalian development and diseases. Exon 1 of H19 carries a mi RNA containing hairpin which has been found to act as the template for mi R-675, and it has been shown that mi R-675 can confer functionality on H19. Aseries of studies have indicated that H19 is always upregulated in hypertrophic hearts, suggesting it might play important roles in cardiac hypertrophy. However, the function and mechanism of H19 in regulating cardiac homeostasis has not been revealed.In this study, we aim to study the role of H19 in the process of cardiac hypertrophy and to explore the underlying mechanism. We firstly detected the expressions of H19 and its encoded mi R-675 in different types of mouse model of cardiac hypertrophy and human diseased hearts, and found they were upregulated in heart samples from pathological cardiac hypertrophy mouse model and human heart failure, while downregulated in exercise-induced physiological cardiac hypertrophy mouse model. To determine the role of H19 in cardiac hypertrophy, we overexpressed H19 in neonatal cardiomyocytes using an adenovirus that contained mouse H19(Ad-H19). Analysis on cardiomyocyte morphology and fetal genes showed that adenovirus-mediated overexpression of H19 reduced cell size both at baseline and in response to PE. To knockdown endogenous H19, a si-H19 was administered in primary cardiomyocytes. The result showed that H19 inhibition could induce cardiomyocyte hypertrophy.In order to understand the mechanism of the inhibitory effect of H19 on cardiac hypertrophy, we detected whether mi R-675 mediated the function of H19 by the following strategies. First, results obtained from overexpression or knockdown of mi R-675 in cardiomyocytes indicated that mi R-675 also inhibited cardiomyocyte hypertrophy. Then we knocked down mi R-675 in H19-overexpressing primary cardiomyocytes and found a reversion of the reduced cardiomyocyte size induced by H19. Furthermore, we constructed adenovirus carrying H19 fragment without pre-mi R-675(H19-Tru) or with mutant sequences of pre-mi R-675(H19-Mut) to infect cardiomyocytes. We found H19 but not mi R-675 was successfully overexpressed in cardiomyocytes infected with these two adenoviruses. Ectopic expression of these two types of H19 fragments both lost the ability to reduce cardiomyocyte size and fetal genes expression. All the results suggested that mi R-675 mediated the inhibitory effect of H19 on cardiomyocyte hypertrophy.Then we studied the mechanism of the function of mi R-675 in cardiomyocytes. Through Target Scan prediction, we chose Ca MKIIδ, a pro-hypertrophic gene, as a candidate. We constructed luciferase reporter gene harboring the normal or mutant type of 3’-UTR of Ca MKIIδ and performed luciferase reporter assays in 293 T cells.Cotransfection of mi R-675 with the luciferase reporter gene linked to the wild-type 3′-UTR of Ca MKIIδ strongly inhibited the luciferase activity, while no effect was observed with the construct harboring a mutant segment of Ca MKIIδ 3′-UTR. Detection of the expression of Ca MKIIδ showed that the Ca MKIIδ m RNA and protein levels were all downregulated by transfection of cardiomyocytes with mi R-675. These results verified that Ca MKIIδ was a direct target of mi R-675. To further investigate that whether Ca MKIIδ could mediate the function of H19 in cardiomyocyte, we knocked down Ca MKIIδ in the primary cardiomyocytes transfected with si-H19, and found a reversion of the cardiomyocyte hypertrophy induced by si-H19, suggesting that Ca MKIIδ partially mediated the effect of H19 on cardiomyocyte hypertrophy.We next evaluated the effect of H19/mi R-675 on cardiac homeostasis in vivo. We injected antagomi R-675 into mice subjected to pressure overload of the left ventricle by TAC and followed the disease for additional three weeks. TAC-induced mi R-675 upregulation was effectively abolished, while TAC-induced cardiac hypertrophy was exacerbated by antagomi R-675 treatments, suggesting that mi R-675 inhibition in vivo would induce cardiac hypertrophy.In summary, all the above findings reveal a novel function of H19/mi R-675 axis targeting Ca MKIIδ as a negative regulator of cardiac hypertrophy, suggesting its potential therapeutic role in cardiac diseases.