| microRNAs (miRNA) are endogenous non-coding single-chain small RNA,which participate in a series of important life processes through regulate gene expression. Researchs had shown that miRNA may be closely related with diseases occurred. miRNA expression and those regulation effects are different in the different types of diseases process. It has been demonstrated that miRNA are closely related to the development of cardiac hypertrophy, arrhythmia and other cardiovascular diseases. An animal study had shown that overexpression of miRNA-1 in the ischemic myocardial tissue of the myocardial infarction rat model could inhibit cardiac connexin 43 (Cx43) and inward rectifier potassium channel 2.1 (Kir2.1) expression and triggered ventricular arrhythmia (VA).Hypertension is a common cardiovascular disease, long-term high blood pressure will lead to left ventricular hypertrophy (LVH). Clinical studies have shown that the rate of VA in hypertensive patients with LVH was several times higher than the normaltensive ones. Currently the mechanisms of VA due to LVH are not yet fully clarified. Therefore, in order to further clarify the molecular mechanism of the occurrence of VA due to hypertensive LVH, and to find new therapeutic targets , we choosed SHR rats as research subjects to practice the following three aspects studies in this study.Part I: The Experimental Research Of Provocative VA In SHRs With LVHThe research had three groups. 17-week-old male SHRs were assigned into LVH group (n=12), 8-week-old SHRs were assigned into non-LVH group(n=12), 17-week-old male WKYs rats were assigned into the control group(n=12). The three groups were used to observe the situation of VA induced by Langendorff perfusion of isolated rat hearts perfused with K+-deficient solution. Results:1. Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; 2. After perfusioning K+-deficient solution, the time of VA occurred in LVH group significantly earlier than non-LVH group and the control group (P<0.01, P<0.05). 3. Arrhythmia score and the VA incidences of LVH group were significantly higher than non-LVH group and the control group (P <0.05). Part II: Analysis Of MicroRNAs Differential Expression Patterns In The Hypertrophic Left Ventricular Tissue Of SHRs . There were two researches in this part: 1.The research of miRNAs differential expression patterns in the hypertrophic left ventricular tissue of SHRs; 2. The research of miRNA-1 expression in SHRs hypertrophic left ventricle by fluorescent quantitative real time polymerase chain reation (RT-PCR) .In the research of the differential expression of miRNA in SHR hypertrophic left ventricular tissuce , 10 seventeen-week-old male SHRs were assigned into LVH group, 10 eight-week-old male SHRs were served as control group. The left ventricular mass index (LVMI), histopathological examination and measurement of myocardial cell diameter (TDM) were used to observe the LVH situation.The total RNA of the two groups were extracted from left ventricle tissue and the differential expression of miRNAs was detected using miRNA microarray chip which containd 924 miRNA microarray probe system.Results: 1. Compared with the control group, systolic and diastolic blood pressure were significantly higher in LVH group and non-LVH group (P<0.05, P<0.01).2.Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; Compared with the control group, LVMI and TDM of the rats in LVH group were significantly bigger (all P<0.05). 3. 13 miRNAs were showed differential expression in LVH rats, 4 were up-regulated and 9 were down-regulated.In the research of miRNA-1 expression in SHR hypertrophic left ventricle by qRT-PCR, we used qRT-PCR to detect miRNA-1 expression in SHR hypertrophied left ventricular tissue. Results: 1. the level of miRNA-1 significantly increased (to 2.08±0.21-fold) higher in the hypertrophic left ventricular tissue of SHRs. 2. miRNA-1 expression in SHR left ventricular myocardium and were positively correlated with LVMI, CVF (r= 0.712 and 0.674, respectively, both P <0.05).Part III: The Regulative Role Of miRNA-1 Working On Myocardium Cx43 And Kir2.1 In SHRs. There were two researches in this part:1. The correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium. 2.The intervention on miRNA-1 expression in SHR hypertrophied left ventricle affecting on Cx43, Kir2.1 expression levels. In the research of the correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium ,we used immunohistochemistry and Western Blot to detect Kir2.1 and Cx43 protein expression in myocardial tissue. The results showed that: Cx43, Kir2.1 relative levels in LVH left ventricular myocardium were significantly lower than the control group (all P<0.05), and significant negatively correlated with the myocardial expression of miRNA-1 level (P <0.05).In the research of the intervention on miRNA-1 expression in SHR hypertrophic left ventricle affecting on Cx43, Kir2.1 expression levels, 17-week-old SHR with the sustainable development of hypertension and LVH as research subjects were randomly divided into the intervention group(n=6) ,blank control group (n=5)and negative control group(n=5). The technology of high-pressure tail vein injection with liposome transiently transfected miRNA-1 inhibitor (Anti-miRNA-1) was use to silence miRNA-1. The results showed: 1.Compared with two control groups, miRNA-1 expression in the left ventricular myocardium of intervention group by being transfected Anti-miRNA-1 was significantly reduced (40±8%, P <0.05). 2. Compared with negative control group and blank control group ,gene silencing by transfection Anti-miRNA-1 can increase Cx43, Kir2.1 protein expression in rat left ventricular myocardium significantly (all P <0.05).The results of this research project show that: 1. The occurred earlier with provocative VA incidences of ventricular arrhythmia increased significantly, which implies that LVH is associated with VA. 2. The expression profile of miRNAs changes in hypertrophic myocardium, suggesting those changed miRNAs may involve in the formation of LVH.3. The level of miRNA-1 increased obviously in the left ventricular hypertrophic tissue of SHRs, its expression was positively correlated with LVMI and CVF in LVH rats. 4. The protein level of Cx43 and Kir2.1 decreased remarkably in the left ventricular hypertrophic tissue of SHRs, and negative correlation with the myocardial expression of miRNA-1. 5. After miRNA-1 was silenced, the level of miRNA-1 significantly decreased, accompanying with the protein level of Cx43 and Kir2.1 increased, suggesting that miRNA-1 might play an important role on the formation of VA due to LVH by inhibiting the expression of Cx43 and Kir2.1. |
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