| Myocardial infarction(MI)is a serious cardiovascular disease that threatens human health.Arrhythmia occurs in most patients with myocardial infarction.Especially ventricular fibrillation is one of the leading causes of death in patients with MI.MicroRNAs(miRNAs)are a class of approximately 20-22 nucleotides long,endogenous non-coding RNAs.The functional strand of mature miRNA could regulate the target gene at the post-transcriptional level.MicroRNAs have been demonstrated as critical factors involved in various cardiovascular diseases including ventricular fibrillation following MI.More and more research show that miRNA is expected to become a new therapeutic target for preventing arrhythmia after MI.However,a comprehensive miRNA expression profile,especially the Gene function and pathways predicted to be targeted by differentially expressed miRNAs and correlation with ventricular fibrillation in ligation-induced MI rats is still unclear.In particular,traditional Chinese medicine has been recognized for its anti-arrhythmic potential.Based on the pharmacodynamic characteristics,the application of traditional Chinese medicine has shown its value in the improvement of existing pathological conditions that have been identified as the mechanisms underlying the generation of arrhythmia.It can be predicted that Chinese medicine will play an increasingly important role in the prevention and treatment of ventricular fibrillation after MI.Wenxin Granule(WXKL)is a traditional Chinese medicine used for the treatment of MI and arrhythmias in China.Several studies have shown that WXKL could prevent significantly the occurrence of malignant arrhythmia and reduce the mortality in MI patients.Whether WXKL has some additional beneficial effects on miRNAs expression and the targeted pathways following MI requires further study.Objective:1.To study the changes of miRNA expression profiles in myocardial tissue of rats with MI,and to analyze the gene functions and pathways of differential miRNA targeting regulation.2.To investigate the correlation between miR-1/miR-133 downstream gap junction,ventricular remodeling and ventricular fibrillation after MI.3.To investigate the mechanism of Wenxin granules in inhibiting ventricular fibrillation after MI from the perspective of miR-1/miR-133 and its downstream gap junctions and ventricular remodeling.Methods:1.MI Rat Model was established by a coronary artery ligation surgery.Then left ventricular tissues were collected for miRNA expression microarray analyses by Agilent Rat miRNA(8*15K,Design ID:070154)4 weeks after operation.Then differentially expressed miRNAs were screened.To assess the pathways predicted to be targeted,the gene ontology and KEGG pathway analysis of differentially expressed miRNAs was performed by using the Targetscan and microRNAorg debases.2.The relative expression of miR-1 and miR-133 were validated by quantitative real-time PCR.The ventricular fibrillation threshold was used to assess the risk of ventricular fibrillation in MI rats.The correlation between cardiac tissue connexin,collagen expression,cardiac structural changes and ventricular fibrillation were investigated through correlation analysis.3.Wenxin granules were used as observation drug.Angiotensin-converting enzyme inhibitor or β-blocker were used as control drug.After 4 weeks of treatment,the ultrastructure of gap junction in myocardial tissue was observed by transmission electron microscopy.The distribution and expression of Cx43,Cx45,type I and type III collagen in myocardial tissue were detected by immunofluorescence or immunohistochemistry.The structure and function of heart were observed by echocardiography.Changes of related protein in the pathways associated with gap junction and ventricular remodeling were detected by western blot.The myocardial tissue pathology,collagen volume and apoptosis were observed by conventional histopathology.Results:1.A total 35 differentially expressed(more than a twofold change)miRNAs were identified.Compared with the control group,17 miRNAs were down-regulated in the model group.And the other 18 miRNAs were up-regulated.A total 5829 potential target genes were predicted.It was found that a large number of targeted gene ontology and 23 predicted pathways.It is noteworthy that gap junctions and extracellular matrix are involved in the three major GO classification panels of the Biological P.rocess,Cellular Component and Molecular Function.Differentially expressed miR-1/miR-133 has been verified to be significantly downregulated in the model group.Gap junctions and extracellular matrix related pathways are most significant in their target regulatory pathways.Wenxin granules and captopril significantly increased the expression of miR-1/miR-133 in rats with MI(P<0.01 or P<0.05).2.Correlation analysis between miR-1/miR-133 downstream gap junction,extracellular matrix collagen remodeling,secondary cardiac structural changes and ventricular fibrillation threshold show:There was a significant positive correlation between Cx43 positive expression area,integrated optical density and ventricular fibrillation threshold,and the correlation coefficients were 0.75 and 0.67(P<0.01).There was no significant correlation between the average optical density of Cx43 and ventricular fibrillation threshold(P>0.05).And There was no significant correlation between the indexes of Cx45 and ventricular fibrillation threshold too(P>0.05).There was a significant negative correlation between collagen volume,type I,type III collagen contents and ventricular fibrillation threshold,and the correlation coefficients were-0.83,-0.84,and-0.84,respectively(P<0.01).There was a significant positive correlation between collagen typeⅠ/Ⅲ collagen ratio and ventricular fibrillation threshold(R=0.79,P<0.01).In terms of the end-diastolic and end-systolic left anterior wall thickness,ventricular septal thickness and ventricular fibrillation threshold,there was a significant positive correlation between each other,and the correlation coefficients were 0.86,0.74,0.84,and 0.77,respectively(P<0.01).There was a significant negative correlation between left ventricular posterior wall thickness and ventricular fibrillation threshold(R=-0.75,P<0.01).There was no significant correlation between left ventricular posterior wall thickness and ventricular fibrillation threshold(P>0.05).As far as the end-diastolic and end-systolic left ventricular diameter and volume,the correlation coefficient was-0.54 at the end of diastole(P<0.01),and the correlation coefficient was-0.74 at the end of systole(P<0.01).In addition,there was also a significant negative correlation between ventricular mass index and ventricular fibrillation threshold(R=-0.65,P<0.01).3.Increased risk of ventricular fibrillation manifests as a significant decrease in ventricular fibrillation threshold in MI rats(P<0.01).Wenxin granules and carvedilol significantly increased the ventricular fibrillation threshold(P<0.05 or P<0.01).4.In terms of in-situ expression of Cx43 and Cx45,the spatial distribution of Cx43 and Cx45 in the myocardial tissue was abnormal in MI rat model.The main manifestation is that they are not concentrated in the disk but are scattered in the cytoplasm.Image analysis showed that the expression area,mean optical density,and integrated optical density of Cx43 in the non-infarct region and the infarct border region of the model group were significantly lower(P<0.05 or P<0.01).The expression level of Cx45 in the non-infarct region was not significantly changed(P>0.05),but the expression area and integral optical density of Cx45 in the infarct border region were significantly increased(P<0.01).Wenxin granules and carvedilol partially improved the spatial distribution of Cx43 and Cx45,significantly increased the expression of Cx43 in the non-infarct region(P<0.05 or P<0.01)and decreased the expression area and integrated optical density of Cx45 in the infarct border region(P<0.01).But there was no significant effect on the expression of Cx43 in infarct border region and the expression of Cx45 in the non-infarct region(P>0.05).5.In terms of gap junction ultrastructure and phosphorylation signal pathways,the ultrastructure of gap junctions was unclear,and there are some breaks in the ultrastructure of the gap junction,and the non-phosphorylated form of Cx43-np was significantly increased,and the Cx43-p/Cx43-np ratio decreased significantly(P<0.01),and the Cx45 did not change significantly(P>0.05)in rat with MI.Wenxin granules and metoprolol were able to protect the ultrastructural of gap junctions to a certain extent,significantly increased Cx43-p,decreased Cx43-np,and could help restore the ratio of Cx43-p/Cx43-np(P<0.05 or P<0.01),but no significant effect on Cx45(P>0.05).In addition,wenxin granules and metoprolol could also regulate the expression of miR-1,PKC,phospho-p44/42 MAPK,phospho-Elk-1 and SRF in the phosphorylation pathway upstream of Cx43 in different degrees.6.When it comes to collagen remodeling of extracellular matrix,the collagen volume,typeⅠ and type Ⅲ collagen content were significantly increased,and the ratio of type Ⅰ/Ⅲcollagen was significantly decreased in rat with MI(P<0.01).Wenxin granules and carvedilol could reduce collagen volume,type Ⅰ and Ⅲ collagen content(P<0.01).And carvedilol could partially restore the ratio of type Ⅰ/Ⅲ collagen(P<0.01).Wenxin granule performance Similar trends but no statistical difference.In addition,both carvedilol and Wenxin granules significantly reduced the expression of TGFβ(P<0.01)but had no significant effect on CTGF(P>0.05).7.As far as secondary changes in ventricular structure and cardiac function,the MI rat model exhibits a structural feature of a centrifugal remodeling with thinning of the infarcted wall and a significant increase in the left ventricular diameter and volume.The left ventricular ejection fraction,short left ventricular shortening fraction and the maximum rate of change of intraventricular pressure were significantly reduced(P<0.01).Wenxin granules and metoprolol could partially improve ventricular remodeling and cardiac function.In addition,both Wenxin Granules and Metoprolol could inhibit myocardial cell apoptosis to some extent.Conclusions:1.MiR-1/miR-133 and its downstream targeted gap junctions and extracellular matrix-related functions and pathways are important components of miRNA expression profile changes after MI.2.Gap junction,extracellular matrix collagen remodeling and secondary cardiac structural changes are closely related to ventricular fibrillation after MI.It is an important way to prevent ventricular fibrillation after MI.3.Wenxin granules can increase ventricular fibrillation threshold,and its mechanism is is related to improvement of gap junction and ventricular remodeling by intervention of myocardial miR-1/miR-133 expression in MI rats. |
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