| Background MiRNAs are a class of small non-coding RNA, consist of 20-23nt, and regulate negatively gene expression at post-transcriptional level in animal, plant and human, involved in a series of physical and pathological process. It has studied that 1/3 genes in human are controlled by miRNAs. MiRNAs exist in serum stablely and could not be degraded. With the increasing development of Molecular biology and the recognition of diseases, the research ranges of miRNAs are wide and miRNAs will be potential therapy targets. MiRNAs have speciality of tissue and space, this demonstrates every miRNA has its own effect. A kind of miRNAs have several target mRNAs and a mRNA is regulated by multiple miRNAs. Among miRNAs, miR-1 and miR-133 are muscle-specific miRNA, which are same transcriptional unit. They are abundant in cardiomyocytes and have myocardium-specific. Both of them are involved in heart development, arrhythmia, cell apoptosis and other pathophysiologic process through regulating targeted mRNAs.Viral myocarditis is a common disease in younger children, of which ECG shows all kinds of arrhythmia. Acute severe myocarditis could cause fatal arrhythmia and heart failure. The invision of viral causes cardiomyocytes apoptosis and cardiac function changes. The experiments indicated that miR-1 and miR-133 are involved in cell apoptosis, and pro-apoptosis gene caspase-9 is the target of miR-133. However, there is no document about the influence of miR-1/133 on the apoptosis of viral myocarditis in mice so far. Electrical remodel and myocardium structural remodel are the main mechanism of arrhythmia, Potassium and Calcium channels are the main ion channels of cardiomyocytes. It has been found in many studies that the miRNAs expression profile in myocardium in the mice model of viral myocarditis has changed, which are involved in different pathological process through different target and pathway. Recent researches have found that miR-1 could stabilize resting membrane potential potassium current Kir2.1 and control Kv4.2 expression. MiR-133 could regulate L-type Calcium channel subunit Cavl.2 expression. It indicated miR-1 and miR-133 have an regulating fuction for electrical activities of cardiomyocytes.Recently, it has been extensive studied that RNA interference treats myocarditis. There are two difficulties for RNA interference:one is that how to resolve virus escapes the drug repression via gene variation, the other is that how to introduce directively miRs to virus infection of cardiomyocytes and have an effect on antivirus. For these, it was found that artificial synthetic miRs mimics have a resistant ability to viral gene variation, and there is an high security. Simultaneous, combined miRs interference could obviously decrease gene escape. The research have found that it could enhance the directive behavior of miR-1/133 mix which labeled by fluorescence that packaged in RNA-LANCEr II reagent and miR-1/133 could arrive at myocardium via tail vein injection.Objective1. To investigate whether miR-1/133 could reduce cardiomyocytes apoptosis and improve cardiac fuction EF, FS of acute viral myocarditis mice.2. To explore whether miR-1/133 are involved in regulating the expressions of potassium and calcium ion channels in mice with acute viral myocarditis, and participating in the process of arrhythmia.Methods1. Built Balb/c mice model of acute viral myocarditis through intraperitoneally injection of Coxsackie virus B3 Nancy strain, The mice were divided randomly into four groups:control group, myocarditis group, myocarditis+miR-1/133 mimics group, myocarditis+miR-1/133 NC group. There were 10 mice in every group and miR-1/133 mimcs and miR-1/133 NC were injected by tail vein at the second day. Observe the general status of mice.2. Echocardiogram was used to detect the left ventricular cardiac function EF and FS.3. HE staining observed the myocardial morphology changes.4. The situation of cardiomyocytes apoptosis was detected by TUNEL staining.5. The real time qRT-PCR was carried out to detect the expressions of miR-1,miR-133; the expressions of apoptosis-related genes Bax, Bcl-2 and Caspase-9; the expressions of transient outward potassium current Ito channel genes Kcnd2, channel transcriptional inhibitor Irx5, and inward rectifying potassium current Iki channel gene Kcnj2; the expression of L-type Calcium channel gene ale mRNA in myocardium of mice in every group.6. Western blot was used to detect the relative expressions of target protein Kv4.2 of Potassium channel gene Kcnd2, target protein Kir2.1 of Potassium channel gene Kcnj2 and target protein Cav1.2 of L-type Calcium channel gene αlc in myocardium of mice in each group.Results1. At the second day after injection intraperitoneally of CVB3, Nancy strain, 104TCED500.1 mL, the hair of mice had no brilliance and were disordered, the mice huddled up and were irritated easily or insensitive to stimulation. They had reduction of sports, lost weight. The symptoms of mice had no change after miR-1/133 NC interference. In the miR-1/133 mimics group, the hair of mice ordered neatly and had brilliance, they had more sports and gained weight.2. The results of echocardiogram showed that: In myocarditis group and myocarditis+miR-1/133 NC group, The cardiac function EF and FS decreased (P< 0.01), compared with control group. They were up-regulated after miR-1/133 mimics injection (P< 0.01).3. HE staining showed in control group, myocardial cells arranged orderly, there were no inflammatory cells infiltrated myocardium matrix; in myocarditis group and myocarditis+miR-1/133 NC group, myocardial cells swelled and arranged disorderly, inflammatory cells infiltrated myocardium matrix; in myocarditis+ miR-1/133 mimics group, myocardial cells arranged orderly, there were no cells edema, few inflammatory cells infiltrated myocardium matrix.4. TUNEL staining results showed There were few cardiomyocytes apoptosis in control and myocarditis+miR-1/133 mimics groups, but in myocarditis and myocarditis+miR-1/133 NC groups there were much cells apoptosis.5. The results of qRT-PCR showed that:(1) The expressions of miR-1, miR-133 in the myocardium of myocarditis group and myocarditis+miR-1/133 NC group decreased obviously (P< 0.01), compared with control group. They were up-regulated after miR-1/133 mimics injection (P< 0.05).(2) Compared with control group, the expressions of Bcl-2 (P<0.05) in myocarditis and myocarditis+miR-1/133 mimics groups were down-regulated, While the expressions of Bax, Caspase-9 were up-regulated (P＜0.01, P＜0.05); After miR-1/133 mimics interference, the expressions of Bcl-2 were up-regulated (P<0.05) and the expressions of Bax, Caspase-9 were down-regulated (P<0.01, P＜0.05).(3) The expressions of Potassium channel genes Kcnd2, Kcnj2 mRNA in the myocardium of myocarditis group and myocarditis+miR-1/133 NC group were down-regulated (P< 0.01), compared with control group. They were up-regulated after miR-1/133 mimics injection (P< 0.05).(4) The expression of Potassium channel transcriptional inhibitor Irx5 mRNA (P< 0.01) increased in myocarditis group and myocarditis+miR-1/133 NC group versus control group. It was down-regulated after miR-1/133 mimics injection (P< 0.05).(5) The expression of L-type Calcium channel gene ale mRNA in the myocardium of myocarditis group and myocarditis+miR-1/133 NC group increased (P< 0.01) versus control group. It was down-regulated after miR-1/133 mimics injection (P< 0.05).6. The results of western blot showed that:(1) The expressions of Potassium channel proteins Kv4.2, Kir2.1 in the myocardium of myocarditis group and myocarditis+miR-1/133 NC group were down-regulated (P< 0.01), compared with control group. They were up-regulated after miR-1/133 mimics injection (P< 0.05).(2) The expression of L-type Calcium channel protein Cavl.2 in the myocardium of myocarditis group and myocarditis+miR-1/133 NC group increased (P< 0.01) versus control group. It was down-regulated after miR-1/133 mimics injection (P < 0.05).Conclusion1. MiR-1/133 mimics complex could reduce cardiomyocytes apoptosis by regulating the expressions of apoptosis-related genes;2. MiR-1/133mimics complex could reduce arrhythmia through regulating the expressions of calcium and potassium ion channels genes in myocarditis, and improve cardiac function. |
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