The Expressions Of Connexin30.2, Connexin43 And Connexin45 In Heart Tissues Of Rats With Acute Myocardial Infarction And The Effect Of Arotinolol Hydrochloride On Their Expressions

BackgroundAcute myocardial infarction(AMI) is one of the severe types of coronary heart disease, the incidence of which is increasingly high year by year.AMI used to be regarded as the chief origin that results in various kinds of arrhythmia, exerting a severely negative influence on the prognosis and the life of patients.The studies in recent years have shown that the incidence and continuity of arrhythmia after myocardial infarction are closely related to the abnormal expression and distribution of gap junction connexin in heart. The cardiac connexins expressed in heart tissues are mainly compsed of Cx30.2, Cx43 and Cx45. Both Cx30.2 and Cx45 play the role of atrioventricular conduction delay, which guarantees the atrial and ventricular in timed and orderly constriction. However, there is no relevant research in animal models yet that focuses on the changes of Cx30.2 and Cx45 expressions. Cx43 is the chief connexin expressed in the ventricular myocardium, where Cx45 is also expressed to some extent. But the change tendency of Cx43 and Cx45 after myocardial infarction is still under heat dispute.It has been proved that beta receptor blocker (Metoprolol) helps decrease the incidence of arrhythmia after myocardial infarction by changing the expression and distribution of the gap junction connexins. Arotinolol hydrochloride, as a kind ofα,beta dual receptor blockers, is helpful for pressure release and anti-arrhythmia. However, no relevant research has ever been done and reported on the expression and distribution of Cx30.2, Cx43 and Cx45 after acute myocardial infarction.ObjectivesThis thesis aims at detecting the expressions of gap junction connexin30.2 (Cx30.2) and connexin45 (Cx45) in node area, the expressions of connexin43 (Cx43) and connexin45 (Cx45) in ventricular myocardium in rats with acute myocardial infarction by Immunohistochemistry and reverse transcription polymerase chain reaction(RT-PCR), as well as the effects of arotinolol hydrochloride on Cx30.2, Cx43 and Cx45 in rats with acute myocardial infarction by Immunohistochemistry and RT-PCR, by creating the following experimental model of rats with acute myocardial infarction. This thesis is intended to provide a theoretical foundation for the positive effects of arotinolol hydrochloride on the prevention and cure of arrhythmia when the acute myocardial infarction happens.Materials and Methods1. Experimental animals and grouping 40pcs healthy and mature male Sprague-Dawley (SD) rats were sampled by the Animal Laboratory Center of Zhengzhou University. The weight and age of the rats is 200g～250g/pc and 8 weeks old respectively. All the rats were randomly divided into four groups:the acute myocardial infarction model group (group MI, n=10), arotinolol hydrochloride treatment group (group A,n=10),sham-operation group (group SH, n=10) and normal control group (group N, n=10).2. Experimental methods and Detected indexes(1) Rats were anesthetized by injecting 10% Chloral hydrate into abdomen after being weighed.The acute myocardial infarction model of rats was induced by ligating the left coronary artery under the help of respirator.Rats in group A were given arotinolol hydrochloride (0.91mg/kg);The other three groups were given the same amount of distilled water;All the rats were given gastric lavage twice a day for 4 weeks.(2) After being stained by hematoxylin-eosin (HE), samples were observed through the olympus optical microscope on the morphological changes of their myocardial cells.(3)The expressions of Cx30.2 and Cx45 in the node area,Cx43 and Cx45 in the area of acute myocardial infarction, non-ischemic area and the fringe of infarction were detected by Immunochemistry. we make use of the micro camera system to analyze the images of Immunochemistry.(4) The expressions of Cx30.2mRNA and Cx45mRNA in the node area, Cx43mRNA and Cx45mRNA in the area of acute myocardial infarction, non-ischemic area and the fringe of infarction were detected by RT-PCR. Quantity one software was used to analyze the results of RT-PCR.3.Statistical Methods The statistical software SSPS17.0 for windows was exploited to analyse data. All the measure data were expressed as means±standard error (x±s).The one way analysis of variance (ANOVA) was used for multiple comparisons, and Least-significant difference(LSD) test was used for each group in every time point for the significance using a value of P<0.05.Results1.The Morphological Changes of Myocardial Tissues The myocardial cells in group SH had less visible changes than those of group N. Large areas of myocardial cells in the infacted area and the fringe of infarction of rats in group MI were necrotic and replaced by extensive proliferation of fiber collagen tissues. The myocardial cells that remained normal were compensatorily hypertrophic and in disorder; There were visible proliferation of fiber collagen tissues and infiltration of the inflammatory cells in group MI; The volume of myocardial cells and the extent of proliferation of myocardial fiber in group A were smaller and less respectively than those in group MI; The arrangement of myocardial fiber in group A was tidier than that in group MI. The degree of the proliferation of fiber collagen tissues in the interstitial and the infiltration of inflammatory cells in group A was less than that in group MI. 2.The expressions of Cx30.2 and Cx45 in the Node Area Consistent were the variation trends concerning the average optical density value and the mRNA grayscale value of Cx30.2 and those of Cx45 respectively.:There was less visible variation in group SH than that in group N, which was statistically meaningless (P>0.05); There were visible increases in group MI, which was statistically meaningful (P<0.05). This indicates that the expressions of Cx30.2 and Cx45 after myocardial infaction were affected to some extent.3. The Expressions of Cx43 and Cx45 in the Ventricular Myocardium The variation trends concerning the average optical density value and the mRNA grayscale value of Cx43 and those of Cx45 were as follows:there was less visible variation concerning the average optical density value and the grayscale value of mRNA for Cx43 and Cx45 respectively in group SH and the normal area of group MI than that in group N, which was statistically meaningless (P>0.05); In the infected area and the fringe of infarction in group MI, the average optical density value and the mRNA grayscale value of Cx43 were visibly less, which was statistically meaningful (P<0.05); while those of Cx45 were visibly more, which was also statistically meaningful(P<0.05). This indicates that the expressions of Cx43 and Cx45 after myocardial infaction were affected to some extent.4. The Effects of Arotinolol hydrochloride on the Expressions of Cx30.2, Cx43 and Cx45 The average optical density value and the mRNA grayscale value of Cx30.2 and those of Cx45 in the node area of rats in group A were all visibly less than those in group MI after 4 weeks of arotinolol hydrochloride treatment, which was statistically meaningful (P<0.05). This demonstrates that arotinolol hydrochloride enable to lighten the reconstruction of Cx30.2 and Cx45 to some extent. The average optical density value and the mRNA grayscale value of Cx43 in the ventricular myocardium both in the fringe area and those in the central area in group A were visibly more than those in group MI respectively, which was statistically meaningful (P<0.05); while those of Cx45were visibly less, which was statistically meaningful (P<0.05). This demonstrates that arotinolol hydrochloride could affact the expressions of Cx43 and Cx45.Conclusions 1. After the acute myocardial infarction happens,the expressions of Cx30.2 and Cx45 protein in the node area increase and are in disorder; the mRNA expressions of them increase.2. After the acute myocardial infarction happens,a great number of Cx43 protein in the ventricular tissue are degradated greatly and are in disorder; the expression of Cx45 protein in the ventricular tissue increases and is in disorder; the mRNA expression of Cx43 decreases,but that of Cx45 increases.3. Arotinolol hydrochloride not only help to decrease the expressions of Cx30.2 and Cx45 in the node area, but also to increase the expression of Cx43 in the ventricular tissue and inhibit the expression of Cx45,which can alleviate ischemic necrosis of the myocardium and protect myocardium.