| BackgroundHypertension is a major risk factor for arteriosclerosis,coronary heart disease and stroke,which may cause irreversible cardiovascular remodeling.Left ventricular hypertrophy(LVH)is one of the early complications of hypertension.LVH is also a independent risk factor of cardiovascular disease.Development of cardiac hypertrophy may be accompanied by heart failure,arrhythmias,and ischaemic heart disease,which increase the risk of sudden death.Gap junctions(GJ),assembled from connexins(Cx),form the cell-to-cell pathways for propagation of precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart.Alterations of gap junction organization and connexin expression are now well established as a consistent feature of human heart disease in which there is an arrhythmic tendency.These alterations may take the form of structural remodeling, involving disturbances in the distribution of gap junctions and/or alteration of the amount or type of connexin(s)expressed.Cx43 is the most prominent connexin expressed by ventricular cardiomyoctyes in mammalian species.In the diseased ventricles,the most consistent quantitative alteration involves heterogenous reduction in Cx43 expression.During the process of cardiac hypertrophic remodeling, myocardial Cx43 also display altered expressions respond to a variety of regulatory processes,including the changes of the amount and distribution of Cx43,which is called GJ remodeling.The 3-hydroxy-3-methylglutaryl coenzyme A(HMO-CoA) reductase inhibitors,or statins,are most widely used clinically to modulate the plasma lipid levels,reducing the plasma total cholesterol and low-density lipoprotein cholesterol levels,and is effective in all types of hyperlipidemia.Several clinical trials have demonstrated statins are not only safe and endurable,a remarkable improvement in motality and morbidity of atherosclerosis related diease,such as coronary atherosclerotic heart disease is also evident in primary and secondary prevention.Many rencent studies have identified numerous pleiotropic effects of statins except for cholesterol lowering.There is evidence that statins induce regression of cardiac hypertrophy.Rat cardiomyocyte hypertrophy induced in vitro by angiotensinⅡ(AngⅡ)was abolished by simvastatin.Cardiac hypertrophy in vivo,induced in rats either by AngⅡinfusion(presence of hypertension)or by transaortic constriction(absence of hypertension),was also inhibited by simvastatin (2 mg·kg-1·d-1for 4 weeks).It has been demonstrated that statins reduce Cx43 expression in atherosclerotic lesions in mouse and rat models.None of these studies has evaluated the effects of statins on Cx43 expression in hypertrophied hearts.ObjectiveIn this study,we evaluated whether the protein expression level and distribution of Cx43 were changed in hypertrophied left ventricular myocardium of spontaneously hypertensive rats(SHR).If Cx43GJ remodeling was to prove to be of general significance in LVH,it would be important to confirm whether statins can reduce Cx43 over-expression and prevent Cx43 displacement.To examine these issues,we observed the effects of atorvastatin administration on LVH in SHR,and whether Cx43 expression had changed to statins.These results provide novel in vivo evidence for the key role of Cx43GJ in LVH and the possible mechanism in anti-hypertrophic effect of statins.MethodsSixteen healthy male nine-week-old SHR weighed 240～265g were randomly divided into two groups:SHR control group(SHR,n=8),and atorvastatin group (SHR-A,30 mg·kg-1d-1,n=8).Sixteen age- and weight-matched male Wistar-Kyoto (WKY)rats were selected as WKY control group(WKY,n=8)and atorvastatin group (WKY-A,30 mg·kg-1d-1,n=8).Atorvastatin was resolved in 1 ml saline and was administered to SHR-A and WKY-A groups by gavage once daily for 8 weeks.Rats in WKY group and SHR group were given the same amount of saline by gavage for 8 weeks.Systolic blood pressure(SBP)was measured at the beginning,2,4,and 8 weeks of the experiment.Echocardiographic measurement was performed at the end of the experiment.Left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular posterior wall thickness at end-systole(LVPWs),left ventricular posterior wall thickness at end-diastole (LVPWd),interventricular septum thickness at end-systole(IVSs)and interventricular septum thickness at end-diastole(IVSd)were measured and left ventricular mass(LVM)was calculated.At the end of the treatment,the rats were anesthesiated by intraperitoneal injection of sodium pentobarbital and 6 ml arterial blood was drawn for the determination of plasma concentrations of total cholesterol (TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C).The plasma brain natriuretic peptide (BNP)level was quantified by ELISA method.Left ventricle isolated along ventricle septum(ventricle septum included)and left ventricle weight to body weight ratio (LVW/BW)was calculated.The myocardium specimen were fixed with 10% formalin,embedded in paraffin,and stained with hematoxylin and eosin(HE).The pathologic changes of cardiomyoctytes were observed with the light microscope and the average transdiameter of cardiomyocyte(TDM)was calculated.Part of the block was sectioned and stained with Masson trichromic staining to assess the myocardial intersitional fibrosis,which was evaluated by collagen volume fraction(CVF). Immunohistochemistry analysis showed the distributions of immunolabeled Cx43 in longitudinally sectioned left ventricular myocardium.The hearts of rats were rapidly excised,weighed,sectioned and snap frozen in liquid nitrogen and then stored at70℃. The expression of Cx43 protein level in the four groups was detected by Western blot analysis.Selected samples were fixed in 2.5%glutaraldehyde solution and transmission electron microscopy investigated ultrastructural changes in cell junctions of cardiocytes.Results1.Compared with WKY and WKY-A groups,SBP was increased significantly in nine-week old SHR and SHR-A rats at the beginning of the experiment.This difference persisted until the end of the observation.SBP elevated progressively in SHR and SHR-A groups.At 8 week of the observation,SBP in SHR and SHR-A groups increased significantly compared with baseline measurement.By the end of the experiment,after atorvastatin treatment,mean SBP of SHR-A group is slightly smaller than that of SHR group,though it is slightly higher at the beginning of the observation,but the difference is not significant.At the meantime,we found SBP in WKY and WKY-A groups was constant over the course.2.M-mode echocardiography showed by the end of the experiment,compared with WKY and WKY-A groups,IVSd,LVPWd,IVSs,LVPWs and LVM increased significantly in SHR group.Compared with SHR,IVSd,LVPWd,IVSs,LVPWs and LVM decreased significantly in SHR-A group.There is no parameter difference between WKY and WKY-A group.3.Compared with WKY and WKY-A groups,plasma TC and HDL-C levels were significantly lower in SHR and SHR-A groups.LDL-C level in SHR-A is lower than that in WKY and WKY-A groups.There was no difference of plasma TG between the four groups.After eight week atorvastatin treatment,there was no difference of plasma TC,HDL-C,LDL-C and TG level between SHR and SHRA groups.There was no difference of plasma lipid level between WKY and WKY-A groups.Atorvastatin didn't show much effects on lipid modulating. 4.Evaluated by histological detection and plasma BNP measurement,LVH was prominent in SHR group by the end of the observation.Compared with WKY group,LVW/BW,TDM,CVF,and plasma BNP level were significantly increased in SHR group.After atorvastatin treatment,they decreased significantly in SHR-A group.These hypertrophic parameters were higher in SHR-A than that in WKY and WKY-A groups,but the differences were not significant.5.Immunohistochemistry analysis showed Cx43 positive spots distributed regularly in WKY and WKY-A groups.Cx43 spots were highly clustered at the intercalated disks and were band-shaped.Compared with WKY and WKY-A groups,Cx43 in SHR group expressed much strongly and less were labeled in disk area.The staining pattern showed varing degrees of dispersion over the cell surface and formed side-to-side contacts,and some were labeled intracardiomyocyte,which manifested Cx43GJ remodeling was prominent in hypertrophied left ventricular myocardium of SHR.Compared with SHR group, Cx43 expression in SHR-A decreased and most of the Cx43 labelling were confined to the intercalated disk structure running transverse to the myocardial fiber orientation,which were similar to WKY and WKY-A groups.This observation indicated a substantial prevention of the Cx43GJ remodeling of atorvastatin.6.Western blot analysis determined the expression of Cx43 protein in left ventricular myocardium in different groups.Cx43 expression was detected in WKY and WKY-A groups,and there was no difference of Cx43 protein level between the two groups.Compared with WKY and WKY-A groups,Cx43 expression increased significantly in left ventricular myocardium of SHR.After eight week treatment with atorvastatin,Cx43 expression of SHR-A decreased significantly compared with SHR,and we found Cx43 protein expression was even lower than that in WKY and WKY-A groups.This indicated that atorvastatin treatment prevented Cx43 over-expression in hypertrophied left ventricular myocardium of SHR.7.Transmission electron microscopy investigated ultrastructural changes in cell junctions of cardiocytes.Electron microscopy revealed GJ was blackly spotted and distributed in the folded region in disk center or nearby places.Compared with WKY and WKY-A groups,GJ in SHR were much abundant and distributed irregularly.They were frequently displaced from their usual locations to other sites at various distances away from the intercalated disks,including those forming side-to-side contact of myocytes.After eight week atorvastatin treatment,we found GJ were least abundant,distributed regularly,and were confined to folded regions of intercalated disk center in SHR-A group.Conclusions1.Eight weeks after the observation,SBP in SHR increased progressively and left ventricular hypertrophy was prominent,including the changes of echocardiographic patameters,the increases of plasma BNP level,LVW/BW, TDM and CVF.SBP of normaltensive rats was constant during the experiment and there was no evidence of left ventricular hypertrophy in normaltensive rats.2.Cx43 distributed regularly and were highly clustered at the intercalated disks in normaltensive rats.In SHR,the expression of Cx43 protein was elevated and Cx43 was no longer confined to the intercalated disks.Instead,it showed varing degrees of dispersion over the cell surface and formed side-to-side contacts of cardiomyocytes.3.After eight week treatment of atorvastatin,SBP and plasma lipid level didn't change much in SHR,but the left ventricular hypertrophy was alleviated,which was demonstrated by echocardiographic parameters,plasma BNP level, LVW/BW,TDM and CVF.The Cx43 protein expression was decreased and it distributed normaly and regularly,mostly in intercalated disks,which indicated atorvastatin prevented Cx43GJ remodeling in hypertrophic ventricular myocardium of SHR.
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