The Expression And Mechanism Of Chromogranin A In Dilated Cardiomyopathy And Its Relationship With Myocardial Fibrosis

Chromogranin A(CGA) is widely distributed in the neuroendocine system and considered as a diagnostic marker of the whole activity of neuroendocrine system.A neuroendocrine activation has been described in the process of Dilated Ccardiomyopathy, which promotes the myocardial cell apoptosis,the stimulation of myocardial interstitial collagen hyperplasia and remodeling,and the imbalance of collagenⅠ(COLⅠ)and collagenⅢ(COLⅢ).Losing the support of collagen,the function of left ventricular coverts from pressure pump to cavity,and eventually leads to heart failure.The CGA is released by sympathetic nerve terminals and counteracts the actions ofβ-adrenergic drugs. It is also showed that CGA can modulate fibroblast cell adhesion and spreading in a differential manner.Catestatin is one of active polypeptides of CGA and an antagon of catecholamine.So it is speculated that CGA,especially catestatin,may also contribute to regulate heart remodeling in patients with DCM.However,the correlation between CGA and myocardial fibrosis and the mechanism of its effects on myocardial fibrosis remained uncertain.In the present study,the expression and location of CGA and myocardial fibrosis in DCM were observed,and also the mechanism of myocardial fibrosis related with CGA and catestatin was studied,so as to give rise to the further study in the role of the neuroendocrine of DCM and clinical evaluation of myocardial fibrosis.PARTⅠ:The expression of chromogranin A in Dilated cardiomyopathyObjective:To evaluate the expression of chromogranin A(CGA) in the peripheral blood and the myocardium of Dilated cardiomyopathy(DCM).Method:Blood serum of normal people and the DCM patients was colleted and the expression of CGA in the peripheral blood was assessed by ELISA.Surgical myocardial specimen from DCM patients who were underwent successful orthotopic cardiac transplantation,and the normal myocardium used for controls were obtained.The expresission of CGA-mRNA was analyzed by RT-PCR.The location and expression of CGA were assessed by immunohistochemistry(INH) with anti-CGA antibody.Results:The level of CGA assessed by ELISA in peripheral blood of DCM patients was 187.6±94.7ng/ml:while that of normal person was 130.6±21.1 ng/ml(P＜0.05).It is performed by RT-PCR that,comparing with normal myocardium,the expression of CGA was significantly increased in myocardium of DCM patients(P＜0.001).Cytoplasic expression of CGA assessed by INH was showed that lots of strong positive granules were produced in DCM slides(P＜0.05),densely arranged in the epicardial and endocardial myocardiocytes;while only few sparse granules were found in the normal myocardium.Conclusion:The expression of CGA,which can be used as the indicator of nenuroendocrine system,is increased in both peripheral blood and myocardium of DCM patients.And CGA may be a potential diagnostic and prognostic indicator of DCM patients.PARTⅡ:The correlation between expression of chromogranin A and myocardial fibrosis in Dilated CardiomyopathyObjective To observe the correlation between expression of chormogranin A(CGA) and myocardial fibrosis in dilated cardiomyopathy(DCM),in order to investigate the role of CGA in the development of myocardial fibrosis of DCM.Methods Surgical myocardial specimen from DCM patients who were underwent successful orthotopic cardiac transplantation,and the normal myocardium used for controls were obtained.The relationship of CGA-mRNA,COLⅠ-mRNA,COLⅢ-mRNA and ADAMTS-1-mRNA were analyzed by real-time PCR.The location and expression of CGA were assessed by immunohistochemistry(INH) with anti-CGA antibody;The collagen specific picrosirus red staining was applied on slides of heart samples from controls and patients,then the collagen volume fraction(CVF) was calculated.And then correlation in morphology was assessed by INH and CVF.Results Cytoplasic expression of CGA assessed by INH was showed that lots of strong positive granules were produced in DCM slides(P＜0.05),densely arranged in the epicardial and endocardial myocardiocytes;while only few sparse granules were found in the normal myocardium.Increase of CVF was obviously observed in DCM myocardial specimen(P＜0.001).It is performed by real-time PCR that CGA-mRNA was correlated with COLⅠ-mRNA,COLⅢ-mRNA and ADAMTS-1-mRNA,and their coefficients respectively were 0.729,0.95,0.665.Also in morphology,the deposition of collagen was surprisingly identical with the strong positive arrangement of CGA.Conclusion It is demonstrated for the first time that the deposition of CGA is related with the myocardial fibrosis of DCM,that is,CGA may influence the process of myocardial remodeling and fibrosis.PARTⅢ:Effect of catestatin on inhibiting the development of myocardial fibrosisObjective:To study the effect and its mechanisms of catestatin,one of the active polypeptides of CGA and an antagon of catecholamine,on inhibiting the development of myocardial fibrosis.Methods:To synthesis the polypeptide of catestatin according to the human catestatin sequence(human CGA_352-372),whose purity was assessed by HPLC.Isoprenaline(ISO) was used as the indicator of catecholamine.SD rat neonatal cardiocytes and cardiac fibroblasts were cultured and treated with ISO or catestatin.To establish the subgroups of ISO and catestatin as follows:ISO:10umol/L,20umol/L,40umol/L;catestatin:1umol/L,2umol/L,4umol/L,10umol/L,25umol/L.The apoptosis ratio of cariocyte was measured by FACS to evaluate the repairing and healing effects of catestatin on cardiocyte damage caused by ISO.The effects of catestatin on inbihiting the generation and immigration of cardiac fibroblasts were respectively detected by cell counting kit-8(CCK-8) and cell scarification experiment.Results:Catestatin purity was 99.21%approved by HPLC.In ISO damage group,the apoptosis ratio of cardiocytes detected by FACS raised with ISO concentration increasing (P＜0.001);in catestatin treatment group,catestatin of all five different concentration group could decrease the cardiocytes apoptosis caused by ISO(20umol/L)(P＜0.05) and its effect was shown by concentration dependence(P＜0.05),that is,the higher concentration of catestatin,the better reparation effects of cadiocytes damage induced by ISO.Comparing to normal group,after 24hous treated with low concentration of catestatin(1umol/l),growth rate of cardiac fibroblasts decreased and the difference was of great statistic significance(P＜0.001);when catestatin reached 4umol/L,that inhibition effect also came to its plauto.As shown in cell scarification experiment, cardiac fibroblasts moved smaller distance in treatment group than normal one,and also this function is related with catestatin concentration(P＜0.001).Conclusions:It is catestatin that,whose effects are concentration dependent,could inhibit the apoptosis of cardiocytes caused by ISO,and also slow down the generation and immigration of cardiac fibroblasts.That is,catestatin may inhibit the genesis and the development of myocardial fibrosis and its different concentrations may also have their variant influences on myocardial fibrosis.