Effects Of Tanshinone IIA On Ventricle-remolding And Its Mechanisms

【Background】Recently some studies suggested that the basic mechanism of the occurrence and development from left ventricular hypertrophy caused by hypertension to congestive heart disease is Ventricle remolding,hypertrophy and apoptosis of cardiomyocyte is the special change of ventricle remolding .Ventricle remolding is due to the changes in structure and function and phenotype of myocardium caused by a series of mechanism on molecule and cell, including hypertrophy , apoptosis and expression of embryo gene and protein in cardiomyocyte; and the change in myocardium exo-matrix and component. Now the emphases of heart failture treatment is to inhibit the stimulus and duidance factor relating with ventricle remolding to improve heart function. Recently some studies suggested AngiotensinII(AngII) is an important stimuli in the hypertrophy and apoptosis of cardiomyocytes induced by pathologic factors,such as pressure load,which can cause myocardial hypertrophy and apoptosis. Cardiomyocyte hypertrophy represent increase of cardiomyocyte volume and myocomma,as well as the change of the type of contract protein in the cardiomyocytes.The degree of cardiomyocyte hypertrophy can be measured by content of cardiomyocyte protein and 3H–Leucine incorporation.Many stimuli factors ,such as mechanical tension,AngⅡand endothelin(endothelin-1,ET-1)can induce myocardial hypertrophy. AngⅡis a kind of important nerve-body fluid factor,which not only has important physiological modulation action to system of heart and blood vessel,but also has important effect on the pathological and physiological process of myocardial hypertrophy and and heart failture. Opposite to mitosis ,apoptosis is the initiative and ordinal process of self-destruction of cell controlled by freedom gene. Recently some studies suggested that capability of cardiomyocyte hyperplasia is limited and the number of cardiomyocyte is relatively fixed. Cardiomyocyte apoptosis will lead to decrease the number of contract units,which is the important cause of depress of heart function .So it plays a important role in the development of heart disease.Lately some research indicate that body fluid factor and cellular factor is the determinant of apoptosis;AngII can induce apoptosis of cardiomyocyte .Tanshinone IIA (STS) is the valid fatsoluble monomer of a traditional Chinese drug -Salvia Miltiorrhiza Bge(SMB) ,which can inhibit ischemia,anoxemia,platelet adhisiveness and thrombosis and improve microcirculation. STS has been widely used in the therapy of coronary heart disease, but its effect of myocardial hypertrophy and apoptosis is rarely reported . In this experiment, the culture of neonatal rat cardiomyocytes were used to study the effect of TanshinoneⅡA(STS) extracted from SMB on the hypertrophic response and apoptosis induced by AngⅡin the cultured neonatal rat cardiomyocytes, and to discuss the possible mechanism of the action of STS.This provides experimental evidences for its clinical use in the prevention and cure of myocardial remolding.【Methods】①To investigate the effect of tanshinoneⅡA(STS) on the hypertrophy induced by AngiotensinⅡ(AngⅡ)in the primary culture of neonatal rat cardiomyocytes. The Cardiomyocytes of Wistar rats were cultured with pancreatin and preplating technique. The hypertrophy of cardiomyocytes was induced with AngiotensinⅡ,and intervened with TanshinoneⅡA and Valsartan . The effect of STS on cardiomyocyte was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay. As the index of cardiomyocyte hypertrophy, the cell size was determined by phase contrast microscope and protein synthesis rate was measured by 3H-Leucine incorporation.②To investigate the effect of tanshinoneⅡA (STS) on the hypertrophy induced by AngiotensinⅡ(AngⅡ)in the primary culture of neonatal rat cardiomyocytes. The Cardiomyocytes of Wistar rats were cultured with pancreatin and preplating technique. The hypertrophy of cardiomyocytes was induced with AngiotensinⅡ, and intervened with TanshinoneⅡA and Valsartan . The proto-oncogene c-fos,c-jun,c-myc mRNA expression was assessed using reverse transcription polymerase chain reaction (RT-PCR).③To study the role of calcineurin (CaN)-dependent signaling pathway in cardiomyocytes hypertrophy by observing the effects of Sodium tanshinone IIA sulfonate (STS) on the hypertrophy induced by AngiotensinⅡ(AngⅡ)in the primary culture of neonatal rat cardiomyocytes, detecting the activities of the cardiomyocytes CaN ;The expression of CaN and NFAT3 was assessed using Western blot or fluorescence microscope.④To investigate the effect of tanshinoneⅡA (STS) on the apoptosis induced by AngiotensinⅡ( AngⅡ)in the primary culture of neonatal rat cardiomyocytes. The Cardiomyocytes of Wistar rats were cultured with pancreatin and preplating technique. The apoptosis of cardiomyocytes was induced with AngiotensinⅡ, and intervened with TanshinoneⅡA and Valsartan.The rate of cardiomyocyte apoptosis were analysed with flow cytometric analysis.⑤To investigate the effect of tanshinoneⅡA (STS) on the apoptosis induced by AngiotensinⅡ( AngⅡ)in the primary culture of neonatal rat cardiomyocytes. The Cardiomyocytes of Wistar rats were cultured with pancreatin and preplating technique. The apoptosis of cardiomyocytes was induced with AngiotensinⅡ,and intervened with TanshinoneⅡA and Valsartan.The protein expression of bcl-2 and bax was assessed using Western blot .⑥To investigate the effect of tanshinoneⅡA (STS) on the apoptosis induced by AngiotensinⅡ( AngⅡ)in the primary culture of neonatal rat cardiomyocytes. The Cardiomyocytes of Wistar rats were cultured with pancreatin and preplating technique. The apoptosis of cardiomyocytes was induced with AngiotensinⅡ, and intervened with TanshinoneⅡA and Valsartan.The expression of Fas/FasL was assessed using Western blot and RT-PCR【Results】①Exposure of cultured cardiomyocytes to STS (0-50μM) for 48h produced marginal cytotoxicity. with treatment of cardiomyocytes by AngII for 7 days, The cell size of cardiomyocyte of the group of AngII increased more prominently than the group of control (P<0.01). STS can restrained the increase of cardiomyocyte size in a dose-dependent manner.,which was induced by AngII(P<0.01); After AngII took effect for 24h, the cardiomyocyte protein synthesis rate of the AngII group increased more prominently than the control group (P<0.01).STS can restrained the increase of cardiomyocyte protein synthesis rate (P<0.01) in a dose-dependent manner,which was induced by AngII.②AngII was added to the culture medium and 30min later c-fos,c-jun,c-myc mRNA expression of cardiomyocyte increased evidently(P<0.01) . If STS was added before that ,they could inhibit the effect of AngII (P<0.01) in a dose-dependent manner.③Stimulated by AngⅡ(1μmol/L) for 12h,the activation of CaN and the expression of CaN,p-CaN,NFAT3 protein in the cardiomyocytes increased significantly in contrast to control, and NFAT3 expressed in nucleus.these effects of AngⅡcan be inhibited by STS in a dose-dependent manner.④With treatment of cardiomyocytes by AngII for 48h, the rate of cardiomyocyte apoptosis increased significantly in contrast to control (P<0.01), the effect of AngⅡcan be inhibited by STS markedly(P<0.01).⑤With treatment of cardiomyocytes by AngII for 12h, the ratio of bcl-2 and bax decreased significantly in contrast to control (P<0.01), the effect of AngⅡcan be inhibited by STS markedly(P<0.01) in a dose-dependent manner.⑥With treatment of cardiomyocytes by AngII for 12h, the protein and mRNA expression of Fas and FasL increased significantly in contrast to control (P<0.01), the effect of AngⅡcan be inhibited by STS markedly in a dose-dependent manner. (P<0.01).【Conclusions】①STS can prevent the hypertrophy of cardiomyocytes induced by AngⅡ.②The results suggest that activation of CaN may play an important role in cardiomyocytes hypertrophy induced by AngⅡ,and the anti-hypertrophic effect of STS on cardiomyocyte hypertrophy induced by Ang II may be associated to it's effect of inhibition to CaN-NFAT3 signaling pathway.③STS can prevent the hypertrophy of cardiomyocytes induced by AngⅡ, and the anti-hypertrophic effect of STS on cardiomyocyte hypertrophy induced by Ang II may be associated to it's effect of inhibition to the increased expression of proto-oncogene c-fos,c-jun,c-myc mRNA induced by Ang II.④STS can prevent the apoptosis of cardiomyocytes induced by Ang II.⑤STS can prevent the apoptosis of cardiomyocytes induced by Ang II, which may relate to it's effect of inhibition to the decreased the ratio of bcl-2 and bax induced by Ang II .⑥STS can prevent the apoptosis of cardiomyocytes induced by AngⅡ, and the anti-apoptosis effect of STS on cardiomyocyte apoptosis induced by Ang II may be associated to it's effect of inhibition to the increased protein and mRNA expression of Fas and FasL induced by Ang II.