Effects Of Simvastatin On Cardiac Myocytes Hypertrophic Induced By Connective Tissue Growth Factor And Its Relation With ERK1/2

Background Cardiac myocytes hypertrophy is regarded as an vital cytopathological founament of left ventricular hypertrophy (LVH), therefore clarifing its mechanism of formation and regulation is very important to the prevention and cure of cardiovascular disease. LVH, whose pathological basis are the cardiac myocytes hypertrophy as well as the abnormal proliferation and collagen synthesis of cardiac fibroblasts,is a significant complication of essential hypertension and a independent risk factor for the mortality and disability of other cardiovascular diseases.Large amount of stadies manifestate that connective tissue growth factor(CTGF) is a kind of growth factor which can induce the abnormal proliferation and collagen synthesis of cardiac fibroblasts. However,it is not clear whether CTGF is invoved in cardiac myocytes hypertrophy and what is its signaling pathway so far.The ultimate targets of curing essential hypertension are avioding cardiovascular events and reducing its mortality and disability, and the crucial goals are regressing LVH,recovering cardiac functions and reducing the untoward efferts. It has been approved that statin,which is 3-hydroxy -3-methylglutaryl coenzyme A reductase inhibitor,can protect cardiovascular system such as inhibiting the proliferation of vascular smoothe muscle cells and then regressing LVH,and its effects is far beyond that of regulate lipidemia.For the past few years, it has become a worldwide hot topic for study that statin inhibit cardiac myocytes hypertrophy and subsequently control cardiac remodeling,but its actual molecular mechanism is still no clear. Extracellular signal- regulated kinase(ERK) is the core of varietal conductive passway on signal of promoting growth.ERK1/2 ,which is the common name of ERK1 and ERK2,is the key point of signal conduction from surface receptor to cell nucleus. Latest studies show that the activating of ERK1/2 is a vital factor which can promote LVH,but whether it is involved in cardiac myocytes hypertrophy induced by CTGF is not well known.Objective This study was designed to observe the effects of simvastatin(Sim) on cardiac myocytes hypertrophy induced by connective tissue growth factor and its relation with ERK1/2 at the cellular and molecular levels . The purpose of the study is to invest the possible signaling pathway mechanism of cardiac myocytes hypertrophy induced by CTGF and inhibited by Sim, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Methods In this study, cultured cardiac myocytes of neonatal SD rats were used as experiment object and proliferative model was constructed by CTGF induction. Upon the model of the primary culture of neonatal Sprague-Dawley(SD) cardiomyocytes, image analysis system was used to measure cell surface area; protein synthesis of myocytes was measured by via[3H]-leucine incorporation rate; Coomassie Brilliant blue method was used to measure cardiac myocytes protein concent ;western blot was used to study the protein expression level of t-ERK1/2 and P-ERK1/2. The following items were observed dynamically: (1) the effect of CTGF on cell surface area; (2) the effect of CTGF on protein synthesis of myocytes; (3) the effect of CTGF on measure cardiac myocytes protein concent; (4) the change of p-ERK and t-ERK expression in cardiac myocytes during the modulation effect of CTGF; (5)the effect of Sim on cell surface area induced by CTGF; (6) the effect of Sim on protein synthesis of myocytes induced by CTGF; (7) the effect of Sim on measure cardiac myocytes protein concent induced by CTGF; (8) the change of p-ERK and t-ERK expression in cardiac myocytes during the modulation effect of Sim.Results (1) CTGF increased cardiac myocytes surface area in a dose dependent manner.In the groups with 10μg/L,25μg/L,50μg/L,100μg/L CTGF,the cell surface of cardiac myocytes was 929.88±132.21μm2,1411.31±129.16μm2,1731.99±152.96μm2,2040.62±205.37μm2 , respectively,both of which were significantly higher than that of control group(P<0.01);PD98059 substantially inhibited the increase of cardiac myocytes surface area induced by CTGF (P<0.01);(2) CTGF increased cardiac myocytes protein synthesis in a dose dependent manner. In the groups with 10μg/L,25μg/L,50μg/L,100μg/L CTGF,the incorporation rate of [3H]-leucine was 1340.99±114.55 cpm/well,1598.81±154.62 cpm/well,2005.65±131.16 cpm/well,2277.28±176.96cpm/well ,respectively,both of which were significantly higher than that of control group(P<0.01);PD98059 substantially inhibited the increase of [3H]-leucine incorporation rate induced by CTGF (P<0.01);(3) CTGF increased cardiac myocytes protein concent in a dose dependent manner. In the groups with 10μg/L,25μg/L,50μg/L,100μg/L CTGF,the cardiac myocytes protein concent was 0.790±0.075 ng/cell,1.054±0.094 ng/cell,1.282±0.105 ng/cell,1.533±0.102ng/cell, respectively,both of which were significantly higher than that of control group(P<0.05～0.01); PD98059 substantially inhibited the increase of cardiac myocytes protein concent induced by CTGF(P<0.01);(4) CTGF increased the protein level of p-ERK1/2 in a dose dependent manner. In the groups with 10μg/L,25μg/L,50μg/L,100μg/L CTGF,the protein level of p-ERK1/2 were significantly higher than that of control group,while the protein level of t-ERK1/2 in all groups were not significantly different; CTGF also increased the protein level of p-ERK1/2 in a time dependent manner. In the groups with 0min,5min,10min,15min time point, the protein level of p-ERK1/2 gradually increased, peaking at 15min,and gradually decreased at 30min,60min,120min,while the protein level of t-ERK1/2 in all groups were not significantly different;(5) As Sim concentration rose, the cardiac myocytes surface area induced by 50μg/L CTGF decreased, the cardiac myocytes surface area in the groups of 10-7mol/L,10-6mol/L,10-5mol/L,10-4mol/L Sim was 1530.58±151.58μm2,1111.46±135.63μm2,901.18±110.47μm2,725.24±92.35μm2,respectively,both of which were significantly lower than that of 50μg/L CTGF group(P<0.01);(6) As Sim concentration rose,the cardiac myocytes protein synthesis induced by 50μg/L CTGF decreased, the incorporation rate of [3H]-leucine was in the groups of 10-7mol/L,10-6mol/L,10-5mol/L,10-4mol/L Sim was 1989.36±101.22 cpm/well,1551.14±102.55 cpm/well,1232.10±93.58cpm/well,1033.46±90.29cpm/well ,respectively,both of which were significantly lower than that of 50μg/L CTGF group(P<0.01); (7) As Sim concentration rose,the cardiac myocytes protein synthesis induced by 50μg/L CTGF decreased, cardiac myocytes protein concent in the groups of 10-7mol/L,10-6mol/L,10-5mol/L,10-4mol/L Sim was 1.206±0.152ng/cell,0.895±0.123 ng/cell,0.722±0.082 ng/cell,0.685±0.065ng/cell ,respectively,both of which were significantly lower than that of 50μg/L CTGF group(P0.01); (8) As Sim concentration rose, the protein level of p-ERK1/2 were significantly lower than that of 50μg/L CTGF group,while the protein level of t-ERK1/2 in all groups were not significantly different.Conclusion (1) CTGF increased cardiac myocytes surface area,protein synthesis,protein concent and the protein level of p-ERK1/2 in a dose dependent manner,suggesting CTGF induces cardiac myocytes hypertrophy. CTGF can induce ERK1/2 of cardiac myocytes phosphorylation in a dose dependent manner and time dependent manner and PD98059 can inhibit the increase of cardiac myocytes surface area,protein synthesis,protein concent and the protein level of p-ERK1/2, suggesting cardiac myocytes hypertrophy induced by CTGF probably realized via ERK1/2 phosphorylation.(2)Simvastatim can inhibit the increase of cardiac myocytes surface area,protein synthesis,protein concent and the protein level of p-ERK1/2 induced by CTGF in a dose dependent manner, suggesting Simvastatin can inhibit cardiac myocytes hypertrophy induced by CTGF and that probably realized via ERK1/2 phosphorylation. In short,CTGF can induce the cardiac myocyte hypertrophy, the latter promote the progression of cardiac remodeling. Sim ,which inhibit the cardiac myocyte hypertrophy induced by CTGF,can interrupt and reverse the cardiac remodeling. The ERK signal pathway is involved in the mechanism of above-metioned processes. However,whether the ERK signal pathway is the only one signal transductive mechanism deserve further exploration.