| Background Cardiac hypertrophy has been proved to be a significant complication of essential hypertension and a independent risk factor for the mortality and disability of other cardiovascular diseases.Cardiomyocyte hypertrophy is regarded as an vital cytopathological foundament of left ventricular hypertrophy(LVH), therefore clarifing its mechanism of formation and regulation is very important to the prevention and cure of cardiovascular disease. The pathological basis of LVH includes cardiomyocyte hypertrophy,proliferation of cardiac fibroblasts and the increase of collagen synthesis. Large amount of studies manifestate that connective tissue growth factor(CTGF) is a kind of growth factor which can induce abnormal proliferation of cardiac fibroblasts and the increase of collagen synthesis. However, it is not clear whether CTGF is invoved in cardiac myocytes hypertrophy and what its signaling pathway is so far. Lacidipine, a new long-acting dihydropyridine calcium antagonist, can decrease blood pressure and efficaciously protect targe organ. It can significantly reduce the mortality of cardiovascular disease and cardiovascular events induced by hypertension. It is regarded as an important antihypertensive drug which has great application prospects and long-term future. Latest studies show that the activating of ERK1/2 is a vital factor which can induce 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 Lacidipine 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 Lacidipine, and provide novel theoretical evidences and strategy for prevention and treatment of hypertensive cardiac remodeling.Methods In this study, cultured cardiac myocytes of neonatal Sprague-Dawley(SD) rats were used as experiment object and proliferative model was constructed by CTGF induction.Upon the model of the primary culture of neonatal 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 Lacidipine on cell surface area induced by CTGF;(6) the effect of Lacidipine on protein synthesis of myocytes induced by CTGF; (7) the effect of Lacidipine 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 Lacidipine.Results (1)CTGF increased cardiac myocytes surface area in a dose dependent manner. In the groups with 10ug/L,25ug/L,50ug/L,100ug/L CTGF,the cell surface of cardiac myocytes was 902.76±127.83um2,1378.44±130.65um2,1729.85±145.24um2,2002.46±197.78um2, respectively,both of which were significantly higher than that of control group(P<0.01); (2) CTGF increased cardiac myocytes protein synthesis in a dose dependent manner. In the groups with 10ug/L,25ug/L,50ug/L,100ug/L CTGF,the incorporation rate of [3H]-leucine was 1329.71±107.19cpm/well,1573.39±149.27cpm/well,1993.84±130.43cpm/well,2247.58±171.35cpm/well, respectively , both of which were significantly higher than that of control group(P<0. 01); (3) CTGF increased cardiac myocytes protein concent in a dose dependent manner. In the groups with10ug/L,25ug/L,50ug/L,100ug/L CTGF,the cardiac myocytes protein concent was 0.805±0.089ng/cell,1.028±0.087ng/cell,1.302±0.109ng/cell,1.540±0.112ng/cell,respectively,both of which were significantly higher than that of control group (P<0.05～0.01); (4) CTGF increased the protein level of p-ERK1/2 in a dose dependent manner.In the groups with 10ug/L,25ug/L,50ug/L,100ug/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,30min,60min,120min 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 Lacidipine concentration rose, the cardiac myocytes surface area induced by 50ug/L CTGF decreased,the cardiac myocytes surface area in the groups of 5μmol/L,10μmol/L,25μmol/L,50μmol/L Lacidipine was 1476.52±156.73μm2,1120.39±149.68μm2,926.10±101.44μm2,739.81±91.55μm2, respectively,both of which were significantly lower than that of 50ug/L CTGF group(P<0.01); (6)As Lacidipine concentration rose,the cardiac myocytes protein synthesis induced by 50ug/L CTGF decreased, the incorporation rate of [3H]-leucine in the groups of 5μmol/L,10μmol/L,25μmol/L,50μmol/L Lacidipine was 2023.12±106.15 cpm/well,1629.15±103.46 cpm/well,1302.19±98.53cpm/well,1055.72±90.96cpm/well, respectively,both of which were significantly lower than that of 50ug/L CTGF group(P<0.01); (7) As Lacidipine concentration rose, the cardiac myocytes protein synthesis induced by 50ug/L CTGF decreased, cardiac myocytes protein concent in the groups of 5μmol/L,10μmol/L,25μmol/L,50μmol/L Lacidipine was 1.269±0.167ng/cell,0.923±0.119 ng/cell,0.766±0.085 ng/cell,0.682±0.063ng/cell,respectively,both of which were significantly lower than that of 50ug/L CTGF group(P<0.01); (8) As Lacidipine concentration rose, the protein level of p-ERK1/2 were significantly lower than that of 50ug/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(2)CTGF can induce phosphorylation of ERK1/2 cardiac myocytes in a dose dependent manner and time dependent manner, suggesting cardiomyocyte hypertrophy induced by CTGF probably realized via ERK1/2 phosphorylation;(3) Lacidipine can inhibit the increase of cardiac myocytes surface area,protein synthesis and protein concent in a dose dependent manner, suggesting Lacidipine can inhibit cardiomyocyte hypertrophy induced by CTGF;(4) Lacidipine can inhibit the protein level of p-ERK1/2 induced by CTGF, suggesting Lacidipine inhibit cardiomyocyte hypertrophy induced by CTGF via the inhibiton of p-ERK1/2.In short, CTGF can induce the cardiac myocyte hypertrophy, the latter promote the progression of cardiac remodeling. Lacidipine, 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. |
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