| Background and ObjectiveCardiac hypertrophy is one of the main ways in which cardiomyocytes respond to mechanical and neurohormonal stimuli.It enables myocytes to increase their work output and to improves cardiac pump function,which is characterized by an increment in cardiomyocyte size,increased protein synthesis and changes in extracellular matrix composition and fibrosis.Although cardiac hypertrophy may initially represent an adaptive response of the myocardium. it often progresses to heart failure. Controlling myocardial hypertrophy and left ventricular (LV) remodeling has been shown to have a beneficial effect, both in anexperimental model and in patients with chronic hypertensive heart disease and hypertrophy. Therefore, numerous studies have focused on characterizing signal transduction pathways and factors that are associated with cardiac hypertrophy. It is known that Ras/Raf/ERK1/2signaling is associated with cardiac hypertrophy.Ras is one member of the small GTP-binding protein. Ras protein act as a molecular switches.Single-induced conversion of the inactive to active state is that stimulated the exchange of GDP for GTP.A marked confomational change caused by GTP binding leads to activation of Ras.The effector domain engages downstream singling molecules only when the protein is in the GTP bound state. Raf is the first direct downstream effector of Ras.Phosphorylation of Raf (p-Raf) leads to activation of Raf. Phosphorylation of Raf is the key point of the Ras signaling pathway that belongs to the ERK transduction cascade, regulates apoptosis and cardiomyocyte growth in response to pressure overload.Extracellular signal-regulated kinase (extracellular regulated protein kinase, ERK)1/2is one of the downstream effectors of p-Raf, p-Raf is required to increase ERK1/2phosphorylation (p-ERK1/2),which is activation of ERK1/2. P-ERK1/2phosphorylates a variety of cytosolic and nuclear target, which is involved into a varirty of growth responses in the heart. Ras,Raf and ERK1/2are involved into Ras/Raf/ERK1/2signaling pathway. A large number of literature data suggest that Ras/Raf/ERK1/2signal pathway is involved into cardiac hypertrophy. The discovery of the human Ras effector Ras-association domainfamily1isoform A (RASSF1A) gene was initiated by the finding that the3p21.3region of the genome. RASSF1A has been reported to bind to active Ras (Ras-GTP) via heterodimerization with Norel/RASSF5(the founding member of the Rassf family), indicating a possible role as a Ras effector. Moreover, the hyperophic response was decreased in RASSF1A-overexpressing cells in phenylephrine indution. RASSF1A may be downregulated under conditions of cardiac hypertrophy, which inhibits Ras/Raf/ERK1/2signaling pathway.Telmisartan is an angiotensin Ⅱ receptor blocker(ARB). ARBs are highly selective for the AT(receptor and block the deleterious effect of Angiotensin Ⅱ (Ang Ⅱ). Ang Ⅱ is a neurohormonal stimuli.It leads to activation of angiotensin Ⅱ receptor.Many singaling pathways linked to angiotensin receptor activation appear to contribute to the hyperophic response, including Ras/Raf/ERK1/2signaling pathway.Therefore, ARBs can inhibit cardiac hypertrophy by inhibition of Ras/Raf/ERK1/2signaling pathway.In the present study, cardiac hypertrophy is promoted following in rats infusions of isoproterenol(ISO), it is related to development of the experimental model of cardiac hypertrophy.It was designed to investigate RASSF1A, p-Raf and p-ERK1/2of expressions. The aim of this study was to elucidate the role of RASSF1A in cardiac hypertrophy and the underlying mechanisms of telmisartan in the reversal of cardiac hypertrophy. The study may provide a novel molecular therapeutic targetor for heart failure.MethodsThirty female Sprague Dawley rats were randomly divided into normal saline (control group), isoproterenol (model group)and isoproterenol with telmisartan (intervention group),10rats in each group.Myocardial hypertrophy models were induced by subcutaneous injection of isoproterenol3mg/(kg·d) for10days,except for conrol group.Control group was administered by subcutaneous injection of normal saline6ml/(kg·d) for10days.The intervention group was fed telmisartan8.33mg/(kg·d) for10days,the control group and model group were fed the same amount distilled water. After the end of the10days treatment,hemodynamic parameters of rats were measured by bio-signal system.Heart wet weight/body weight (HW/BW) and Left ventricular wet weight/body weight (LVW/BW) were measured.By HE staining and Masson’s trichrome staining,myocardial tissue changes and myocardial fibers changes were analyzed.Collagen volume fraction(CVF) was measured by Masson’s trichrome staining. The expressions of atrial natriuretic factor (ANF) and RASSF1A mRNA were detected by reverse transcriptase poly merasechain reaction(RT-PCR). The expressions of RASSF1A,p-Raf and p-ERK1/2protein were observed by immunohistochemistry respectively. Differences were considered to be statistically significant when P<0.05.Results1Hemodynamic changesLeft ventricular systolic pressure (LVSP) was significantly lower in model group and intervention group than in control group(130.55±5.73mmHg vs123.96±7.45mmHg vs145.93±6.830mmHg,P<0.05).Left ventricular systolic pressure (LVSP) was greater in intervention group than in model group(123.96±7.45mmHg vs130.55±5.73mmHg,P<0.05).Left ventricular end-diastolic pressure (LVEDP) was significantly greater in model group and intervention group than in control group (6.63±0.32mmHg vs5.67±0.36mmHg vs4.49±0.36mmHg,P<0.05).Left ventricular end diastolic pressure (LVEDP) was significantly lower in intervention group than in model group(5.67±0.36mmHg vs6.63±0.32mmHg,P<0.05).+dp/dtmax was significantly lower in model group and intervention group than in control group(5681.04±92.19mmHg/s vs6237.42±37.50mmHg/s vs6723.09±82.78mmHg/s,P<0.05).+dp/dtmax was greater in intervention group than in model group(6237.42±37.50mmHg/s vs5681.04±92.19mmHg/s,P<0.05).-dp/dtmaxwas significantly lower in model group and intervention group than in control group (4632.04±138.70mmHg/s vs4938.84±105.46mmHg/s vs5256.85±145.11mmHg/s,P<0.05).-dp/dtmax was greater in intervention group than in model group(938.84±105.46mmHg/s vs4632.04±138.70mmHg/s,,P<0.05)2Heart wet weight/body weight (HW/BW) and Left ventricular wet weight/body weight (LVW/BW)Heart wet weight/body weight (HW/BW) was significantly greater in model group and intervention group than in control group (3.69±0.03mg/g vs3.44±0.01mg/g vs3.04±0.02mg/g,P<0.05).Heart wet weight/body weight (HW/BW) was significantly lower in intervention group than in model group(3.44±0.01mg/g vs3.69±0.03mg/g,P<0.05).Left ventricular wet weight/body weigh was significantly greater in model group and intervention group than in control group (2.82±0.09mg/g vs2.57±0.07mg/g vs2.31±0.01mg/g,P<0.05).Left ventricular wet weight/body weigh was significantly lower in intervention group than in model group (2.57±0.07mg/g vs2.82±0.09mg/g,P<0.05)3HE stainingMyocardial structure in control group is neat, no abnormal morphology. In model group, there were abnormal morphology,myocardial fiber hyperplasia and inflammatory exudate.In intervention group,damage of myocardial is lower than in model group, but the abnormal cell morphology were seen. 4Masson’s trichrome staining and collagen volume fraction(CVF)There were a large number of blue-stained collagen fibers among derangement of myocardial cells in model group.But there was rarely the blue-stained region in control group. Blue-stained region in intervention group was more than in control group,but was lower than in model group.Collagen volume fraction(CVF) in model group was the hightest.It in control group was the lowest.CVF in intervention group was more than in control group,but was lower than in model group.There was significant difference among the three groups(8.43±5.25vs14.02±0.95vs4.27±0.84,P<0.05).5Reverse transcriptase polymerase chain reaction (RT-PCR)The expression of ANF mRNA in Left ventricular was significantly greater in model group and intervention group than in control group (1.60±0.18vs1.03±0.23vs0.80±0.11,P<0.05). The expression of ANF mRNA in Left ventricular was significantly lower in intervention group than in model group(1.03±0.23vs1.60±0.18,P<0.05). The expression of RASSF1A mRNA in Left ventricular was significantly lower in model group and intervention group than in control group (0.77±0.11vs1.15±0.25vs1.76±0.34,P<0.05). The expression of RASSF1A mRNA in Left ventricular was significantly greater inintervention group than in model group (1.15±0.25vs0.77±0.11,P<0.05)6ImmunohistochemistryThe expression of p-Raf protein in left ventricle of model group was significantly higher than in control group and intervention group,It in intervention group is higher than in control group.There was significant difference among the three groups(160.72±11.49vs133.28±2.73vs115.94±4.06,P<0.05). The expression of p-ERK1/2protein in left ventricle of model group was significantly higher than in control group and intervention group, it in intervention group is higher than in control group. There was significant difference among the three groups (154.23±4.02vs129.24±3.49vs92.03±7.34,P<0.05). The expression of RASSF1A protein in left ventricle of model group and intervention group were less than in control group. The expression of RASSF1A protein in left ventricle of intervention group was higher than in model group. There was significant difference among the three group (116.66±3.94vs133.8±1.17vs168.29±7.49,P<0.05)Conclusions1. The expressions of p-Raf and p-ERK1/2in hpertrophic myocardial tissues are elevated confirmed to participate in the process of cardiac hypertrophy induced by isoproterenol. Ras/Raf/ERK1/2signal pathway regulates the isoproterenol-induced myocardial hypertrophy.2. The expression of RASSF1A in isoproterenol-induced myocardial hypertrophy is low, while expressions of p-Raf and p-ERK1/2are lower. RASSF1A may inhibit Ras/Raf/ERK1/2signal pathway, is a cardiac hypertrophy negative regulatory factor.3. To compared with model group, the expressions of p-Raf and p-ERK1/2in intervention group are lower, telmisartan makes expression of RASSF1A elevated in myocardial tissue.Telmisartan inhibits cardiac hypertrophy by up-regulated RASSF1A and down-regulated Ras/Raf/ERK1/2signal pathway. |
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