The Effect Of P53 Overexpression On Cardiomyocytes Hypertrophy Induced By Mechanical Stress

Clinically,myocardiohypertrophy is a common complication of cardiovascular diseases.in the early phase the compensated hypertrohy can relieve the mechanical stress of the ventricular wall and increase the cardiac output.Persistent hypertrophy would eventually develops into heart dilation and in turn its disadvantagious effect would results in the incidence of heart failure and sudden death.It is well established that hypertrophy is an independent risk factor for prediction of morbidity and mortality in cardiovascular diseases.Overload in circulation,which exerts mechanical stress to heart,is the primary factor involved in myocardiohypertrophy.The protien kinase ERK transduction pathway plays an pivotal role in myocardial hypertrophy.In transgenic mice overexpressing MEK1,ERK1/2 is abnormally activated and the myocardium appears marked hypertrophic feature.ERK^1/2 can be activated in the myocardium after the aorta is transversally coarctated.Based on these experimental results,we conclude that ERK_1/2 are important molecular regulators in intracellular hypertrophic signal transduction pathway triggered by mechanical stress.As a classical tumor suppressor gene,p53 is highlightened in the area of malignant tumors.Researches about the role of P53 in cardiovascular field are mainly focused on cardiomyocyte apoptosis triggered by a variety of stimulating factors.It was reported that p53 impaired angiogenesis by reducing hif-1 to inhibit the expression of angiogenic factors including VEGF et al and consequently p53 could inhibit myocardiohypertrophy on the whole heart and accelerate the transition to cardiac functional impairment.The myocytes itself also express a small amount of p53,which is possibly involved in DNA synthesis and the control of cell dividing cycle.So far,it is not yet clear whether and how p53 is directly invoved in the initiation and progression of the myocardiohypertrophy.The purpose in the experiment is to observe whether there is some direct relationships between p53 and the hypertrophy of the isolated cardiomyocytes induced by stretch.As mentioned above,ERK_1/2 is an important molecule involved in the intracellular hypertrophic signal transduction,so we consider p-erk_1/2 as a marker of cardiomyocyte hypertrophy.We divided the experiment into 2 parts.In the first part,we measured p-erk_1/2 expression by Western Blot to reveal the p-erk expression alternation after stretching the cardiomyocytes for varying time.In the second part,considering the rarity of p53 in cadiomyocytes and the difficulty in measurement,we firstly transfected cardiomyocytes with p53-Adv to induce the high expression of p53,then stretched the cardiomyocytes for varying time and measured p-erk_1/2 by western Blot to observe the changes in p-ERK expression.Finally we attempt to analysis the possible role of p53 in the initiation and progression of stretch-induced cardiomyocytes hypertrophy by comparing the resuls of 2 parts.PartⅠEffect of different stretching time on expression of p-erk_1/2 in cardiomyocytesObjective:To observe the alteration of p-ERK expression in cultured cardiomyocytes after being stretched for varying time.Methods:Cardiomyocytes derived from neonatal Sprague-Dawley rats were seeded on silicone plates and divided into 6 groups.After cultured in 10%- DMEM for 24 hours,the culture medium was replaced by serum-free DMEM.Among the 6 groups,5 groups were individually stretched for 5m,10m,30m,1h,2h,and the other one group was non-stretched.Finally,the whole protein in each group was extracted,and p-ERK1/2 was measured by western Blot.Results:The level of p-ERK in 5m group was significantly elevated compared with non-stretch group(P＜0.05).The level ofp-ERK_1/2 expression increased as stretching time extended.Conclusions:ERK_1/2 can be activated by mechanical stretch time-dependantly. ERK system is an important signal transduction pathway involved in the initiation and progression of cardiomyocytes hypertrophy induced by mechanical stretch. PartⅡThe effect of p53 overexpression on p-erk_{1/ 2} expression in cardiomyocytes stretched for varying time.Objective:To observe the effect of p53 overexpression on p-erk_1/2 expression in cardiomyocytes stretched for varying time after p53-Adv transfection.Methods:(1) Cardiomyocytes derived from neonatal Sprague-Dawley rats were seeded on sillicon plates and divided into 6 groups.Five groups of the cardiomyocytes were transfected with p53-Adv,and the MOI of each transfected group was 10,20,40, 80 and 160,leaving one group untransfected.Finally,the levels of p53 in each group were measured seperately by Western Blot.(2) Cardiomyocytes seeded on sillicon plates were divided into six groups.Firstly the cardiomyocytes were transfected with p53-Adv for 24 hours at 80 MOI.Then 5 groups were individually stretched for 5m,10m,30m, 1h and 2h,leaving one group unstretched.Finally,the levels of p-ERK_1/2 in each group were measured by western Blot.Results:(1) P53 expression was hardly detected when transfected at lower MOI, but obviously increased after being transfected at 80 MOI,indicating the cardiomyocytes can be effectively transfected with p53-Adv at 80 MOI.(2) Compared with non-stretched group,the level of p-erk expression in 5m group was significantly elevated despite of the overexpression of p53(P＜0.05).Expression of p-ERK_1/2 in other four stretched groups were all inhibited by p53 overexpression and lower than the 5min group(P＜0.05),and the level in 2h group was the lowest.Conclusions:The elevation of p-ERK_1/2 in cardiomyocytes after being stretched for 5 minutes can not be inhibited by p53 overexpression,however,p-ERK_1/2 elevation can be significantly inhibited when being stretched for longer time,implying that,in later phase,p53 may directly attenuate the degree of myocardiohypertrophy induced by mechanical stress and is invovled in the transition of the myocardium from the compensated state to the decompensated state.