Effect Of 5-AZA-2’-dC On Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy

【Background】According to the China Cardiovascular Disease Report published in 2011,the mortality of cardiovascular disease endangering human health increased every year,and had the youth oriented tendency.Cardiac hypertrophy is the common pathophysiology of many cardiovascular diseases,such as hypertension,coronary heart disease,atherosclerosis,valve disease.Previous studies have shown that abnormal expression of genes related to the calcium signaling pathway in cardiomyocytes can lead to intracellular calcium homeostasis,then through the activation of calcineurin(CaN)and calmodulin protein kinase(CaMKs),resulting in cardiac hypertrophy.However,the mechanism of calcium level change and calcium signaling pathway genes regulation are not yet fully understood.It has been found that changes in the gene promoter region methylation levels modulating abnormal expression of genes can lead to disease.Gene therapy is the hot spot in recent year.In our previous study,we found that the promotor region of sarcoplasmic reticulum Ca2+-ATPase(SERCA2a)has the CpG island methylation regulatory sites,and other studies also shows that the methylation level has great different between the normal group and cardiac disease group.SERCA2 a is a key protein involved in sequestration of Ca2+ into the sarcoplasmic reticulum(SR)during diastole.There is a reduction of SERCA2 a protein level and function in cardiac dieases.This study aims to investigate whether the DNA methyltransferase inhibitor 5-AZA-2’-dC can inhibit cardiomyocyte hypertrophy through the regulation of sarcoplasmic reticulum Ca2+-ATPase(SERCA2a)gene expression on AngiotensinⅡ-induced cardiomyocyte hypertrophy.We conducted the following experiment.【Objective】To investigate the effect of 5-AZA-2’-dC on AngiotensinⅡ(AngⅡ)-induced cardiomyocyte hypertrophy.【Methods】Cultured cells derived from neonatal heart were divided into 5 groups: normal control;hypertrophic group;5-AZA-2’-dC group;treatment group;pretreatment group.Neonatal rat cardiomyocyte hypertrophic response was assayed by the size of cardiomyocytes and ANP expressive level.The expression of sarcoplasmic reticulum Ca2+-ATPase(SERCA2a),total calmodulin kinase Ⅱ(CaMK Ⅱ)and phospho-CaMK Ⅱ(p-CaMK Ⅱ)detected by Western blot.The intracellular calcium changes of cardiomyocytes were imaged by confocal fluorescent microscopy.【Results】Cells treated with Ang Ⅱ at 10-6mol/L for 48 h were chosen as hypertrophic cardiomyocyte model.The mRNA and protein level of ANP was significantly decreased in the treatment and pretreatment groups compared with hypertrophic group.The protein level of SERCA2 a was significantly decreased in the hypertrophic group,and increased in the treatment and pretreatment group compared with hypertrophic group,in the meanwhile,phospho-CaMK Ⅱ shown an opposite change tendency.The time required for increasing and declining to half of the intracellular calcium peak value were both delayed in hypertrophic group,as the treatment and pretreatment groups shown shorter timing compared with hypertrophic group.【Conclusions】5-AZA-2’-dC could inhibit AngⅡ-induced cardiomyocyte hypertrophy which might be related to regulate SERCA2 a expression.Increased SERCA2 a expression may maintain the calcium homeostasis through shortening the time of transfer Ca2+ from the cytosol of the cell to the lumen of the sarcoplasmic reticulum.