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Aims: Maternal nicotine exposure caused alteration of gene expression patterns and programming of cardiovascular dysfunction.The present study is to investigate the effect of nicotine on cardiac myogenic gene expression together with relevant epigenetic regulation during in vitro cardiac differentiation and in vivo cardiac development.Methods: To study the effect of nicotine on cardiomyogenesis,an in vitro cardiac differentiation model was generated by murine embryonic bodies(EBs).EBs derived from mouse embryonic stem cells(mESCs)were induced to 12 days cardiac differentiation with or without nicotine treatment.Differentiated EBs at 12-day maturation were obtained to study cardiac myogenic gene expression and relevant promoter DNA methylation pattern.As in vivo cardiac myogenesis model,pregnant Sprague– Dawley rats were exposed to nicotine through gestation,hearts were isolated from neonatal offspring for echocardiography testing heart function and further molecular study.Results: In vitro studies on cardiac differentiation model found no significant effect on mESCs proliferation and two embryonic biomarkers(Oct4 and Nanog)mRNA expression with nicotine treatment.However,nicotine exposure significantly decreased the percentage of spontaneous beating EBs as well as the amount of GATA4 positive cells at 12 days of maturation.Persistent nicotine exposure resulted in up-regulation of 5-methylcytosine,DNMT1 and DNMT3A but decreased GATA4 and Tbx5 gene expression due to promoter DNA hypermethylation.These effects of nicotine were reversed by general nicotinic acetylcholine receptors(nAChRs)inhibitor,suggesting the involvement of nAChRs in the direct adverse impact of nicotine on cardiac differentiation.Consistent results of GATA4 and Tbx5 gene suppression and promoter DNA hypermethylation by maternal nicotine treatment were obtained from in vivo cardiac development model.Echocardiography showed reduced ejection fraction(EF%),systolic and diastolic left ventricular anterolateral wall(LVAW;s and LVAW;d)as well as systolic and diastolic left ventricular posterior wall(LVPW;s and LVPW;d)in offspring hearts with maternal nicotine treatment.Conclusion: Persistent nicotine exposure induces GATA4 and Tbx5 gene repression by promoter DNA hypermethylation during in vitro cardiac differentiation and in vivo cardiac myogenesis.Nicotine treatment results in a reduction of spontaneous beating EBs in cardiac differentiation and cardiac malfunction in offspring heart.This adverse impact on cardiomyogenesis is directly mediated by nicotine and nAChRs.