Epidemiology studies suggest that maternal smoking and passive smoking have strongly resulted in the occurrence of congenital heart defects (CHD) in offspring.Cigarette smoke (CS) can be divided into mainstream smoke (MS) and sidestream smoke (SS); CS chemistry study indicates that significant differences exist in the composition of MS and SS.Therefore, MS and SS were suspected to process toxicity dissimilarly.However, much less was known about the difference in the developmental effects induced by MS and SS.In the current study, heart development was mimicked by mouse embryonic stem cells (ESCs) differentiation.After MS and SS exposure, by tracing the bone morphogenetic protein (BMP)-Smad4 signaling pathway, interruption of downstream gene expression was observed, including Gata4,Mef2c and Nkx2.5, as well as myosin heavy chain and myosin light chain.Specifically, SS caused inhibition of Gata4 expression, even at non-cytotoxic concentration.Further, SS-induced hypoacetylation in promoter regions of Gata4 reflected the orchestration of CS-gene modulation-epigenetic regulation.Even though SS induced apoptosis in ESC-derived cardiomyocytes, the partial clearance in cells with down-regulated Gata4 caused these cells to survive and undergo further differentiation, which laid potential risk for abnormal heart development.These data uncovered the difference between MS and SS on heart development preliminarily.