Recently, the increasing significance of the epicardium in cardiac development and regeneration is beginning to be recognized. However, because of the small proportion of primary epicardial cells and the limited cell culture time, further research on the mechanism of epicardial cells is hindered. Here, we transfected simian virus 40 Large T (SV40‐LT) into primary epicardial cells to establish an immortalized cell line, named EpiSV40. We further demonstrated that EpiSV40 can be easy to culture and has the proliferation, migration and differentiation capacities comparable to primary epicardial cells. EpiSV40 can serve as an ideal in vitro model for epicardial cell research, which will booster the study of the epicardium in cardiac development and heart regeneration.

Congenital heart disease (CHD) is the major cause of morbidity/mortality in infancy and childhood. Using a mouse model to uncover the mechanism of CHD is essential to understand its pathogenesis. However, conventional 2D phenotyping methods cannot comprehensively exhibit and accurately distinguish various 3D cardiac malformations for the complicated structure of heart cavity. Here, a new automated tool based on microcomputed tomography (micro‐CT) image data sets known as computer‐assisted cardiac cavity tracking (CACCT) is presented, which can detect the connections between cardiac cavities and identify complicated cardiac malformations in mouse hearts automatically. With CACCT, researchers, even those without expert training or diagnostic experience of CHD, can identify complicated cardiac malformations in mice conveniently and precisely, including transposition of the great arteries, double‐outlet right ventricle and atypical ventricular septal defect, whose accuracy is equivalent to senior fetal cardiologists. CACCT provides an effective approach to accurately identify heterogeneous cardiac malformations, which will facilitate the mechanistic studies into CHD and heart development.

Enhancer of zeste homolog 2 (EZH2) is an important transcriptional regulator in development that catalyzes H3K27me3. The role of EZH2 in epicardial development is still unknown. In this study, we show that EZH2 is expressed in epicardial cells during both human and mouse heart development.Ezh2epicardial deletion resulted in impaired epicardial cell migration, myocardial hypoplasia, and defective coronary plexus development, leading to embryonic lethality. By using RNA sequencing, we identified that EZH2 controls the transcription of tissue inhibitor of metalloproteinase 3 (TIMP3) in epicardial cells during heart development. Loss-of-function studies revealed that EZH2 promotes epicardial cell migration by suppressing TIMP3 expression. We also found that epicardialEzh2deficiency–induced TIMP3 up-regulation leads to extracellular matrix reconstruction in the embryonic myocardium by mass spectrometry. In conclusion, our results demonstrate that EZH2 is required for epicardial cell migration because it blocksTimp3transcription, which is vital for heart development. Our study provides new insight into the function of EZH2 in cell migration and epicardial development.