Abstract Human heart development is governed by transcription factor (TF) networks controlling dynamic and temporal gene expression alterations. Therefore, to comprehensively characterize these transcriptional regulations, day-to-day transcriptomic profiles were generated throughout the directed cardiac differentiation, starting from three distinct human induced pluripotent stem cell lines from healthy donors (32 days). We applied an expression-based correlation score to the chronological expression profiles of the TF genes, and clustered them into 12 sequential gene expression waves. We then identified a regulatory network of more than 23 000 activation and inhibition links between 216 TFs. Within this network, we observed previously unknown inferred transcriptional activations linking IRX3 and IRX5 TFs to three master cardiac TFs: GATA4, NKX2-5 and TBX5. Luciferase and co-immunoprecipitation assays demonstrated that these 5 TFs could (1) activate each other’s expression, (2) interact physically as multiprotein complexes and (3) together, finely regulate the expression of SCN5A , encoding the major cardiac sodium channel. Altogether, these results unveiled thousands of interactions between TFs, generating multiple robust hypotheses governing human cardiac development.

Deciphering transcriptional networks during human cardiac development