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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD); however, the underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics-in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5-sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c, manifesting as ventricular septation defects, was validated in mice. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.