Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes

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, 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. On day 25 of the cardiac differentiation protocol, hiPSC-CMs were incubated with 10μM of mirdametinib (PD0325901, Cayman Chemical), a selective and non ATP-competitive dual MEK1 and MEK2 inhibitor, and 5μM of SB203580 (Cayman Chemical), a p38 and PDK1 inhibitor, in RPMI1640 supplemented with B27 (plus insulin). The media with cocktail of inhibitors (SP) or DMSO vehicle control (0.01%) was changed on day 28 of the differentiation protocol. On day 30, the hiPSC-CMs were harvested for further analyses. Untreated day ~90 hiPSC-CMs were used as a reference for cardiac maturation. Total RNA was isolated using TRIzol reagent (Invitrogen) and the PureLink RNA mini kit (Invitrogen, #12183025) with on-column DNase treatment following the manufacturer’s recommendations. Reverse transcription was performed using SuperScript™ VILO™ cDNA synthesis kit (Invitrogen, #11754250). Two-μg of cDNA were used for further QC and RNA-seq experiments.