SummaryCardiac maturation is an important developmental phase culminating in profound biological and functional changes to adapt to the high demand environment after birth1,2. Maturation of human pluripotent stem cell-derived human cardiac organoids (hCO) to more closely resemble human heart tissue is critical for understanding disease pathology. Herein, we profile human heart maturationin vivo3to identify key signalling pathways that drive maturation in hCOs4,5. Transient activation of both the 5’ AMP-activated kinase (AMPK) and estrogen-related receptor (ERR) promoted hCO maturation by mimicking the increased functional demands of post-natal development. hCOs cultured under these directed maturation (DM) conditions (DM-hCOs) display robust transcriptional maturation including increased expression of mature sarcomeric and oxidative phosphorylation genes resulting in enhanced metabolic capacity. DM-hCOs have functionally mature properties such as sarcoplasmic reticulum-dependent calcium handling, accurate responses to drug treatments perturbing the excitation-coupling process and ability to detect ectopy CASQ2 and RYR2 mutants. Importantly, DM- hCOs permit modelling of complex human disease processes such as desmoplakin (DSP) cardiomyopathy, which is driven by multiple cell types. Subsequently, we deploy DM-hCOs to demonstrate that bromodomain extra-terminal inhibitor INCB054329 rescues theDSPphenotype. Together, this study demonstrates that recapitulatingin vivodevelopment promotes advanced maturation enabling disease modelling and the identification of a therapeutic strategy forDSP-cardiomyopathy.
