The mammalian heart undergoes major transitions during postnatal life to acquire the physiological properties of an adult organ. Postnatal life imposes numerous adaptations including electrophysiological, structural and metabolic maturation of cardiomyocytes1, which occur coincident with loss of proliferative capacity and regenerative potential2,3. The discovery of key upstream drivers of cardiomyocyte maturation and cell cycle arrest remains one of the most important unanswered questions in cardiac biology. Discovery of these drivers would facilitate current attempts to promote cardiomyocyte maturation in vitro for drug discovery and to de-differentiate adult cardiomyocytes in vivo for regenerative medicine. A recent study has suggested that the shift from a low oxygen environment in utero towards a high oxygen environment after birth acts as a key trigger for cardiomyocyte cell cycle exit4. Moreover, it was recently demonstrated that proliferative adult cardiomyocytes reside in a hypoxic niche5 and that exposure of adult mice to gradual hypoxemia is sufficient to drive cell cycle re-entry and regeneration following infarction6. However, it is currently unclear whether postnatal changes in oxygen tension or the associated shifts in cardiomyocyte metabolism are sufficient to promote maturation and cell cycle arrest as human pluripotent stem cell (hPSC)-derived cardiomyocytes fail to mature when cultured at 21% oxygen7,8. There are considerable changes in metabolic substrate provision during early postnatal life. The mammalian heart relies on high concentrations of carbohydrates and the presence of insulin in utero but later switches to fatty acid dominated substrates present in milk and low insulin levels post-birth9. In order to adapt to these changes in substrates, cardiomyocytes upregulate the genes required for fatty acid oxidation after birth10. The importance of these metabolic adaptations for cardiomyocyte maturation has been difficult to study because genetic disruption of fatty acid oxidation components in vivo can have a broad range of negative health impacts11. Therefore, there is a need to develop alternative approaches for studying the impact of cardiomyocyte metabolism on the maturation process. hPSCs are now widely used for the generation of defined human somatic cell types, including cardiomyocytes. These cardiomyocytes have now been used extensively for developmental studies, drug screening, disease modeling, and heart repair. However, lack of maturity and inappropriate responses to pharmacological agents have been identified as limitations in 2D or embryoid body based differentiation strategies12. To improve maturity of hPSC-derived cardiomyocytes, long-term culture can be used13, although long-term cultures may not be amenable to high-throughput screening applications and adult-like maturity is still not achieved14. In an effort to better simulate heart muscle structure and function, cardiac tissue engineering to form 3D engineered heart tissue has been used15-19. However, despite these recent advances in human cardiac tissue engineering, cardiac tissues derived from hPSC still lack many features of fully mature adult heart tissue20. Moreover, engineered heart tissue fabrication, culture, mechanical loading and pacing protocols, and analysis methods using organ baths are costly, labor intensive, and the multiple handling steps induce variability. In order to facilitate higher-throughput experiments, platforms for engineered heart tissue production have been miniaturized, however, screening experiments using semi-automated force of contraction analyses have only been published in 24-well plate formats21. Therefore, we developed a novel 96-well device, the heart dynamometer (Heart-Dyno), for high-throughput functional screening of human cardiac organoids (hCOs) to facilitate screening on a larger scale. The Heart-Dyno is designed to facilitate automated formation of dense muscle bundles from minimal cells and reagents while also facilitating culture and automated force of contraction analysis without any tissue handling. Using the Heart-Dyno, we define serum-free 3D culture conditions that promote structural, electrophysiological, metabolic and proliferative maturation of hPSC-derived cardiac organoids. Furthermore, we uncover a metabolic mechanism governing cardiomyocyte cell cycle arrest through repression of a β-catenin and YAP1 dependent signalling.