Modeling myocardial physiological growth using human pluripotent stem cell derived cardiomyocytes and 3D cardiac microtissues

Prior work suggests that exercise-responsive molecules may mediate exercise-induced myocardial physiological growth and promote functional recovery after ischemia- reperfusion injury in adult mice. Based on these findings, multiple mouse models of myocardial physiological growth have been successfully established. However, an in vitro model of physiological growth in cardiomyocytes derived from human embryonic stem cells (hESC-CMs) has not been successfully developed. To address this gap, we generated an inducible hESC cell line with forced Cbp/P300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain 4 (CITED4) gene expression, and differentiated those hESCs towards cardiomyocytes. The results showed that CITED4 expression increased cell size and proliferation in hESC-CMs, and promoted cardiomyocyte proliferation in 3D cardiac microtissues. Forced expression of CITED4 induced activation of Protein kinase B (also known as AKT1) signaling, which was necessary for CITED4-induced proliferation of hESC-CMs and 3D cardiac microtissues, while mTOR signaling mediated both proliferation and physiological hypertrophy induced by CITED4. In an in vitro model mimicking ischemia-reperfusion, CITED4 expression inhibited cardiomyocyte apoptosis in hESC-CMs and 3D cardiac microtissues, and this effect was mediated by activation of mTOR signaling. In conclusion, we successfully generate a physiological growth model in hESC-CMs and 3D cardiac microtissues. Moreover, physiological growth induced by CITED4 is mediated by activation of the mTOR signaling, which is necessary to promote proliferation and physiological hypertrophy, and to alleviate apoptosis after ischemia- reperfusion injury in hESC-CMs and 3D cardiac microtissues.