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

Cardiac physiological growth is needed for increased demands of heart function after exercise. Prior work suggests that exercise-responsive molecules, including Cbp/P300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain 4 (CITED4), can mediate exercise-induced myocardial physiological growth and promote functional recovery after ischemia-reperfusion injury in adult mice. Moreover, forced expression of CITED4 induces physiological cardiac growth in rodent model. Multiple mouse models of myocardial physiological growth have been established by activating genes such as CITED4, IGF1R and AKT. However, an in vitro model of physiological growth in cardiomyocytes derived from human embryonic stem cells (hESC-CMs) has not yet been developed. To provide clinically relevant models for exploring the molecular mechanism of physiological growth, we generated an inducible hESC cell line with forced CITED4 gene expression and differentiated those hESCs towards cardiomyocytes. The results showed that forced CITED4 expression increased cell size and proliferation in hESC-CMs, and promoted cardiomyocyte proliferation in 3D cardiac microtissues. Activation of protein kinase B (also known as AKT1) signaling was necessary for CITED4-induced proliferation in 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 injury, CITED4 expression inhibited cardiomyocyte apoptosis in hESC-CMs and 3D cardiac microtissues, and this effect was mediated by activation of the 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 both proliferation and physiological hypertrophy, and to alleviate apoptosis after ischemia-reperfusion injury in hESC-CMs and 3D cardiac microtissues.