Abstract Body: Background: Current protocols can generate highly-pure populations of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate key characteristics of mature in vivo cardiomyocytes. Yet, there exists a risk of arrhythmias when hiPSC-CMs are injected into large animal models. This prompts further investigation into the mechanisms of hiPSC-CM maturation to facilitate clinical translation.

Hypothesis: The forkhead box (FOX) family of transcription factors can regulate maturation in neonatal cardiomyocytes through a balance between FOXO and FOXM1. We also previously found that p53 activation could enhance hiPSC-CM maturation. Therefore, we hypothesized that p53 activation increases FOXO and decreases FOXM1 to promote hiPSC-CM maturation in three-dimensional (3D) suspension culture.

Methodology: 3D cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator), LOM612 (FOXO activator), AS1842856 (FOXO inhibitor), or RCM-1 (FOXM1 inhibitor), starting 2 days after onset of beating. The hiPSC-CMs were assessed for maturation in metabolic, contractile, and electrophysiological properties, by Seahorse mito stress test, Multi electrode array, and VALA kinetic image cytometer respectively.

Results: P53 activation promoted FOXO upregulation and FOXM1 downregulation in hiPSC-CMs, measured by RT-qPCR and immunostaining. Alongside this, p53 activation also promoted hiPSC-CM metabolic and contractile maturational characteristics, seen by increase in oxygen consumption and beat amplitude respectively. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2 and eliminated spontaneous beating. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics of hiPSC-CMs such as increase in contractility, oxygen consumption, and voltage peak maximum upstroke velocity. Further, by single-cell RNAseq, FOXO activated groups showed increase in cardiac maturational pathways compared against DMSO control groups.

Conclusions: These results show that p53 activation promotes FOXO and suppresses FOXM1, which modulate hiPSC-CM maturation in 3D suspension. Our study expands current understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3D culture system.