Abstract 10415: Dysregulation of FOXO-FOXM1 Signaling Inhibits Maturation of IPSC-Derived Cardiomyocytes in 3D Suspension Culture

Abstract

Directed differentiation of human pluripotent stem cells (PSC) into cardiomyocytes via manipulation of Wnt signaling leads to generation of immature cardiomyocytes, more closely resembling a fetal state. It has become increasingly apparent that metabolic parameters regulate of cardiomyocyte maturation. The forkhead box (FOX) family of transcription factors has previously been shown to regulate metabolic phenotype in neonatal cardiomyocytes through a balance between FOXO and FOXM proteins. Therefore, we hypothesized that dysregulation of FOXO-FOXM1 signaling inhibits maturation of iPSC-derived cardiomyocytes (iPSC-CMs). We cultured iPSC-CMs in 3D suspension culture using an orbital shaker. iPSC-CMs were treated with RCM-1 (FOXM1 inhibitor), LOM612 (FOXO nuclear translocator), or AS1842856 (FOXO inhibitor) starting 2 days after onset of beating. We found that inhibition of FOXO with AS1842856 resulted in loss of expression of cardiac-specific markers such as cardiac troponin T as well as loss of spontaneous beating. In contrast, inhibition of FOXM1 with RCM-1 or activation of FOXO with LOM612 resulted in retention of a cardiomyocyte phenotype with continued expression of cardiac troponin T but with significantly increased expression membrane protein expression of Kir2.1 (Figure), the protein largely responsible for maintaining the resting membrane potential in cardiomyocytes. These results suggest that inhibition of FOXM1 and/or activation of FOXO signaling may facilitate maturation of iPSC-derived cardiomyocytes. Figure. (A) % of TNNT2+ iPSC-derived cardiomyocytes that express Kir2.1 and (B) Mean fluorescence intensity of Kir2.1 in iPSC-derived cardiomyocytes after treatment with DMSO (vehicle control), RCM-1, LOM612, or AS1842856. *p<0.05, ***p<0.001, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test.

Unlocking the Molecular Cues for Cardiomyocyte Proliferation

“…Inhibiting FOXO1 also results in a reduction in the expression of maturation-associated hallmarks of CMs, including MYH7, MYL2, and NPPA, in 2D-cultured hiPSC-CMs . Moreover, the inhibition of FOXO1 leads to the loss of expression of the cardiac-specific marker cTnT and the loss of spontaneous beating in 3D suspension-cultured hiPSC-CMs (Garbern et al, 2021).…”