Pharmacological modulation of GLI signaling promotes human iPSC-derived cardiomyocyte maturation via PGC1α-associated mitochondrial programs.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a scalable platform for cardiovascular research but remain structurally and functionally immature, limiting their utility for studying cardiac biology, disease mechanisms, and clinical translation. Developmental signaling pathways that coordinate cardiomyocyte maturation remain incompletely understood. Here, we investigated whether pharmacological modulation of GLI signaling promotes a shift toward a more mature hiPSC-CM phenotype through PGC1α-linked mitochondrial and metabolic programs using both 2D and 3D engineered models. Treatment of hiPSC-CMs with GANT61 led to coordinated changes consistent with relative cardiomyocyte maturation, including increased cellular anisotropy, improved sarcomeric organization, reduced spontaneous beating rate, and advanced action potential characteristics. In contrast, pharmacological or genetic activation of GLI signaling suppressed maturation-associated phenotypes. In 3D-engineered heart tissues (EHTs), GLI modulation improved cellular alignment, tissue organization and contractile function. Transcriptomic and gene expression analyses revealed enrichment of mitochondrial and metabolic pathways, including increased expression of PGC1α, NRF1, and MFN2, together with upregulation of fatty acid oxidation-related gene programs. Confocal imaging demonstrated more extensive mitochondrial networks with increased branching and connectivity. Supporting these findings, we found elevated mtND1 and mtND5 levels, consistent with increased mitochondrial DNA copy number and mitochondrial biogenesis. Mechanistically, GANT61 increased MEF2A transcripts with a concomitant induction in protein levels, and motif analysis identified MEF2A binding sites in the proximal PGC1α promoter, suggesting a putative role for MEF2A in mediating PGC1α expression. Furthermore, pharmacological inhibition of PGC1α attenuated GANT61-induced structural and functional changes, supporting a PGC1α-dependent mechanism. Collectively, these findings identify GLI signaling as a modulatory pathway influencing the structural, electrophysiological, and mitochondrial gene programs associated with hiPSC-CM maturation and highlight GLI modulation as a strategy to enhance the physiological relevance of hiPSC-derived cardiac models.