Sustained β-catenin activation via GSK3 inhibition promotes direct fibroblast-to-cardiomyocyte reprogramming

During iPSC differentiation, cardiomyocyte formation requires transient activation of canonical Wnt/β-catenin signaling followed by pathway inhibition. Inspired by the mechanisms governing iPSC differentiation into cardiomyocytes, this study sought to explore whether these pathways similarly affect direct fibroblast-to-cardiomyocyte reprogramming. In contrast to iPSC differentiation, direct reprogramming required early inhibition of glycogen synthase kinase-3 (GSK3), which leads to activation β-catenin signaling, without a subsequent requirement for pathway suppression. Pharmacological GSK3 inhibition enhanced cardiomyocyte yield and improved functional maturation, as demonstrated by optical calcium mapping. Reporter assays confirmed increased β-catenin transcriptional activity, while non-canonical Wnt signaling was unaffected. Transcriptomic analysis revealed enhanced enrichment of muscle-associated gene ontology terms and modulation of immune and signaling pathways following GSK3 inhibition. Together, these findings suggest that although indirect and direct cardiac reprogramming share elements of Wnt pathway involvement, their temporal and mechanistic requirements differ substantially.