Berberine Attenuates Hypertrophic Phenotype in RAF1-Mutant Induced Pluripotent Stem Cell-Derived Cardiomyocytes through Suppression of ERK5-Cyclin D1 Signaling.

BACKGROUND: Noonan syndrome-associated hypertrophic cardiomyopathy (NS-HCM) occurs frequently in patients with RAF1 mutations, but the signaling mechanisms underlying the hypertrophic cardiomyocyte phenotype remain incompletely understood, and effective therapeutic targets remain to be defined. This study aimed to characterize MAPK signaling dysregulation in RAF1-mutant induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and to determine whether berberine (BBR) attenuates the hypertrophic phenotype through modulation of ERK5-Cyclin D1 signaling. METHODS: An in vitro NS-HCM cardiomyocyte model was established using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a patient harboring the RAF1 p.Ser257Leu mutation. Wild-type iPSC-CMs were generated from healthy donors. Immunofluorescence was used to assess cardiomyocyte surface area, sarcomeric organization, and Ki-67/EdU-based cell-cycle activity. Transcriptomic analyses of public NS-HCM datasets were performed to identify differentially expressed genes and enriched pathways. Western blotting was performed to evaluate the activation of MEK1/2-ERK1/2 and ERK5 signaling pathways, downstream Cyclin D1 expression, and hypertrophic markers including ANP and BNP. RAF1S257L/+ iPSC-CMs were treated with BBR, a MEK1/2 inhibitor (U0126), or a MEK5 inhibitor (BIX02189) to evaluate pathway modulation and hypertrophic phenotype. RESULTS: RAF1S257L/+ iPSC-CMs exhibited hallmark features of NS-HCM, including increased cell surface area, disrupted sarcomeric organization, and elevated expression of hypertrophic markers ANP and BNP. Transcriptomic analysis revealed significant enrichment of the MAPK pathway. Both MEK1/2-ERK1/2 and ERK5-Cyclin D1 pathways were markedly activated in RAF1S257L/+ iPSC-CMs, and treatment with U0126 or BIX02189 attenuated this activation and reduced cardiomyocyte hypertrophy. BBR treatment suppressed ERK5 phosphorylation and Cyclin D1 expression, decreased Ki-67 positivity, and similarly attenuated the hypertrophic phenotype. CONCLUSION: RAF1-mutant iPSC-CMs exhibited concurrent activation of the MEK1/2-ERK1/2 and ERK5-Cyclin D1 signaling pathways. BBR attenuated the hypertrophic phenotype while reducing ERK5 phosphorylation and Cyclin D1 expression without significantly altering MEK1/2-ERK1/2 signaling. These findings suggest that ERK5-Cyclin D1 signaling contributes to the hypertrophic phenotype of RAF1-mutant cardiomyocytes and support further investigation of BBR as a potential therapeutic candidate for RAF1-associated NS-HCM.