Abstract Body: Mutations in sarcomeric proteins such as MYH7 and MYBPC3 cause familial hypertrophic cardiomyopathy, yet the signaling pathways linking sarcomere dysfunction to cardiomyocyte hypertrophy remain poorly understood. We hypothesized that dysregulated kinase signaling cooperates with O-GlcNAcylation to regulate hypertrophic remodeling in cardiomyocytes carrying truncating MYBPC3 mutations.
Human iPSC–derived cardiomyocytes (hiPSC-CMs) from wild-type cells (WTC11) and cells carrying a truncating MYBPC3 mutation (c.1577_1580dupCACT; p.Cys528Thrfs*4) were analyzed by multi-electrode array contractility assays. MYBPC3-truncated hiPSC-CMs paced at 3 Hz exhibited increased beat amplitude at baseline (1.10±0.04% vs 0.92±0.04%, p=0.0056) and during β-adrenergic stimulation compared with wild-type cells (1.35±0.04% vs 1.14±0.04%, p≤0.005). In addition, prolonged excitation–contraction delay was observed during β-adrenergic stimulation (216.7±5.7 vs 178.7±7.4 ms, p≤0.05; n=24 wells per group).
Wild-type and MYBPC3-mutant hiPSC-CMs were exposed to phenylephrine to induce hypertrophy. Cellular hypertrophy was quantified using confocal microscopy and automated image analysis. In wild-type hiPSC-CMs, phenylephrine increased nuclear area by 12.4% compared with vehicle (p≤0.0001). Inhibition of SRC, ERK1/2, MEK1/2, or JAK2 reduced hypertrophy by 10–18% (p≤0.0001). In MYBPC3-mutant hiPSC-CMs, phenylephrine increased nuclear area by 9% compared with vehicle (111.3±2.6 μm² vs 101.3±2.5 μm² p≤0.036), and kinase inhibition alone did not suppress hypertrophic remodeling. Increasing O-GlcNAc signaling with the O-GlcNAcase inhibitor Thiamet G enhanced hypertrophy by 16.9% compared with phenylephrine alone (p≤0.0001). In contrast, combined inhibition of O-GlcNAc and kinase signaling reduced hypertrophy by 23.4% (SRC inhibition, p≤0.0001) and 10.7% (MEK1/2 inhibition, p≤0.0031).
These findings identify kinase and O-GlcNAc signaling as a cooperative regulatory axis driving hypertrophic remodeling and impaired stress adaptation in MYBPC3-mutant cardiomyocytes, supporting combined targeting of these pathways as a strategy to modulate disease-relevant phenotypes in hypertrophic cardiomyopathy.