Abstract Body: Introduction: Inherited cardiomyopathies caused by mutations in calcium-handling proteins frequently produce arrhythmogenic substrates with unpredictable responses to antiarrhythmic drugs. The phospholamban R14del mutation is strongly associated with dilated cardiomyopathy, heart failure, and malignant ventricular arrhythmias, yet the electrophysiological phenotype and pharmacologic responses at the single cardiomyocyte level remain incompletely defined.

Methods: Human induced pluripotent stem cell-derived cardiomyocytes were generated from an isogenic pair consisting of wild-type cells and a CRISPR-engineered phospholamban R14del mutant line. High-throughput optical voltage imaging was used to record action potentials from thousands of individual cardiomyocytes and classify electrophysiological phenotypes using blinded morphology-based categorization. Pharmacological perturbations targeted late sodium current using GS967 and transient outward potassium current using NS5806 and acacetin. Action potential morphologies, including normal waveforms, prolonged action potentials, and early afterdepolarizations were quantified across treatment conditions.

Results: Phospholamban R14del cardiomyocytes exhibited a baseline arrhythmogenic phenotype characterized by a reduced proportion of normal action potentials and increased prolonged action potentials and early afterdepolarizations compared with wild-type cells. Pharmacological perturbations revealed striking genotype-dependent responses. GS967 produced moderate electrophysiological remodeling in wild-type cardiomyocytes but markedly exacerbated arrhythmogenic phenotypes in phospholamban R14del cells, including progressive loss of normal action potentials and increased prolonged morphologies. NS5806 induced severe electrophysiological destabilization in the mutant genotype with near-complete loss of normal action potentials at high concentrations. In contrast, acacetin preserved electrophysiological stability in wild-type cardiomyocytes but failed to restore normal electrophysiology in phospholamban R14del cells.

Conclusions: Phospholamban R14del cardiomyopathy creates a distinct electrophysiological substrate that fundamentally alters antiarrhythmic drug responsiveness. These findings highlight the importance of genotype-specific drug evaluation and demonstrate the value of patient-derived cardiomyocyte models for precision medicine approaches in inherited cardiomyopathies.