Substrate stiffness attenuates cardiomyocyte depolarization slope via sodium channel kinetics modulation

The excessive deposition of extracellular matrix (ECM) components in cardiac tissue increases myocardial stiffness, contributing to diastolic dysfunction and arrhythmia risk. While this clinical association is established, the underlying electromechanical mechanisms remain elusive. Using human and neonatal rat cardiomyocytes cultured on tunable 3D PDMS substrates replicating infarcted rat heart decellularized ECM (dECM) stiffness, we systematically investigated how ECM-mediated mechanical changes alter action potentials and sodium current dynamics, as well as Nav1.5 expression. 3D PDMS substrates were fabricated with stiffness values spanning the mechanical range of infarcted rat heart dECM (Young's modulus: ∼20-400 kPa; elastic modulus: ∼5-30 pN/nm). Human induced pluripotent stem cell-derived cardiomyocytes (15-day culture), neonatal rat cardiomyocytes (3-day culture) and human embryonic kidney 293 (24-hour culture) cells stably expressing human Nav1.5 were cultured on the these PDMS substrates. Stiffness-dependent cellular electrophysiological effects were assessed using patch-clamp recordings. The gene and protein level changes were assessed by real-time quantitative polymerase chain reaction and immune blot. We found that evaluated substrate stiffness levels progressively reduced the AP upstroke slope, resulted in proarrhythmic AP morphologies characterized by increased AP instability and triangulation. Electrophysiological analysis revealed this mechanical modulation occurred through distinct sodium channel kinetic alterations: (1) a significant rightward-shifted voltage-dependent activation curve, and (2) a faster transition from a closed state to inactivation occurred, while maintaining unchanged current density, steady-state inactivation properties, and recovery time across stiffness conditions. This effect may arise from a conformation change in Nav1.5, since the expression and distribution of Nav1.5 has not altered across three PDMS groups. In summary, substrate stiffening impaired cardiomyocyte depolarization and promoted conduction abnormalities via Nav1.5 dysfunction, suggesting a mechanoelectrical mechanism in ECM deposition related arrhythmogenesis. Engineered substrate stiffness caused Nav1.5 dysfunction and promoted arrhythmia risk.Abbreviations: PDMS: polydimethylsiloxane; hiPSC-CMs: human induced pluripotent stem cell–derived cardiomyocytes; HEK293: human embryonic kidney 293 cells. • Increased stiffness of the substrate causes the dysfunction of Na v 1.5 kinetics. • Substrate stiffness increased arrhythmia risks of cardiomyocytes. • Substrate stiffness did not change Nav1.5 expression or localization. • Stiffness-induced Nav1.5 dysfunction may be mechanosensitive.