The RyR2-P2328S mutation downregulates Na_v1.5 producing arrhythmic substrate in murine ventricles

Catecholaminergic polymorphic ventricular tachycardia (CPVT) predisposes to ventricular arrhythmia due to altered Ca²⁺ homeostasis and can arise from ryanodine receptor (RyR2) mutations including RyR2-P2328S. Previous reports established that homozygotic murine RyR2-P2328S (RyR2^S/S) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Na_v1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2^S/S hearts to connexin-43 (Cx43) and Na_v1.5 expression and Na⁺ current (I_Na). Stimulation protocols applying extrasystolic S2 stimulation following 8 Hz S1 pacing at progressively decremented S1S2 intervals confirmed an arrhythmic tendency despite unchanged ventricular effective refractory periods (VERPs) in Langendorff-perfused RyR2^S/S hearts. Dynamic pacing imposing S1 stimuli then demonstrated that progressive reductions of basic cycle lengths (BCLs) produced greater reductions in conduction velocity at equivalent BCLs and diastolic intervals in RyR2^S/S than WT, but comparable changes in AP durations (APD₉₀) and their alternans. Western blot analyses demonstrated that Cx43 protein expression in whole ventricles was similar, but Na_v1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2^S/S compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced I_Na in RyR2^S/S ventricles. We thus attribute arrhythmogenesis in RyR2^S/S ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Na_v1.5 reducing I_Na, despite normal determinants of repolarization and passive conduction. The measured changes were quantitatively compatible with earlier predictions of linear relationships between conduction velocity and the peak I_Na of the AP but nonlinear relationships between peak I_Na and maximum Na⁺ permeability.