This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

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 (RyR2S/S) hearts show an atrial arrhythmic phenotype associated with reduced action potential (AP) conduction velocity and sodium channel (Nav1.5) expression. We now relate ventricular arrhythmogenicity and slowed AP conduction in RyR2S/S hearts to connexin-43 (Cx43) and Nav1.5 expression and Na+ current (INa). 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 RyR2S/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 RyR2S/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 Nav1.5 expression in both whole tissue and membrane fractions were significantly reduced in RyR2S/S compared to wild-type (WT). Loose patch-clamp studies similarly demonstrated reduced INa in RyR2S/S ventricles. We thus attribute arrhythmogenesis in RyR2S/S ventricles resulting from arrhythmic substrate produced by reduced conduction velocity to downregulated Nav1.5 reducing INa, 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 INa of the AP but nonlinear relationships between peak INa and maximum Na+ permeability.

AimsCardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav1.5 currents in homozygous RyR2-P2328S (RyR2S/S) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2S/S mice and their wild-type (WT) littermates.MethodsWe explored effects of 1 μm flecainide on WT and RyR2S/S atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na+ currents (INa) were recorded using loose patch clamping of superfused atria.ResultsRyR2S/S showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2S/S but pro-arrhythmic in WT. It increased INa in RyR2S/S atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2S/S, but reduced CV while sparing AERP in WT. Thus, RyR2S/S hearts have low λ relative to WT; flecainide then increases λ in RyR2S/S but decreases λ in WT.ConclusionsFlecainide (1 μm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca2+ release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

Calcium-mediated increases in Nedd4-2 downregulate Nav1.5 by ubiquitination. Nav1.5 is downregulated and co-localizes with Nedd4-2 and ubiquitin in failing rat heart. These data suggest a role of Nedd4-2 in Nav1.5 downregulation in HF.

Voltage-gated sodium (Nav) channels drive the rising phase of the action potential, essential for electrical signalling in nerves and muscles. The Nav channel α-subunit contains the ion-selective pore. In the cardiomyocyte, Nav1.5 is the main Nav channel α-subunit isoform, with a smaller expression of neuronal Nav channels. Four distinct regulatory β-subunits (β1–4) bind to the Nav channel α-subunits. Previous work has emphasised the β-subunits as direct Nav channel gating modulators. However, there is now increasing appreciation of additional roles played by these subunits. In this review, we focus on β-subunits as homophilic and heterophilic cell-adhesion molecules and the implications for cardiomyocyte function. Based on recent cryogenic electron microscopy (cryo-EM) data, we suggest that the β-subunits interact with Nav1.5 in a different way from their binding to other Nav channel isoforms. We believe this feature may facilitate trans-cell-adhesion between β1-associated Nav1.5 subunits on the intercalated disc and promote ephaptic conduction between cardiomyocytes.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels.

Mutations in the cardiac ryanodine receptor Ca 2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2 P2328S/P2328S (denoted RyR2 S/S ) mice. We have now examined P2328S RyR2 channels from RyR2 S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca 2+ ] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca 2+ ] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC 50 ) for Ca 2+ activation, from ∼3.5 µM Ca 2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC 50 ) for inactivation from ∼50 mM in WT channels to ≤7 μM in P2328S channels, which is into systolic [Ca 2+ ] levels. Unexpectedly, the shift in Ca 2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca 2+ homeostasis in myocytes from RyR2 S/S mice and the CPVT and AF phenotypes.

SummaryRecent papers have attributed arrhythmic substrate in murine RyR2‐P2328S hearts to reduced action potential (AP) conduction velocities (CV), reflecting acute functional inhibition and/or reduced expression of sodium channels. We explored for acute effects of direct exchange protein directly activated by cAMP (Epac)‐mediated ryanodine receptor‐2 (RyR2) activation on arrhythmic substrate and CV. Monophasic action potential (MAP) recordings demonstrated that initial steady (8 Hz) extrinsic pacing elicited ventricular tachycardia (VT) in 0 of 18 Langendorff‐perfused wild‐type mouse ventricles before pharmacological intervention. The Epac activator 8‐CPT (8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate) (VT in 1 of 7 hearts), and the RyR2 blocker dantrolene, either alone (0 of 11) or with 8‐CPT (0 of 9) did not then increase VT incidence (P>.05). Both progressively increased pacing rates and programmed extrasystolic (S2) stimuli similarly produced no VT in untreated hearts (n=20 and n=9 respectively). 8‐CPT challenge then increased VT incidences (5 of 7 and 4 of 8 hearts respectively; P<.05). However, dantrolene, whether alone (0 of 10 and 1 of 13) or combined with 8‐CPT (0 of 10 and 0 of 13) did not increase VT incidence relative to those observed in untreated hearts (P>.05). 8‐CPT but not dantrolene, whether alone or combined with 8‐CPT, correspondingly increased AP latencies (1.14±0.04 (n=7), 1.04±0.03 (n=10), 1.09±0.05 (n=8) relative to respective control values). In contrast, AP durations, conditions for 2:1 conduction block and ventricular effective refractory periods remained unchanged throughout. We thus demonstrate for the first time that acute RyR2 activation reversibly induces VT in specific association with reduced CV.