Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action

Abstract

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.

How does flecainide impact RyR2 channel function?

“…Voltage-independent RyR2 inhibition by flecainide was observed in a series of experiments on RyR2 channels from WT and homozygous RyR2-P2328S +/+ mouse hearts (Table 1; Salvage et al, 2021a;Dulhunty et al, 2022). These experiments were performed using channels weakly activated to end-diastolic levels by a physiological [Ca 2+ ] of 1 µM on the cytoplasmic side and 1 mM on the luminal side, notably in the absence of ATP or Mg 2+ , or other channel modulators.…”

“…In addition to its inhibition of RyR2 and its classical IC inhibitory actions on peak I Na (Na V 1.5), flecainide inhibits the skeletal muscle I Na (Na V 1.4; Desaphy et al, 2004) and a number of potassium channels including K V 1.5 (I Kur ), K V 4.2 (I Tof ), and K V 11.1/hERG (I Kr ) as detailed in Salvage et al (2018), as well as directly activating NCX1 (Kuroda et al, 2017). As such it is not surprising that there may be several binding sites on RyR2, which is the largest of the known ion channels with a vast exposed cytoplasmic surface, which could convey multiple contrasting inhibitory and activating effects (Mehra et al, 2014;Salvage et al, 2021a;Dulhunty et al, 2022). This section, along The positively charged flecainide when added to the cytoplasmic solution moves into the pore where it is too large to pass through the selectivity filter and remains trapped (Bannister et al, 2021).…”

Molecular Changes in the Cardiac RyR2 With Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)

“…This blocking action of flecainide would presumably reduce current flowing through RyR2 during systole and diastole and be independent of the location of the mutation. Flecainide can also activate RyR2 channels at low concentrations, or inhibit at higher concentrations, independently of the direction of current flow ( Salvage et al, 2021 ; Dulhunty et al, 2022 ). This indicates binding to RyR2 at cytoplasmic sites distant from the pore, however, the location of these sites is as yet unknown.…”

The Antiarrhythmic Mechanisms of Flecainide in Catecholaminergic Polymorphic Ventricular Tachycardia

“…More recently, the study by Salvage et al showed that low cytoplasmic concentrations (0.5–10 μM) of flecainide activated isolated mouse RyR2 channels. In contrast, high cytoplasmic concentrations (50–100 μM) of flecainide showed an inhibitory action ( Salvage et al, 2021 ).…”