Heart World Conference 2026

Abstract

Cardiac output and rhythm depend on the release and the take-up of Ca²⁺ from the sarcoplasmic reticulum (SR) [1]. Excessive diastolic Ca²⁺ leak from the SR due to dysfunctional Ca²⁺ release channels (Ryanodine channels, RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias [2]. Diastolic SR Ca²⁺ leak has been linked to arrhythmogenesis in both the inherited arrhythmia syndrome 'catecholaminergic polymorphic ventricular tachycardia' (CPVT) and acquired forms of heart disease such as heart failure (HF) [3]. Advanced HF fosters an environment of arrhythmogenicity from increased sympathetic nervous system tone, noradrenaline concentration and activation of β₁- and β₂-adrenoceptors (ARs) [4]. The hyperadrenergic state of HF results in leaky RyR2 channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6 [5]. Hence, stabilization of the RyR2 closed state during diastole, resulting in suppression of abnormal SR Ca²⁺ leak, is a promising therapeutic strategy against HF and lethal arrhythmias [6].

Phenytoin, commonly used to treat epilepsy, has shown potential as an antiarrhythmic by inhibiting diastolic Ca²⁺ leak in failing human cardiomyocytes [7]. Importantly, its inhibitory effect appears selective for RyR2 channels from failing hearts and not from healthy tissue, suggesting that its action may depend on RyR2's phosphorylation status [7]. Similar behaviour has been observed with other RyR2 modulators like dantrolene and calmodulin inhibitors [8].

This study explored phenytoin's inhibitory mechanism using single-channel recordings and Western blotting. RyR2 was isolated from β-blocked (n=4) and non-β-blocked (n=5) failing human hearts as described previously (9). RyR2s from β-blocked hearts (pooled from 4 hearts) were also stimulated with 10 mM noradrenaline for 15 mins to mimic adrenergic stress. RyR2 were incorporated into lipid bilayers and the channel gating was measured at diastolic [Ca²⁺]. Under these conditions, Phenytoin at 30 μM produced ~20% inhibitory effect within non-β-Blocked (p=0.006) and Stimulated β-Blocked (p ≤ 0.0001) cohorts, where activation of RyR2 was shown in the β-Blocked cohort (p=0.005). The inhibition was associated with higher relative phosphorylation levels in non-β-Blocked (S2808: 84.28 ± 2.05%, S2814: 82.96 ± 2.00%) and stimulated β-Blocked (S2808: 97.49 ± 3.90%, S2814: 90.74 ± 4.29%) hearts, whereas β-Blocked hearts exhibit lower phosphorylation levels (S2808: 61.89 ± 1.78%, S2814: 62.77 ± 1.83%). To further test this dependency, RyR2s were dephosphorylated using PP1 or rephosphorylated using PKA or endogenous CaMKII. Dephosphorylation abolished phenytoin’s effect in non-β-blocked hearts (p=0.36), while phosphorylation at S2808 and S2814 restored its inhibitory action (p=0.0007 and p=0.0002, respectively). In β-blocked hearts, phosphorylation reinstated phenytoin’s inhibition by ~20% (p=0.004).

These findings demonstrate that phenytoin’s ability to inhibit RyR2 is strongly dependent on the receptor's phosphorylation state. Thus, while phenytoin may be effective in HF patients with elevated adrenergic activity, it may be unsuitable for those receiving β-blockers, where RyR2 phosphorylation is reduced and the drug may paradoxically increase channel activity.