Loss-of-function RyR2 mutations cause various types of arrhythmias through divergent abnormal Ca²⁺ signaling

Abstract

Loss-of-function (LOF) mutations in type 2 ryanodine receptor (RyR2) have been linked to various types of cardiac arrhythmias including idiopathic ventricular fibrillation (IVF), short-coupled variant of Torsades de Pointes (scTdP) and long QT syndrome (LQTS). However, cellular mechanisms of divergent phenotypes remain unclear. We investigated Ca²⁺ signaling by four LOF RyR2 mutants, E4146D (ED), S4168P (SP), K4594Q (KQ) and S4938F (SF), in HEK293 cells and HL-1 cardiomyocytes. Patients with ED and SF showed VF and scTdP, whereas those with SP and KQ showed LQTS without life-threatening events. ED and SF moderately suppressed [³H]ryanodine binding with reduced Ca²⁺ sensitivity, and SP and KQ almost completely suppressed the binding. In HEK293 cells which express homotetrameric RyR2s, WT cells showed cytosolic ([Ca²⁺]_cyt) oscillations with concomitant decrease in ER Ca²⁺ ([Ca²⁺]_ER), but all the LOF mutant cells showed elevated [Ca²⁺]_ER with no [Ca²⁺]_cyt oscillations. In HL-1 cells, which have endogenous WT RyR2s, expression of SP or KQ exerted no Ca²⁺ waves but only reduced Ca²⁺ transients. In contrast, ED and SF exerted frequent localized Ca²⁺ waves. These results suggest that moderate suppression of RyR2 may cause abnormal Ca²⁺ signaling leading to scTdP and VF, whereas severe suppression results in less arrhythmic events. Ca²⁺ signaling by heterotetrameric channels composed of WT and mutant RyR2s need to be further clarified.