Identification of loss-of-function RyR2 mutations associated with idiopathic ventricular fibrillation and sudden death

Abstract

Mutations in cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). Most CPVT RyR2 mutations characterized are gain-of-function (GOF), indicating enhanced RyR2 function as a major cause of CPVT. Loss-of-function (LOF) RyR2 mutations have also been identified and are linked to a distinct entity of cardiac arrhythmia termed RyR2 Ca2+ release deficiency syndrome (CRDS). Exercise stress testing (EST) is routinely used to diagnose CPVT, but it is ineffective for CRDS. There is currently no effective diagnostic tool for CRDS in humans. An alternative strategy to assess the risk for CRDS is to directly determine the functional impact of the associated RyR2 mutations. To this end, we have functionally screened 18 RyR2 mutations that are associated with idiopathic ventricular fibrillation (IVF) or sudden death. We found two additional RyR2 LOF mutations E4146K and G4935R. The E4146K mutation markedly suppressed caffeine activation of RyR2 and abolished store overload induced Ca2+ release in HEK293 cells. E4146K also severely reduced cytosolic Ca2+ activation and abolished luminal Ca2+ activation of single RyR2 channels. The G4935R mutation completely abolished caffeine activation of and [3H]ryanodine binding to RyR2. Co-expression studies showed that the G4935R mutation exerted dominant negative impact on the RyR2 wildtype channel. Interestingly, the RyR2-G4935R mutant carrier had a negative EST, and the E4146K carrier had a family history of sudden death during sleep, which are different from phenotypes of typical CPVT. Thus, our data further support the link between RyR2 LOF and a new entity of cardiac arrhythmias distinct from CPVT.

The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca2+ and Mg2+

“…CPVT mutations can sometimes lead to a reduction in the expression levels of RyR2 protein (for example, Zhong et al . 2021) and so we examined V2475F(+/‐) RyR2 levels by immunoblot analysis (Figs 6 and 6). The V2475F mutation did not cause a reduction in RyR2 levels, suggesting that all possible combinations of mutated monomers shown in Fig.…”

A Type 2 Ryanodine Receptor Variant in the Helical Domain 2 Associated with an Impairment of the Adrenergic Response

“…Interestingly, reported LOF RyR2 mutations resulted in a loss or a reduction in luminal Ca 2+ activation as occurs in our variant [ 32 , 33 ]. Furthermore, two murine models with heterozygous LOF RyR2 mutations inducing a loss in luminal Ca 2+ activation, A4860G+/− and D4646A +/−, have been studied [ 31 , 63 ].…”

“…These gain of function (GOF) mutations are mostly located in four specific domains [ 28 , 29 ]. In addition, loss of function (LOF) RyR2 mutations have also been identified and recently linked to a distinct entity of cardiac arrhythmia termed ‘RyR2 Ca 2+ release deficiency syndrome’ (CRDS) [ 27 , 30 , 31 , 32 , 33 , 34 ]). The CRDS patients show a normal exercise stress test (EST) and display syncope and sudden death due to ventricular fibrillation [ 31 ]).…”

“Ryanopathies” and RyR2 dysfunctions: can we further decipher them using in vitro human disease models?

“…He was successfully treated with Metoprolol and Levetiracetam. [ 51 ] C2277R The RyR2-C2277R variant, located in the calstabin-binding domain, was identified in 8 members of the same family. The proband and her other family members presented ventricular extrasystoles (VE), bigeminy and/or trigeminy, doublets and non-sustained VT upon exercise stress test and adrenaline test.…”

“…To compare with CPVT RyR2 GOF mutations, RyR2-E4146K and -G4935R mutations induce cardiac arrhythmias under non-stressful conditions and are silent under exercise stress testing. Therefore, RyR2 LOF mutations are associated with a distinct phenotype than CPVT and constitute a new entity of cardiac arrythmias [ 51 ].…”