Eleonora Savio-Galimberti

Role of common and rare variants in SCN10A: results from the Brugada syndrome QRS locus gene discovery collaborative study

AIMS Brugada syndrome (BrS) remains genetically heterogeneous and is associated with slowed cardiac conduction. We aimed to identify genetic variation in BrS cases at loci associated with QRS duration. METHODS AND RESULTS A multi-centre study sequenced seven candidate genes (SCN10A, HAND1, PLN, CASQ2, TKT, TBX3, and TBX5) in 156 Caucasian SCN5A mutation-negative BrS patients (80% male; mean age 48) with symptoms (64%) and/or a family history of sudden death (47%) or BrS (18%). Forty-nine variants were identified: 18 were rare (MAF <1%) and non-synonymous; and 11/18 (61.1%), mostly in SCN10A, were predicted as pathogenic using multiple bioinformatics tools. Allele frequencies were compared with the Exome Sequencing and UK10K Projects. SKAT methods tested rare variation in SCN10A finding no statistically significant difference between cases and controls. Co-segregation analysis was possible for four of seven probands carrying a novel pathogenic variant. Only one pedigree (I671V/G1299A in SCN10A) showed co-segregation. The SCN10A SNP V1073 was, however, associated strongly with BrS [66.9 vs. 40.1% (UK10K) OR (95% CI) = 3.02 (2.35-3.87), P = 8.07 × 10-19]. Voltage-clamp experiments for NaV1.8 were performed for SCN10A common variants V1073, A1073, and rare variants of interest: A200V and I671V. V1073, A200V and I671V, demonstrated significant reductions in peak INa compared with ancestral allele A1073 (rs6795970). CONCLUSION Rare variants in the screened QRS-associated genes (including SCN10A) are not responsible for a significant proportion of SCN5A mutation negative BrS. The common SNP SCN10A V1073 was strongly associated with BrS and demonstrated loss of NaV1.8 function, as did rare variants in isolated patients.

Voltage-gated sodium channels: biophysics, pharmacology, and related channelopathies

Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. They are primarily formed by a pore-forming multi-spanning integral membrane glycoprotein (α-subunit) that can be associated with one or more regulatory β-subunits. The latter are single-span integral membrane proteins that modulate the sodium current (INa) and can also function as cell adhesion molecules. In vitro some of the cell-adhesive functions of the β-subunits may play important physiological roles independently of the α-subunits. Other endogenous regulatory proteins named “channel partners” or “channel interacting proteins” (ChiPs) like caveolin-3 and calmodulin/calmodulin kinase II (CaMKII) can also interact and modulate the expression and/or function of VGSC. In addition to their physiological roles in cell excitability and cell adhesion, VGSC are the site of action of toxins (like tetrodotoxin and saxitoxin), and pharmacologic agents (like antiarrhythmic drugs, local anesthetics, antiepileptic drugs, and newly developed analgesics). Mutations in genes that encode α- and/or β-subunits as well as the ChiPs can affect the structure and biophysical properties of VGSC, leading to the development of diseases termed sodium “channelopathies”.  This review will outline the structure, function, and biophysical properties of VGSC as well as their pharmacology and associated channelopathies and highlight some of the recent advances in this field.

SCN10A/Nav1.8 modulation of peak and late sodium currents in patients with early onset atrial fibrillation

AIMS To test the hypothesis that vulnerability to atrial fibrillation (AF) is associated with rare coding sequence variation in the SCN10A gene, which encodes the voltage-gated sodium channel isoform NaV1.8 found primarily in peripheral nerves and to identify potentially disease-related mechanisms in high-priority rare variants using in-vitro electrophysiology. METHODS AND RESULTS We re-sequenced SCN10A in 274 patients with early onset AF from the Vanderbilt AF Registry to identify rare coding variants. Engineered variants were transiently expressed in ND7/23 cells and whole-cell voltage clamp experiments were conducted to elucidate their functional properties. Resequencing SCN10A identified 18 heterozygous rare coding variants (minor allele frequency ≤1%) in 18 (6.6%) AF probands. Four probands were carriers of two rare variants each and 14 were carriers of one coding variant. Based on evidence of co-segregation, initial assessment of functional importance, and presence in ≥1 AF proband, three variants (417delK, A1886V, and the compound variant Y158D-R814H) were selected for functional studies. The 417delK variant displayed near absent current while A1886V and Y158D-R814H exhibited enhanced peak and late (INa-L) sodium currents; both Y158D and R818H individually contributed to this phenotype. CONCLUSION Rare SCN10A variants encoding Nav1.8 were identified in 6.6% of patients with early onset AF. In-vitro electrophysiological studies demonstrated profoundly altered function in 3/3 high-priority variants. Collectively, these data strongly support the hypothesis that rare SCN10A variants may contribute to AF susceptibility.

Suppression of Spontaneous Ca Elevations Prevents Atrial Fibrillation in Calsequestrin 2-Null Hearts

Background—Atrial fibrillation (AF) risk has been associated with leaky ryanodine receptor 2 (RyR2) Ca release channels. Patients with mutations in RyR2 or in the sarcoplasmic reticulum Ca-binding protein calsequestrin 2 (Casq2) display an increased risk for AF. Here, we examine the underlying mechanisms of AF associated with loss of Casq2 and test mechanism-based drug therapy. Methods and Results—Compared with wild-type Casq2+/+ mice, atrial burst pacing consistently induced atrial flutter or AF in Casq2−/− mice and in isolated Casq2−/− hearts. Atrial optical voltage maps obtained from isolated hearts revealed multiple independent activation sites arising predominantly from the pulmonary vein region. Ca and voltage mapping demonstrated diastolic subthreshold spontaneous Ca elevations (SCaEs) and delayed afterdepolarizations whenever the pacing train failed to induce AF. The dual RyR2 and Na channel inhibitor R-propafenone (3 &mgr;mol/L) significantly reduced frequency and amplitude of SCaEs and delayed afterdepolarizations in atrial myocytes and intact atria and prevented induction of AF. In contrast, the S-enantiomer of propafenone, an equipotent Na channel blocker but much weaker RyR2 inhibitor, did not reduce SCaEs and delayed afterdepolarizations and failed to prevent AF. Conclusions—Loss of Casq2 increases risk of AF by promoting regional SCaEs and delayed afterdepolarizations in atrial tissue, which can be prevented by RyR2 inhibition with R-propafenone. Targeting AF caused by leaky RyR2 Ca channels with R-propafenone may be a more mechanism-based approach to treating this common arrhythmia.

Channel Activity of Cardiac Ryanodine Receptors (RyR2) Determines Potency and Efficacy of Flecainide and R-Propafenone against Arrhythmogenic Calcium Waves in Ventricular Cardiomyocytes.

Flecainide blocks ryanodine receptor type 2 (RyR2) channels in the open state, suppresses arrhythmogenic Ca2+ waves and prevents catecholaminergic polymorphic ventricular tachycardia (CPVT) in mice and humans. We hypothesized that differences in RyR2 activity induced by CPVT mutations determines the potency of open-state RyR2 blockers like flecainide (FLEC) and R-propafenone (RPROP) against Ca2+ waves in cardiomyocytes. Using confocal microscopy, we studied Ca2+ sparks and waves in isolated saponin-permeabilized ventricular myocytes from two CPVT mouse models (Casq2-/-, RyR2-R4496C+/-), wild-type (c57bl/6, WT) mice, and WT rabbits (New Zealand white rabbits). Consistent with increased RyR2 activity, Ca2+ spark and wave frequencies were significantly higher in CPVT compared to WT mouse myocytes. We next obtained concentration-response curves of Ca2+ wave inhibition for FLEC, RPROP (another open-state RyR2 blocker), and tetracaine (TET) (a state-independent RyR2 blocker). Both FLEC and RPROP inhibited Ca2+ waves with significantly higher potency (lower IC50) and efficacy in CPVT compared to WT. In contrast, TET had similar potency in all groups studied. Increasing RyR2 activity of permeabilized WT myocytes by exposure to caffeine (150 µM) increased the potency of FLEC and RPROP but not of TET. RPROP and FLEC were also significantly more potent in rabbit ventricular myocytes that intrinsically exhibit higher Ca2+ spark rates than WT mouse ventricular myocytes. In conclusion, RyR2 activity determines the potency of open-state blockers FLEC and RPROP for suppressing arrhythmogenic Ca2+ waves in cardiomyocytes, a mechanism likely relevant to antiarrhythmic drug efficacy in CPVT.