A truncating SCN5A mutation combined with genetic variability causes sick sinus syndrome and early atrial fibrillation

Abstract

Background: Mutations in the SCN5A gene, encoding the α subunit of the cardiac Na(+) channel, Nav1.5, can result in several life-threatening arrhythmias.

Objective: To characterize a distal truncating SCN5A mutation, R1860Gfs*12, identified in a family with different phenotypes including sick sinus syndrome, atrial fibrillation (AF), atrial flutter, and atrioventricular block.

Methods: Patch-clamp and biochemical analyses were performed in human embryonic kidney 293 cells transfected with wild-type (WT) and/or mutant channels.

Results: The mutant channel expressed alone caused a 70% reduction in inward sodium current (INa) density compared to WT currents, which was consistent with its partial proteasomal degradation. It also led to a negative shift of steady-state inactivation and to a persistent current. When mimicking the heterozygous state of the patients by coexpressing WT and R1860Gfs*12 channels, the biophysical properties of INa were still altered and the mutant channel α subunits still interacted with the WT channels. Since the proband developed paroxysmal AF at a young age, we screened 17 polymorphisms associated with AF risk in this family and showed that the proband carries at-risk polymorphisms upstream of PITX2, a gene widely associated with AF development. In addition, when mimicking the difference in resting membrane potentials between cardiac atria and ventricles in human embryonic kidney 293 cells or when using computer model simulations, R1860Gfs*12 induced a more drastic decrease in INa at the atrial potential.

Conclusion: We have identified a distal truncated SCN5A mutant associated with gain- and loss-of-function effects, leading to sick sinus syndrome and atrial arrhythmias. A constitutively higher susceptibility to arrhythmias of atrial tissues and genetic variability could explain the complex phenotype observed in this family.

Keywords: Arrhythmia; Atrial fibrillation; Na(v)1.5; PITX2; Polymorphism; SCN5A; SNP; Sodium.

Figure 1. Pedigree and ECG recordings of the family

(A) Pedigree of the family. The proband (III.2) is indicated by an arrow. Squares represent males, circles females, + and − signs, mutation carriers and non-carriers. The haplotypes for 5 SNPs located upstream of the PITX2 gene are indicated under each subject. At-risk alleles are written in red and protective ones in blue. (B) ECG recordings of the proband. The pacemaker spikes are indicated by arrows. The proband's ECG showed prolongation of the PR interval, a sign of first degree AV block. (C) ECG recordings of the father (II.2) showing the atrial flutter that he developed at 42 (a), and his normal sinus rhythm after cardioversion with left axis deviation (b). (c and d) ECG recordings of II-2 during the ajmaline test (1mg/kg over 5 min) he underwent at 64. At basal condition (c, time 0), the patient has a regular sinus rhythm with a heart rate (HR) of 60 beats/min, a prolongation of the PR interval of 240 ms, and a QRS duration of 120 ms. After the injection of ajmaline (d, time 7 min), bradycardia was observed (HR= 40 beats/min) with a prolongation of the PR interval (300 ms) and of the QRS duration (240 ms), without sign of BrS. Rapid sodium-bicarbonate infusion was then immediately started.

Figure 2. Electrophysiological characterization of Na_v1.5 channels (1)

(A) Representative families of Na⁺ current traces of WT, R1860Gfs*12 and WT + R1860Gfs*12 channels. (B) Current density-voltage relationships. R1860Gfs*12 current density is significantly reduced compared to WT and WT + R1860Gfs*12 channels. (C) Activation-V_m relationship of R1860Gfs*12 was shifted to more positive potentials compared to WT and WT + R1860Gfs*12 channels. (D) Steady-state inactivation-V_m relationships were shifted towards negative potential for R1860Gfs*12 and WT + R1860Gfs*12 channels compared to WT channels. Numbers of cells and statistical analysis are reported in Table 1.

Figure 3. Electrophysiological characterization of Na_v1.5 channels (2)

(A) and (B) Fast and slow time constants (τ_f, and τ_s, respectively) for WT, R1860Gfs*12 and WT + R1860Gfs*12 channels were plotted against the test potential. R1860Gfs*12 and WT + R1860Gfs*12 showed an overall slowing of the fast and slow rates of inactivation compared to the WT. (C) Three superimposed representative I_Na traces corresponding to the above conditions. The R1860Gfs*12 and the WT + R1860Gfs*12 current traces were normalized to the peak of the WT current (≈ 1200 pA). (D) Ratios of the persistent Na⁺ current amplitude (end of pulse) over the peak I_Na amplitude for WT, WT + R1860Gfs*12 and R1860Gfs*12 channels, reported in percentage. (E) Ratios of the I_Na loss between HPs of -86 and -83 mV for WT, WT+ R1860Gfs*12 and R1860Gfs*12. Peak current density was calculated at -20 mV. * indicates P≤0.05, ** P≤0.01, *** P≤0.001, and n the number of tested cells.

Figure 4. Computer model simulations of single atrial cell and ventricular cell membrane action potentials

Computed AP time courses and expanded time-scale inset exhibiting first time derivatives (dV/dt) of the AP upstroke, in A: atrium and in B: ventricle. Computed dV/dt time courses exhibit a higher decrease in [dV/dt]_max for the “heterozygous atrium” condition than for WT (respectively: 68 V/s vs. 212 V/s) whereas the decrease was smaller for “heterozygous ventricle” condition than for WT (respectively: 147 V/s vs. 220 V/s).

Figure 5. Biochemical analysis

HEK cells were transfected with GFP-Na_v1.5 (A) and with GFP-Na_v1.5 and/or Flag-Na_v1.5 (B). Data are represented as arbitrary units. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as total protein loading control. (A) Western blots of total cell lysates show that the R1860Gfs*12 protein quantity is significantly lower than the WT. Incubation of transfected cells with MG132 prevented the degradation of the C-terminal mutant; ***P≤0.001. (B) Co-immunoprecipitation of Na_v1.5 α-subunits tagged with either Flag or GFP. Immunoprecipitation was performed with the anti-GFP antibody, and western blot revealed with the anti-Flag antibody. These results demonstrated an interaction between R1860Gfs*12 α-subunits, and between WT and R1860Gfs*12 α-subunits.