PMC7396896_01
(A) Timeline of diagnostic interventions for the described case. ECG traces of (B) the proband on the day of diagnosis with absent atrial activity with junctional escape, (C) repeated during fever at 39 C which unmasked BrS ECG pattern with elevated ST segment, (D) the father carrying the SCN5A founder mutation with absent BrS ECG type-1 pattern after ajmaline challenge, but (E) present during fever at 39 C (visible in lead V1), and (F) the asymptomatic aunt carrying theSCN5A founder mutation with ECG type-1 pattern after ajmaline challenge. (G) Pedigree of the family showing the segregation of the identified variants. Full symbols indicate the affected individuals. The red plus and minus symbols indicate, respectively, the presence or the absence of the variant indicated on the left side of the pedigree.
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F1
Based on the ECG, he was diagnosed with sick sinus syndrome (SSS) with junctional escape (Figure 1B). Although during follow-up no ventricular tachyarrhythmias were documented, an ECG taken during fever (39 C) unmasked the characteristic BrS type-1 pattern (Figure 1C), and the boy received an endovascular VVI-ICD as there is a high estimated risk of developing ventricular arrhythmias (timeline: Figure 1A). His ajmaline challenge test was negative, but an ECG taken during a fever episode showed a BrS coved-shape ECG pattern (Figures 1D,E). The sister of the father was diagnosed with an ajmaline-induced BrS type-1 ECG pattern (Figure 1F). A familial segregation analysis revealed the founder mutation in the proband's father (II:3) and paternal aunt (II:2) and de novo occurrence of the p.Phe1571Leu variant in the index patient (III:1) (Figure 1G).
(A) Schematic representation of the Nav1.5 structure with location of the p.Phe1571Leu variant (big full red circle) and p.Arg1632His (small red circle). The alignment shows the conservation of Phe at position 1571 in SCN5A amino acid sequence between species. The accession numbers for the UniProt database are indicated on the left side. (B) Electrophysiological properties of WT Nav1.5 and Nav1.5-F1571L alone or co-expressed with beta1. Displayed from left to right are the representative whole-cell ionic current recordings of WT Nav1.5, Nav1.5-F1571L, WT Nav1.5 + beta1, and Nav1.5-F1571L + beta1. The Na+-selective currents were elicited with the pulse protocol shown on the left above the traces. The horizontal bar at the beginning of the traces indicates zero current level. (C) Current density of WT Nav1.5 (black circles, n = 8), WT Nav1.5 + beta1 (gray circles, n = 9), Nav1.5-F1571L (full red inverted triangles, n = 7), and Nav1.5-F1571L + beta1 (open red inverted triangles, n = 9) were obtained by normalizing the peak current amplitudes from pulse protocols shown in (B) to the cell capacitance. (D) Voltage dependence of channel activation, G-V curves, are represented for WT Nav1.5, Nav1.5-F1571L, WT Nav1.5 + beta1, and Nav1.5-F1571L + beta1. (E) The voltage-dependent kinetics of channel activation are shown as means +- SEM.
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F2
Phenylalanine at position 1,571 is highly conserved between species (Figure 2A), and in silico prediction on the functional impact of the variant is possibly damaging (MutationTaster, SIFT, PolyPhen; ENST00000413689.5). The current-voltage (I-V) relationship showed that the current densities for the Nav1.5 and Nav1.5-F1571L, either expressed alone or in presence of beta1, did not differ (Figures 2B,C). The activation kinetics as well as the voltage dependence of channel activation (G-V curves) did not show significant differences, yielding a V1/2 of -13.8 +- 1.7 and -14.9 +- 0.9 mV, with slope factors of 14.5 +- 2.4 and 14 +- 1.3 mV for Nav1.5 and Nav1.5-F1571L, respectively (Figures 2D,E).
Inactivation properties of WT Nav1.5 and Nav1.5-F1571L alone or co-expressed with beta1. (A) Displayed from left to right are the representative whole-cell inactivation recordings of WT Nav1.5 + beta1 and Nav1.5-F1571L + beta1. The INa were elicited with the pulse protocol shown underneath the traces. (B) The time constants of channel inactivation for WT Nav1.5 (black circles, n = 5), WT Nav1.5 + beta1 (gray circles, n = 12), Nav1.5-F1571L (full red inverted triangles, n = 9), and Nav1.5-F1571L + beta1 (open red inverted triangles, n = 12). The values shown are means +- SEM. (C) Voltage dependence of channel inactivation obtained by plotting the normalized current amplitudes at -10 mV, elicited after 500 ms of conditioning pre-pulse depolarization, as a function of the pre-pulse potential. (D) Displayed from left to right are the representative whole-cell recordings of recovery from inactivation of WT Nav1.5 + beta1 and Nav1.5-F1571L + beta1. The protocol used is shown underneath the traces. (E) Graph representing the recovery from inactivation, sampled after 500 ms from induction of inactivation.
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F3
We subsequently analyzed the kinetics of inactivation, the voltage dependence of inactivation, and the recovery from inactivation (Figures 3A-E). From the inactivation time constant, we observed a 3-fold slower inactivation (P < 0.001) for the variant (1.96 +- 0.12 ms at 0 mV, n = 7) compared to Nav1.5 WT (0.67 +- 0.08 ms at 0 mV, n = 8) (Figure 3B). Interestingly, the Nav1.5-F1571L variant displayed a significant 18-mV hyperpolarizing shift in the voltage dependence of inactivation compared to WT (P = 0.001), yielding inactivation curves with a V1/2 of -104 +- 3 mV (n = 9) and -86 +- 3 mV (n = 5), combined with a slope factor of 7.6 +-0.8 and 5.4 +-0.7 mV for Nav1.5-F1571L and Nav1.5, respectively (Figure 3C). The time constants of recovery from inactivation revealed a slower recovery for the variant channel compared to WT (P < 0.001), which was not rescued by co-expression with the beta1 subunit (P < 0.001) [Nav1.5: 7.4 +- 0.1 ms (n = 6); Nav1.5-F1571L: 15.2 +- 0.1 ms (n = 12); Nav1.5+ beta1: 5.2 +- 0. 2 ms (n = 10); Nav1.5-F1571L + beta1: 13 +- 0.1 ms (n = 15)] (Figures 3D,E).
Inactivation properties of WT Nav1.5 + beta1 and Nav1.5-F1571L +beta1 in the presence of nemertide alpha-1. (A) Displayed from left to right are the representative whole-cell recordings of WT Nav1.5 + beta1 and Nav1.5-F1571L + beta1 in control conditions (black tracings) and upon steady-state modification by 5 muM nemertide alpha-1 (red tracings). The currents were elicited with the pulse protocol shown underneath the traces. Steady-state toxin modulation was achieved after ~2 min. (B) Kinetics of channel inactivation for WT Nav1.5 + beta1 (circles) and Nav1.5-F1571L + beta1 (inverted triangles) in control conditions (black) and upon steady-state modification by nemertide alpha-1 (red) are shown. The values are means +- SEM. (C) The voltage dependence of channel inactivation for WT Nav1.5 + beta1 (black symbols) and Nav1.5-F1571L + beta1 (red) in control conditions (circles) and upon steady-state modification by nemertide alpha-1 (inverted triangles) is shown. (D) Scaled-up view of the voltage dependence of channel inactivation in the physiological range of resting membrane potentials, with a focus on the comparison to the WT Nav1.5 + beta1. Note that, in the presence of nemertide alpha-1, the amount of inactivated Nav1.5-F1571L + beta1 channels (red inverted triangles) is similar to the WT in control conditions (black circles).
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F4
Two minutes of perfusion of 5 muM of nemertide alpha-1 induced a steady-state modification of INa inactivation of both Nav1.5 + beta1 and the variant p.F1571L + beta1 (Figure 4A). Nav1.5-F1571L + beta1 also reported a significant 3-fold slowing of inactivation (P = 0.002) displaying at 0 mV a time constant of 2.22 +- 0.57 ms in the absence of nemertide alpha-1 and 5.82 +- 0.68 ms in its presence (n = 6) (Figure 4B). Next, we investigated the voltage dependence of inactivation and observed no significant changes in the V1/2 upon toxin addition for both Nav1.5+beta1 and Nav1.5-F1571L+ beta1 (P > 0.5). In contrast, the slope factors became significantly shallower and amounted, after toxin modification, to 10.6 +- 0.8 mV (n = 6) and 12.3 +- 1.3 mV (n = 6) for Nav1.5 + beta1 and Nav1.5-F1571L + beta1, respectively (Figures 4C,D).