Complex interactions in a novel SCN5A compound mutation associated with long QT and Brugada syndrome: Implications for Na+ channel blocking pharmacotherapy for de novo conduction disease

Fig 1. ECG and intracardiac recordings. A. Resting 12-lead ECG showing sinus tachycardia, mild QTc prolongation (470ms), atypical ST elevation in lead V2, rate-independent varying QRS aberrancy (solid arrow), and rate-independent changes in P wave morphology (dashed arrow). B. Surface ECG and intracardiac electrograms during sinus rhythm showing varying QRS aberrancy and normal HV interval of 55ms. Abbreviations: surface ECG leads I, II, aVL, V1, V2; bipolar electrograms from high right atrium (HRA), His bundle-proximal (HIS-P), His bundle-distal (HIS-D), right ventricular apex (RVA).

Fig 2. Varying atrial activation in sinus rhythm. A. Surface ECG and intracardiac electrograms showing varying P wave morphology and CS activation during sinus rhythm without changing heart rate. Note CS activation is distal to proximal on the first two beats, but then reverses by the fourth beat. B. Surface ECG and intracardiac electrograms showing varying P wave morphology and right atrial activation during sinus rhythm without a change in heart rate. Note the difference in high right atrial to His bundle activation time between the first and second beats. Abbreviations are the same as in Fig 1. CS indicates bipolar electrograms recorded from the proximal CS (CS 9–10) to distal CS (CS 1–2).

Fig 3. ECG response to drug intervention. Baseline resting ECG shows sinus tachycardia, normal QTc interval, atypical ST elevation in V3, and one right bundle branch block (RBBB) aberrant sinus beat. Procainamide resulted in sinus slowing, marked ST elevation in V1 and V2, but no Brugada type 1 ECG pattern. Mexilitine did not change the resting sinus rate, nor shorten the QTc interval. QRS aberrancy in one sinus beat is still present. Nadolol produced sinus slowing without marked QTc prolongation (i.e. >500 ms).

Fig 4. Location of A647D and P1332L mutation. A. Schematic representation of the membrane topology of SCN5A and the location of the A647D and P1332L mutations. The Na+ channel consists of 4 homologous domains (labelled I to IV), each with 6 transmembrane segments. B. Pedigree of patient’s family. The proband is the son with both the P1332L and A647D SCN5A mutations.

Fig 5. A647D mutation decreases INa of WT and P1332L when co-expressed. A. Typical whole-cell traces of INa recorded in CHO-K1 cells expressing WT or P1332L channels with or without A647D channels. B. Summary of peak INa density measurements as a function of voltage in CHO-K1 cells (n = 7 in each group). C. Summary of the whole-cell Na+ channel conductance (GNa) as a function of voltage with the lines showing the best fits to a Boltzmann function (n = 7 in each group). D. Comparison of Gmax (estimated by Boltzmann fits to the data in Panel C) for the indicated channels expressed in CHO-K1 cells. #: P = 0.036 versus WT alone; *: P = 0.039 versus P1332L alone.

Fig 6. Biophysical features of INa in CHO-K1 over-expressed with WT, P1332L and co-expressed with A647D. A. The time for 50% decay of INa measured from its peak as a function of membrane voltage. B. The whole-cell Na+ channel conductance is plotted as a function of the membrane potential for the voltage protocols shown. Conductance was estimated as described in the Methods. The lines were generated from a non-linear least-squares fit to the Boltzmann equation and the parameters for the voltage for 50% activation and the slope factor are summarized in Table 2. C. Steady-state inactivation curve, whole-cell Na+ channel conductance; the 50% inactivation and slope are listed in Table 2. D. INa recovery from inactivation curve; the recovery time constants and sample size are listed in Table 2.

Fig 7. Effects of A647D channels on late currents. A. Typical current traces normalized for membrane capacitance recorded from CHO-K1 expressed with WT or P1332L channels with or without A647D channels. B. Same currents (as in A) beginning after the peak INa at a higher magnification, late INa. C. Summarizes the ratio of INa measured at 150ms to peak INa (*: P<0.05 for P1332L (n = 14) compared to WT (n = 8), A647D (n = 5), WT+A647D (n = 7), A647D-P1334L (n = 7) and P1332L+A647D (n = 10)). D. Typical traces normalized for cell capacitance recorded with voltage protocol in the inset. E. The same traces (as shown in D) displayed at a higher current amplitude resolution to more clearly illustrate late INa. The broken line represents the zero current level with all currents being leak-corrected. F. Summarized the integral of absolute (not normalized) inward Na+ charge movement during the ramp protocol (*: P<0.05 for P1332L (n = 6) when compared to WT (n = 6), A647D (n = 5), WT+A647D (n = 6), A647D-P1332L (n = 6) and P1332L+A647D (n = 9)).

Fig 8. A647D decreased mexiletine sensitivity of P1332L when coexpressed. A. The blockage of mexiletine (80μM) on SCN5A Na+ current was estimated by recovery from inactivation using the protocol shown in the inset with 8 s start-to-start interval. B. The dose-response for mexiletine block was determined by plotting the relative amplitude of the slower component of the INa recovery from inactivation as a function of mexiletine concentration.

Fig 9. Mexiletine use-dependent block of WT and mutations Na+ currents. A Normalized representative recordings of INa in CHO-K1 cells over-expressed with WT and mutant Na+ channels using the protocol shown in insets; B. Comparison of the ratio of INapeak of the 7th trace in 20μM mexiletine to INapeak of baseline; *: P<0.05, compared to all other groups.

Fig 10. Biophysical features of INa in the OVVR model simulating WT, P1332L, WT+A647D and P1332L+A647D Na+ channels. A. 50% peak INa decay. B. Steady-state activation. C. Steady-state inactivation. D. Recovery from inactivation. In each panel, the kinetics matches the respective experimental data shown in Fig 6.

Fig 11. Action potential dynamics in the OVVR model simulating WT, P1332L, WT+A647D and P1332L+A647D Na+ channels during S1S2 pacing. A. Transmembrane potential for the last S1 beat during S1 pacing. B. APD restitution for S2 during the S1S2 pacing protocol with the maximal slope listed for each curve. C. Transmembrane potential during the action potential upstroke for the last S1 beat during S1 pacing. D. The maximum dVm/dt during the action potential upstroke for S2 during the S1S2 pacing protocol.

Fig 12. Whole-cell INa in the OVVR model simulating WT, P1332L, WT+A647D and P1332L+A647D Na+ channels during S1S2 pacing. A. Whole-cell INa for the last S1 beat during S1 pacing. B. Rate-dependent peak INa for each S2 in the S1S2 pacing protocol. C. The percentage of the baseline WT peak INa during S1 pacing for each S2 in the S1S2 pacing protocol for the four models. D. Early component of INa for the last S1 beat during S1 pacing. E. Late component of INa for the last S1 beat during S1 pacing.