3.1. Clinical case
In 2001, a 20-year-old otherwise healthy male suddenly lost consciousness while playing basketball as his sentinel cardiac event. The emergency response team found him in ventricular fibrillation, and he was defibrillated rapidly and successfully on the basketball court and recovered fully with no neurological sequelae. He had no prior syncopal spells, and there was no history of sudden death in his family. After comprehensive cardiological evaluation both locally and after referral to Mayo Clinic, the only peculiarity noted was an epsilon wave on 12-lead ECG (Figure ). However, the T waves were not inverted in the right precordial leads, and there was no other evidence to suggest ARVC/D. Specifically, there was no ectopy on ambulatory recording or late potentials on signal-averaged ECG, and the echocardiogram was normal with no structural cardiac disease. In addition, a cardiac CT scan with and without contrast was unremarkable with no fatty infiltration of the right ventricle, aneurysmal dilatation, right ventricular enlargement, or dysfunction noted. With these negative findings, a right ventricular biopsy for AVRC was considered to be low yield for the risk. Besides ARVC/D and other cardiomyopathies, other possible cardiac conditions (anomalous coronary arteries, long QT syndrome, BrS, and catecholaminergic polymorphic ventricular tachycardia) were evaluated and excluded by a negative coronary angiogram, negative epinephrine QT stress test, negative procainamide challenge, and negative electrophysiology study with and without isoproterenol. An ICD was implanted without complication. The patient returned for follow-up 1 year later for repeat imaging studies and once again the contrast CT scan was unremarkable. No subsequent events have occurred after 8 years of follow-up.
Twelve-lead ECG of the 20-year-old index case showing sinus rhythm, QRS axis within normal limits. Epsilon waves are evident in leads V1, V2, and V3.
3.2. Mutational analysis
Mutational analysis of the RYR2 and LQTS genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) did not yield putative arrhythmia mutations. A T to G base substitution at position 161 was identified in SCN3B which yielded a missense mutation V54G [valine (V) to glycine (G) at position 54] (Figure A). This mutation was absent in 800 references alleles. V54G was localized to the extracellular region of the Navβ3 subunit and involves a highly conserved residue across species (Figure B and D). The proband's mother hosted the mutation and was asymptomatic, but notably also displayed ‘J’ point elevation on his 12-lead ECG (Figure C).
Figure 2 (A) Left: Proband DNA sequence chromatogram showing a T > G substitution generating a valine (V) to glycine (G) substitution at residue 54 of Navβ3. Right: Wild-type DHPLC profile in blue colour, mutant, and aberrant profile in red colour. (more ...)
3.3. Navβ3-V54G and INa
Representative INa traces from HEK-293 cells expressing SCN5A co-expressed with a blank vector control, Navβ3-WT, or Navβ3-V54G (Figure A) show that the mutation markedly decreased peak INa density, and summary data (Figure B) show that this decrease was statistically significant. Summary plots of the current–voltage relationship normalized to peak (Figure A and Table ) show that Navβ3-V54G caused a depolarizing shift of the voltage dependence of activation compared with Navβ3-WT, but not to SCN5A + vector; this effect may have contributed to the decreased INa density. In HEK-293 cells, but not COS cells, Navβ3-WT caused a 4 mV negative shift in the midpoint of inactivation compared with SCN5A + vector, and this effect was lost in the presence of Navβ3-V54G (Figure B and Table ). These data are consistent with changes in kinetics that result in a ‘loss of function’ by Navβ3-V54G. No changes were observed for late persistent INa (Table ).
Figure 3 Navβ3-V54G reduced INa in HEK-293 cells (A) Whole-cell current traces from representative cells expressing SCN5A + vector, SCN5A co-expressed with β3-WT or Navβ3-V54G 24 h after transfection. INa was elicited from a step depolarization (more ...)
Figure 4 Navβ3-WT shifted inactivation kinetics, but Navβ3-V54G did not. (A) Peak current–voltage plot of summary INa normalized to the maximal INa in response to a series of depolarizations from a holding potential of −140 mV. (more ...)
Kinetics of SCN5A + β3 in HEK-293 cells
3.4. Navβ3 associates with Nav1.5
We developed a Navβ3 antibody with the epitope targeted to the extracellular domain of Navβ3. The antibody gave bands at the expected molecular weight for Navβ3-WT and Navβ3-V54G expressed in COS cells, but not for the Navβ1, Navβ2, or Navβ4 subunits of the sodium channel (data not shown), suggesting that the antibody was specific for the Navβ3 subunit over other highly homologous sodium channel β subunits. Interestingly, we detected a band for Navβ3 in non-transfected HEK-293 cells, the standard cell line used for studies of SCN5A, but not in COS cells (data not shown). To determine whether SCN5A and Navβ3 are associated, we performed co-IP experiments by immunoprecipitating with the Navβ3 antibody and probing for SCN5A with Nav1.5 antibody or anti-Flag antibody (Figure
). In HEK-293 cells co-expressing SCN5A and Navβ3-WT (Figure A
, lane 4), the Navβ3 subunit antibody co-immunoprecipitated SCN5A, and it also co-immunoprecipitated SCN5A when SCN5A was expressed alone (Figure A
, lane 3), consistent with the finding that HEK-293 cells have endogenous Navβ3 subunits. SCN5A was also co-immunoprecipitated by Navβ3 in homogenates obtained from adult cardiac myocytes from mouse (Figure A
, lane 1), but the signal for SCN5A in neonatal mouse myocytes was very weak, consistent with a reported absence of Navβ3 subunits at this stage.7
Co-expressing Navβ3-WT or Nav3-V54G and the Flag-tagged SCN5A in COS cells also co-immunoprecipitated the complex.
Figure 5 Co-IP of SCN5A and Navβ3 subunits. (A) Heart homogenates were obtained from adult or neonatal mouse hearts, and from cell lysates obtained from HEK-293 cells expressing SCN5A alone or cells co-expressing SCN5A and Navβ3. (B and C) Cell (more ...)
3.5. Navβ3-V54G caused an SCN5A trafficking defect
The location of co-expressed SCN5A and Navβ3 subunits was investigated in HEK-293 cells by immunocytochemistry and confocal microscopy. The SCN5A location was visualized by expressing an SCN5A-Flag construct and probing with a Flag antibody (green signal) (Figure D, b and f), and the Navβ3 subunit was probed using the native antibody (red signal) (Figure D, c and g). For Navβ3-WT (Figure D, top panels), both SCN5A and Navβ3-WT localized at the plasma membrane. However, Navβ3 -V54G caused a marked decrease in the cell surface signal for both SCN5A and Navβ3 (Figure D, bottom panels). These results suggest that Navβ3-V54G caused retention of the two subunits and accounted for the decrease in INa density in the presence of Navβ3-V54G.
3.6. β3-V54G and INa in COS cells
Although HEK-293 cells are the standard heterologous cell line for the study of SCN5A, our observation that HEK-293 cells have endogenously expressing Navβ3 prompted us to also study the effects of the Navβ3 subunit on SCN5A in COS cells which we have showed lack the endogenous Navβ3 subunit. Representative INa traces from COS cells co-expressing SCN5A and one of the following plasmids: a blank vector control, Navβ3-WT, Navβ3-V54G, or both Navβ3-WT and Navβ3-V54G at 1 : 1 ratio (Figure A and Table ). In these cells, Navβ3-WT caused an increase in INa density, non-significant compared with SCN5A alone, and Navβ3-V54G had a profound and significant suppressive effect on INa density. When Navβ3-WT was co-expressed with NaVβ3-V54G, the INa density was not significantly different from Navβ3-WT. Summary of the INa density in each group is shown (Figure B and Table ). In addition, co-expression with SCN5A, Navβ1 and Navβ3-WT, or Navβ3-V54G had no significant additional effects on kinetics (Table ). In COS cells co-expressing the Navβ1 and Navβ3-WT, the levels of INa density were similar than in the absence of Navβ1. However, for Navβ3-V54G, the presence of the Navβ1 ‘partially rescues’ the decrease in INa density (Figure A and B).
Figure 6 Navβ3-V54G reduced INa in COS cells. Equal amounts of blank vector, Navβ3-WT or Navβ3-V54G, and in some cells Navβ1-WT were co-transfected with SCN5A in COS cells for a total amount of 1.5 µg DNA. (A) Whole-cell (more ...)
Kinetics of SCN5A + β1-WT and β3 in COS cells
3.7. SCN5A cell surface expression in COS cells by live-cell western technique
This relatively new technique provided a tool to quantitatively measure cell surface expression of SCN5A by imaging living cells attached at the bottom of 35 mm tissue culture plates. Here, COS cells co-expressing SCN5A Flag-tagged and Navβ3 plasmids as in Figure A and B were subjected to live-cell western blot using the anti-Flag antibody, then the signal was detected by a secondary anti-mouse antibody labelled with an infrared dye (IRDye 800), and detected on an infrared imaging system (Odyssey). Figure C shows the infrared signal at 800 nm wave length, and each plate has a confluent layer of COS cells with good transfection efficiency as indicated by GFP expression. A non-transfected plate was used to subtract the background from each plate containing cells expressing the SCN5A and the Navβ3 constructs. The infrared imaging analysis software quantifies the infrared signal (pixels count) within a pre-defined area (mm2), and Figure C shows the relative mean infrared signal intensities. The live-cell western technique is consistent with and corroborates the loss-of-function/trafficking-defective findings from the whole cell patch clamp and immunocytochemical analyses.