Fig 1.
Pedigree and Holter (25 mm/s, 10 mm = 1 mV) results of the family.
(A) Phenotypic and genotypic traits are represented by specific symbols in the pedigree. (B) Terminal QRS notch in leads II and aVF (arrows) without ST-segment elevation shown in the Holter of the proband (III: 5). (C) J-point elevation from 1 mm to 3 mm in leads II, III, aVF, and V3—V5 (arrows), which behaved as a terminal QRS notch without ST-segment elevation in III: 7. (d) Terminal QRS notch in leads III and aVF and concave ST-segment elevation (1 mm) in leads II, III, aVF, and V4-V6 (arrows) is presented for IV: 3. (E) Terminal QRS slur with ST-segment elevation in leads II, III, aVF, and V4, and a terminal QRS notch with ST-segment elevation in lead V3 with J-point elevation from 1.5 mm to 2 mm (arrows) is presented for IV: 4.
Table 1.
ECG characteristics of affected individuals.
Fig 2.
Gene variants of CACNA1C and SCN5A identified in the family.
(A) Direct sequencing reveals a heterozygous mutation (c.5747A>G, p.Q1916R) in CACNA1C. (B) Amino acid sequencing alignments of CANCA1C indicate that Q1916 is highly conserved across mammals (red font). (C) Topology model of the α-subunit of LTCC. The localization of the mutation is indicated by a red dot, and polymorphisms are indicated by green dots. (D) A variant (c.3578G>A, p.R1193Q) in SCN5A. E. SCN5A-R1193Q is highly conserved among different species (red font). (F) Topology model of the sodium channel α-subunit. The localization of the polymorphism is indicated by a green dot.
Table 2.
The feature of the variants that identified.
Table 3.
The correlation between genotype and phenotype.
Fig 3.
Macroscopic and kinetic characteristics of the mutant LTCC.
(A) Representative whole-cell Ca2+ currents recorded from HEK293T cells transfected with WT or Q1916R mutation. Currents were elicited with the pulse protocol illustrated in the inset. (B) Current-voltage relationship for WT (n = 28) and Q1916R (n = 17) calcium channels. The peak Q1916R mutant ICa at 0 mV was decreased by 60%, when compared with the WT ICa; values are the means ± SEMs (*P<0.05, **P<0.01). (C-D) The voltage-dependence of steady state activation (SSA) and steady state inactivation (SSI) did not significantly differ between the WT and Q1916R calcium channels.
Fig 4.
Effects of testosterone and isoproterenol on the WT and mutant LTCC.
(A-B) The representative whole-cell Ca2+ current traces and average current-voltage relationship show a decrease in current density in both the WT (n = 6) and Q1916R (n = 8) LTCC after treatment with testosterone (*P<0.05, Q1916R or WT+tes versus WT-vehicle; #P<0.05, Q1916R+tes versus Q1916R-vehicle). (C-D) The voltage-dependence of SSA and SSI were not significantly altered among the 4 groups. (E-F) The representative whole-cell Ca2+ current traces and average current-voltage relationship show an increase in current density in the WT (n = 12) LTCC but not the Q1916R (n = 10) LTCC after treatment with isoproterenol (*P<0.05, **P<0.01, Q1916R or WT+ISO versus WT-vehicle). (G-H) The voltage-dependence of SSA displayed a left-shift in both the WT and Q1916R LTCC after treatment with isoproterenol, whereas no change was observed in SSI. SSA: steady state activation, SSI: steady state inactivation, tes: testosterone, ISO: isoproterenol.
Fig 5.
The expression and localization of the WT and CACNA1C-Q1916R channels in HEK293T cells.
(A-B) Expression levels of WT and mutant of CaV1.2α1C in the intact cell, membrane fraction and cytoplasmic fraction of the HEK293T cells. The left two lanes are blank controls with or without empty vector for CACNA1C. The expression of mutant CACNA1C was decreased in the intact cell, membrane and cytoplasmic lysates synchronously. The relative expression levels were normalized to GAPDH for both the intact cell and cytoplasmic lysate and were normalized to ATP1A2 for the membrane lysate. (n = 5, values are the means ± SEMs, *P<0.05, CACNA1C-Q1916R versus WT). (C) Confocal microscopy images of immunofluorescent staining in HEK293T cells. The red fluorescence indicated CACNA1C, and blue fluorescence indicated staining of the nucleus. The results showed that the intensity was reduced in the CACNA1C-Q1916R group.
Fig 6.
Single-cell ventricle AP modeling of three different cell types of myocardial layers.
(A) Simulated AP of midmyocardial cell (M cell) in WT (red dashed line) and CACNA1C-Q1916R mutant condition (black solid line). (B-C) AP shape contrast between endocardial cell and epicardial cell in WT (B) or mutant condition (C). (D) Curves of the transmural voltage gradient between endocardial cell (endo) and epicardial cell (epi) in the WT condition (black) and the mutant condition (red) in the early repolarization stage (red shade region). (E-G) Effects of testosterone (Tes) on AP of M cell (E), epi (F) and endo (G). (H) Curves of transmural voltage gradient between endo and epi in the early repolarization stage (red shade region).
Fig 7.
A graphical illustration of the balances represented by the relationships of three genetic contributors linked to the ERS phenotype in this study.
(A) The disequilibrium represented the male patients with the CACNA1C mutation and SCN5A variation, the protective effect of the SCN5A variation was less than the pathogenic effects of CACNA1C mutation plus male sex, so the indicator turned pointed left toward the ERS phenotype. (B) The disequilibrium represented the female patients with the CACNA1C mutation. The pathogenic effect of the CACNA1C mutation was greater than the protective effect of female sex; therefore, the indicator pointed left toward the ERS phenotype. (C) The equilibrium state represented the female case with the CACNA1C mutation plus the SCN5A variation. The pathogenic effect of the CACNA1C mutation was neutralized by the SCN5A variation plus female sex, so the indicator consequently stopped in the middle pointing to the normal phenotype.