Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model

Abstract

Background: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/principal findings: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS< or = 18 ms; QRS in wild-type littermates: 10-18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a(+/-) mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones.

Conclusions: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression.

Figure 1. Variable degrees of ventricular conduction defects in Scn5a ^+/− mice.

A. Representative lead I ECGs from 10 week-old wild-type (WT) mice and Scn5a ^+/− mice with mild and severe conduction defects. Scale bar, 100 ms. B. Distribution of QRS interval duration with corresponding Gaussian fits in 10 week-old WT mice (white bars) and Scn5a ^+/− mice with mild (grey bars) and severe (black bars) phenotype. C. Effects of age (X-axis) on QRS interval duration (Y-axis) in WT mice (open symbols) and Scn5a ^+/− mice with mild (filled circles) and severe (filled triangles) phenotype. See Table 1 for statistics.

Figure 2. Variable effects of Na⁺ channel blockade in Scn5a ^+/− mice and SCN5A-mutated patients.

Ajmaline-induced increase in QRS interval (Y-axes) in 10 week- and >53 week-old WT (n = 11 and n = 10; open bars), mildly (n = 10 and n = 10; grey bars) and severely (n = 11 and n = 10; black bars) affected Scn5a ^+/− mice (left panel), and in young (6–31 years; mean = 22 years) and older (41–61 years; mean = 50 years) non-mutated (n = 18 and n = 17, open bars) and SCN5A-mutated patients with either mild (n = 12 and n = 11; grey filled bars) or severe (n = 7 and n = 12; black filled bars) QRS interval prolongation (right panel). *, ***, p<0.05 and p<0.001 respectively versus WT mice or non-mutated patients. †, p<0.05 versus Scn5a ^+/− mice or SCN5A-mutated patients with a mild phenotype. The difference between old mildly affected mice and old WT mice did not reach significance (p = 0.07). Same comment for the older patients.

Figure 3. Variable levels of fibrosis in Scn5a ^+/− mice.

A. Sirius red staining of ventricle from 85 week-old WT, mildly and severely affected Scn5a ^+/− mice. Fibrosis appears in red. A score of 0 was attributed to the WT mouse shown, 1 to the mild Scn5a ^+/− mouse and respectively 2 and 3 to left and right severe Scn5a ^+/− mice. B. Atf3 and Egr1 mRNA ventricular levels (in arbitrary units) in WT (open bars), mildly (grey bars) and severely (black bars) affected Scn5a ^+/− mice as a function of age. ***, p<0.001 versus WT and mild.

Figure 4. Cardiac arrhythmias in Scn5a ^+/− mice with severe phenotype.

Representative episodes of spontaneous ventricular arrhythmias recorded in two different Scn5a ^+/− mice with a severe phenotype under anesthesia. An ECG recorded in an age-matched WT mouse is given for comparison. Scale bar, 500 ms.

Figure 5. Moderate ionic remodeling in Scn5a ^+/− mice.

Percentage of variation in ventricular expression (Y-axes) of 46 genes encoding ion channel subunits (ch) and connexins (Cx) in Scn5a ^+/− mice with mild (grey bars; n = 5) and severe (black bars; n = 7) phenotype versus WT mice (n = 10). Sub, subunits. *, **, ***, p<0.05, p<0.01 and p<0.001 respectively versus WT; †, †††, p<0.05 and p<0.001 respectively versus mild phenotype.

Figure 6. Decreased Na_v1.5 expression in Scn5a ^+/− mice.

A. Representative Western blots showing the expression levels of Na_v1.5 in WT mice and Scn5a ^+/− mice (mean age = 18±1 weeks) with a mild or a severe phenotype. Protein loading was controlled by anti-actin immunoblotting. B. Quantification of Na_v1.5 expression in WT mice (open bars) and Scn5a ^+/− mice with mild (grey bars) and severe (black bars) phenotype was performed on Western blots from 10 mice in each group by normalizing the intensities of the Na_v1.5 bands to the actin bands. ***, p<0.001 versus WT; †, p<0.05 versus mild Scn5a ^+/− mice. C. Confocal images of Na_v1.5 in ventricular cardiomyocytes isolated from WT mice (left panel) and Scn5a ^+/− mice with mild (middle panel) and severe (right panel) phenotype. Scale bar, 20 µm. In Scn5a ^+/− images presented, a WT cardiomyocyte is also included in the image frame (yellow arrows) to illustrate the clear difference in WT versus Scn5a ^+/− intercalated disc staining intensities.

Figure 7. Variable I_Na densities in Scn5a ^+/− single ventricular cardiomyocytes.

A. Representative I_Na traces (protocol in inset) obtained from ventricular myocytes in a 12 week-old WT mouse and Scn5a ^+/− mice with mild and severe phenotype. Horizontal bar, 2 ms; vertical bar, 40 pA/pF. B. I_Na density in myocytes from WT and Scn5a ^+/− mice with mild and severe phenotype (4 mice in each group, 4 to 6 cells for each mouse). **, p<0.01 versus WT mice.

Figure 8. Action potential amplitude (AP amp; top) and maximum upstroke velocity (Vmax; bottom) as a function of pacing frequency.

White circles, wild type mice (n = 7); black circles, Scn5a ^+/− mice with a mild phenotype (n = 6); black triangles, Scn5a ^+/− mice with a severe phenotype (n = 5). At 800 bpm, 4 preparations from Scn5a ^+/− mice with a severe phenotype were periodically (for 1 of them) or continuously (for the 3 others) in 2∶1 conduction block. *, p<0.05.