Striking In vivo phenotype of a disease-associated human SCN5A mutation producing minimal changes in vitro

Abstract

Background: The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain.

Methods and results: We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose-dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at -30 mV; DN/H, 19.2±3.1 pA/pF, P<0.001 vs. H/H; DN/DN, 9.3±1.1 pA/pF, P<0.001 versus H/H).

Conclusions: Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.

Figure 1

D1275N SCN5A mutation in a patient with sinus node dysfunction, atrial flutter, and conduction disease. A, Pedigree. The proband is indicated by the arrow. Individuals carrying the mutation are indicated (+). Individuals tested negative for the mutation are indicated (−). A filled symbol indicates phenotype positive. B, Electrocardiogram and rhythm strips in the proband. C, Heterozygous single-nucleotide change in SCN5A (c.3823G→A) resulting in p.D1275N.

Figure 2

Electrocardiography in mice. A, Representative ECG traces in leads I at 3 weeks. B, Representative signal-averaged ECG traces in leads I (black) and II (gray) at 3 weeks. See Table 1 for detailed results. C, Arrhythmias recorded in DN/DN mice at 12 weeks.

Figure 3

Dilated cardiomyopathy phenotype. A, Representative echocardiograms showing prominent increased end-systolic dimensions in DN/H and DN/DN mice at 12 weeks. See Table 2 for summary results. B, Masson’s trichrome staining in mice hearts. Scale bars indicate 1 mm.

Figure 4

Wild-type and D1275N sodium current in Chinese hamster ovary cells (A, B, C) and tsA201 cells (D, E, F). Wild-type or D1275N channels were coexpressed with β1 subunits in tsA201 cells. A and D, Representative current traces. See Table 3 for summary results. B and E, Current voltage relationships. C and F, Voltage dependence of activation and inactivation. The pulse protocols are shown in the inset.

Figure 5

Sodium current in male ventricular cardiomyocytes at 3 weeks showing altered sodium channel function by the DN allele. A, Representative current traces in H/H, DN/H, and DN/DN cells. See Table 3 for summary results. B, Current voltage relationships. C, Late sodium current at −30 mV. Late current amplitude was normalized to peak current amplitude. D, Voltage dependence of activation and inactivation. E and F, Inactivation time constant (τ) at −30 mV. *P<0.001 versus H/H. ^†P<0.001 versus DN/H. N indicates the number of cardiomyocytes from 3 mice.

Figure 6

Action potential in male ventricular cardiomyocytes at 3 weeks. A, Representative action potential traces. B, Action potential duration at 50% and 90% repolarization. C, Action potential amplitude. *P<0.05 versus H/H. ^†P<0.01 versus H/H. ^‡P<0.01 versus DN/H. N indicates the number of cardiomyocytes from 3 mice.

Figure 7

Sodium channel expression levels at 3 weeks. A, Representative Western blots in ventricles. B, Sodium channel expression levels normalized to those of H/H. Calnexin was used as the loading control. C, Relative expression levels of SCN5A transcript normalized to those of β-actin in ventricle. *P<0.05 versus H/H.

Figure 8

Immunostaining for sodium channel (Nav1.5) at 3 weeks. Heart sections from the ventricles were stained with anti Nav1.5 (green). Note the obvious lateral staining in the H/H heart and its absence in the DN/DN heart.