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. 2015 Mar 15;593(6):1389-407.
doi: 10.1113/jphysiol.2014.277699. Epub 2014 Sep 17.

Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current, altered intracellular calcium homeostasis and arrhythmias in murine hearts

Xianming Lin  1 , Heather O'Malley, Chunling Chen, David Auerbach, Monique Foster, Akshay Shekhar, Mingliang Zhang, William Coetzee, José Jalife, Glenn I Fishman, Lori Isom, Mario Delmar
Affiliations

Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current, altered intracellular calcium homeostasis and arrhythmias in murine hearts

Xianming Lin et al. J Physiol. .

Abstract

Key points: Na(+) current (INa) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa, delayed after-depolarizations, triggered beats, delayed Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis.

Abstract: Na(+) current (INa) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) α subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes.

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Figures

Figure 1
Figure 1
Characterization of cardiac-specific Scn1b null mice A, schematic of breeding and genotyping strategy. The original targeting construct for generation of Scn1bflox mice contained exons 1, 2 and 3 (E1–E3) from the endogenous Scn1b allele, with E2 flanked by loxP sites (arrows) and an inverted PGK-neo-cassette flanked by FRT sites (pentagons). An inverted Neo cassette was flanked by FRT sites (pentagons). Scn1bflox mice, with the neo cassette intact, were generated previously. Crossing of Scn1bflox mice with FLPe transgenic mice (vertical arrow) resulted in removal of the Neo cassette. B, PCR of genomic DNA (obtained from mouse tails) from a typical litter was performed with two sets of primers. Top, loxP-5′ (5′-GTT ACT CAC CAC AGT GAC ATC CTC-3′) and loxP-3′ (5′-CAT CCA GCG TAT CAC ATC CTC ATC-3′) amplified a 720 bp band corresponding to the endogenousE allele, a 1114 bp band corresponding to the floxed allele, or a 492 bp band corresponding to the deleted allele. Note: none of the mice analysed in this litter contained the deleted allele in tail DNA. Bottom, PCR primers CreF-2 (5′-TCCAATTTACTGACCGTACACC-3′) and CreR-5′ (GGTATCTCTGACCAGAGTCATC-3′) amplified a 850 bp band corresponding to Cre recombinase. Lanes 1 and 4: Fl/Fl/Cre–; lanes 2, 3 and 8: Fl/E/Cre–; lane 5: Fl/Fl/Cre+; lanes 6, 9 and 10: Fl/E/Cre+; lane 7: E/E/Cre+; lane 11: E/E/Cre–. C, Western blot analysis of heart and brain membranes. Left, brain or heart membranes from cardiac-specific Scn1b null mice (65 μg protein/lane). Right, brain membranes from Scn1b global null or WT mice shown to demonstrate specificity of the anti-β1 antibody (65 μg protein/lane). Anti-β1 dilution, 1:3000. E, endogenous allele; Fl, floxed allele; WT, wild-type.
Figure 2
Figure 2
Region-specific recordings of Na+ current in juvenile cardiac ventricular myocytes Cells were obtained from mice with global knockout of the Scn1b gene (Scn1b–/–; red circles) or littermate controls (WT; black circles), Data collected from macropatch recordings of the cell midsection (A–C) or from the cell end, in the region previously occupied by the ID (D–F). Peak current–voltage relations (A and D), voltage dependence of steady-state inactivation (B and E) and time course of recovery from inactivation (C and F). Quantitative analysis reported in Table1. ID, intercalated disc; M, midsection; WT, wild-type.
Figure 3
Figure 3
Effect of 100 nm TTX on region-specific INa recorded from cardiac ventricular myocytes dissociated from juvenile global Scn1b null mice (Scn1b–/–) Average peak current–voltage relations (A and C) and voltage dependence of steady-state inactivation curves (B and D). Recordings were obtained from the cell midsection (A and B) or from the region previously occupied by the ID. Quantitative analysis reported in Table2. ID, intercalated disc; M, midsection; TTX, tetrodotoxin.
Figure 4
Figure 4
TTX-resistant INa recorded from the midsection of juvenile cardiac ventricular myocytes Cells were dissociated from global Scn1b null mice (Scn1b–/–; data in red symbols) or littermate controls (WT; black symbols). Peak current–voltage relations (A) and voltage dependence of steady-state inactivation (B) recorded in the presence of 100 nm TTX. Quantitative analysis reported in Table3. M, midsection; TTX, tetrodotoxin; WT, wild-type.
Figure 5
Figure 5
Gene expression levels in juvenile hearts Transcript levels for Scn1a, Scn3a, Scn5a, Scn8a and Scn10a were examined in hearts of control animals (Scn1b+/+) or Scn1b nulls (Scn1b–/–). Each gene expression was normalized to the CT of GAPDH. ‘Normalized RQ’ refers to the normalized value of expression of a given gene, estimated from 2−ΔΔCT. Normalized RQ values were 1.88 ± 0.27 for Scn3a (P = 0.03 vs. wild-type) and 1.26 ± 0.05 for Scn5a (P = 0.008 vs. wild-type). CT, threshold cycle.
Figure 6
Figure 6
Surface topology characteristics of cardiac myocytes resolved by scanning ion conductance microscopy Surface of a juvenile (A) and of an adult (B) Scn1b null myocyte. C and D, topology profiles along the red dotted lines in (A) and (B), respectively. Average depths (measured from top of crest to bottom of valley) in juvenile cells (E) or in adult cells (F) either control or null for Scn1b (Scn1b–/–). G and F, distance between valleys. Notice that juvenile cells have a rather smooth topology, with minimum variations in depth, but the periodicity of the ridges is the same as that observed in adult cells.
Figure 7
Figure 7
Fraction of super-resolution scanning patch clamp seals in which we detected the number of active channels expressed in the abscissae The number of separate recordings reported in each histogram is indicated in the top left of each panel. The pipette was placed on the crest (A) or on the opening of the T-tubules (B). Adult ventricular myocytes were obtained from cardiac-specific Scn1b null animals (Scn1b–/–) or from its control. In a separate group, Na+ channels in cardiac-specific Scn1b null cells were recorded in the presence of 100 nm TTX in the internal pipette solution. TTX, tetrodotoxin.
Figure 8
Figure 8
Action potential duration and delayed after-depolarizations in cardiac-specific Scn1b null adult ventricular myocytes Action potentials recorded from ventricular myocytes isolated from control (black trace in A), and from cardiac-specific Scn1b null (Scn1b–/–) mice (red traces in A and in B). Notice the presence of a delayed after-depolarization recorded from the Scn1b null cell (A) and a triggered action potential (B). Average measurements of APD50 (C) and APD70 (D). Number of cells studied were 18 for Scn1b null and 12 for wild-type.
Figure 9
Figure 9
Ca2+ transients in adult ventricular myocytes dissociated from cardiac-specific Scn1b null mice, or control A, Ca2+ transients recorded from a control adult myocyte (top), from a cardiac-specific Scn1b null (Scn1b–/–) myocyte (middle) and from an Scn1b–/– myocyte in the presence of 100 nm TTX (bottom). The cells were paced for 3 min at a rate of 1 Hz. The traces inside the boxed insets labelled ‘1’, ‘2’ and ‘3’ are displayed at an expanded time scale in (B). Red dots in the bottom traces in (B) mark the pacing event. Red arrowheads in trace ‘1’ point to the presence of Ca2+ oscillations occurring after the relaxation of the Ca2+ spike. C, average fractional increase in diastolic Ca2+ (percentage from baseline) after 3 min of regular pacing at a frequency of 1 Hz. D, time to first Ca2+ oscillation recorded from control cells, those from cardiac-specific Scn1b null mice (Scn1b–/–) and Scn1b–/– cells exposed to 100 nm TTX. Number of cells studied was 14, 14 and 11 for wild-type, Scn1b null and Scn1b null with TTX, respectively. TTX, tetrodotoxin.
Figure 10
Figure 10
Characteristics of Ca2+ transients recorded from cells dissociated from cardiac-specific Scn1b null (Scn1b–/–) hearts, or their control ‘Scn1b–/– + TTX’ refers to data obtained from Scn1b–/– cells in the presence of 100 nm TTX. A, example of Ca2+ transients recorded under the three different conditions. The fluorescent transient was normalized to its maximum amplitude for comparison. B–D, each presents average data obtained at pacing frequencies of 0.5 and 1 Hz. Significance (P) values indicated for the respective brackets. Variables measured were relaxation time constant (B), total amplitude of the Ca2+ transient (C) and time to peak (D). Number of cells studied was 15, 19 and 11 for WT, Scn1b null and Scn1b null with TTX, respectively. TTX, tetrodotoxin.
Figure 11
Figure 11
Arrhythmia events in cardiac-specific Scn1b null hearts Spontaneous initiation of NSVT in an isolated cardiac-specific Scn1b null heart. A and B, time–space plot showing the sinus (S) activation followed by a PVC and the initiation of NSVT (R, re-entry 21.1 Hz). B, time–space plot zoom of the red box in (A) shows in detail the rapid propagation of the sinus impulse followed by a PVC leading to the first (R1) and second (R2) re-entrant impulses. C, activation map of sinus activation of the ventricles (dual ventricular breakthrough in the RV and LV). D, Early afterdepolarization formation in the LV (*), leading to a propagated response. The impulse propagated fast through the RV, while it propagated slowly and eventually resulted in a wavebreak at the LV, leading to the formation of a rotor (R1). E, second rotation indicates that the rotor meandered, with subsequent changes in the direction of propagation. F, volume-conducted ECG recording showing the sinus activation followed by a PVC and the initiation of NSVT. G, Torsade de pointes ECG morphology (21.9 Hz) during NSVT in a volume-conducted ECG from a second Langendorff-perfused Scn1b-MHC heart. LV, left ventricle; NSVT, non-sustained ventricular tachycardia; PVC, premature ventricular complex; RV, right ventricle; S, sinus.
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