Mice with an NaV1.4 sodium channel null allele have latent myasthenia, without susceptibility to periodic paralysis

Abstract

Over 60 mutations of SCN4A encoding the NaV1.4 sodium channel of skeletal muscle have been identified in patients with myotonia, periodic paralysis, myasthenia, or congenital myopathy. Most mutations are missense with gain-of-function defects that cause susceptibility to myotonia or periodic paralysis. Loss-of-function from enhanced inactivation or null alleles is rare and has been associated with myasthenia and congenital myopathy, while a mix of loss and gain of function changes has an uncertain relation to hypokalaemic periodic paralysis. To better define the functional consequences for a loss-of-function, we generated NaV1.4 null mice by deletion of exon 12. Heterozygous null mice have latent myasthenia and a right shift of the force-stimulus relation, without evidence of periodic paralysis. Sodium current density was half that of wild-type muscle and no compensation by retained expression of the foetal NaV1.5 isoform was detected. Mice null for NaV1.4 did not survive beyond the second postnatal day. This mouse model shows remarkable preservation of muscle function and viability for haploinsufficiency of NaV1.4, as has been reported in humans, with a propensity for pseudo-myasthenia caused by a marginal Na(+) current density to support sustained high-frequency action potentials in muscle.

Keywords: SCN4A; channelopathy; congenital myasthenic syndrome; skeletal muscle; weakness.

Mutations in Na_V1.4 are most often associated with myotonia or periodic paralysis. Loss of function changes are rare, and are seen in congenital myasthenia, congenital myopathy, and hypokalemic periodic paralysis. Using an NaV1.4 knock-out mouse, Wu et al. show that haploinsufficiency gives rise to latent myasthenia, but not periodic paralysis.

Figure 1

Generation of the Na_V1.4 null allele. (A) A knock-in mutation for mR663H in exon 12 (denoted R669H in reference to the human mutation) was previously generated by homologous recombination (Wu et al., 2011) and also contained loxP sites flanking exon 12 (top). Crossing with a Cre expressing line deleted exon 12 and 254 bp of intronic DNA. (B) Genotyping by PCR amplification produced a 525 bp product from the wild-type allele and a 212 bp product from the Cre-deleted allele.

Figure 2

Sodium current density is reduced in +/Δ_Ex12 muscle. (A) Peak Na⁺ current amplitude normalized to whole-cell capacitance is plotted as a function of the test depolarization from a holding potential of −100 mV. (B) Sodium current recorded from a +/Δ_Ex12 fibre in response to a test depolarization from −120 mV to −40 mV is shown before (control) and after exposure to 200 nM TTX. Background ionic currents and capacitance current have been subtracted, as described previously (Fu et al., 2011).

Figure 3

Isometric contractile force of the soleus muscle, in vitro. (A) Force transients elicited by field stimulation with a 100 Hz train of 80 mA current pulses applied for 400 ms. Each record is the response to a single trial. (B) Steady-state isometric force is plotted as a function of the stimulus current intensity, relative to the maximal force observed with an 80 mA stimulus. Symbols are average values for wild-type (+/+) n = 7 and +/Δ_Ex12 n = 5 soleus muscle preparations. Dashed line shows that to produce force at 50% of the maximal value, a higher stimulus current was required for (+/Δ_Ex12) compared to wild-type soleus. (C) A box plot of the steady-state force produced by 80 mA current stimulation. No difference in maximal steady-state force was observed in a comparison of wild-type (+/+; n = 12), +/Δ_Ex12 (n = 18), or Na_V1.4-R669H heterozygote (+/m; n = 8) muscles; whereas the homozygous mutant (m/m; n = 6) had reduced force (P < 0.01 ANOVA). Data for Na_V1.4-R669H mice reproduced from our prior report (Wu et al., 2011).

Figure 4

Pseudo-myasthenic decline in force for +/Δ_Ex12 soleus. (A) Force transients elicited by field stimulation over a range of submaximal current stimulus intensities. Each trace is a single trial [black for wild-type (+/+), red for +/Δ_Ex12] in response to a 100 Hz pulse train of 400-ms duration. Current intensity was increased from 10, 15, 20, 30, to 40 mA (smallest to greatest response). A prominent decline in force occurred ∼100 ms from stimulus onset (arrows) for +/Δ_Ex12 soleus. (B) The amplitude of the decline in force, relative to the early peak (see inset), is shown as a function of stimulus intensity. The decline was much larger for (+/Δ_Ex12) (n = 6), than for wild-type (+/+) soleus (n = 8), even if a compensatory rightward shift was introduced (dashed line) to offset the difference stimulus-force relationship shown in Fig. 3B. (C) The relative decline in force is plotted as a function of the early peak amplitude, normalized to the maximum observed for an 80 mA stimulus. This transformation shows the decline in force for +/Δ_Ex12 soleus was substantial even when the force level was >50% of the maximal value. Each symbol is the response for a single trial.

Figure 5

Pseudo-myasthenic CMAP response to repetitive nerve stimulation. The relative amplitude of the CMAP during repetitive nerve stimulation is plotted as a function of pulse number. At baseline (control, top row) a small decrease in CMAP amplitude occurred at higher frequencies of stimulation for wild-type (+/+) soleus (n = 3) and the decrease tended to be slightly larger for +/Δ_Ex12 soleus (n = 3), but was not statistically different. Partial block of nicotinic acetylcholine receptors with curare (bottom row) revealed a latent defect of neuromuscular transmission shown by the more prominent decline of +/Δ_Ex12 CMAP during repetitive nerve stimulation.

Figure 6

Potassium challenge during in vitro contraction. (A) Tetanic force is normalized to the control response in 4.7 mM K⁺, and a hypokalaemic challenge of 2 mM was applied for 20 min. With a supramaximal stimulus intensity of 80 mA (left), soleus from wild-type (+/+) and +/Δ_Ex12 had a small decrease of 10% to 15% that was indistinguishable. Responses from HypoKPP soleus muscle with the Na_V1.4-R669H mutation, however, exhibited larger decrease of 30% decrease for heterozygous (+/m) and 85% for homozygous mutants (m/m); data reproduced from our previous study (Wu et al., 2011). With a submaximal stimulus intensity of 40 mA (right), a larger decrease in force occurred for both wild-type (+/+) and +/Δ_Ex12 soleus in 2 mM K⁺. In addition, the early peak force for +/Δ_Ex12 muscle (open symbols, red) was followed by a sag to a lower steady state (filled symbol); whereas no detectable sag was observed from wild-type muscle (black). (B) A hyperkalemic challenge with 10 mM K⁺ did not elicit a reduction of tetanic force for wild-type or +/Δ_Ex12 soleus muscle.

Figure 7

CMAP during a glucose plus insulin challenge. Muscle excitability in vivo was monitored by the amplitude of the gastrocnemius and soleus CMAP elicited by stimulation of the sciatic nerve. A continuous infusion of glucose plus insulin started at time 0 and consistently produced a decrease in CMAP for HypoKPP susceptible mice [Na_V1.4-R669H heterozygotes, (+/m) reproduced from our prior study (Wu et al., 2011)], but not for controls, wild-type (+/+) or +/Δ_Ex12.