Generation of NaV1.4-R669H mice.
The R669H mutation in human NaV1.4 associated with HypoPP was introduced to the mouse ortholog (mNaV1.4-R663H) by homologous recombination using a 22.4-kb targeting vector, pNAR663H, containing exons 8–15 (Figure A). The arginine-to-histidine HypoPP mutation in exon 13 was paired with silent polymorphisms at codons 661 and 662 to aid in genotyping by PCR and restriction digest. Resistance to neomycin was used to screen for recombination in 129/Sv ES cells, and blastocyst injection was performed by the UT Southwestern Transgenic Core Facility. Founder mice had a high degree of chimerism, and germline transmission with recombination of the targeting construct at the correct site in F1 progeny was confirmed by PCR amplification of an 8.5-kb product from splenic DNA using a forward primer at a unique site in pNAR663H and a reverse primer in exon 16 located downstream from the targeting sequence (Figure B). Sequence analysis confirmed the presence of the murine R663H mutation and correct alignment of the recombination event upstream of exon 16. For consistency with the literature on HypoPP in humans, the heterozygous SCN4A+/R669H and homozygous SCN4AR669H/R669H mutant mice are referred to herein as R669H+/m and R669Hm/m, respectively.
The F1 R669H+/m mice were viable, developed normally, and bred successfully. To excise the FRT-flanked neomycin resistance gene from intron 12, we crossed a R669H+/m male with a female 129/Svflip1 mouse that constitutively expresses Flp recombinase. All subsequent breeding was in the 129/Sv strain, with the neomycin-deleted line. Genotyping was performed by PCR amplification of genomic DNA with a primer pair that spanned the intron 12/exon 13 boundary (Figure C). The retained intronic FRT and loxP sites in the mutant allele produced a 480-bp product that was distinguishable from the 400-bp WT amplimer. Genotyping at 4 weeks of age revealed the mutant allele at the expected Mendelian frequencies. In matings between WT and R669H+/m mice, the offspring were 52% WT and 48% R669H+/m (n = 151). When heterozygote pairs were bred, the offspring were 21% WT, 30% R669Hm/m, and 49% R669H+/m (n = 139).
Expression of the mutant allele was detected by RT-PCR amplification with a primer pair that spanned the exon 13/14 junction. Allele-specific forward primers were designed to selectively amplify first-strand cDNAs synthesized from WT or R669H transcripts. Specificity was demonstrated in Figure D; the mutant allele primer H failed to amplify a product from WT cDNA, whereas the WT primer R failed to generate a product from R669Hm/m cDNA. The abundance of each transcript was estimated from the OD of each allele-specific product after 24 cycles of amplification, normalized to a control product amplified from β-actin (Figure E). Transcript levels for WT and R669H alleles in R669H+/m mice were 0.71 and 0.72, respectively, relative to NaV1.4 transcript expression in WT mice, and that for the mutant allele in R669Hm/m mice was 0.98.
R669H mice appear normal.
Viability of R669H+/m
mice was indistinguishable from that of WT animals, consistent with the frequency distribution of genotypes observed at 4 weeks of age. Body mass was measured weekly from 4 to 52 weeks of age for WT and R669H+/m
mice, and no difference was observed (P
> 0.3, n
= 9 per genotype; Supplemental Figure 1; supplemental material available online with this article; doi:
). Mutant mice, R669H+/m
, had normal locomotor activity, without visibly apparent myotonic stiffness or spontaneous attacks of weakness. Quantitative grip strength testing showed a modest trend for hindlimb weakness, with approximately 10% reduction in maximal force for R669H+/m
versus WT, for both male and female mice ages 8–12 months, but this was not statistically significant (n
= 8; P
> 0.1; Supplemental Figure 1). No difference was observed in forelimb grip strength.
In vitro contraction testing reveals a HypoPP phenotype.
Spontaneous attacks of weakness in human periodic paralysis occur with high variability in frequency and severity, and even provocative maneuvers do not always trigger an attack (1
). In vitro contraction testing by hypokalemic challenge is a more reliable method to provoke a reduction of peak force in HypoPP muscle. Moreover, local extracellular K+
concentration can be controlled more easily and with greater accuracy in the tissue bath than can be achieved in vivo.
Isometric tetanic contractions were measured for soleus muscle maintained at 37°C in a tissue bath. Contractions were elicited by direct field stimulation of muscle fibers using parallel wire electrodes (2-ms pulses, 100 Hz; n = 40), and the bath contained curare (0.25 μM) to block neuromuscular transmission from activation of terminal branches of motor axons. In the standard bath containing 4.75 mM K+, the baseline force was reduced for R669Hm/m mice (12.3 ± 0.49 g; n = 33; P < 0.0001) compared with R669H+/m (16.3 ± 0.38 g; n = 36) or WT (15.7 ± 0.37 g; n = 41). Force transients recorded in the standard bath, after 10 minutes in a 2 mM K+ challenge, and then 10 minutes after return to control are shown for representative individual soleus muscles from each genotype in Figure A. Muscle from the R669H mice had increased susceptibility to loss of force generation in 2 mM K+ that was more severe for R669Hm/m than R669H+/m mice. The kinetics for the rise and decay of force transients were mildly slowed for R669Hm/m soleus during episodes of weakness in low K+, but there was no evidence of prolonged after contractions that are characteristic of myotonia.
In vitro contraction testing demonstrates a HypoPP phenotype.
The time course of the onset and recovery from a loss of muscle force during a 30-minute exposure to 2 mM K+ is shown as averaged responses from 8–10 muscle preparations per genotype in Figure B. The effect of R669H gene dosage is reflected by the nadir in force during the hypokalemic challenge, with that for R669Hm/m less than that for R669H+/m. Partial recovery in force for R669Hm/m muscle occurred during the 2 mM K+ interval, as shown by the force increase after 15 minutes. In 4 of 9 R669Hm/m fibers, the force recovered to greater than 50% of baseline during the hypokalemic challenge, but the asynchronous timing of recovery caused an attenuation of the mean response with an increased SEM. With a 3 mM K+ challenge, severe loss of force was again triggered for R669Hm/m soleus, but the recovery during hypokalemia was dramatically more prominent than that with 2 mM K+ (Figure C). All 10 R669Hm/m muscles tested showed pronounced oscillations in force with recovery and then recurrent loss during a 30-minute challenge in 3 mM K+. The period of the oscillations was similar for all muscles, with a mean of 16.8 ± 1.1 minutes (n = 10), whereas the amplitude was more variable (Figure C). In all cases, the behavior was remarkably similar for paired left and right muscles from the same animal (albeit tested in separate tissue baths) compared with responses in muscles from other animals. This pattern suggests that the response to hypokalemia is highly consistent, but that the basal state of susceptible muscles varied from animal to animal in an unpredictable fashion, possibly related to the recent level of motor activity or food ingestion immediately prior to the study.
The dose-response relation for K+
-induced weakness is shown in Figure D. The force measured 10 minutes after initiation of the K+
challenge was selected to define the dose-response relation, since this time point reflects the extent of force reduction before recovery or oscillations ensued. WT soleus muscle tolerated a wide range in extracellular K+
from 2 to 8 mM with less than a 10% reduction in tetanic isometric force. For R669H+/m
soleus, a model for the mutant allele dosage in human HypoPP, susceptibility to loss of force was markedly increased by hypokalemia at K+
of 2 mM (P
< 0.0005) and 1 mM (P
< 0.0001) compared with WT. Conversely, the responses at 8 and 12 mM K+
were not different from WT. The K+
sensitivity of R669H+/m
muscle showed a clear HypoPP phenotype. These data provide the first comprehensive dose-response relation to our knowledge in support of HypoPP arising from a mutation in NaV
1.4. Importantly, these data also show that R669H+/m
mice do not have an increased susceptibility to force reduction in high K+
. This distinction is notable because other disease-associated mutations in NaV
1.4 with gain-of-function alterations in channel activity will predispose muscle to attacks of hyperkalemic periodic paralysis (HyperPP), often accompanied with myotonia (22
muscle was more susceptible to a severe loss of force, in either low or high K+
. Because spontaneous oscillations in muscle force were so prominent for R669Hm/m
soleus in 3 mM K+
, we depicted the response in Figure D as the mean value recorded at 10 minutes (n
= 6), with dashed lines denoting the average maximum and minimum forces observed during the 30-minute exposure.
The carbonic anhydrase inhibitor acetazolamide (ACTZ) reduces the frequency and severity of paralytic attacks in some patients with periodic paralysis. Moreover, we have previously demonstrated the efficacy of 100 μM ACTZ in suppressing the reduction of force by hyperkalemic challenge in the NaV
1.4-M1592V HyperPP mouse (23
). Paired testing of control or pretreatment with 200 μM ACTZ for 30 minutes in R669H+/m
muscles from the EDL (n
= 4) or soleus (n
= 5) failed to reveal protection from weakness during a 2 mM K+
challenge in 8 of 9 trials.
Recovery from loss of force is ouabain sensitive.
The robust occurrence of oscillations in muscle force during a 3 mM K+
challenge for R669Hm/m
muscle (Figure C) provided an opportunity to investigate the basis for recovery from an attack of periodic paralysis, independent of shifts in serum K+
. Because depolarization of Vrest
is the proximate cause of weakness during an attack of HypoPP (4
), we reasoned that compensation from the Na+
-ATPase pump might be required to repolarize fibers. Pilot experiments showed that normal mouse soleus can tolerate the pump inhibitor ouabain up to a concentration of 1 μM without a loss of muscle force over a 30-minute observation period. Therefore, we tested whether pretreatment with 1 μM ouabain would block recovery in muscle force for R669Hm/m
soleus during a subsequent exposure to 3 mM K+
. Responses for a pair of WT and R669Hm/m
muscles from 2 animals are shown in Figure A. Unlike WT muscle, the R669Hm/m
soleus did not tolerate 1 μM ouabain in the standard bath containing 4.75 mM K+
(20- to 40-minute interval). This suggested that R669Hm/m
soleus has an increased dependency on pump activity to maintain Vrest
and excitability, even in normal K+
. Hypokalemic challenge with 3 mM K+
produced a further reduction in force for R669Hm/m
muscle and dramatically suppressed the oscillation in force; some fibers failed to recover even after the K+
was returned to 4.75 mM (Figure A). Recovery ensued only after the ouabain was washed out (90–110 minutes). To isolate the K+
-dependent component of the force for R669Hm/m
fibers in the presence of ouabain, we subtracted the linear decay of force in 1 μM ouabain while in standard bath solution. The average behavior recorded from 4 R669Hm/m
fibers demonstrated that the hypokalemia-induced reduction in force was still discernable in the presence of 1 μM ouabain, but that recovery did not occur over a 30-minute observation period in 3 mM K+
(Figure B). The variability in force responses from individual fibers is illustrated in Figure C. Large amplitude peak-to-peak oscillations in relative force were observed for all 10 R669Hm/m
fibers exposed to 3 mM K+
(relative value 0.44 ± 0.043), whereas in 4 other R669Hm/m
muscles tested, the presence of 1 μM ouabain the amplitude was greatly diminished (relative value 0.073 ± 0.037; P
Recovery from weakness was ouabain sensitive.
In vivo loss of muscle excitability and force from glucose plus insulin challenge.
Susceptibility to an attack of HypoPP in vivo was investigated by inducing hypokalemia with a continuous infusion of glucose plus insulin. Pilot experiments revealed a large variability in responses, with onset of weakness varying from 10 to 60 minutes after the start of the infusion and with the severity of force reduction ranging from 50% to 90% or even some failures. In an effort to achieve greater consistency in the responses, we used a pharmacologic approach to establish a common starting point for the extracellular K+. Basal hypokalemia was induced by pretreatment for 48 hours with an oral K-binding resin, sodium polystyrene sulfonate, which reduced serum K+ to 2.79 ± 0.21 mM (WT), 2.65 ± 0.07 mM (R669H+/m), and 2.24 ± 0.08 mM (R669Hm/m), but did not result in attacks of weakness detectable by observing motor behavior. These mice were then stably anesthetized for hours by isoflurane inhalation, and the glucose plus insulin mixture was administered intravenously (0.5 ml/h) at the jugular vein. Contraction was elicited by a single shock applied to the sciatic nerve. Muscle excitability was monitored as the compound muscle action potential (CMAP), recorded extracellularly with an electromyography needle positioned in the gastrocnemius or soleus muscles. The Achilles tendon was severed and attached to a force transducer to monitor muscle contraction. At the end of the 2-hour infusion, serum K+ decreased within each genotype and was significantly lower for R669Hm/m compared with WT mice (1.51 ± 0.14 mM vs. 2.25 ± 0.13 mM; P < 0.05), whereas R669H+/m mice showed a similar trend that did not reach statistical difference (1.97 ± 0.34 mM; P = 0.07, ANOVA with Bonferroni correction).
Figure A shows an example of the primary data, with a simultaneous measurement of CMAP amplitude and peak muscle force. The baseline CMAP amplitude, after sodium polystyrene sulfonate but before insulin infusion, was reduced for R669Hm/m compared with WT or R669H+/m mice (P < 0.05; Figure B). Basal twitch force, however, did not differ among the various genotypes. The CMAP and peak twitch force were measured every minute over a 1-hour period of glucose plus insulin infusion. In R669H mutant muscle, the relative amplitudes for these measures of muscle electrical excitability and mechanical contraction decreased markedly within minutes of starting the infusion (Figure , C and D), whereas WT muscle was relatively unaffected. As with the in vitro contraction test (Figure ), a gene dosage effect was apparent, with the decline in CMAP and force being more rapid and more extensive for R669Hm/m than for R669H+/m soleus.
In vivo reduction of muscle excitability and force from glucose plus insulin challenge.
In addition to the R669H effects on amplitude, the shape of the CMAP waveform was altered for HypoPP muscle. CMAP duration was increased at baseline, before the glucose plus insulin infusion (WT, 1.06 ± 0.09 ms, n = 17; R669H+/m, 1.43 ± 0.13 ms, n = 18; R669Hm/m, 1.88 ± 0.09 ms, n = 8; P < 0.001). During the infusion, CMAP duration became further prolonged in R669H mice, whereas the CMAP waveform was stable in WT animals (Figure E). The time course for the prolongation of CMAP duration over the first 20 minutes of the infusion is shown in Figure F. The increase in CMAP duration occurred with a lag of about 8 minutes, similar to the lag for the decrement in CMAP amplitude and twitch force (Figure , B and C). On average, nearly a 3-fold increase in CMAP duration was observed during glucose plus insulin infusion for R669Hm/m muscle.
Muscle fiber excitability of R669H mice.
Attacks of weakness in HypoPP are caused by transient impairment of muscle fiber excitability, which in turn is derived from a failure to maintain Vrest
was measured by impalement of fibers from whole soleus muscle maintained in vitro at 37°C. In 4.75 mM K+
was comparable for 501 WT and 456 R669H+/m
fibers (–72.6 ± 0.66 and –73.6 ± 0.41 mV, respectively; P
= 0.21). Upon reducing the bath K+
to 2 mM, however, the values diverged: WT fibers hyperpolarized by –9 mV to –81.7 ± 0.82 mV, as expected, whereas R669H+/m
fibers depolarized mildly by +3 mV to –70.6 ± 0.40 mV, with a net result that in 2 mM K+
fibers were depolarized by 11 mV compared with WT (P
In addition to the effect on Vrest, the intrinsic excitability of R669H HypoPP fibers was reduced. Current clamp recording with 2 microelectrodes was used to elicit action potentials from a fixed holding potential of –85 mV. Action potentials in R669H+/m fibers often failed to overshoot 0 mV, in contrast to responses in WT fibers, for which every trial had a peak greater than 0 mV (Figure A). Responses were compared quantitatively as the amplitude of the action potential elicited by a 2-ms current injection of 1.5× threshold intensity. The action potential amplitude was larger for WT than for R669H+/m fibers (115 ± 2.9 mV vs. 86.3 ± 7.3 mV; n = 15 per group; P < 0.005; Figure B). The half-amplitude duration was prolonged in R669H+/m fibers (1.3 ± 0.2 ms vs. 0.78 ± 0.05 ms in WT; P < 0.005). The maximum rate of rise for the action potential (dV/dt) was reduced in R669H+/m fibers (253 ± 49 mV/ms vs. 389 ± 24 mV/ms in WT; P < 0.05). There was no difference in the voltage threshold (R669H+/m, –56.4 ± 1.4 mV; WT, –55.0 ± 1.0 mV) or stimulus current threshold (R669H+/m, 141 ± 10 nA; WT, 141 ± 11 nA).
Reduced muscle excitability in R669H fibers.
To assess the susceptibility to firing bursts of myotonic discharges, current clamp recordings were performed with a 100-ms stimulus (Figure C). For both WT and R669H+/m fibers, only a single action potential was elicited as the current intensity was increased up to 1.5× threshold. The absence of myotonic bursts of discharges was consistent with the normal relaxation times for the in vitro contraction measurements (Figure B), which demonstrated that the R669H mutation does not cause myotonia.
Expression studies of NaV
1.4-R669H (rat isoform) in frog oocytes have demonstrated an anomalous inward current at potentials more negative than –20 mV, in which the voltage sensor is biased toward the inward conformation that favors channel closure (15
). This gating pore current flows through an accessory pathway created by the misfit between the voltage sensor and the channel complex and has been proposed to be the cause of aberrant depolarization during an attack of paralysis. To confirm the presence of the gating pore current in affected mouse fibers, we recorded steady-state currents in voltage-clamp studies of fibers dissociated from the footpad (flexor digitorum brevis and lumbricales). Blockers were used to suppress Na+
, and Ca2+
currents, and a chloride-free extracellular solution was used to suppress the Cl–
current. The residual current was still large (~5 nA/nF at –100 mV) compared with the predicted amplitude of the gating pore current (~1 nA/nF), and so the sensitivity for detection was further increased by extracting the component of current blocked by 3.5 mM lanthanum, which produces approximately 65% block of the gating pore current (24
). Fibers from R669Hm/m
mice showed an increased inward current at test potentials more negative than –20 mV compared with those from WT mice (Figure D). The slope conductance was increased for R669Hm/m
compared with WT fibers (15.2 ± 1.2 nS/nF vs. 8.0 ± 1.6 nS/nF; n
= 14 per group; P
= 0.011), suggestive of a gating pore conductance of 7 nS/nF at Vrest
of –90 mV. The difference between these 2 current-voltage curves revealed the typical current-voltage profile of a gating pore current activated at hyperpolarized potentials (Figure E).
Histological features of R669H mice.
Quadriceps muscles from WT, R669H+/m
, and R669Hm/m
adult mice aged 8–12 months were evaluated histologically (Figure ). Muscles from both R669H+/m
mutants revealed modest, nonspecific changes compared with WT fibers in the form of occasional red-staining subsarcolemmal inclusions of uncertain origin in the Gomori trichrome stain (Figure , F and J). Similar subsarcolemmal accumulations were observed in type 2 fibers from 2 patients with HypoPP caused by the NaV
1.4-R672G mutation and were attributed to transverse tubular aggregates (9
). Ultrastructural evaluation revealed very slight dilatation of sarcoplasmic triads in R669Hm/m
animals (data not shown), but no other abnormalities. Vacuolar changes were not found in the R669H mouse fibers, nor in the human HypoPP NaV
1.4-R672G specimens, which was remarkable because vacuoles are commonly a prominent feature of biopsies from patients with HypoPP caused by mutations in CaV
Histological analysis of quadriceps muscles.