Retigabine suppresses loss of force in mouse models of hypokalaemic periodic paralysis.

Recurrent episodes of weakness in periodic paralysis are caused by intermittent loss of muscle fibre excitability, as a consequence of sustained depolarization of the resting potential. Repolarization is favoured by increasing the fibre permeability to potassium. Based on this principle, we tested the efficacy of retigabine, a potassium channel opener, to suppress the loss of force induced by a low-K+ challenge in hypokalaemic periodic paralysis (HypoPP). Retigabine can prevent the episodic loss of force in HypoPP. Knock-in mutant mouse models of HypoPP (Cacna1s p.R528H and Scn4a p.R669H) were used to determine whether pre-treatment with retigabine prevented the loss of force, or post-treatment hastened recovery of force for a low-K+ challenge in an ex vivo contraction assay. Retigabine completely prevents the loss of force induced by a 2 mM K+ challenge (protection) in our mouse models of HypoPP, with 50% inhibitory concentrations of 0.8 ± 0.13 µM and 2.2 ± 0.42 µM for NaV1.4-R669H and CaV1.1-R528H, respectively. In comparison, the effective concentration for the KATP channel opener pinacidil was 10-fold higher. Application of retigabine also reversed the loss of force (rescue) for HypoPP muscle maintained in 2 mM K+. Our findings show that retigabine, a selective agonist of the KV7 family of potassium channels, is effective for the prevention of low-K+ induced attacks of weakness and to enhance recovery from an ongoing loss of force in mouse models of type 1 (Cacna1s) and type 2 (Scn4a) HypoPP. Substantial protection from the loss of force occurred in the low micromolar range, well within the therapeutic window for retigabine.

Voltage-dependent Ca2+ release is impaired in hypokalemic periodic paralysis caused by CaV1.1-R528H but not by NaV1.4-R669H.

Hypokalemic periodic paralysis (HypoPP) is a channelopathy of skeletal muscle caused by missense mutations in the voltage sensor domains (usually at an arginine of the S4 segment) of the CaV1.1 calcium channel or of the NaV1.4 sodium channel. The primary clinical manifestation is recurrent attacks of weakness, resulting from impaired excitability of anomalously depolarized fibers containing leaky mutant channels. Although the ictal loss of fiber excitability is sufficient to explain the acute episodes of weakness, a deleterious change in voltage sensor function for CaV1.1 mutant channels may also compromise excitation-contraction coupling (EC-coupling). We used the low-affinity Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) to assess voltage-dependent Ca2+-release as a measure of EC-coupling for our knock-in mutant mouse models of HypoPP. The peak ΔF/F0 in fibers isolated from CaV1.1-R528H mice was about two-thirds of the amplitude observed in WT mice; whereas in HypoPP fibers from NaV1.4-R669H mice the ΔF/F0 was indistinguishable from WT. No difference in the voltage dependence of ΔF/F0 from WT was observed for fibers from either HypoPP mouse model. Because late-onset permanent muscle weakness is more severe for CaV1.1-associated HypoPP than for NaV1.4, we propose that the reduced Ca2+-release for CaV1.1-R528H mutant channels may increase the susceptibility to fixed myopathic weakness. In contrast, the episodes of transient weakness are similar for CaV1.1- and NaV1.4-associated HypoPP, consistent with the notion that acute attacks of weakness are primarily caused by leaky channels and are not a consequence of reduced Ca2+-release.

Kv4.2 autism and epilepsy mutation enhances inactivation of closed channels but impairs access to inactivated state after opening.

A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K+ channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca2+ influx into dendrites and regulating spike timing-dependent plasticity. Here, we show that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to our data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. Our results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, our data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. Our results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins.

Guidelines on clinical presentation and management of nondystrophic myotonias.

The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation. In the absence of genetic confirmation, the diagnosis is supported by detailed electrophysiological testing, exclusion of other related disorders, and analysis of a variant of uncertain significance if present. Symptomatic treatment with a sodium channel blocker, such as mexiletine, is usually the first step in management, as well as educating patients about potential anesthetic complications.

Review of the Diagnosis and Treatment of Periodic Paralysis.

Periodic paralyses (PPs) are rare neuromuscular disorders caused by mutations in skeletal muscle sodium, calcium, and potassium channel genes. PPs include hypokalemic paralysis, hyperkalemic paralysis, and Andersen-Tawil syndrome. Common features of PP include autosomal dominant inheritance, onset typically in the first or second decades, episodic attacks of flaccid weakness, which are often triggered by diet or rest after exercise. Diagnosis is based on the characteristic clinic presentation then confirmed by genetic testing. In the absence of an identified genetic mutation, documented low or high potassium levels during attacks or a decrement on long exercise testing support diagnosis. The treatment approach should include both management of acute attacks and prevention of attacks. Treatments include behavioral interventions directed at avoidance of triggers, modification of potassium levels, diuretics, and carbonic anhydrase inhibitors. Muscle Nerve 57: 522-530, 2018.

Sodium Channelopathies of Skeletal Muscle.

The NaV1.4 sodium channel is highly expressed in skeletal muscle, where it carries almost all of the inward Na+ current that generates the action potential, but is not present at significant levels in other tissues. Consequently, mutations of SCN4A encoding NaV1.4 produce pure skeletal muscle phenotypes that now include six allelic disorders: sodium channel myotonia, paramyotonia congenita, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, congenital myasthenia, and congenital myopathy with hypotonia. Mutation-specific alternations of NaV1.4 function explain the mechanistic basis for the diverse phenotypes and identify opportunities for strategic intervention to modify the burden of disease.

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

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.

Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility.

Excitation-contraction (EC) coupling comprises events in muscle that convert electrical signals to Ca(2+) transients, which then trigger contraction of the sarcomere. Defects in these processes cause a spectrum of muscle diseases. We report that STAC3, a skeletal muscle-specific protein that localizes to T tubules, is essential for coupling membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum (SR). Consequently, homozygous deletion of src homology 3 and cysteine rich domain 3 (Stac3) in mice results in complete paralysis and perinatal lethality with a range of musculoskeletal defects that reflect a blockade of EC coupling. Muscle contractility and Ca(2+) release from the SR of cultured myotubes from Stac3 mutant mice could be restored by application of 4-chloro-m-cresol, a ryanodine receptor agonist, indicating that the sarcomeres, SR Ca(2+) store, and ryanodine receptors are functional in Stac3 mutant skeletal muscle. These findings reveal a previously uncharacterized, but required, component of the EC coupling machinery of skeletal muscle and introduce a candidate for consideration in myopathic disorders.

Hydroxyapatite-coated collars reduce radiolucent line progression in cemented distal femoral bone tumor implants.

BACKGROUND: Aseptic loosening of massive bone tumor implants is a major cause of prosthesis failure. Evidence suggests that an osteointegrated hydroxyapatite (HA)-coated collar would reduce the incidence of aseptic loosening around the cemented intramedullary stem in distal femoral bone tumor prostheses. Because these implants often are used in young patients with a tumor, such treatment might extend the longevity of tumor implants. Questions/purposes We asked whether (1) HA-coated collars were more likely to osteointegrate; (2) HA collars were associated with fewer progressive radiolucent lines around the stem-cement interface; and (3) HA-coated collars were associated with less bone loss at the bone-shoulder implant junction? METHODS: Twenty-two patients were pair-matched to one of two groups--either (1) implants with a HA-coated ingrowth collar (HA Collar Group); or (2) implants without an ingrowth collar (Noncollar Group). Age, sex, and length of followup were similar in both groups. HA-coated collars were developed and used at our institution from 1992 to address the high failure rate attributable to aseptic loosening in patients with massive bone tumor implants. Before this, smooth titanium shafts were used routinely adjacent to bone at the transection site. The minimum followup was 2 years (mean, 7 years; range, 2-12 years). Radiographs obtained throughout the followup period were analyzed and osteointegration at the shaft of the implant quantified. Radiolucent line progression around the cemented stem was semi-quantitatively assessed and cortical bone loss at the bone-shoulder implant junction was measured during the followup period. RESULTS: Comparison of the most recent radiographs showed nine of 11 patients had osteointegrated HA collars, whereas only one patient in the Noncollar Group had osteointegration (p > 0.001). The radiolucent line score quantified around the cemented stem was lower in the HA Collar Group when compared with the Noncollar Group (p = 0.001). Results showed an increase in cortical bone loss at the bone-shoulder implant junction in the Noncollar Group when compared with the HA Collar Group (p < 0.001). CONCLUSIONS: Osteointegration at the implant collar resulted in fewer radiolucent lines adjacent to the intramedullary cemented stem and decreased cortical bone loss immediately adjacent to the transection site. These results suggest that the HA collar may help reduce the risk of aseptic loosening in patients with this type of implant, but longer followup and a larger prospective comparison series are necessary to prove this more definitively.

The use of megaprosthesis in the treatment of periprosthetic knee fractures.

PURPOSE: The optimum treatment for periprosthetic fractures in the region of the distal femur is undefined. Although internal fixation for prostheses which are stable is commonly utilised, this can lead to very prolonged morbidity and failure of the fracture to unite. Where the prosthesis is either loose or infected, revision surgery is required and a 'tumour type' prosthesis can be successfully utilised. METHODS: The outcome of 27 patients treated by an endoprosthetic replacement for periprosthetic fracture of the distal femur between 1988 and 2013 are reported. Two cases were treated by two-stage revision due to persistent infection. Clinical outcomes were assessed by Knee Society score and Knee Society pain scores. RESULTS: All 27 patients mobilised rapidly in the post-operative period and infection where present was eradicated by the technique. There have been no cases of further revision in the patients where clinical follow-up is available. Knee Society scores following treatment averaged 88 and pain scores 43 at six months post-operatively. Eight patients have died during the long study period reflecting the age of the study population. CONCLUSIONS: The use of a tumour-type endoprosthesis in situations of comminuted periprosthetic fractures associated with a loose prosthesis shows favourable results with low complication rates and rapid mobilisation.

Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis.

Transient attacks of weakness in hypokalaemic periodic paralysis are caused by reduced fibre excitability from paradoxical depolarization of the resting potential in low potassium. Mutations of calcium channel and sodium channel genes have been identified as the underlying molecular defects that cause instability of the resting potential. Despite these scientific advances, therapeutic options remain limited. In a mouse model of hypokalaemic periodic paralysis from a sodium channel mutation (NaV1.4-R669H), we recently showed that inhibition of chloride influx with bumetanide reduced the susceptibility to attacks of weakness, in vitro. The R528H mutation in the calcium channel gene (CACNA1S encoding CaV1.1) is the most common cause of hypokalaemic periodic paralysis. We developed a CaV1.1-R528H knock-in mouse model of hypokalaemic periodic paralysis and show herein that bumetanide protects against both muscle weakness from low K+ challenge in vitro and loss of muscle excitability in vivo from a glucose plus insulin infusion. This work demonstrates the critical role of the chloride gradient in modulating the susceptibility to ictal weakness and establishes bumetanide as a potential therapy for hypokalaemic periodic paralysis arising from either NaV1.4 or CaV1.1 mutations.

Non-dystrophic myotonia: prospective study of objective and patient reported outcomes.

Non-dystrophic myotonias are rare diseases caused by mutations in skeletal muscle chloride and sodium ion channels with considerable phenotypic overlap between diseases. Few prospective studies have evaluated the sensitivity of symptoms and signs of myotonia in a large cohort of patients. We performed a prospective observational study of 95 participants with definite or clinically suspected non-dystrophic myotonia recruited from six sites in the USA, UK and Canada between March 2006 and March 2009. We used the common infrastructure and data elements provided by the NIH-funded Rare Disease Clinical Research Network. Outcomes included a standardized symptom interview and physical exam; the Short Form-36 and the Individualized Neuromuscular Quality of Life instruments; electrophysiological short and prolonged exercise tests; manual muscle testing; and a modified get-up-and-go test. Thirty-two participants had chloride channel mutations, 34 had sodium channel mutations, nine had myotonic dystrophy type 2, one had myotonic dystrophy type 1, and 17 had no identified mutation. Phenotype comparisons were restricted to those with sodium channel mutations, chloride channel mutations, and myotonic dystrophy type 2. Muscle stiffness was the most prominent symptom overall, seen in 66.7% to 100% of participants. In comparison with chloride channel mutations, participants with sodium mutations had an earlier age of onset of stiffness (5 years versus 10 years), frequent eye closure myotonia (73.5% versus 25%), more impairment on the Individualized Neuromuscular Quality of Life summary score (20.0 versus 9.44), and paradoxical eye closure myotonia (50% versus 0%). Handgrip myotonia was seen in three-quarters of participants, with warm up of myotonia in 75% chloride channel mutations, but also 35.3% of sodium channel mutations. The short exercise test showed ≥10% decrement in the compound muscle action potential amplitude in 59.3% of chloride channel participants compared with 27.6% of sodium channel participants, which increased post-cooling to 57.6% in sodium channel mutations. In evaluation of patients with clinical and electrical myotonia, despite considerable phenotypic overlap, the presence of eye closure myotonia, paradoxical myotonia, and an increase in short exercise test sensitivity post-cooling suggest sodium channel mutations. Outcomes designed to measure stiffness or the electrophysiological correlates of stiffness may prove useful for future clinical trials, regardless of underlying mutation, and include patient-reported stiffness, bedside manoeuvres to evaluate myotonia, muscle specific quality of life instruments and short exercise testing.

Periodic paralysis.

Periodic paralysis is a rare, dominantly inherited disorder of skeletal muscle in which episodic attacks of weakness are caused by a transient impairment of fiber excitability. Attacks of weakness are often elicited by characteristic environmental triggers, which were the basis for clinically delineating subtypes of periodic paralysis and are an important distinction for optimal disease management. All forms of familial periodic paralysis are caused by mutations of ion channels, often selectively expressed in skeletal muscle, that destabilize the resting potential. The missense mutations usually alter channel function through gain-of-function changes rather than producing a complete loss-of-function null. The knowledge of which channel gene harbors a variant, whether that variant is expected to (or known to) alter function, and how altered function impairs fiber excitability aides in the interpretation of patient signs and symptoms, the interpretation of gene test results, and how to optimize therapeutic intervention for symptom management and improve quality of life.

The common missense mutation D489N in TRIM32 causing limb girdle muscular dystrophy 2H leads to loss of the mutated protein in knock-in mice resulting in a Trim32-null phenotype.

Mutations in tripartite motif protein 32 (TRIM32) are responsible for several hereditary disorders that include limb girdle muscular dystrophy type 2H (LGMD2H), sarcotubular myopathy (STM) and Bardet Biedl syndrome. Most LGMD2H mutations in TRIM32 are clustered in the NHL ß-propeller domain at the C-terminus and are predicted to interfere with homodimerization. To get insight into TRIM32's role in the pathogenesis of LGMD2H and to create an accurate model of disease, we have generated a knock-in mouse (T32KI) carrying the c.1465G > A (p.D489N) mutation in murine Trim32 corresponding to the human LGMD2H/STM pathogenic mutation c.1459G > A (p.D487N). Our data indicate that T32KI mice have both a myopathic and a neurogenic phenotype, very similar to the one described in the Trim32-null mice that we created previously. Analysis of Trim32 gene expression in T32KI mice revealed normal mRNA levels, but a severe reduction in mutant TRIM32 (D489N) at the protein level. Our results suggest that the D489N pathogenic mutation destabilizes the protein, leading to its degradation, and results in the same mild myopathic and neurogenic phenotype as that found in Trim32-null mice. Thus, one potential mechanism of LGMD2H might be destabilization of mutated TRIM32 protein leading to a null phenotype.

Leukocyte common antigen-related phosphatase is a functional receptor for chondroitin sulfate proteoglycan axon growth inhibitors.

Chondroitin sulfate proteoglycans (CSPGs) are a family of extracellular matrix molecules with various functions in regulating tissue morphogenesis, cell division, and axon guidance. A number of CSPGs are highly upregulated by reactive glial scar tissues after injuries and form a strong barrier for axonal regeneration in the adult vertebrate CNS. Although CSPGs may negatively regulate axonal growth via binding and altering activity of other growth-regulating factors, the molecular mechanisms by which CSPGs restrict axonal elongation are not well understood. Here, we identified a novel receptor mechanism whereby CSPGs inhibit axonal growth via interactions with neuronal transmembrane leukocyte common antigen-related phosphatase (LAR). CSPGs bind LAR with high affinity in transfected COS-7 cells and coimmunoprecipitate with LAR expressed in various tissues including the brain and spinal cord. CSPG stimulation enhances activity of LAR phosphatase in vitro. Deletion of LAR in knock-out mice or blockade of LAR with sequence-selective peptides significantly overcomes neurite growth restrictions of CSPGs in neuronal cultures. Intracellularly, CSPG-LAR interaction mediates axonal growth inhibition of neurons partially via inactivating Akt and activating RhoA signals. Systemic treatments with LAR-targeting peptides in mice with thoracic spinal cord transection injuries induce significant axon growth of descending serotonergic fibers in the vicinity of the lesion and beyond in the caudal spinal cord and promote locomotor functional recovery. Identification of LAR as a novel CSPG functional receptor provides a therapeutic basis for enhancing axonal regeneration and functional recovery after CNS injuries in adult mammals.

Identification and functional characterization of Kir2.6 mutations associated with non-familial hypokalemic periodic paralysis.

Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by ∼78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.

Proximal ulna endoprosthetic replacement for bone tumours in young patients.

PURPOSE: The optimal reconstructive method after resection of malignant bone tumours of the proximal ulna is unknown.We report the outcome of endoprosthetic replacement in a young patient population. METHODS: This was a retrospective review of four patients[three males and one female; mean age 17.5 (range 11­31)years] who underwent limb salvage with a proximal ulnar endoprosthetic replacement following excision of malignant bone tumour. Mean follow-up was 85 (range 14­194) months. RESULTS: All patients were alive at final follow-up and reported an improvement in pain. One patient required transhumeral amputation for intralesional excision complicating a local recurrence at one month. Two patients developed fixed flexion deformities of the elbow, one of whom required radial-head excision. Mean Musculoskeletal Tumour Society (MSTS)score and Toronto Extremity Salvage Score (TESS) were 27(range 25­28) and 81 (73­88), respectively. CONCLUSIONS: Custom-made proximal ulna endoprosthetic replacement following resection of malignant bone tumours in young patients provides a stable reconstruction option with satisfactory function and without apparent compromise in patient survival.