A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurologic disease characterized by progressive weakness that results in death within a few years of onset by respiratory failure. Myostatin is a member of the TGF-β superfamily that is predominantly expressed in muscle and acts as a negative regulator of muscle growth. Attenuating myostatin has previously been shown to produce increased muscle mass and strength in normal and disease animal models. In this study, a mouse model of ALS (SOD1^G93A transgenic mice) was treated with a soluble activin receptor, type IIB (ActRIIB.mFc) which is a putative endogenous signaling receptor for myostatin in addition to other ligands of the TGF-β superfamily. ActRIIB.mFc treatment produces a delay in the onset of weakness, an increase in body weight and grip strength, and an enlargement of muscle size whether initiated pre-symptomatically or after symptom onset. Treatment with ActRIIB.mFc did not increase survival or neuromuscular junction innervation in SOD1^G93A transgenic mice. Pharmacologic treatment with ActRIIB.mFc was superior in all measurements to genetic deletion of myostatin in SOD1^G93A transgenic mice. The improved function of SOD1^G93A transgenic mice following treatment with ActRIIB.mFc is encouraging for the development of TGF-β pathway inhibitors to increase muscle strength in patients with ALS.     

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse

Introduction: In this study we investigated the action of RAP‐031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF‐κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. Methods: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. Results: RAP‐031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP‐031 with UDCA produced increases in FPT that were associated with an increase in WBT. Conclusions: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF‐κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice. Muscle Nerve, 2011     

The therapeutic potential of soluble activin type IIB receptor treatment in a limb girdle muscular dystrophy type 2D mouse model

Limb girdle muscular dystrophy type 2D (LGMD2D) is characterized by progressive weakening of muscles in the hip and shoulder girdles. It is caused by a mutation in the α-sarcoglycan gene and results in absence of α-sarcoglycan in the dystrophin-glycoprotein complex. The activin type IIB receptor is involved in the activin/myostatin pathway, with myostatin being a negative regulator of muscle growth. In this study, we investigated the effects of sequestering myostatin by a soluble activin type IIB receptor (sActRIIB) on muscle growth in Sgca-null mice, modelling LGMD2D. Treatment was initiated at 3 weeks of age, prior to the disease onset, or at 9 weeks of age when already in an advanced stage of the disease. We found that early sActRIIB treatment resulted in increased muscle size. However, this led to more rapid decline of muscle function than in saline-treated Sgca-null mice. Furthermore, no histopathological improvements were seen after sActRIIB treatment. When initiated at 9 weeks of age, sActRIIB treatment resulted in increased muscle mass too, but to a lesser extent. No effect of the treatment was observed on muscle function or histopathology. These data show that sActRIIB treatment as a stand-alone therapy does not improve muscle function or histopathology in Sgca-null mice.     

In vivo measurement of hindlimb neuromuscular function in mice

Introduction: In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles. Methods: An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short‐ and long‐latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice. Results: PF did not change with one‐ and three‐times‐per‐week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT. Conclusions: The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models. Muscle Nerve, 2012     

In vivo treatment with the NF‐κB inhibitor ursodeoxycholic acid (UDCA) improves tension development in the isolated mdx costal diaphragm

Introduction: Previous experiments have indicated that in vivo administration of ursodeoxycholic acid (UDCA) inhibits nuclear NF‐κB activation and has beneficial effects on the structure and function of dystrophic (mdx) muscle. We examined the effect of UDCA on tension development in dystrophic muscle. Methods: Isometric tension development was examined in costal diaphragms that were freshly isolated from vehicle and UDCA treated mdx mice. Percent recovery scores were obtained by directly comparing these measurements to those obtained from age‐matched nondystrophic mice. Results: Vehicle treated mdx mice exhibited significantly reduced optimal muscle lengths (l_o) and specific twitch and tetanic tensions compared with age‐matched nondystrophic mice. UDCA treated preparations exhibited significantly improved tension development with a 33% recovery score. Conclusions: Because UDCA is used in treating certain clinical disorders, these results provide a rationale for human clinical trials using this and related drugs for treatment of Duchenne and related muscular dystrophies. Muscle Nerve 53: 431–437, 2016     

Progressive motor unit loss in the G93A mouse model of amyotrophic lateral sclerosis is unaffected by gender

We examined whether there are gender differences in the progressive loss of functional motor units in SOD1^G93A transgenic mice. Isometric muscle and motor unit twitch contractions were recorded in fast‐ and slow‐twitch muscles in response to stimulation of the sciatic nerve. Using a modified motor unit number estimation technique (ITS‐MUNE), we found that motor unit numbers declined rapidly from 40 to 90 days of age during the asymptomatic phase of ALS in fast‐ but not slow‐twitch hindlimb muscles of both male and female mice. There was a corresponding decline in twitch and tetanic contractile forces of the fast‐twitch muscles. Gender did not affect the progressive loss of motor units and associated decline in force production. We conclude that gender does not alter progressive, muscle‐specific motor unit loss in ALS, even though gender does influence disease onset. Muscle Nerve 39: 318–327, 2009     

Combined XIL‐6R and urocortin‐2 treatment restores MDX diaphragm muscle force

Introduction: Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration leading to immobility, respiratory failure, and premature death. As chronic inflammation and stress are implicated in DMD pathology, the efficacy of an anti‐inflammatory and anti‐stress intervention strategy in ameliorating diaphragm dysfunction was investigated. Methods: Diaphragm muscle contractile function was compared in wild‐type and dystrophin‐deficient mdx mice treated with saline, anti‐interleukin‐6 receptor antibodies (xIL‐6R), the corticotrophin‐releasing factor receptor 2 (CRFR2) agonist, urocortin 2, or both xIL‐6R and urocortin 2. Results: Combined treatment with xIL‐6R and urocortin 2 rescued impaired force in mdx diaphragms. Mechanical work production and muscle shortening was also improved by combined drug treatment. Discussion: Treatment which neutralizes peripheral IL‐6 signaling and stimulates CRFR2 recovers force‐generating capacity and the ability to perform mechanical work in mdx diaphragm muscle. These findings may be important in the search for therapeutic targets in DMD. Muscle Nerve 56 : E134–E140, 2017     

Contractile properties of slow and fast skeletal muscles from protease activated receptor‐1 null mice

Introduction: Protease‐activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow‐twitch soleus muscles and fast‐twitch extensor digitorum longus (EDL) muscles from PAR‐1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR‐1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR‐1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR‐1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR‐1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014     

Effect of voluntary physical activity initiated at age 7 months on skeletal hindlimb and cardiac muscle function in mdx mice of both genders

Introduction: The effects of voluntary activity initiated in adult mdx (C57BL/10ScSc‐DMD^mdx/J) mice on skeletal and cardiac muscle function have not been studied extensively. Methods: We studied the effects of 3 months of voluntary wheel running initiated at age 7 months on hindlimb muscle weakness, increased susceptibility to muscle contraction?induced injury, and left ventricular function in mdx mice. Results: We found that voluntary wheel running did not worsen the deficit in force‐generating capacity and the force drop after lengthening contractions in either mdx mouse gender. It increased the absolute maximal force of skeletal muscle in female mdx mice. Moreover, it did not affect left ventricular function, structural heart dimensions, cardiac gene expression of inflammation, fibrosis, or remodeling markers. Conclusion: These results indicate that voluntary activity initiated at age 7 months had no detrimental effects on skeletal or cardiac muscles in either mdx mouse gender. Muscle Nerve 52 : 788–794, 2015     

Interaction of posttetanic potentiation and the catchlike property in mouse skeletal muscle

Introduction: Posttetanic potentiation (PTP) and the catchlike property (CLP) enhance contractile function in skeletal muscle. We investigated the CLP during dynamic performance in mouse hindlimb muscles with (wild‐type) and without (skMLCK^‐/‐) the primary mechanism for PTP (myosin phosphorylation) (in vitro, 25°C). Methods: Extensor digitorum longus muscles of both genotypes were stimulated with constant frequency and catchlike trains (CFT and CLT), before and after a potentiating stimulus (PS). Results: Before the PS, the CLT increased concentric force/work relative to the CFT, but this effect was greater for skMLCK^‐/‐ than wild‐type muscles. After the PS, the catchlike effect was reduced in wild‐type muscles but unchanged in skMLCK^‐/‐ muscles that did not display PTP. Conclusions: These data suggest that PTP interferes with the CLP during concentric force development at moderate speeds of shortening. We conclude that the physiological utility of each mechanism and their interactions provide important modulations to fast skeletal muscle function. Muscle Nerve 54 : 308–316, 2016     

Metformin increases peroxisome proliferator–activated receptor γ Co‐activator‐1α and utrophin a expression in dystrophic skeletal muscle

Introduction: Metformin (MET) stimulates skeletal muscle AMP‐activated protein kinase (AMPK), a key phenotype remodeling protein with emerging therapeutic relevance for Duchenne muscular dystrophy (DMD). Our aim was to identify the mechanism of impact of MET on dystrophic muscle. Methods: We investigated the effects of MET in cultured C₂C₁₂ muscle cells and mdx mouse skeletal muscle. Expression of potent phenotypic modifiers was assessed, including peroxisome proliferator–activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and receptor‐interacting protein 140 (RIP140), as well as that of the dystrophin‐homolog, utrophin A. Results: In C₂C₁₂ cells, MET augmented expression of PGC‐1α, PPARδ, and utrophin A, whereas RIP140 content was reciprocally downregulated. MET treatment of mdx mice increased PGC‐1α and utrophin A and normalized RIP140 levels. Conclusions: In this study we identify the impact of MET on skeletal muscle and underscore the timeliness and importance of investigating MET and other AMPK activators as relevant therapeutics for DMD. Muscle Nerve 52 : 139–142, 2015     

Oesophageal morphometry and residual strain in a mouse model of osteogenesis imperfecta

Recently, it was demonstrated in the oesophagus that the zero-stress state is not a closed cylinder but an open circular cylindrical sector. The closed cylinder with no external loads applied is called the no-load state and residual strain is the difference in strain between the no-load state and the zero-stress state. To understand the physiology and pathology of the oesophagus, it is necessary to know the zero-stress state and the stress-strain relationships of the tissues in the oesophagus, and the changes of these states and relationships due to biological remodelling of the tissues under stress. The aim of this study was to investigate the morphological and biomechanical remodelling at the no-load and zero-stress states in mutant osteogenesis imperfecta murine (oim) mice with collagen deficiency. The oesophagi of seven oim and seven normal wild-type mice were excised, cleaned, and sectioned into rings in an organ bath containing calcium-free Krebs solution with dextran and EGTA. The rings were photographed in the no-load state and cut radially to obtain the zero-stress state. Equilibrium was awaited for 30 min and the specimens were photographed again. Circumferences, submucosa and muscle layer thicknesses and areas, and the opening angle were measured from the digitized images. The oesophagi in oim mice had smaller layer thicknesses and areas compared to the wild types. The largest reduction in layer thickness in oim mice was found in the submucosa (approximately 36%). Oim mice had significantly larger opening angles (120.2 +/- 4.5 degrees ) than wild-type mice (93.0 +/- 11.2 degrees ). The residual strain was compressive at the mucosal surface and tensile at the serosal surface in both oim and wild types. In the oim mice, the residual strains at the serosal and mucosal surfaces and the mucosa-submucosal-muscle layer interface were higher than in the wild types (P < 0.05). The gradient of residual strain per unit thickness was higher in oim mice than in wild-type mice, and was highest in submucosa (P < 0.05). The only morphometric measure that was similar in oim and wild-type mice was the inner circumference in the no-load state. In conclusion, our data show significant differences in the residual strain distribution and morphometry between oim mice and wild-type mice. The data suggest that the residual stress in oesophagus is caused by the tension in the muscle layer rather than the stiffness of the submucosa in compression and that the remodelling process in the oim oesophagus is due mainly to morphometric and biomechanical alterations in the submucosa.     

CYFIP2 p.Arg87Cys Causes Neurological Defects and Degradation of CYFIP2

Here, we report the generation and comprehensive characterization of a knockin mouse model for the hotspot p.Arg87Cys variant of the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene, which was recently identified in individuals diagnosed with West syndrome, a developmental and epileptic encephalopathy. The Cyfip2^+/R87C mice recapitulated many neurological and neurobehavioral phenotypes of the patients, including spasmlike movements, microcephaly, and impaired social communication. Age-progressive cytoarchitectural disorganization and gliosis were also identified in the hippocampus of Cyfip2^+/R87C mice. Beyond identifying a decrease in CYFIP2 protein levels in the Cyfip2^+/R87C brains, we demonstrated that the p.Arg87Cys variant enhances ubiquitination and proteasomal degradation of CYFIP2. ANN NEUROL 2023;93:155–163     

Inflammation in muscular dystrophy and the beneficial effects of non‐steroidal anti‐inflammatory drugs

Introduction: Glucocorticoids are the only drugs available for the treatment of Duchenne muscular dystrophy (DMD), but it is unclear whether their efficacy is dependent on their anti‐inflammatory activity. Methods: To address this issue, mdx mice were treated daily with methylprednisolone and non‐steroidal anti‐inflammatory drugs (NSAIDs: aspirin, ibuprofen, parecoxib). Results: NSAID treatment was effective in ameliorating muscle morphology and reducing macrophage infiltration and necrosis. The percentage of regenerating myofibers was not modified by the treatments. The drugs were effective in reducing COX‐2 expression and inflammatory cytokines, but they did not affect utrophin levels. The effects of the treatments on contractile performance were analyzed. Isometric tension did not differ in treated and untreated muscle, but the resistance to fatigue was decreased by treatment with methylprednisolone and aspirin. Conclusions: NSAIDs have a beneficial effect on mdx muscle morphology, pointing to a crucial role of inflammation in the progression of DMD. Muscle Nerve, 2012     

Age‐dependent neuromuscular impairment in prion protein knockout mice

Introduction: The cellular prion protein (PrP^C) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP^C is still ambiguous. Evidence has suggested that PrP^C is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. Methods: We analyzed the age‐dependent influence of PrP^C on treadmill test–based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild‐type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP^C. Results: We found that aged PrP‐KO mice displayed a reduction in treadmill performance compared with PrP‐expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. Conclusion: PrP‐KO mice have an age‐dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations. Muscle Nerve 53: 269–279, 2016     

Reductions in muscle quality and quantity in chronic inflammatory demyelinating polyneuropathy patients assessed by magnetic resonance imaging

Introduction: Weakness in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) may be caused by decreases in muscle quantity and quality, but this has not been explored. Methods: Twelve patients with CIDP (mean age 61 years) and 10 age‐matched (mean age 59 years) control subjects were assessed for ankle dorsiflexion strength, and two different MRI scans (T1 and T2) of leg musculature. Results: Isometric strength was 36% lower in CIDP patients compared with controls. Tibialis anterior muscle volumes of CIDP patients were smaller by ～17% compared with controls, and non‐contractile tissue volume was ～58% greater in CIDP patients. When normalized to total muscle or corrected contractile volume, strength was ～29% and ～18% lower, respectively, in CIDP patients. Discussion: These results provide insight into the structural integrity of muscle contractile proteins and pathologic changes to whole‐muscle tissue composition that contribute to impaired muscle function in CIDP. Muscle Nerve 58 : 396–401, 2018     

Role for Toll-like receptor 3 in muscle regeneration after cardiotoxin injury

Introduction: Sterile tissue injury induces an inflammatory response involving cytokines that have crucial roles in the tissue repair that follows. Methods: MyH3 and type 1 interferon (IFN) were assessed by qPCR after cardiotoxin (CTX)‐induced muscle injury. Results: CTX‐induced injury increased expression of IFN‐regulated genes, IFIT1 and MX‐2, which was blocked in type 1 IFN receptor (IFNR)‐deficient mice. However, IFNR‐deficient mice showed no significant differences in muscle regeneration as assessed by MyH3 expression. MyH3 was significantly reduced in TLR3‐deficient but not MyD88‐deficient mice. TLR3‐deficient mice also showed altered expression of proinflammatory cytokines, IL‐6, IL‐1β, and TNF‐α. Conclusions: CTX‐induced muscle injury increased markers of innate immune activation, but blocking type 1 IFN signaling had no effect on muscle regeneration. Taken together, these results suggest a role for TLR3, and perhaps other innate immune signals, in the inflammatory response to CTX‐induced muscle injury and consequent muscle regeneration. Muscle Nerve, 2011     

Electrophysiological subtypes and prognosis of Guillain–Barré syndrome in Northeastern China

Introduction: The dystrophic features in hindlimb skeletal muscles of female mdx mice are unclear. Methods: We analyzed force‐generating capacity and force decline after lengthening contraction‐induced damage (fragility). Results: Young (6‐month‐old) female mdx mice displayed reduced force‐generating capacity (?18%) and higher fragility (23% force decline) compared with female age‐matched wild‐type mice. These 2 dystrophic features were less accentuated in young female than in young male mdx mice (?32% and 42% force drop). With advancing age, force‐generating capacity decreased and fragility increased in old (20 month) female mdx mice (?21% and 57% force decline), but they were unchanged in old male mdx mice. Moreover, estradiol treatment had no effect in old female mdx mice. Conclusions: Female gender–related factors mitigate dystrophic features in young but not old mdx mice. Further studies are warranted to identify the beneficial gender‐related factor in dystrophic muscle. Muscle Nerve, 2013     

Effect of Oestrogen Receptors on Brain NMDA Receptors of 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine Mice

Parkinson’s disease (PD) is characterised by the loss of nigrostriatal dopamine (DA) neurones and glutamate overactivity. There is substantial evidence to suggest that oestrogens prevent or delay the disease. 17β‐oestradiol has neuroprotective effects in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD and modulates brain NMDA receptors. In MPTP‐lesioned mice, oestrogen receptor (ER)α and ERβ are important in 17β‐oestradiol‐induced neuroprotection. To evaluate the role of ERs in the response of NMDA receptors to lesion, we compared wild‐type (WT) with ER knockout (KO) C57Bl/6 male mice that received 7, 9 or 11 mg/kg of MPTP. These mice were also treated with MPTP (9 mg/kg) and 17β‐oestradiol. [³H]Ro 25‐6981 specific binding autoradiography was used to label NMDA receptors containing NR2B subunits. In the frontal and cingulate cortex and striatum, vehicle‐treated WT mice had higher [³H]Ro 25‐6981 specific binding compared to ERKO mice. Cortical [³H]Ro 25‐6981 specific binding decreased with increasing doses of MPTP in WT and ERKOα but not ERKOβ mice, whereas a dose‐related decrease was only observed in the striatum of WT mice remaining low in ERKOα and ERKOβ mice. No effect of 17β‐oestradiol treatment in intact or MPTP‐lesioned mice of all three genotypes was observed in the cortex, whereas it increased striatal specific binding of intact ERKOβ and MPTP‐lesioned WT mice. Striatal [³H]Ro 25‐6981 specific binding positively correlated with striatal DA concentrations only in WT mice. MPTP and 17β‐oestradiol treatments had more limited effects in the hippocampus. Only in the CA3 and dentate gyrus did vehicle and 17β‐oestradiol‐treated ERKOα mice have higher [³H]Ro 25‐6981 specific binding than WT and ERKOβ mice, whereas MPTP decreased this specific binding only in the CA1, CA2 and CA3 of ERKOα mice. Hence, brain NMDA receptors were affected by the deletion of ERs, which affect the response to MPTP and 17β‐oestradiol treatments with brain region specificity.