A marginal level of dystrophin partially ameliorates hindlimb muscle passive mechanical properties in dystrophin‐null mice

Introduction: The goal of this study was to determine whether a minimal level of dystrophin expression improves the passive mechanical properties of skeletal muscle in the murine Duchenne muscular dystrophy model. Methods: We compared the elastic and viscous properties of the extensor digitorum longus muscle (EDL) in mdx3cv and mdx4cv mice at 6, 14, and 20 months of age. Both strains are on the C57Bl/6 background, and both lose the full‐length dystrophin protein. Interestingly, mdx3cv mice express a near full‐length dystrophin at ∽5% of the normal level. Results: We found that the stress–strain profile and the stress relaxation rate of the EDL in mdx3cv mice were partially preserved in all age groups compared with age‐matched mdx4cv mice. Conclusion: Our results suggest that a low level of dystrophin expression may treat muscle stiffness in Duchenne muscular dystrophy. Muscle Nerve, 2012     

Sarcoplasmic–endoplasmic–reticulum Ca2+‐ATPase and calsequestrin are overexpressed in spared intrinsic laryngeal muscles of dystrophin‐deficient mdx mice

In the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. We investigated whether this protection is related to an increased expression of calcium‐binding proteins, which may protect against the elevated calcium levels seen in dystrophic fibers. The expression of sarcoplasmic–endoplasmic–reticulum Ca²⁺‐ATPase and calsequestrin was examined in ILM and in nonspared limb muscles of control and mdx mice using immunofluorescence and immunoblotting. Dystrophic ILM presented a significant increase in the proteins studied when compared to controls. The increase of Ca²⁺‐handling proteins in dystrophic ILM may permit better maintenance of calcium homeostasis, with the consequent absence of myonecrosis. The results further support the concept that abnormal Ca²⁺‐handling is involved in dystrophinopathies. Muscle Nerve, 2009     

Weekly oral prednisolone improves survival and strength in male mdx mice

Although corticosteroids alleviate weakness in mdx mice, no long-term treatment has determined whether this benefit is maintained. We studied mdx mice forelimb grip strength and fatigue from 3 through 84 weeks and followed survival through 104 weeks. The mdx mice were given twice weekly oral prednisolone (5 mg/kg) beginning at 3 or 4 weeks. Treated mdx mice survived longer than untreated mice. Between 3 and 10 weeks, treated and untreated mdx mice had similar strength. Between 10 and 24 weeks, strength and strength per gram body weight declined more slowly in treated than untreated mdx mice. Between 24 and 84 weeks, treated and untreated mdx mice declined in strength at the same rate, although treated mice remained stronger. Forelimb grip fatigue was present in untreated mdx mice at all time-points compared to wild-type and was not changed significantly by treatment. We have demonstrated long-term benefit of oral prednisolone in the mdx mouse model of Duchenne muscular dystrophy (DMD). As corticosteroids remain the most validated long-term treatment of DMD, this work may allow for better prediction of synergistic treatments likely to translate to effective improvement for boys with this progressive muscular dystrophy.     

Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb‐girdle muscular dystrophy type 2E

Introduction: Limb‐girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the β‐sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. β‐Sarcoglycan‐deficient (Sgcb‐null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. Methods: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1‐month‐old (clinical onset of the disease) and 7‐month‐old control mice (C57Bl/6J, Rag2/γc‐null) and immunocompetent and immunodeficient dystrophic mice (Sgcb‐null and Sgcb/Rag2/γc‐null, respectively). Results: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc‐null muscles showed a significant reduction of alkaline phosphate‐positive mesoangioblasts. Discussion: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve 58 : 133–144, 2018     

Aging normal and dystrophic mouse muscle: Analysis of myogenicity in cultures of living single fibers

In the early stages of Duchenne muscular dystrophy, chronic muscle degeneration is counterbalanced by regeneration whose progressive failure beyond the fifth year is attributed to an accelerated senescence associated with excessive myogenic cell division. This idea was tested by counting the numbers of myogenic cells accumulating over 90 h around individual living fibers isolated from muscles of dystrophic (mdx) and normal mice, 14–550 days of age. In cultures of normal muscle fibers, the number of myogenic cells per fiber decreased with increasing age of the animal. Cultures from young mdx mice exhibited an age-related increase in myogenic cell number, attaining at 50 days three times the number in control cultures. Thereafter, myogenic cell number progressively declined with animal age to control values. Thus, there is no evidence that extensive myoblast proliferation in young dystrophic mice—and, by association, in Duchenne muscular dystrophy patients—depletes their myogenic responsiveness in later life when they become weak and wasted. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 173–183, 1998     

LAMA2‐related myopathy: Frequency among congenital and limb‐girdle muscular dystrophies

Introduction: Muscular dystrophy caused by LAMA2‐gene mutations is an autosomal recessive disease typically presenting as a severe, early‐onset congenital muscular dystrophy (CMD). However, milder cases with a limb‐girdle type muscular dystrophy (LGMD) have been described. Methods: In this study, we assessed the frequency and phenotypic spectrum of LAMA2‐related muscular dystrophy in CMD (n = 18) and LGMD2 (n = 128) cohorts identified in the last 15 years in eastern Denmark. The medical history, brain‐MRI, muscle pathology, muscle laminin‐α2 expression, and genetic analyses were assessed. Results: Molecular genetics revealed 2 pathogenic LAMA2 mutations in 5 of 18 CMD and 3 of 128 LGMD patients, corresponding to a LAMA2‐mutation frequency of 28% in the CMD and 2.3% in the LGMD cohorts, respectively. Conclusions: This study demonstrates a wide clinical spectrum of LAMA2‐related muscular dystrophy and its prevalence in an LGMD2 cohort, which indicates that LAMA2 muscular dystrophy should be included in the LGMD2 nomenclature. Muscle Nerve 52: 547–553, 2015     

Localized expression of specific P2X receptors in dystrophin-deficient DMD and mdx muscle

Using a combination of molecular and immunohistochemical methods, we have obtained evidence for a distinctive change in the expression patterns of ATP-gated (P2X) receptor subunits in dystrophic muscle from both Duchenne muscular dystrophy (DMD) patients and the mdx mouse model. In control myofibres there was no staining for any P2X subtype studied here, although P2X1 stained the smooth muscle of the blood vessels and P2X6 nerves and the tunica intima in small arteries. In contrast, P2X1 and P2X6 were co-expressed strongly in small regenerating muscle fibres in the dystrophic muscles, whereas this expression decreased in fully regenerated fibres. Moreover, immunoreactivity for the P2X2 receptor re-appeared in dystrophic muscle, where it co-localised with the Type 1 fibres. There is, thus, a burst of production of several P2X receptor subtypes in regenerating dystrophic muscle, which may have implications for drug targets for this muscle pathology.     

Expression of intercellular adhesion molecule‐1 by myofibers in mdx mice

Introduction: We investigated the extent to which intercellular adhesion molecule‐1 (ICAM‐1), a critical protein of the inflammatory response, is expressed in skeletal muscles of mdx mice (a murine model of Duchenne muscular dystrophy). Methods: Muscles were collected from control and mdx mice at 2‐24 weeks of age and analyzed for ICAM‐1 expression by means of Western blot and immunofluorescence. Results: Western blot revealed higher expression of ICAM‐1 in mdx compared with control muscles through 24 weeks of age. In contrast to control muscles, ICAM‐1 was expressed on the membrane of damaged, regenerating, and normal myofibers of mdx mice. CD11b+ myeloid cells also expressed ICAM‐1 in mdx muscles, and CD11b+ cells were closely associated with the membrane of myofibers expressing ICAM‐1. Conclusions: These findings support a paradigm in which ICAM‐1 and its localization to myofibers in muscles of mdx mice contributes to the dystrophic pathology. Muscle Nerve 52: 795–802, 2015     

Fukutin gene mutations in an Italian patient with early onset muscular dystrophy but no central nervous system involvement

Hypoglycosylation of α‐dystroglycan characterizes a subgroup of muscular dystrophies of variable severity, including Fukuyama congenital muscular dystrophy. We found fukutin gene mutations in a 4.5‐year‐old Italian patient, with reduced α‐dystroglycan expression, dystrophic features on muscle biopsy, hypotonia since birth, mild myopathy, but no brain involvement. Mutations in the fukutin gene can be associated with much milder phenotypes than classical Fukuyama congenital muscular dystrophy, and, although rare, can occur in non‐Japanese. Muscle Nerve, 2009     

Effects of prednisolone on skeletal muscle contractility in mdx mice

Current treatment for Duchenne muscular dystrophy (DMD) is chronic administration of the glucocorticoid prednisolone. Prednisolone improves muscle strength in boys with DMD, but the mechanism is unknown. The purpose of this study was to determine how prednisolone improves muscle strength by examining muscle contractility in dystrophic mice over time and in conjunction with eccentric injury. Mdx mice began receiving prednisolone (n = 23) or placebo (n = 16) at 5 weeks of age. Eight weeks of prednisolone increased specific force of the extensor digitorum longus muscle 26%, but other parameters of contractility were not affected. Prednisolone also improved the histological appearance of muscle by decreasing the number of centrally nucleated fibers. Prednisolone treatment did not affect force loss during eccentric contractions or recovery of force following injury. These data are of clinical relevance, because the increase in muscle strength in boys with DMD taking prednisolone does not appear to occur via the same mechanism in dystrophic mice. Muscle Nerve, 2009     

Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF−/mdx). Body mass and muscle mass of the TNF−/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.     

Noninvasive in vivo assessment of muscle impairment in the mdx mouse model--a comparison of two common wire hanging methods with two different results

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disorder that arises from a mutation in the gene for the cytoskeletal protein dystrophin, normally expressed in the myofibres. The most widely applied animal model in DMD basic research is the C57BL/10ScSn-mdx/J mouse, commonly referred to as the “mdx mouse”. The potential benefit of novel interventions in this in vivo model is often assessed by functioning tests, as the improvement of muscle impairment is the final goal of all approaches to treat DMD.In this study we compared two (TWHT) and four limb wire hanging tests (FWHT) for utility in evaluating muscle impairment in the mdx-mouse relative to its C57BL/10 wild-type counterpart. Our objective was to determine an optimal approach to perform wire hanging measurements in this model system such that latency to fall is indicative of the dystrophic phenotype that provides a quantitative measure of its presentation, and can be used to assess functional improvements that result from therapeutic intervention.Surprisingly the results of the latency times in the TWHT did not allow discrimination between the mdx population and their healthy counterparts, whereas hanging times in the FWHT enabled ready discrimination between the muscle function of mutant and wild-type animals. Furthermore, we analyzed confounding factors that explain the strengths and weaknesses of each wire hanging test configuration.The results of this study are of relevance for investigators who rely on pre clinical function tests to assess potential therapies in DMD.     

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models

Duchenne muscular dystrophy (DMD) is an X‐linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin‐associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn^+/? and mdx/utrn^?/? mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn^?/? mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation‐evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high‐rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn^?/? mice. Overall, we showed mild ‘myasthenia‐like’ neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.     

Tenascin-C expression in dystrophin-related muscular dystrophy

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration. © 1996 John Wiley & Sons, Inc.     

Timeline of cardiac dystrophy in 3–18‐month‐old MDX mice

The dystrophin‐deficient (mdx) mouse remains the most commonly used model for Duchenne muscular dystrophy (DMD). Mdx mice show a predominantly covert cardiomyopathy, the hallmark of which is fibrosis. We compared mdx and normal mice at six ages (3, 6, 9, 12, 15, and 18 months) using in vivo assessment of cardiac function, selective collagen staining, and measures of TGF‐β mRNA, Evans blue dye infiltration, macrophage infiltration, and aortic wall thickness. Clear temporal progression was demonstrated, including early fragility of cardiomyocyte membranes, which has an unrelated impact on cardiac function but is associated with macrophage infiltration and fibrosis. Aortic wall thickness is less in older mdx mice. Mdx mice display impaired responses to inotropic challenge from a young age; this is indicative of altered adrenoreceptor function. We draw attention to the paradox of ongoing fibrosis in mdx hearts without a strong molecular signature (in the form of TGF‐β mRNA expression). Muscle Nerve, 2010     

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     

Serum CK,calcium, magnesium,and oxidative phosphorylation in mdx mouse muscular dystrophy

Serum creatine kinase (CK) activity, calcium (Ca) and magnesium (Mg) contents of skeletal muscle and isolated mitochondria, as well as oxidative phosphorylation of X-linked muscular dystrophic (mdx) mice were compared with normal control animals at ages 5, 10, and 23 weeks. Serum CK is elevated in mdx mice at all ages, with highest activities at 5 weeks. The Ca content of dystrophic skeletal muscle is increased at all ages, whereas no clearly abnormal trend in muscle Mg levels was observed. Noncollagen protein (NCP), which was used as a reference base, is significantly diminished in muscle from 10- and 23-week-old mdx animals. Isolated mitochondria from mdx mice have elevated calcium content and decreased respiratory control ratios with NAD-linked substrates pyruvate/malate. The findings are distinct from those in dystrophic mice, strain 129/ReJ, but similar to observations in dystrophic hamsters and Duchenne muscular dystrophy and reflect the occurrence of overt muscle cell necrosis.     

Cardiac pathology exceeds skeletal muscle pathology in two cases of limb‐girdle muscular dystrophy type 2I

Limb‐girdle muscular dystrophy type 2I (LGMD‐2I) is caused by mutations in the fukutin‐related protein gene (FKRP) that lead to abnormal glycosylation of α‐dystroglycan in skeletal muscle. Heart involvement in LGMD‐2I is common, but little is known about a underlying cardiac pathology. Herein we describe two patients with LGMD‐2I (homozygous FKRP mutation c.826C>A, p.Leu276Ile) who developed severe congestive heart failure that required cardiac transplantation. The dystrophic pathology and impairment of α‐dystroglycan glycosylation were severe in the heart but mild in skeletal muscle, underscoring the lack of correlation between cardiac and skeletal muscle involvement in some LGMD‐2I patients. Muscle Nerve, 2009     

Reduced muscle necrosis and long-term benefits in dystrophic mdx mice after cV1q (blockade of TNF) treatment

Tumour necrosis factor (TNF) is a potent inflammatory cytokine that appears to exacerbate damage of dystrophic muscle in vivo. The monoclonal murine specific antibody cV1q that specifically neutralises murine TNF demonstrated significant anti-inflammatory effects in dystrophic mdx mice. cV1q administration protected dystrophic skeletal myofibres against necrosis in both young and adult mdx mice and in adult mdx mice subjected to 48 h voluntary wheel exercise. Long-term studies (up to 90 days) in voluntarily exercised mdx mice showed beneficial effects of cV1q treatment with reduced histological evidence of myofibre damage and a striking decrease in serum creatine kinase levels. However, in the absence of exercise long-term cV1q treatment did not reduce necrosis or background pathology in mdx mice. An additional measure of well-being in the cV1q treated mice was that they ran significantly more than control mdx mice.