Dystrophic Muscle Disease Research Articles

Defining the structure-activity relationship for a novel class of allosteric MKP5 inhibitors

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease.

A Spotlight on T Lymphocytes in Duchenne Muscular Dystrophy-Not Just a Muscle Defect.

The lack of dystrophin in Duchenne muscular dystrophy (DMD) results in membrane fragility resulting in contraction-induced muscle damage and subsequent inflammation. The impact of inflammation is profound, resulting in fibrosis of skeletal muscle, the diaphragm and heart, which contributes to muscle weakness, reduced quality of life and premature death. To date, the innate immune system has been the major focus in individuals with DMD, and our understanding of the adaptive immune system, specifically T cells, is limited. Targeting the immune system has been the focus of multiple clinical trials for DMD and is considered a vital step in the development of better treatments. However, we must first have a complete picture of the involvement of the immune systems in dystrophic muscle disease to better understand how inflammation influences disease progression and severity. This review focuses on the role of T cells in DMD, highlighting the importance of looking beyond skeletal muscle when considering how the loss of dystrophin impacts disease progression. Finally, we propose that targeting T cells is a potential novel therapeutic in the treatment of DMD.

Macrophage plasticity in Duchenne muscular dystrophy: a nexus of pathological remodelling with therapeutic implications.

Duchenne muscular dystrophy (DMD) is characterized by chronic skeletal muscle necrosis, leading to muscle regeneration failure and fibrosis. Although macrophages (MPs) are normally essential for muscle regeneration, dysregulated MP function promotes pathological muscle remodelling. Infiltrating MPs can be predominantly pro-inflammatory (M1 biased), anti-inflammatory (M2 biased) or of a mixed phenotype and can originate from the adult bone marrow (monocyte dependent) or embryonic precursors (monocyte independent). In mdx mice (genetic model of DMD) lacking either Toll-like receptor (Tlr) 2 or Tlr4, it is found that MP infiltration of dystrophic muscle is significantly reduced and that the MP phenotype is shifted toward a more anti-inflammatory profile. This is accompanied by significant improvements in muscle histology and force production. Lack of the chemokine receptor CCR2, which impedes monocyte release from the bone marrow, leads to similar beneficial effects in mdx mice. Evidence was also found for Tlr4-regulated induction of trained innate immunity in MPs cultured from the bone marrow of mdx mice before their entry into the muscle. These MPs exhibit epigenetic and metabolic alterations, accompanied by non-specific hyper-responsiveness to multiple stimuli, which is manifested by potentiated upregulation of both pro- and anti-inflammatory genes. In summary, exaggerated recruitment of monocyte-derived MPs and signs of trained innate immunity at the level of the bone marrow are features of the immunophenotype associated with dystrophic muscle disease. These phenomena are regulated by Toll-like receptors that bind endogenous damage-associated molecular pattern (DAMP) molecules, suggesting that DAMP release from dystrophic muscles modulates MP plasticity at the bone marrow level through Toll-like receptor-driven mechanisms.

Drugging an “undruggable” phosphatase

Pharmacology Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have roles in many diseases but have been considered “undruggable” because of their high degree of conservation. Gannam et al. identified and characterized a small molecule that inhibited MKP5 but did not affect the activity of related phosphatases. In muscle cells, the inhibitor blocked MKP5 from inactivating the MAPKs and inhibited a signaling pathway that promotes fibrosis in dystrophic muscle disease. These results demonstrate that it is possible to target MKPs with a high degree of specificity. Sci. Signal. 13 , eaba3043 (2020).

An allosteric site on MKP5 reveals a strategy for small-molecule inhibition.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered "undruggable," but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK-derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-β1 signaling in muscle and that the inhibitor blocked TGF-β1-mediated Smad2 phosphorylation. TGF-β1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

Skeletal muscle fibrosis: an overview.

Extracellular matrix (ECM) is an essential component of skeletal muscle. It provides a framework structure that holds myofibers and blood capillaries and nerves supplying the muscle. In addition, it has a principal role in force transmission, maintenance and repair of muscle fibers. Excessive accumulation of ECM components, especially collagens, either due to excessive ECM production, alteration in ECM-degrading activities, or a combination of both is defined as fibrosis. Skeletal muscle fibrosis impairs muscle function, negatively affects muscle regeneration after injury and increases muscle susceptibility to re-injury, therefore, it is considered a major cause of muscle weakness. Fibrosis of skeletal muscle is a hallmark of muscular dystrophies, aging and severe muscle injuries. Thus, a better understanding of the mechanisms of muscle fibrosis will help to advance our knowledge of the events that occur in dystrophic muscle diseases and develop innovative anti-fibrotic therapies to reverse fibrosis in such pathologic conditions. This paper explores an overview of the process of muscle fibrosis, as well as different murine models for studying fibrosis in skeletal muscles. In addition, factors regulating fibrosis and strategies to inhibit muscle fibrosis are discussed.

Imaging Patterns of Muscle Atrophy.

The role of muscle imaging in the diagnosis of inherited and acquired muscle diseases has gained clinical relevance. In particular, magnetic resonance imaging (MRI) is increasingly being used for diagnostic purposes, especially with its capability of whole-body musculature assessment. The assessment and quantification of muscle involvement in muscle diseases can be of diagnostic value by identifying a certain involvement pattern and thus narrowing the differential diagnosis and supporting the clinical diagnosis. In addition, more recently the role of imaging has gone beyond diagnostic purposes and includes disease as well as treatment monitoring. Conventional and quantitative muscle MRI techniques allow for the detection of subclinical disease progression (e.g., in muscular dystrophies) and is a powerful surrogate outcome measure in clinical trials. We present and discuss recent data on the role of conventional and quantitative MRI in the diagnosis and monitoring of inherited dystrophic muscle diseases as well as muscle denervation.

Glycerol-induced injury as a new model of muscle regeneration.

Skeletal muscle regenerates efficiently following injuries and diseases. However, muscle regeneration is compromised in several conditions by adipocyte infiltration and excessive collagen deposition. Adipocyte infiltration is a characteristic feature of sarcopenia, diabetes, cachexia, muscular dystrophies and advanced cases of Duchenne muscular dystrophy (DMD), while excessive collagen deposition is a hallmark of muscular dystrophies and severe muscle injuries, such as lacerations, contusions and strains. Muscle adipogenesis and fibrosis are major causes of muscle weakness that impairs muscle function. Therefore, it is essential to develop an appropriate injury model to understand the mechanisms of adipocyte formation and fibrosis during muscle regeneration. This will help to advance our knowledge regarding the events that occur in dystrophic muscle diseases and develop innovative therapies for such pathological conditions. To date, several experimental injury models have been used to investigate skeletal muscle regeneration. Intramuscular injection of glycerol has been used as a new model to induce muscle injury and regeneration. It was reported that degenerative changes following glycerol-induced injury resemble those seen in DMD suggesting a similarity in their mechanism. The objective of the current review is to provide an overview of the current knowledge of using glycerol as a new model to induce muscle injury and regeneration.

P 42 Congenital myasthenia in adult patients – a diagnostic and therapeutic challenge

Congenital myasthenic syndromes (CMS) are a heterogenic group of mainly recessive inherited loss-of-function mutations of different molecular structures of the motor endplate leading to a defective neuromuscular transmission. They typically present in early childhood with ocular, bulbar and fatigable proximal muscle weakness. Occasionally symptoms may occur first in late childhood or even early adolescence. Here we report two patients who were first diagnosed CMS in early adulthood. In the first patient dysphagia was present in early childhood seen as perinatal hypoxic brain injury despite normal cranial MRI and remarkable cognitive development. By the age of 10 years the patient began to suffer from a fatigable general and ocular muscle weakness as well as bilateral ptosis. Electromyography was myopathic in absence of a decrement after repetitive stimulation. Furthermore, creatin kinase was normal and myasthenia-specific auto-antibodies were absent. Considering a mitochondriopathy a muscle biopsy was performed showing pronounced atrophy of single muscle fibers. By the age of 26 the patient was evaluated again and the differential diagnosis CMS was considered. Genetic diagnostics revealed a homozygous mutation in the CHRNA1-gen. Symptomatic treatment with pyridostigmine led to a significant clinical improvement. In the second case a 25 yo patient reported a fatigable proximal muscle weakness, dysarthria and impaired ocular muscle function since the age of 10. Furthermore, a high palate and mild dysmorphic signs were suspicious. Electromyography showed myopathic potentials. Blood tests and a muscle biopsy were normal. Finally, genetic testing revealed mutation in the COLQ-gen. Initiation of 3,4-diaminopyridine treatment led to clinical improvement whereas salbutamol showed no supporting effect. These two patients show that the diagnosis CMS may be challenging. Clinical and diagnostic results may mimic primary myopathy or dystrophic muscle disease as well as mitochondriopathy. Furthermore pathological myasthenia-specific auto-antibodies targeting the motor endplate are absent. Finally, it is import to take the differential diagnosis CMS into account. Because of different treatment strategies the underlying genetic changes should be identified.

Adipose Tissue-Derived Stem Cell Secreted IGF-1 Protects Myoblasts from the Negative Effect of Myostatin

Myostatin, a TGF-β family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

Facioscapulohumeral Muscular Dystrophy: A Radiologic and Manometric Study of the Pharynx and Esophagus

Facioscapulohumeral muscular dystrophy (FSHD) is not a recognized neuromuscular cause of dysphagia. However, a study of pharyngoesophageal function in FSHD has not been performed or reported. The aim of this study was to ascertain by relatively noninvasive techniques whether the dystrophic muscle disease that underlies FSHD involves the pharyngeal and/or the esophageal striated and smooth muscles. We used conventional cineradiography and intraluminal esophageal manometry on separate occasions to study pharyngeal and esophageal function in 20 patients with FSHD at various stages of disease, with or without complaints of deglutition. Age- and sex-matched control data were used for comparison of the manometric component of the study. Twelve men and eight women with FSHD were studied. The mean patient age was 38.1 years (41.9 years for controls), and the age range was 19-61 years (22-55 years for controls). The mean disease duration was 16.7 years (range = 4-39 years).Five patients admitted to having intermittent oropharyngeal dysphagia (difficulty to initiate swallowing, cough after swallowing, sensation of food stuck in throat, or nasal regurgitation), and three patients admitted to intermittent esophageal dysphagia (difficulty swallowing both liquids and solids). Chest roentgengrams showed a hiatal hernia in four patients, but no active cardiopulmonary disease. Abnormal instrumental results were documented in eight patients: Cineradiography detected ineffectual pharyngeal contractions (2 patients), pharyngeal diverticula but normal pharyngeal motility (2 patients), and decreased cricopharyngeal and upper esophageal relaxation (2 patients). The mean manometric pressure of the patient group was not significantly different from the control data. However, manometry detected motility abnormalities that were not reflected in the mean data and included increased lower esophageal sphincter resting pressure with normal or abnormal relaxation (2 patients) and inconsistent, high-amplitude, long-duration, primary peristaltic contractions (1 patient). Patients with FSHD did not spontaneously volunteer intermittent complaints of deglutition. This study did not definitely establish that the cause of abnormal pharyngeal and cervical esophageal function was related to the dystrophic process that underlies FSHD. Any esophageal dysmotility was nonspecific and insignificant and was caused by an undetermined, probably neuropathic, process unrelated to the muscular dystrophy.

Muscle Diseases: The Muscular Dystrophies

Dystrophic muscle disease can occur at any age. Early- or childhood-onset muscular dystrophies may be associated with profound loss of muscle function, affecting ambulation, posture, and cardiac and respiratory function. Late-onset muscular dystrophies or myopathies may be mild and associated with slight weakness and an inability to increase muscle mass. The phenotype of muscular dystrophy is an endpoint that arises from a diverse set of genetic pathways. Genes associated with muscular dystrophies encode proteins of the plasma membrane and extracellular matrix, and the sarcomere and Z band, as well as nuclear membrane components. Because muscle has such distinctive structural and regenerative properties, many of the genes implicated in these disorders target pathways unique to muscle or more highly expressed in muscle. This chapter reviews the basic structural properties of muscle and genetic mechanisms that lead to myopathy and muscular dystrophies that affect all age groups.

Movement, Singing, and Instrument Playing Strategies for a Child with Myotonic Dystrophy

ABSTRACT: Myotonic dystrophy, a type of muscular dystrophy, is defined as a genetic disorder with progressive muscle deterioration and weakness. The subject in this study, a 10-year-old boy, was diagnosed with congenital myotonic dystrophy with gross and fine motor deficits and articulation disorders. The purpose of this case study was to investigate the transfer of gross motor, articulation, and instrument playing skills during small group music therapy sessions to regular physical education classes and regular music education classes. Results indicated that the frequency of accurate task performances were significantly correlated in music therapy and physical education classes and music therapy and music education classes. Although the results of this study may not substantiate the transfer of skills from the music therapy group sessions to regular physical education and music education classes, the findings may suggest the efficacy of practicing gross motor, articulation, and instrument playing skills in small group music therapy settings as supplemental strategies that may enhance regular classroom skills. According to Emery (2000), there are at least 40,000 individuals affected with a severe form of muscular dystrophy in the United States of America. Muscular dystrophy is defined in various literatures as a hereditary and progressive condition that results in increasing degeneration of muscle cells (AhIstrom & Natterlund, 1999), increasing loss of muscle power (Willard & Spackman, 1971), and muscle weakness and wasting (Emery, 2000; Porr & Rainville, 1999). Different types of muscular disorders involve different groups of muscles (AhIstrom & Natterlund, 1999). Myotonic dystrophy is a distinct type of muscular dystrophy in which the congenital onset type is considered the most severe diagnosis (Harper, 2001; Moxley, 1997; Ruben & Macciomei, 1986). Harper (2001 (defined myotonic dystrophy as a genetically determined disorder where a characteristic pattern of dystrophic muscle disease is associated with myotonia and with specific abnormalities of a variety of other systems. All myotonic disorders appear to result 1) from either specific mutations in genes that encode muscle chloride or sodium channels or 2) from mutations, such as the protein kinase in myotonic dystrophy (Moxley, 1997). The affected muscles enlarge due to fat and connective tissue deposit, but muscle fibers degenerate and atrophy. The various affected areas associated with myotonic dystrophy include the face, fingers, hands, forearms, legs, and feet (Marieb, 2002). Weakness in the muscles of the face is a prominent feature of myotonic dystrophy (Porr & Rainville, 1999), which is especially severe in children with congenital myotonic dystrophy (Emery, 2000; Harper, 2001; Moxley, 1997; Ruben & Macciomei, 1986). Harper (2001) further pointed out that the jaw may hang open in children with congenital myotonic dystrophy that results in mouth breathing and speech disturbance and difficulties. Delayed motor development is another striking characteristic in myotonic dystrophy (Harper, 2001). Children with congenital myotonic dystrophy exhibit muscle wasting and weakness of lower leg and forearm muscles as well as the small muscles of the hands (Emery, 2000; Moxley, 1997; Ruben & Macciomei, 1986) and imbalance during gross motor skills (Harper, 2001). In addition to various physical deficits, mental retardation, usually moderate to mild in degree, is associated with congenital myotonic dystrophy (Harper, 2001 ; Jamero & Dundore, 1982; Moxley, 1997). Children with congenital myotonic dystrophy exhibit an increased frequency of hearing and speech disorders (Moxley, 1997), attention deficiencies (Ambrosini & Nurnberg, 1979; Moxley, 1997), mental deterioration (Ambrosini & Nurnberg, 1979; Jamero & Dundore, 1982), and memory disturbance (Ambrosini & Nurnberg, 1979) as they grow older. Other associated psychological aspects include attention deficit hyperactivity disorder and problem behaviors (Harper, 2001), somnolence, and apathy (Ambrosini & Nurnberg, 1979; Harper, 2001), and depression (Harper, 2001). …

Increased expression of δCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.

Muscle relaxation in patients with neuromuscular diseases

The classification of neuromuscular diseases with regard to the use of muscle relaxants is based on the localisation of the particular abnormality. Three types of syndromes can be differentiated: (1) denervation states; (2) disturbances of neuromuscular transmission; and (3) intracellular disease. Succinylcholine should be avoided in all types of denervation syndrome due to the possibility of life-threatening hyperkalaemia. The time frame during which succinylcholine must be avoided following a traumatic denervation or burn begins 24 h after the event. The exact period of risk is unknown, but a duration of 6 months can be considered the absolute minimum. Patients may display increased sensitivity to non-depolarising muscle relaxants following damage to the second motoneuron (e.g., amyotrophic lateral sclerosis), whereas in diseases of the first motoneuron (e.g., cerebral apoplexy), increased resistance to muscle relaxants may be observed in the affected parts of the body. In diseases of the neuromuscular junction (myasthenia gravis) there is increased sensitivity to non-depolarising muscle relaxants. No complications have been described following the use of succinylcholine in these patients, however, the use of reversal agents may lead to prolongation of the effect of succinylcholine. Patients with a primary myopathy may display increased sensitivity to non-depolarising muscle relaxants. The use of drugs with acetylcholine-like actions (succinylcholine, reversal agents) should be avoided due to the danger of triggering muscle spasms in patients with myotonic disease and the risk of rhabdomyolysis in patients with dystrophic muscle disease. Irrespective of the type of muscle disease present, titration of the dose of muscle relaxant should always be done using a nerve stimulator.(ABSTRACT TRUNCATED AT 250 WORDS)

Vecuronium titration for muscle relaxation in myotonic dystrophy

SummaryA 14‐year‐old boy with pronounced myotonic dystrophy and cardiac malformation had a normal ED95 (53 μg·kg−1) of vecuronium, whereas the recovery index (25‐75% twitch recovery) was three times as long as in the absence of neuromuscular disease. We conclude that in young patients with dystrophic muscle disease individual titration of the muscle relaxant with the aid of a nerve stimulator does not preclude a considerable delay in the recovery of neuromuscular transmission.

Morphologic and morphometric analysis of muscle in X-linked myotubular myopathy

The X-linked form of myotubular myopathy is highly lethal in neonates. Several autopsy-derived muscles from two probands of a new kindred who survived for 100 days because of intensive supportive care were analyzed by light microscopy, morphometry, enzyme histochemistry, and electron microscopy. The results were compared with a similar analysis of muscle from control fetal and neonatal subjects. The findings, in addition to the characteristic centronucleated hypotrophic myofibers, included widespread myofiber degeneration and focal contraction band necrosis that differed from the types seen in other myopathic and dystrophic muscle diseases. A high frequency of degenerating nuclei that often contained large nucleoli was observed. Because of the paradoxic nuclear morphology, nuclear failure (in migration and myofibrillogenesis) is believed to be of central importance in the pathogenesis of this disease.