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     

Utrophin is a calpain substrate in muscle cells

Calpains are Ca2+ -dependent cytosolic cysteine proteases that participate in the pathology of Duchenne muscular dystrophy (DMD). Utrophin is a functional homolog of dystrophin that partially compensates for dystrophin deficiency in myofibers of mdx mice. In this study, we investigated the susceptibility of utrophin to cleavage by calpain in vitro and in muscle cells. We found that utrophin is a direct in vitro substrate of purified calpain I and II. Cleavage of utrophin by calpain I or II generates specific degradation products that are also found in cultured control and DMD myotubes under conditions with elevated intracellular Ca2+ levels. In addition, we showed that activation of cellular calpains by Ca2+ ionophore treatment reduces utrophin protein levels in muscle cells and that calpain inhibition prevents this Ca2+ -induced reduction in utrophin levels. These observations suggest that, beside its known effect on general muscle protein degradation, calpain contributes to DMD pathology by specifically degrading the compensatory protein utrophin.     

The abnormal expression of utrophin in Duchenne and Becker muscular dystrophy is age related

J. Taylor, F. Muntoni, V. Dubowitz and C. A. Sewry (1997) Neuropathology and Applied Neurobiology 23, 399–405 The abnormal expression of utrophin in Duchenne and Becker muscular dystrophy is age related Utrophin is a 395 kDa protein with considerable homology to dystrophin. It is highly expressed in the sarcolemma of normal fetal muscle fibres but is confined to neuromuscular and myotendinous junctions, and blood vessels in adult muscle. Sarcolemmal expression occurs on regenerating fibres, irrespective of the disease, and is also seen on mature fibres in Duchenne and Becker muscular dystrophies (DMD, BMD), and inflammatory myopathies. The reasons for the abnormal expression in DMD and BMD are unclear. We have studied this expression of utrophin immunocytochemically on mature fibres in 42 cases of DMD and BMD, aged 3 months–24 years of age. All cases had some mature fibres, with no detectable fetal myosin, that showed sarcolemmal expression of utrophin. The number of these fibres and the intensity of fluorescence was low in young cases before the age of 2 years and increased with age. The fluorescence was graded on a scale of 0 to ++++ and there were significantly more cases under 2 years of age (10/12) with a grading of utrophin of only +, compared with those over 2 years (4/30, P < 0.001). Some revertant fibres, but not all, expressed utrophin and dystrophin. Our data show that the abnormal expression of utrophin on mature muscle fibres in DMD and BMD is not a continuation of the expression that occurs in fetal or regenerating muscle, but is a secondary event caused by unknown factors. The immunocytochemical intensity of utrophin is variable between cases and there is no correlation with clinical severity. As all cases studied had some expression of utrophin on mature fibres, this may be a useful additional tool for distinguishing BMD from other dystrophies, especially in cases with minimal abnormalities in dystrophin expression and/or no detectable mutation in the gene.     

CXCR4 enhances engraftment of muscle progenitor cells

Cell‐based therapy is a possible avenue for the treatment of Duchenne muscular dystrophy (DMD), an X‐linked skeletal muscle‐wasting disease. We have demonstrated that cultured myogenic progenitors derived from the adult skeletal muscle side population can engraft into dystrophic fibers of non‐irradiated, non–chemically injured mouse models of DMD (mdx^5cv) after intravenous and intraarterial transplantation, with engraftment rates approaching 10%. In an effort to elucidate the cell‐surface markers that promote progenitor cell extravasation and engraftment after systemic transplantation, we found that expression of the chemokine receptor CXCR4, whose ligand SDF‐1 is overexpressed in dystrophic muscle, enhances the extravasation of these cultured progenitor cells into skeletal muscle after intraarterial transplantation. At 1 day post‐transplantation, mice that received CXCR4‐positive enhanced green fluorescent protein (eGFP)‐positive cultured cells derived from the skeletal muscle side population displayed significantly higher amounts of eGFP‐positive mononuclear cells in quadriceps and tibialis anterior than mice that received CXCR4‐negative eGFP‐positive cells derived from the same cultured population. At 30 days posttransplantation, significantly higher engraftment rates of donor cells were observed in mice that received CXCR4‐positive cells compared with mice transplanted with CXCR4‐negative fractions. Our data suggest that CXCR4 expression by muscle progenitor cells increases their extravasation into skeletal muscle shortly after transplantation. Furthermore, this enhanced extravasation likely promotes higher donor cell engraftment rates over time. Muscle Nerve 40: 562–572, 2009     

Exercise-induced muscle growth is muscle-specific and age-dependent

Introduction: Sarcopenia, and the importance of satellite cells (SCs) in muscle growth led us to examine the effects of exercise and age on SC activation and gene expression. Methods: Eight‐ and 18‐month‐old mice were either sedentary or underwent 3 weeks of exercise (N = 24). Body mass, distance traveled, and grip strength were recorded at weekly intervals. The extensor digitorum longus (EDL), tibialis anterior (TA), gastrocnemius (GAST), and quadriceps (QUAD) muscles were analyzed along with muscle fiber area, SC activation, neuronal nitric oxide synthase (NOS‐I), MyoD, and myostatin protein content. Results: Older mice demonstrated decreased body mass, grip strength, and fiber area, but these changes were not affected by exercise. The QUAD muscle from young mice demonstrated an exercise‐induced increase in SC activation and NOS‐I and downregulation of myostatin. Conclusions: Exercise‐induced activation of SCs and regulation of gene expression are muscle‐specific and age‐dependent. Perturbed sensitivity to exercise in older mice provides insight into sarcopenia and potential treatments. Muscle Nerve, 2011     

Non-toxic ubiquitous over-expression of utrophin in the mdx mouse

Duchenne muscular dystrophy (DMD) is an inherited, severe muscle wasting disease caused by the loss of the cytoskeletal protein, dystrophin. Patients usually die in their late teens or early twenties of cardiac or respiratory failure. We have previously demonstrated that the dystrophin related protein, utrophin is able to compensate for the loss of dystrophin in the mdx mouse, the mouse model of the disease. Expression of a utrophin transgene under the control of an HSA promoter results in localization of utrophin to the sarcolemma and prevents the muscle pathology. Here we show that the over-expression of full-length utrophin in a broad range of tissues is not detrimental in the mdx mouse. These findings have important implications for the feasibility of the up-regulation of utrophin in therapy for DMD since they suggest that tissue specific up-regulation may not be necessary.     

Emerging Strategies in the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a progressive X-linked degenerative muscle disease due to mutations in the DMD gene. Genetic confirmation has become standard in recent years. Improvements in the standard of care for DMD have led to improved survival. Novel treatments for DMD have focused on reducing the dystrophic mechanism of the muscle disease, modulating utrophin protein expression, and restoring dystrophin protein expression. Among the strategies to reduce the dystrophic mechanisms are 1) inhibiting inflammation, 2) promoting muscle growth and regeneration, 3) reducing fibrosis, and 4) facilitating mitochondrial function. The agents under investigation include a novel steroid, myostatin inhibitors, idebenone, an anti-CTGF antibody, a histone deacetylase inhibitor, and cardiosphere-derived cells. For utrophin modulation, AAV-mediated gene therapy with GALGT2 is currently being investigated to upregulate utrophin expression. Finally, the strategies for dystrophin protein restoration include 1) nonsense readthrough, 2) synthetic antisense oligonucleotides for exon skipping, and 3) AAV-mediated micro/minidystrophin gene delivery. With newer agents, we are witnessing the use of more advanced biotechnological methods. Although these potential breakthroughs provide significant promise, they may also raise new questions regarding treatment effect and safety.     

Actin sliding velocity on pure myosin isoforms from dystrophic mouse muscles

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by skeletal muscle wasting and atrophy. Recent evidence suggests that the impaired skeletal muscle performance in DMD is not solely dependent on a loss of contractile muscle mass. In this study the myosin motor function of mdx and control (wildtype, WT) mice was compared using pure myosin isoforms in an “in vitro motility assay” (IVMA). Actin sliding velocity (Vf) on myosin 2B extracted from single muscle fibers of gastrocnemius muscles was significantly lower in mdx mice (3.48 ± 0.13 μm/s, n = 18) than in WT mice (4.02 ± 0.19 μm/s, n = 10). No difference in Vf was found between myosin 1 extracted from soleus muscles of mdx (0.84 ± 0.04 μm/s, n = 13) and of WT (0.89 ± 0.04 μm/s, n = 10). The results suggest that the dystrophic process alters myosin molecular function, and this contributes to the functional impairment in dystrophic muscles. Muscle Nerve 40: 249–256, 2009     

Dystrophin-associated protein abnormalities in dystrophin-deficient muscle fibers from symptomatic and asymptomatic Duchenne/Becker muscular dystrophy carriers

The absence of dystrophin in muscle fibers is associated with a major reduction in dystrophin-associated proteins (DAPs) and disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. We investigated the expression of the DAPs β-dystroglycan, α-sarcoglycan, γ-sarcoglycan and syntrophin as well as utrophin in the muscles of 13 Duchenne muscular dystrophy (DMD) carriers (with variable percentages of dystrophin-deficient fibers and with a range of clinical symptoms), 2 Becker muscular dystrophy (BMD) carriers (expressing a highly truncated protein in some fibers), 2 girls with a DMD-like phenotype, and 11 BMD carriers with almost normal dystrophin expression (reduced or patchy distribution in a few fibers only and rare dystrophin-deficient fibers). DAPs were highly reduced in all fibers lacking dystrophin in the DMD carriers, but were almost normal in the dystrophin-deficient fibers of the 2 BMD carriers with highly truncated dystrophin. In the 11 BMD carriers with nearly normal dystrophin, the few fibers with reduced or patchy dystrophin immunostaining also showed reduced DAP expression in correlation with dystrophin expression. Immunoblot for β-dystroglycan and α-sarcoglycan confirmed the immunohistochemical findings. Utrophin expression was slightly increased in a proportion of fibers in the DMD and BMD carriers with dystrophin mosaicism. We found no correlation between utrophin expression and DAP expression. We conclude that absence or reduction of dystrophin in muscle fibers of DMD and BMD carriers causes a reduction of DAPs in the same fibers, as observed in DMD and BMD patients, while utrophin does not seem to play a role in DAP expression in adult muscle. Received: 11 January 1996 / Revised, accepted: 16 April 1996     

Physical exercise during muscle regeneration improves recovery of the slow/oxidative phenotype

Introduction: As skeletal muscle mass recovery after extensive injury is improved by contractile activity, we explored whether concomitant exercise accelerates recovery of the contractile and metabolic phenotypes after muscle injury. Methods: After notexin‐induced degeneration of a soleus muscle, Wistar rats were assigned to active (running exercise) or sedentary groups. Myosin heavy chains (MHC), metabolic enzymes, and calcineurin were studied during muscle regeneration at different time points. Results: The mature MHC profile recovered earlier in active rats (21 days after injury) than in sedentary rats (42 days). Calcineurin was higher in the active degenerated than in the sedentary degenerated muscles at day 14. Citrate synthase and total lactate dehydrogenase (LDH) activity decreased after injury and were similarly recovered in both active and sedentary groups at 14 or 42 days, respectively. H‐LDH isozyme activity recovered earlier in the active rats. Conclusions: Exercise improved recovery of the slow/oxidative phenotype after soleus muscle injury. Muscle Nerve 55 : 91–100, 2017     

Activity of Krebs cycle enzymes in mdx mice

Introduction: Duchenne muscular dystrophy (DMD) is a degenerative disease of skeletal, respiratory, and cardiac muscles caused by defects in the dystrophin gene. More recently, brain involvement has been verified. Mitochondrial dysfunction and oxidative stress may underlie the pathophysiology of DMD. In this study we evaluate Krebs cycle enzymes activity in the cerebral cortex, diaphragm, and quadriceps muscles of mdx mice. Methods: Cortex, diaphragm, and quadriceps tissues from male dystrophic mdx and control mice were used. Results: We observed increased malate dehydrogenase activity in the cortex; increased malate dehydrogenase and succinate dehydrogenase activities in the diaphragm; and increased citrate synthase, isocitrate dehydrogenase, and malate dehydrogenase activities in the quadriceps of mdx mice. Conclusion: This study showed increased activity of Krebs cycle enzymes in cortex, quadriceps, and diaphragm in mdx mice. Muscle Nerve 53 : 91–95, 2016     

Dystrophinopathy muscle biopsies in the genetic testing ERA: One center's data

Introduction: Comprehensive genetic testing for dystrophinopathy can detect ～95% of pathogenic variants in the dystrophin gene (DMD) and is often the preferred diagnostic approach. Methods: We reviewed pathology reports for muscle biopsies evaluated at the University of Iowa with a pathological diagnosis of dystrophinopathy based on dystrophic histopathology and abnormal immunofluorescence staining: reduced to absent dystrophin, expression of utrophin, and loss of neuronal nitric oxide synthase. Results: The percentage of muscle biopsies with dystrophinopathy has been stable since 1997. Among 2,298 biopsies evaluated between 2011 and 2016, 72 (3.1%) had pathologic features of dystrophinopathy. Median age at biopsy was 8 years (range, 0.66–84). Half had undergone DMD genetic testing prior to biopsy. Clinical phenotypes recorded on requisitions were typical of muscular dystrophy for 57 (79%) biopsies. Discussion: Muscle biopsy continues to play an important role in the diagnosis of dystrophinopathy, particularly in patients with later symptom onset, comorbidities, or normal DMD genetic testing results. Muscle Nerve 58 : 148–152, 2018     

Statin-associated changes in skeletal muscle function and stress response after novel or accustomed exercise

Introduction: The most common side effect of statins, myopathy, is more likely in exercisers. We investigated the interaction of statin treatment with novel vs. accustomed exercise on muscle function, heat shock protein (Hsp) expression, and caspase activation. Methods: Mice received daily cerivastatin or saline for 2 weeks, with/without wheel running (RW) (novel/sedentary). Accustomed groups completed 2 weeks of RW before statins. At 4 weeks, plantarflexor isometric force, Hsp25, αB‐crystallin, caspase‐3 and ‐9, and plasma creatine kinase (CK) were quantified. Results: Statins reduced force in sedentary and novel groups, compared with saline, by 15% and 27%, respectively. Muscle fatigability increased 21% and 30% with statins compared with saline in sedentary and novel groups, respectively. Accustomed exercise prevented statin‐associated force loss and increased fatigability. CK did not correlate with functional outcomes. RW increased Hsp protein in all groups. Conclusion: Our results suggest that exercise prior to statin treatment can protect against decrements in muscle function. Muscle Nerve 2011     

Utility of cystatin C to monitor renal function in duchenne muscular dystrophy

Creatinine as a marker of renal function has limited value in Duchenne muscular dystrophy (DMD) because of reduced muscle mass. Alternative methods of assessing renal function are sorely needed. Cystatin C, a nonglycosylated protein unaffected by muscle mass, is potentially an ideal biomarker of nephrotoxicity for this population but requires validation. In all, 75 subjects were recruited: 35 DMD (mean age 10.8 ± 5.4 years, corticosteroids n = 19, ambulatory n = 26), 29 healthy controls, 10 with renal disease, and one DMD with renal failure. Cystatin C levels in DMD were normal irrespective of age, ambulation, or corticosteroid treatment. Serum cystatin C was 0.67 ± 0.11 mg/l compared to normal controls 0.69 ± 0.09. mg/l. In these same individuals serum creatinine was severely reduced (0.27 ± 0.12 mg/dl) versus normals (0.75 ± 0.15 mg/dl, P < 0.01). In one DMD subject in renal failure, cystatin C was elevated. This study demonstrates the potential value of cystatin C as a biomarker for monitoring renal function in DMD. Its applicability extends to other neuromuscular diseases. Muscle Nerve, 2009     

Expression of utrophin (dystrophin–related protein) during regeneration and maturation of skeletal muscle in canine X–linked muscular dystrophy

The regulation of utrophin, the autosomal homologue of dystrophin, has been studied in the canine X–linked model of Duchenne muscular dystrophy. Dystrophic muscle has been shown to exhibit abnormal sarcolemmal expression of utrophin, in addition to the normal expression at the neuromuscular junction, in peripheral nerves, vascular tissues and regenerating fibres. To establish whether this abnormal presence of utrophin in dystrophic muscle is a consequence of continued expression following regeneration, or is attributable to a disease related up–regulation, the expression of utrophin was compared immunocytochemically with that of dystrophin, β–spectrin and neonatal myosin in regenerating normal and dystrophic canine muscle, following necrosis induced by the injection of venom from the snake Notechis scutatis. In normal regenerating muscle, sarcolemmal utrophin and dystrophin were detected concomitantly from 2–3 d post–injection, prior to the expression of β–spectrin. Down–regulation of utrophin was apparent in some fibres from 7 d, and it was no longer present on the extra–junctional sarcolemma by 14 d. Neonatal myosin was still present in all fibres at this stage, but dystrophin and β–spectrin had been fully restored. In dystrophic regenerating muscle, downregulation of utrophin occurred from 7 d, although it persisted on some fibres until 28 d, longer than in normal muscle. At 42 d, however, utrophin in dystrophic muscle was only detected in a population of small fibres thought to represent a second cycle of regeneration, with no immunolabelling of mature fibres. The results show that most utrophin is down–regulated in regenerating dystrophic fibres, prior to neonatal myosin, thus abnormal sarcolemmal expression of utrophin in dystrophic muscle is unlikely to be a continuation of the maturational process. Persistence of both utrophin and neonatal myosin, however, suggest a delay in the maturation of dystrophic muscle. In addition, a second cycle of degeneration and regeneration in dystrophic muscle does not occur whilst utrophin is still present, suggesting it may have a protective role against fibre damage and necrosis.     

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     

Low‐intensity running exercise enhances the capillary volume and pro‐angiogenic factors in the soleus muscle of type 2 diabetic rats

Introduction: We determined the effects of low‐intensity exercise on the three‐dimensional capillary structure and associated angiogenic factors in the soleus muscle of Goto‐Kakizaki (GK) diabetic rats. Methods: Four groups of male rats were studied: sedentary nondiabetic (Con), exercised nondiabetic control (Ex), sedentary GK, and exercised GK (GK+Ex). Rats in the Ex and GK+Ex groups were subjected to chronic low‐intensity running on a treadmill (15 m/min, 60 min/session, 5 sessions/week for 3 weeks). Results: Although mean capillary volume and diameter were lower in the GK compared with all other groups, low‐intensity exercise increased both of these measures in GK rats. Mitochondrial markers, i.e., SDH activity and PGC‐1α expression, and the levels of angiogenic factors were higher in the GK+Ex than all other groups. Exercise increased vascular endothelial growth factor (VEGF) protein levels and the VEGF‐to‐TSP‐1 ratio, an indicator of angiogenesis, in GK rats. Conclusions: Combined, the results indicate that low‐intensity exercise reduces some of the microcirculatory complications in type 2 diabetic muscles. Muscle Nerve 51: 391–399, 2015     

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenetic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin, and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p<0.0001), and by 50% in skeletal muscle (p<0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labeling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.