Hsp72 preserves muscle function and slows progression of severe muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilizing protein dystrophin. Dystrophin-deficient muscle fibres are fragile and susceptible to an influx of Ca(2+), which activates inflammatory and muscle degenerative pathways. At present there is no cure for DMD, and existing therapies are ineffective. Here we show that increasing the expression of intramuscular heat shock protein 72 (Hsp72) preserves muscle strength and ameliorates the dystrophic pathology in two mouse models of muscular dystrophy. Treatment with BGP-15 (a pharmacological inducer of Hsp72 currently in clinical trials for diabetes) improved muscle architecture, strength and contractile function in severely affected diaphragm muscles in mdx dystrophic mice. In dko mice, a phenocopy of DMD that results in severe spinal curvature (kyphosis), muscle weakness and premature death, BGP-15 decreased kyphosis, improved the dystrophic pathophysiology in limb and diaphragm muscles and extended lifespan. We found that the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA, the main protein responsible for the removal of intracellular Ca(2+)) is dysfunctional in severely affected muscles of mdx and dko mice, and that Hsp72 interacts with SERCA to preserve its function under conditions of stress, ultimately contributing to the decreased muscle degeneration seen with Hsp72 upregulation. Treatment with BGP-15 similarly increased SERCA activity in dystrophic skeletal muscles. Our results provide evidence that increasing the expression of Hsp72 in muscle (through the administration of BGP-15) has significant therapeutic potential for DMD and related conditions, either as a self-contained therapy or as an adjuvant with other potential treatments, including gene, cell and pharmacological therapies.

BGP-15 Improves Aspects of the Dystrophic Pathology in mdx and dko Mice with Differing Efficacies in Heart and Skeletal Muscle.

Duchenne muscular dystrophy is a severe and progressive striated muscle wasting disorder that leads to premature death from respiratory and/or cardiac failure. We have previously shown that treatment of young dystrophic mdx and dystrophin/utrophin null (dko) mice with BGP-15, a coinducer of heat shock protein 72, ameliorated the dystrophic pathology. We therefore tested the hypothesis that later-stage BGP-15 treatment would similarly benefit older mdx and dko mice when the dystrophic pathology was already well established. Later stage treatment of mdx or dko mice with BGP-15 did not improve maximal force of tibialis anterior (TA) muscles (in situ) or diaphragm muscle strips (in vitro). However, collagen deposition (fibrosis) was reduced in TA muscles of BGP-15-treated dko mice but unchanged in TA muscles of treated mdx mice and diaphragm of treated mdx and dko mice. We also examined whether BGP-15 treatment could ameliorate aspects of the cardiac pathology, and in young dko mice it reduced collagen deposition and improved both membrane integrity and systolic function. These results confirm BGP-15's ability to improve aspects of the dystrophic pathology but with differing efficacies in heart and skeletal muscles at different stages of the disease progression. These findings support a role for BGP-15 among a suite of pharmacological therapies for Duchenne muscular dystrophy and related disorders.

Scriptaid enhances skeletal muscle insulin action and cardiac function in obese mice.

AIM: To determine the effect of Scriptaid, a compound that can replicate aspects of the exercise adaptive response through disruption of the class IIa histone deacetylase (HDAC) corepressor complex, on muscle insulin action in obesity. MATERIALS AND METHODS: Diet-induced obese mice were administered Scriptaid (1 mg/kg) via daily intraperitoneal injection for 4 weeks. Whole-body and skeletal muscle metabolic phenotyping of mice was performed, in addition to echocardiography, to assess cardiac morphology and function. RESULTS: Scriptaid treatment had no effect on body weight or composition, but did increase energy expenditure, supported by increased lipid oxidation, while food intake was also increased. Scriptaid enhanced the expression of oxidative genes and proteins, increased fatty acid oxidation and reduced triglycerides and diacylglycerides in skeletal muscle. Furthermore, ex vivo insulin-stimulated glucose uptake by skeletal muscle was enhanced. Surprisingly, heart weight was reduced in Scriptaid-treated mice and was associated with enhanced expression of genes involved in oxidative metabolism in the heart. Scriptaid also improved indices of both diastolic and systolic cardiac function. CONCLUSION: These data show that pharmacological targeting of the class IIa HDAC corepressor complex with Scriptaid could be used to enhance muscle insulin action and cardiac function in obesity.

Identification of FHL1 as a therapeutic target for Duchenne muscular dystrophy.

Utrophin is a potential therapeutic target for the fatal muscle disease, Duchenne muscular dystrophy (DMD). In adult skeletal muscle, utrophin is restricted to the neuromuscular and myotendinous junctions and can compensate for dystrophin loss in mdx mice, a mouse model of DMD, but requires sarcolemmal localization. NFATc1-mediated transcription regulates utrophin expression and the LIM protein, FHL1 which promotes muscle hypertrophy, is a transcriptional activator of NFATc1. By generating mdx/FHL1-transgenic mice, we demonstrate that FHL1 potentiates NFATc1 activation of utrophin to ameliorate the dystrophic pathology. Transgenic FHL1 expression increased sarcolemmal membrane stability, reduced muscle degeneration, decreased inflammation and conferred protection from contraction-induced injury in mdx mice. Significantly, FHL1 expression also reduced progressive muscle degeneration and fibrosis in the diaphragm of aged mdx mice. FHL1 enhanced NFATc1 activation of the utrophin promoter and increased sarcolemmal expression of utrophin in muscles of mdx mice, directing the assembly of a substitute utrophin-glycoprotein complex, and revealing a novel FHL1-NFATc1-utrophin signaling axis that can functionally compensate for dystrophin.

Alterations in Notch signalling in skeletal muscles from mdx and dko dystrophic mice and patients with Duchenne muscular dystrophy.

New Findings What is the central question of this study? The Notch signalling pathway plays an important role in muscle regeneration, and activation of the pathway has been shown to enhance muscle regeneration in aged mice. It is unknown whether Notch activation will have a similarly beneficial effect on muscle regeneration in the context of Duchenne muscular dystrophy (DMD). What is the main finding and its importance? Although expression of Notch signalling components is altered in both mouse models of DMD and in human DMD patients, activation of the Notch signalling pathway does not confer any functional benefit on muscles from dystrophic mice, suggesting that other signalling pathways may be more fruitful targets for manipulation in treating DMD. Abstract In Duchenne muscular dystrophy (DMD), muscle damage and impaired regeneration lead to progressive muscle wasting, weakness and premature death. The Notch signalling pathway represents a central regulator of gene expression and is critical for cellular proliferation, differentiation and apoptotic signalling during all stages of embryonic muscle development. Notch activation improves muscle regeneration in aged mice, but its potential to restore regeneration and function in muscular dystrophy is unknown. We performed a comprehensive examination of several genes involved in Notch signalling in muscles from dystrophin-deficient mdx and dko (utrophin- and dystrophin-null) mice and DMD patients. A reduction of Notch1 and Hes1 mRNA in tibialis anterior muscles of dko mice and quadriceps muscles of DMD patients and a reduction of Hes1 mRNA in the diaphragm of the mdx mice were observed, with other targets being inconsistent across species. Activation and inhibition of Notch signalling, followed by measures of muscle regeneration and function, were performed in the mouse models of DMD. Notch activation had no effect on functional regeneration in C57BL/10, mdx or dko mice. Notch inhibition significantly depressed the frequency-force relationship in regenerating muscles of C57BL/10 and mdx mice after injury, indicating reduced force at each stimulation frequency, but enhanced the frequency-force relationship in muscles from dko mice. We conclude that while Notch inhibition produces slight functional defects in dystrophic muscle, Notch activation does not significantly improve muscle regeneration in murine models of muscular dystrophy. Furthermore, the inconsistent expression of Notch targets between murine models and DMD patients suggests caution when making interspecies comparisons.

Making fast-twitch dystrophic muscles bigger protects them from contraction injury and attenuates the dystrophic pathology.

The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) renders muscle fibers highly fragile and susceptible to damage during contractions. Contraction-mediated injury is a major contributor to the progressive degeneration and etiology of muscle wasting in DMD. The prevailing understanding is that large fibers are highly susceptible to contraction damage and are affected preferentially, whereas smaller fibers are relatively spared in DMD. We tested the hypothesis that a pharmacological treatment that caused myofiber hypertrophy would increase the susceptibility of muscles from dystrophin-deficient mdx mice to contraction-induced injury, and thus aggravate the dystrophic pathology. The beta-agonist formoterol (100 microg/kg per day, i.p.) was administered to mdx mice for 28 days. Formoterol increased muscle mass, fiber cross-sectional area, and maximum force producing capacity by 30%, 23%, and 21%, respectively, in fast-twitch tibialis anterior muscles of mdx mice. Myofiber hypertrophy and increased maximum force producing capacity were also observed in the predominantly slow-twitch soleus muscles of mdx mice. Our original hypothesis was rejected since tibialis anterior muscles from formoterol-treated mdx mice had lower cumulative force deficits, indicating that they were less susceptible to contraction-induced injury. Formoterol treatment did not affect injury susceptibility in soleus muscles. These findings indicate that making dystrophic muscles bigger protects them from contraction damage and does not aggravate the dystrophic pathophysiology. These novel results further support the contention that anabolic agents have therapeutic potential for muscle wasting conditions including DMD.

Early functional muscle regeneration after myotoxic injury in mice is unaffected by nNOS absence.

Nitric oxide (NO) is an important signaling molecule produced in skeletal muscle primarily via the neuronal subtype of NO synthase (NOS1, or nNOS). While many studies have reported NO production to be important in muscle regeneration, none have examined the contribution of nNOS-derived NO to functional muscle regeneration (i.e., restoration of the muscle's ability to produce force) after acute myotoxic injury. In the present study, we tested the hypothesis that genetic deletion of nNOS would impair functional muscle regeneration after myotoxic injury in nNOS(-/-) mice. We found that nNOS(-/-) mice had lower body mass, lower muscle mass, and smaller myofiber cross-sectional area and that their tibialis anterior (TA) muscles produced lower absolute tetanic forces than those of wild-type littermate controls but that normalized or specific force was identical between the strains. In addition, muscles from nNOS(-/-) mice were more resistant to fatigue than those of wild-type littermates (P < 0.05). To determine whether deletion of nNOS affected muscle regeneration, TA muscles from nNOS(-/-) mice and wild-type littermates were injected with the myotoxin notexin to cause complete fiber degeneration, and muscle structure and function were assessed at 7 and 10 days postinjury. Myofiber cross-sectional area was lower in regenerating nNOS(-/-) mice than wild-type controls at 7 and 10 days postinjury; however, contrary to our original hypothesis, no difference in force-producing capacity of the TA muscle was evident between the two groups at either time point. Our findings reveal that nNOS is not essential for functional muscle regeneration after acute myotoxic damage.

Emerging drugs for treating skeletal muscle injury and promoting muscle repair.

INTRODUCTION: Musculoskeletal injuries represent a major global public health problem and muscle injury contributes significantly to the burden of disability and suffering. Drugs that can attenuate muscle trauma and/or hasten muscle repair to restore function can help reduce the economic burden and alleviate personal suffering and financial hardship. AREAS COVERED: This review provides an update on some emerging drugs with therapeutic potential for muscle injury including those that could attenuate damage or improve regeneration. Although there are few (if any) drugs in development specifically for muscle injury, there are numerous drugs in development for cardiovascular complications, such as ischemia-reperfusion, that might also have efficacy for promoting regeneration after similar events in skeletal muscle. EXPERT OPINION: Drugs in development for muscle wasting or inflammatory diseases should also be considered within the context of modulating the events associated with muscle degeneration and regeneration. More rigorous pre-clinical evaluations, especially of a drug's efficacy for improving function, would help minimize false leads and hasten development of effective approaches for treating muscle damage and promoting repair after injury.

Defective lysosome reformation during autophagy causes skeletal muscle disease.

The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy.

Metabolic remodeling of dystrophic skeletal muscle reveals biological roles for dystrophin and utrophin in adaptation and plasticity.

OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.

Cellular mechanisms underlying temporal changes in skeletal muscle protein synthesis and breakdown during chronic {beta}-adrenoceptor stimulation in mice.

Chronic stimulation of ß-adrenoceptors with ß-adrenoceptor agonists (ß-agonists) can induce substantial skeletal muscle hypertrophy, but the mechanisms mediating this muscle growth have yet to be elucidated. We investigated whether chronic ß-adrenoceptor stimulation in mice with the ß-agonist formoterol alters the muscle anabolic response following ß-adrenoceptor stimulation. Twelve-week-old C57BL/6 mice were treated for up to 28 days with a once-daily injection of either saline (control, n = 9) or formoterol (100 µg kg⁻¹; n = 9). Rates of muscle protein synthesis were assessed at either 1, 7 or 28 days of treatment, 6 h after injection. Protein synthesis rates were higher in formoterol-treated mice at day 7 (∼1.5-fold, P < 0.05), but not at day 1 or 28. The increased muscle protein synthesis was associated with increased phosphorylation of S6K1 (r = 0.49, P < 0.01). Formoterol treatment acutely reduced maximal calpain activity by ∼25% (P < 0.05) but did not affect atrogin-1 protein levels and proteasome-mediated proteolytic activity, despite significantly enhanced phosphorylation of Akt (P < 0.05). Formoterol increased CREB phosphorylation by ∼30% (P < 0.05) and PPARγ coactivator-1α (PGC-1α) by 11-fold (P < 0.05) on day 1 only. These observations identify that formoterol treatment induces muscle anabolism, by reducing calpain activity and by enhancing protein synthesis via increased PI-3 kinase/Akt signalling.

Intramuscular beta2-agonist administration enhances early regeneration and functional repair in rat skeletal muscle after myotoxic injury.

Systemic administration of beta(2)-adrenoceptor agonists (beta(2)-agonists) can improve skeletal muscle regeneration after injury. However, therapeutic application of beta(2)-agonists for muscle injury has been limited by detrimental cardiovascular side effects. Intramuscular administration may obviate some of these side effects. To test this hypothesis, the right extensor digitorum longus (EDL) muscle from rats was injected with bupivacaine hydrochloride to cause complete muscle fiber degeneration. Five days after injury, half of the injured muscles received an intramuscular injection of formoterol (100 mug). Muscle function was assessed at 7, 10, and 14 days after injury. A single intramuscular injection of formoterol increased muscle mass and force-producing capacity at day 7 by 17 and 91%, respectively, but this effect was transient because these values were not different from control levels at day 10. A second intramuscular injection of formoterol at day 7 prolonged the increase in muscle mass and force-producing capacity. Importantly, single or multiple intramuscular injections of formoterol did not elicit cardiac hypertrophy. To characterize any potential cardiovascular effects of intramuscular formoterol administration, we instrumented a separate group of rats with indwelling radio telemeters. Following an intramuscular injection of formoterol, heart rate increased by 18%, whereas systolic and diastolic blood pressure decreased by 31 and 44%, respectively. These results indicate that intramuscular injection can enhance functional muscle recovery after injury without causing cardiac hypertrophy. Therefore, if the transient cardiovascular effects associated with intramuscular formoterol administration can be minimized, this form of treatment may have significant therapeutic potential for muscle-wasting conditions.

FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1).

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

Dysfunctional muscle and liver glycogen metabolism in mdx dystrophic mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice. RESULTS: Dystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype. CONCLUSION: We identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.

Tranilast administration reduces fibrosis and improves fatigue resistance in muscles of mdx dystrophic mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disorder caused by mutations in the dystrophin gene that result in the absence of the membrane-stabilising protein dystrophin. Dystrophic muscle fibres are susceptible to injury and degeneration, and impaired muscle regeneration is associated with fibrotic deposition that limits the efficacy of potential pharmacological, cell- and gene-based therapies. Novel treatments that can prevent or attenuate fibrosis have important clinical merit for DMD and related neuromuscular diseases. We investigated the therapeutic potential for tranilast, an orally bioavailable anti-allergic agent, to prevent fibrosis in skeletal muscles of mdx dystrophic mice. RESULTS: Three-week-old C57Bl/10 and mdx mice received tranilast (~300 mg/kg) in their food for 9 weeks, after which fibrosis was assessed through histological analyses, and functional properties of tibialis anterior muscles were assessed in situ and diaphragm muscle strips in vitro. Tranilast administration did not significantly alter the mass of any muscles in control or mdx mice, but it decreased fibrosis in the severely affected diaphragm muscle by 31% compared with untreated mdx mice (P < 0.05). A similar trend of decreased fibrosis was observed in the tibialis anterior muscles of mdx mice (P = 0.10). These reductions in fibrotic deposition were not associated with improvements in maximum force-producing capacity, but we did observe small but significant improvements in the resistance to fatigue in both the diaphragm and TA muscles of mdx mice treated with tranilast. CONCLUSION: Together these findings demonstrate that administration of potent antifibrotic compounds such as tranilast could help preserve skeletal muscle structure, which could ultimately increase the efficacy of pharmacological, cell and gene replacement/correction therapies for muscular dystrophy and related disorders.

Intramuscular administration of PEGylated IGF-I improves skeletal muscle regeneration after myotoxic injury.

OBJECTIVE: Musculoskeletal injuries represent a major public health problem and drugs that can improve muscle repair and restore function are needed for patients with these conditions and other related muscular pathologies. Increasing insulin-like growth factor-I (IGF-I) levels in skeletal muscle improves regeneration after myotoxic injury and while administration of IGF-I has a potential for accelerating healing after trauma, optimizing its method of delivery and obviating potential side-effects currently associated with recombinant human (rh) IGF-I, remain a hurdle. DESIGN: We compared the treatment efficacy of rhIGF-I with a polyethylene glycol modified IGF-I (PEG-IGF-I) analog to improve functional repair of mouse tibialis anterior muscles after myotoxic injury, testing the hypothesis that PEG-IGF-I would exert greater beneficial effects on regenerating skeletal muscles than rhIGF-I due to improved pharmacokinetic properties. We also examined the relative efficacy of systemic versus local delivery of these IGF-I variants for improving functional muscle regeneration. RESULTS: Local delivery of PEG-IGF-I, but not rhIGF-I, at 4 days post-injury significantly improved early functional recovery as evident by a 27% increase in normalized force compared with saline control (P<0.05), whereas systemic application of either IGF-I variant was not effective. The improved function with intramuscular PEG-IGF-I administration was attributed to a greater and prolonged residence within the regenerating muscles, resulting in increased Akt activation and a 13% larger fiber cross-sectional area compared with rhIGF-I (P<0.05). CONCLUSIONS: These data support the hypothesis that PEG-IGF-I is more efficacious than rhIGF-I in hastening early fiber regeneration and improving muscle function after injury, highlighting its therapeutic potential for muscular pathologies.

Therapeutic potential of PEGylated insulin-like growth factor I for skeletal muscle disease evaluated in two murine models of muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a fatal monogenetic disease with affected males displaying severe and progressive muscle wasting and weakness eventually leading to premature death. Possible therapeutic benefits of insulin-like growth factor I (IGF-I) have been studied extensively in various models of muscle disease and DMD with IGF-I as a mediator of improved skeletal muscle regeneration by enhancing myoblast proliferation and differentiation. DESIGN: We tested the efficacy of a novel IGF-I analogue, a polyethylene glycol modified IGF-I (PEG-IGF-I), to ameliorate the pathophysiology of muscular dystrophy in two mouse models of DMD. We used mdx mice which lack dystrophin (as in DMD) but exhibit only a relatively mild phenotype, and the dko mouse which is a transgenic model lacking utrophin in addition to dystrophin, and which exhibits a more severe, lethal phenotype like that in DMD. RESULTS: In young mdx mice, twice-weekly PEG-IGF-I s.c. injections for 6 weeks protected the diaphragm muscle against fatigue and the tibialis anterior (TA) muscle against contraction-induced injury. However, this beneficial effect of PEG-IGF-I was less pronounced in mdx mice when treatment was initiated later in adulthood. In severely affected dko mice PEG-IGF-I treatment did not affect pathophysiological parameters including animal survival. CONCLUSIONS: These data suggest a therapeutic benefit with PEG-IGF-I treatment only in mild muscle pathologies, since its potential to ameliorate the pathophysiology in models of severe muscular dystrophies was limited. Treatment should be initiated only for mild muscle pathologies if functional benefits are to be realised and therefore may be relevant as a short-term therapy to hasten the functional repair of otherwise healthy muscles after injury.

Insulin-like growth factor-I analogue protects muscles of dystrophic mdx mice from contraction-mediated damage.

Contraction-mediated injury is a major contributing factor to the pathophysiology of muscular dystrophy and therefore therapies that can attenuate this type of injury have clinical relevance. Systemic administration of insulin-like growth factor-I (IGF-I) has been shown to improve muscle function in dystrophic mdx mice, an effect associated with a shift towards a more oxidative muscle phenotype and a reduced susceptibility to contraction-mediated damage. The actions of IGF-I in vivo are modulated by IGF binding proteins (IGFBPs), which generally act to inhibit IGF-I signalling. We tested the hypothesis that an analogue of IGF-I (LR IGF-I), which has significantly reduced binding affinity for IGFBPs, would improve the dystrophic pathology by reducing the susceptibility to muscle injury. Dystrophic mdx and wild-type (C57BL/10) mice were administered LR IGF-I continuously ( approximately 1.5 mg kg(-1) day(-1)) via osmotic mini-pump for 4 weeks. Administration of LR IGF-I reduced the susceptibility of extensor digitorum longus, soleus and diaphragm muscles to contraction damage, as evident from lower force deficits after a protocol of lengthening contractions. In contrast to the mechanism of protection conferred by administration of IGF-I, the protection conferred by LR IGF-I was independent of changes in muscle fatigue and oxidative metabolism. This study further indicates that modulation of IGF-I signalling has therapeutic potential for muscular diseases.

Modulation of insulin-like growth factor (IGF)-I and IGF-binding protein interactions enhances skeletal muscle regeneration and ameliorates the dystrophic pathology in mdx mice.

Administration of recombinant human insulin-like growth factor-I (rhIGF-I) has beneficial effects in animal models of muscle injury and muscular dystrophy. However, the results of these studies may have been confounded by interactions of rhIGF-I with endogenous IGF-binding proteins (IGFBPs). To date, no study has examined whether inhibiting IGFBP interactions with endogenous IGF-I can improve muscle fiber regeneration or muscular pathologies. We tested the hypothesis that reducing IGFBP interactions with endogenous IGF-I would enhance muscle regeneration after myotoxic injury and improve the dystrophic pathology in mdx mice. We administered an IGF-I aptamer (NBI-31772; 6 mg/kg per day, continuous infusion) to C57BL/10 mice undergoing regeneration after myotoxic injury or to mdx dystrophic mice. NBI-31772 binds all six IGFBPs with high affinity and releases "free" endogenous IGF-I. NBI-31772 treatment increased the rate of functional repair in fast-twitch tibialis anterior muscles after notexin-induced injury as evidenced by an increase in maximum force producing capacity (P(o)) at 10 days after injury. In contrast, NBI-31772 administration for 28 days did not alter P(o) of extensor digitorum longus (EDL) and soleus muscles or normalized force of diaphragm muscle strips from mdx mice. Although IGFBP inhibition reduced the susceptibility of the fast-twitch EDL and the diaphragm muscle to contraction-mediated damage, it increased muscle fatigability during repeated maximal contractions. Although the results in the myotoxic injury model suggest IGF-I signaling is important in this model, the results in the mdx model are mixed.