Transcriptomics reveals transient and dynamic muscle fibrosis and atrophy differences following spinal cord injury in rats.

BACKGROUND: The rate and magnitude of skeletal muscle wasting after severe spinal cord injury (SCI) exceeds most other disuse conditions. Assessing the time course of molecular changes can provide insight into the progression of muscle wasting post-SCI. The goals of this study were (1) to identify potential targets that may prevent the pathologic features of SCI in soleus muscles and (2) to establish therapeutic windows for treating these pathologic changes. METHODS: Four-month-old Sprague-Dawley male rats received T9 laminectomy (SHAM surgery) or severe contusion SCI. Hindlimb locomotor function was assessed weekly, with soleus muscles obtained 1 week, 2 weeks, 1 month and 3 months post-surgery (n = 6-7 per group per timepoint). RNA was extracted from muscles for bulk RNA-sequencing analysis (n = 3-5 per group per timepoint). Differentially expressed genes (DEGs) were evaluated between age-matched SHAM and SCI animals. Myofiber size, muscle fibre type and fibrosis were assessed on contralateral muscles. RESULTS: SCI produced immediate and persistent hindlimb paralysis, with Basso-Beattie-Bresnahan locomotor scores remaining below 7 throughout the study, contributing to a progressive 25-50% lower soleus mass and myofiber atrophy versus SHAM (P < 0.05 at all timepoints). Transcriptional comparisons of SCI versus SHAM resulted in 184 DEGs (1 week), 436 DEGs (2 weeks), 133 DEGs (1 month) and 1200 DEGs (3 months). Upregulated atrophy-related genes included those associated with cell senescence, nuclear factor kappa B, ubiquitin proteasome and unfolded protein response pathways, along with upregulated genes that negatively influence muscle growth through the transforming growth factor beta pathway and inhibition of insulin-like growth factor-I/Akt/mechanistic target of rapamycin and p38/mitogen-activated protein kinase signalling. Genes associated with extracellular matrix (ECM), including collagens, collagen crosslinkers, proteoglycans and those regulating ECM integrity, were enriched within upregulated DEGs at 1 week but subsequently downregulated at 2 weeks and 3 months and were accompanied by >50% higher ECM areas and hydroxyproline levels in SCI muscles (P < 0.05). Myofiber remodelling genes were enriched in upregulated DEGs at 2 weeks and 1 month and were downregulated at 3 months. Genes that regulate neuromuscular junction remodelling were evident in muscles post-SCI, along with slow-to-fast fibre-type shifts: 1 week and 2 weeks SCI muscles were composed of 90% myosin heavy chain (MHC) type I fibres, which decreased to only 16% at 3 months and were accompanied by 50% fibres containing MHC IIX (P < 0.05). Metabolism genes were enriched in upregulated DEGs at 1 month and were further enriched at 3 months. CONCLUSIONS: Our results substantiate many known pathologic features of SCI-induced wasting in rat skeletal muscle and identify a progressive and dynamic transcriptional landscape within the post-SCI soleus. Future studies are warranted to consider these therapeutic treatment windows when countering SCI muscle pathology.

Ablation of specific insulin-like growth factor I forms reveals the importance of cleavage for regenerative capacity and glycosylation for skeletal muscle storage.

Insulin-like growth factor-I (IGF-I) facilitates mitotic and anabolic actions in all tissues. In skeletal muscle, IGF-I can promote growth and resolution of damage by promoting satellite cell proliferation and differentiation, suppressing inflammation, and enhancing fiber formation. While the most well-characterized form of IGF-I is the mature protein, alternative splicing and post-translational modification complexity lead to several additional forms of IGF-I. Previous studies showed muscle efficiently stores glycosylated pro-IGF-I. However, non-glycosylated forms display more efficient IGF-I receptor activation in vitro, suggesting that the removal of the glycosylated C terminus is a necessary step to enable increased activity. We employed CRISPR-Cas9 gene editing to ablate IGF-I glycosylation sites (2ND) or its cleavage site (3RA) in mice to determine the necessity of glycosylation or cleavage for IGF-I function in postnatal growth and during muscle regeneration. 3RA mice had the highest circulating and muscle IGF-I content, whereas 2ND mice had the lowest levels compared to wild-type mice. After weaning, 4-week-old 2ND mice exhibited higher body and skeletal muscle mass than other strains. However, by 16 weeks of age, muscle and body size differences disappeared. Even though 3RA mice had more IGF-I stored in muscle in homeostatic conditions, regeneration was delayed after cardiotoxin-induced injury, with prolonged necrosis most evident at 5 days post injury (dpi). In contrast, 2ND displayed improved regeneration with reduced necrosis, and greater fiber size and muscle mass at 11 and 21 dpi. Overall, these results demonstrate that while IGF-I glycosylation may be important for storage, cleavage is needed to enable IGF-I to be used for efficient activity in postnatal growth and following acute injury.

Loss of calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wildtype (WT) mice, we determined if loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD2A pathology.

Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain.

Proinsulin Like Growth Factor I (prolGF-I) and myostatin (Mstn) regulate muscle regeneration and mass when intravenously delivered. We tested if chloroplast bioencapsulated forms of these proteins may serve as a non-invasive means of drug delivery through the digestive system. We created tobacco (Nicotiana tabacum) plants carrying GFP-Fc1, proIGF-I-Fc1, and Mstn-Fc1 fusion genes, in which fusion with the immunoglobulin G Fc domain improved both protein stability and absorption in the small intestine. No transplastomic plants were obtained with the Mstn-Fc1 gene, suggesting that the protein is toxic to plant cells. proIGF-I-Fc1 protein levels were too low to enable in vivo testing. However, GFP-Fc1 accumulated at a high level, enabling evaluation of chloroplast-made Fc fusion proteins for oral delivery. Tobacco leaves were lyophilized for testing in a mouse system. We report that the orally administered GFP-Fc1 fusion protein (5.45 µg/g GFP-Fc1) has been taken up by the intestinal epithelium cells, evidenced by confocal microscopy. GFP-Fc1 subsequently entered the circulation where it was detected by ELISA. Data reported here confirm that chloroplast expression and oral administration of lyophilized leaves is a potential delivery system of therapeutic proteins fused with Fc1, with the advantage that the proteins may be stored at room temperature.

Chloroplast transformation for bioencapsulation and oral delivery using the immunoglobulin G fragment crystallizable (Fc) domain.

Proinsulin Like Growth Factor (prolGF1) and myostatin (Mstn) regulate muscle regeneration when intravenously delivered. We set out to test if chloroplast bioencapsulated forms of these proteins may serve as a non-invasive means of drug delivery through the digestive system. We created tobacco (Nicotiana tabacum) plants carrying GFP-Fc1, proIGF-I-Fc1, and Mstn-Fc1 fusion genes, in which fusion with the immunoglobulin G Fc domain improved both protein stability and absorption in the small intestine. No transplastomic plants were obtained with the Mstn-Fc1 gene, suggesting that the protein is toxic to plant cells. proIGF-I-Fc1 protein levels were too law to enable in vivo testing. However, GFP-Fc1 accumulated at a high level, enabling evaluation of chloroplast-made Fc fusion proteins for oral delivery. Tobacco leaves were lyophilized for testing in a mouse system. We report that the orally administered GFP-Fc fusion protein (5.45 µg/g GFP-Fc) has been taken up by the intestinal epithelium cells, evidenced by confocal microscopy. GFP-Fc subsequently entered the circulation where it was detected by ELISA. Data reported here confirm that chloroplast expression and oral administration of lyophilized leaves is a potential delivery system of therapeutic proteins fused with Fc, with the advantage that the proteins may be stored at room temperature.

Phosphorylation of AMPKα at Ser485/491 Is Dependent on Muscle Contraction and Not Muscle-Specific IGF-I Overexpression.

Glucose is an important fuel for highly active skeletal muscles. Increased adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratios during repetitive contractions trigger AMP-activated protein kinase (AMPK), indicated by phosphorylation of AMPKαThr172, which promotes glucose uptake to support heightened energy needs, but it also suppresses anabolic processes. Inhibition of AMPK can occur by protein kinase B (AKT)-mediated phosphorylation of AMPKαSer485/491, releasing its brake on growth. The influence of insulin-like growth factor I (IGF-I) on glucose uptake and its interplay with AMPK activation is not well understood. Thus, the goal of this study was to determine if increased muscle IGF-I altered AMPKα phosphorylation and activity during muscle contraction. Adult male mice harboring the rat Igf1a cDNA regulated by the fast myosin light chain promoter (mIgf1+/+) and wildtype littermates (WT) were used in the study. mIgf1+/+ mice had enhanced glucose tolerance and insulin-stimulated glucose uptake, but similar exercise capacity. Fatiguing stimulations of extensor digitorum longus (EDL) muscles resulted in upregulated AMPKα phosphorylation at both Thr172 and Ser485/491 in WT and mIgf1+/+ muscles. No differences in the phosphorylation response of the downstream AMPK target TBC1D1 were observed, but phosphorylation of raptor was significantly higher only in WT muscles. Further, total raptor content was elevated in mIgf1+/+ muscles. The results show that high muscle IGF-I can enhance glucose uptake under resting conditions; however, in contracting muscle, it is not sufficient to inhibit AMPK activity.

Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.

Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation.

INTRODUCTION: Fuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates. OBJECTIVES: The goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism. METHODS: Isolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles. RESULTS: Pyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation. CONCLUSION: Isotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Novel γ-sarcoglycan interactors in murine muscle membranes.

BACKGROUND: The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5. METHODS: To gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells. RESULTS: We identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl--co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25. CONCLUSIONS: Results support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.

Pharmacologic approaches to prevent skeletal muscle atrophy after spinal cord injury.

Skeletal muscle atrophy is a hallmark of severe spinal cord injury (SCI) that is precipitated by the neural insult and paralysis. Additionally, other factors may influence muscle loss, including systemic inflammation, low testosterone, low insulin-like growth factor (IGF)-1, and high-dose glucocorticoid treatment. The signaling cascades that drive SCI-induced muscle loss are common among most forms of disuse atrophy and include ubiquitin-proteasome signaling and others. However, differing magnitudes and patterns of atrophic signals exist after SCI versus other disuse conditions and are accompanied by endogenous inhibition of IGF-1/PI3K/Akt signaling, which combine to produce exceedingly rapid atrophy. Several well-established anabolic agents, including androgens and myostatin inhibitors, display diminished ability to prevent SCI-induced atrophy, while ursolic acid and ß2-agonists more effectively attenuate muscle loss. Strategies combining physical rehabilitation regimens to reload the paralyzed limbs with drugs targeting the underlying molecular pathways hold the greatest potential to improve muscle recovery after severe SCI.

The impact of hindlimb disuse on sepsis-induced myopathy in mice.

Sepsis induces a myopathy characterized by loss of muscle mass and weakness. Septic patients undergo prolonged periods of limb muscle disuse due to bed rest. The contribution of limb muscle disuse to the myopathy phenotype remains poorly described. To characterize sepsis-induced myopathy with hindlimb disuse, we combined the classic sepsis model via cecal ligation and puncture (CLP) with the disuse model of hindlimb suspension (HLS) in mice. Male C57bl/6j mice underwent CLP or SHAM surgeries. Four days after surgeries, mice underwent HLS or normal ambulation (NA) for 7 days. Soleus (SOL) and extensor digitorum longus (EDL) were dissected for in vitro muscle mechanics, morphological, and histological assessments. In SOL muscles, both CLP+NA and SHAM+HLS conditions elicited ~20% reduction in specific force (p < 0.05). When combined, CLP+HLS elicited ~35% decrease in specific force (p < 0.05). Loss of maximal specific force (~8%) was evident in EDL muscles only in CLP+HLS mice (p < 0.05). CLP+HLS reduced muscle fiber cross-sectional area (CSA) and mass in SOL (p < 0.05). In EDL muscles, CLP+HLS decreased absolute mass to a smaller extent (p < 0.05) with no changes in CSA. Immunohistochemistry revealed substantial myeloid cell infiltration (CD68+) in SOL, but not in EDL muscles, of CLP+HLS mice (p < 0.05). Combining CLP with HLS is a feasible model to study sepsis-induced myopathy in mice. Hindlimb disuse combined with sepsis induced muscle dysfunction and immune cell infiltration in a muscle dependent manner. These findings highlight the importance of rehabilitative interventions in septic hosts to prevent muscle disuse and help attenuate the myopathy.

Deletion of muscle Igf1 exacerbates disuse atrophy weakness in mice.

Muscle atrophy occurs as a result of prolonged periods of reduced mechanical stimulation associated with injury or disease. The growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and load sensing pathways can both aid in recovery from disuse through their shared downstream signaling, but their relative contributions to these processes are not fully understood. The goal of this study was to determine whether reduced muscle IGF-1 altered the response to disuse and reloading. Adult male mice with inducible muscle-specific IGF-1 deletion (MID) induced 1 wk before suspension and age-matched controls (CON) were subjected to hindlimb suspension and reloading. Analysis of muscle force, morphology, gene expression, signaling, and tissue weights was performed in nonsuspended (NS) mice, and those suspended for 7 days or reloaded following suspension for 3, 7, and 14 days. MID mice displayed diminished IGF-1 protein levels and muscle atrophy before suspension. Muscles from suspended CON mice displayed a similar extent of atrophy and depletion of IGF-1, yet combined loss of load and IGF-1 was not additive with respect to muscle mass. In contrast, soleus force generation capacity was diminished to the greatest extent when both suspension and IGF-1 deletion occurred. Recovery of mass, force, and gene expression patterns following suspension were similar in CON and MID mice, even though IGF-1 levels increased only in muscles from CON mice. Diminished strength in disuse atrophy is exacerbated with the loss of muscle IGF-1 production, whereas recovery of mass and strength upon reloading can occur even IGF-1 is low.NEW & NOTEWORTHY A mouse model with skeletal muscle-specific inducible deletion of Igf1 was used to address the importance of this growth factor for the consequences of disuse atrophy. Rapid and equivalent loss of IGF-I and mass occurred with deletion or disuse. Decrements in strength were most severe with combined loss of load and IGF-1. Return of mass and strength upon reloading was independent of IGF-1.

Actions and interactions of IGF-I and MMPs during muscle regeneration.

Muscle regeneration requires the coordination of several factors to mobilize satellite cells and macrophages, remodel the extracellular matrix surrounding muscle fibers, and repair existing and/or form new muscle fibers. In this review, we focus on insulin-like growth factor I and the matrix metalloproteinases, which are secreted proteins that act on cells and the matrix to resolve damage. While their actions appear independent, their interactions occur at the transcriptional and post-translational levels to promote feed-forward activation of each other. Together, these proteins assist at virtually every step of the repair process, and contribute significantly to muscle regenerative capacity.

Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo.

This study evaluated the direct effect of a phytochemical, hesperidin, on pre-osteoblast cell function as well as osteogenesis and collagen matrix quality, as there is little known about hesperidin's influence in mineralized tissue formation and regeneration. Hesperidin was added to a culture of MC3T3-E1 cells at various concentrations. Cell proliferation, viability, osteogenic gene expression and deposited collagen matrix analyses were performed. Treatment with hesperidin showed significant upregulation of osteogenic markers, particularly with lower doses. Mature and compact collagen fibrils in hesperidin-treated cultures were observed by picrosirius red staining (PSR), although a thinner matrix layer was present for the higher dose of hesperidin compared to osteogenic media alone. Fourier-transform infrared spectroscopy indicated a better mineral-to-matrix ratio and matrix distribution in cultures exposed to hesperidin and confirmed less collagen deposited with the 100-µM dose of hesperidin. In vivo, hesperidin combined with a suboptimal dose of bone morphogenetic protein 2 (BMP2) (dose unable to promote healing of a rat mandible critical-sized bone defect) in a collagenous scaffold promoted a well-controlled (not ectopic) pattern of bone formation as compared to a large dose of BMP2 (previously defined as optimal in healing the critical-sized defect, although of ectopic nature). PSR staining of newly formed bone demonstrated that hesperidin can promote maturation of bone organic matrix. Our findings show, for the first time, that hesperidin has a modulatory role in mineralized tissue formation via not only osteoblast cell differentiation but also matrix organization and matrix-to-mineral ratio and could be a potential adjunct in regenerative bone therapies.

Antagonistic control of myofiber size and muscle protein quality control by the ubiquitin ligase UBR4 during aging.

Sarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. Here, we find that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome. Importantly, muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4/poe and of ESCRT members (HGS/Hrs, STAM, USP8) that degrade ubiquitinated membrane proteins compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that these ubiquitin ligases antithetically regulate myofiber size and muscle protein quality control.

The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle degenerative disease caused by loss of dystrophin protein. DMD has no cure and few treatment options. Preclinical efforts to identify potential DMD therapeutics have been hampered by lack of a small animal model that recapitulates key features of the human disease. While the dystrophin-deficient mdx mouse on the C57BL/10 genetic background (B10.mdx) is mildly affected, a more severe muscle disease is observed when the mdx mutation is crossed onto the DBA/2J genetic background (D2.mdx). In this study, the functional and histological progression of the D2.mdx skeletal muscle pathology was evaluated to determine the distinguishing features of disease. Data herein details the muscular weakness and wasting exhibited by D2.mdx skeletal muscle, as well as severe histopathological features, which include the rapid progression of fibrosis and calcifications in the diaphragm and progressive fibrosis accumulation in limb muscles. Furthermore, a timeline of D2.mdx progression is provided that details distinct stages of disease progression. These data support the D2.mdx as a superior small animal model for DMD, as compared to the B10.mdx model. The insights provided in this report should facilitate the design of preclinical evaluations for potential DMD therapeutics.

The ties that bind: functional clusters in limb-girdle muscular dystrophy.

The limb-girdle muscular dystrophies (LGMDs) are a genetically pleiomorphic class of inherited muscle diseases that are known to share phenotypic features. Selected LGMD genetic subtypes have been studied extensively in affected humans and various animal models. In some cases, these investigations have led to human clinical trials of potential disease-modifying therapies, including gene replacement strategies for individual subtypes using adeno-associated virus (AAV) vectors. The cellular localizations of most proteins associated with LGMD have been determined. However, the functions of these proteins are less uniformly characterized, thus limiting our knowledge of potential common disease mechanisms across subtype boundaries. Correspondingly, broad therapeutic strategies that could each target multiple LGMD subtypes remain less developed. We believe that three major "functional clusters" of subcellular activities relevant to LGMD merit further investigation. The best known of these is the glycosylation modifications associated with the dystroglycan complex. The other two, mechanical signaling and mitochondrial dysfunction, have been studied less systematically but are just as promising with respect to the identification of significant mechanistic subgroups of LGMD. A deeper understanding of these disease pathways could yield a new generation of precision therapies that would each be expected to treat a broader range of LGMD patients than a single subtype, thus expanding the scope of the molecular medicines that may be developed for this complex array of muscular dystrophies.

Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration.

BACKGROUND/AIMS: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. METHODS: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. RESULTS: Under basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. CONCLUSION: These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

A Key Role for the Ubiquitin Ligase UBR4 in Myofiber Hypertrophy in Drosophila and Mice.

Skeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, we have used RNAi screening in Drosophila to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo. We find that whereas RNAi for some ubiquitin ligases induces myofiber atrophy, loss of others (including the N-end rule ubiquitin ligase UBR4) promotes hypertrophy. In Drosophila and mouse myofibers, loss of UBR4 induces hypertrophy via decreased ubiquitination and degradation of a core set of target proteins, including the HAT1/RBBP4/RBBP7 histone-binding complex. Together, this study defines the repertoire of ubiquitin ligases that regulate myofiber size and the role of UBR4 in myofiber hypertrophy.

Functional muscle hypertrophy by increased insulin-like growth factor 1 does not require dysferlin.

INTRODUCTION: Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice. METHODS: Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. RESULTS: Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles. DISCUSSION: We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies.