Cachexia is a debilitating condition characterized by extreme skeletal muscle wasting that contributes significantly to morbidity and mortality. Efforts to elucidate the underlying mechanisms of muscle loss have predominantly focused on events intrinsic to the myofiber. In contrast, less regard has been given to potential contributory factors outside the fiber within the muscle microenvironment. In tumor-bearing mice and patients with pancreatic cancer, we found that cachexia was associated with a type of muscle damage resulting in activation of both satellite and nonsatellite muscle progenitor cells. These muscle progenitors committed to a myogenic program, but were inhibited from completing differentiation by an event linked with persistent expression of the self-renewing factor Pax7. Overexpression of Pax7 was sufficient to induce atrophy in normal muscle, while under tumor conditions, the reduction of Pax7 or exogenous addition of its downstream target, MyoD, reversed wasting by restoring cell differentiation and fusion with injured fibers. Furthermore, Pax7 was induced by serum factors from cachectic mice and patients, in an NF-κB-dependent manner, both in vitro and in vivo. Together, these results suggest that Pax7 responds to NF-κB by impairing the regenerative capacity of myogenic cells in the muscle microenvironment to drive muscle wasting in cancer.

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Skeletal muscle differentiation is a complex, highly coordinated process that relies on precise temporal gene expression patterns. To better understand this cascade of transcriptional events, we used expression profiling to analyze gene expression in a 12-day time course of differentiating C2C12 myoblasts. Cluster analysis specific for time-ordered microarray experiments classified 2895 genes and ESTs with variable expression levels between proliferating and differentiating cells into 22 clusters with distinct expression patterns during myogenesis. Expression patterns for several known and novel genes were independently confirmed by real-time quantitative RT-PCR and/or Western blotting and immunofluorescence. MyoD and MEF family members exhibited unique expression kinetics that were highly coordinated with cell-cycle withdrawal regulators. Among genes with peak expression levels during cell cycle withdrawal were Vcam1, Itgb3, Itga5, Vcl, as well as Ptger4, a gene not previously associated with the process of myogenesis. One interesting uncharacterized transcript that is highly induced during myogenesis encodes several immunoglobulin repeats with sequence similarity to titin, a large sarcomeric protein. These data sets identify many additional uncharacterized transcripts that may play important functions in muscle cell proliferation and differentiation and provide a baseline for comparison with C2C12 cells expressing various mutant genes involved in myopathic disorders.

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Specific isoforms of laminin (LN) are concentrated at neuromuscular junctions (NMJs) where they may participate in synaptic organization or function. In myotubes from C2 cells, LN is concentrated within the majority of spontaneous acetylcholine receptor (AChR) aggregates. Neural agrin substantially increases this colocalization, suggesting that agrin can recruit LN into AChR aggregates. Addition of LN to C2 myotubes induces a more than twofold increase in the number of AChR aggregates. These aggregates have a larger size and are more dense than are those induced by agrin, suggesting that LN is involved in the growth and/or stabilization of AChR aggregates. Consistent with this hypothesis, an antiserum to LN reduces the size of individual AChR aggregates but increases their number. In C2 myotubes, extracellular matrix receptors containing the integrin beta1 subunit are poorly colocalized with AChR aggregates, suggesting that integrins may not be involved in LN-induced aggregation. In contrast, almost all AChR aggregates are associated with dystroglycan immunoreactivity, and monoclonal antibody (mAb) IIH6 against alpha-dystroglycan (alpha-DG), a LN and agrin receptor, causes a concentration-dependent inhibition of LN-induced aggregation. Moreover, S27 cells, which lack a functional alpha-DG, and two C2-derived cell lines expressing antisense DG mRNA fail to aggregate AChRs in response to LN. Finally, LN-induced AChR aggregation does not involve the phosphorylation of the muscle-specific tyrosine kinase receptor (MuSK) or the AChR beta subunit. We hypothesize that the interaction of LN with alpha-DG contributes to the growth and/or stabilization of AChR microaggregates into macroaggregates at the developing NMJ via a MuSK-independent mechanism.

Background-Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy often result from deletion mutations in the dystrophin gene that may lead to expression of an altered dystrophin protein in cardiac muscle. Cardiac involvement is present in Ϸ70% of BMD and all X-linked dilated cardiomyopathy cases. To date, the timing of cardiomyopathy development remains unpredictable. We analyzed 78 BMD and X-linked dilated cardiomyopathy patients with common deletion mutations predicted to alter the dystrophin protein and correlated their mutations to cardiomyopathy age of onset. This approach was chosen to connect dystrophin structure with function in the heart. Methods and Results-Detailed cardiac information was collected for BMD and X-linked dilated cardiomyopathy patients with defined dystrophin gene deletion mutations. Patients were grouped based on the dystrophin protein domain affected by the deletion. Deletions affecting the amino-terminal domain are associated with early-onset dilated cardiomyopathy (DCM; mid-20s), whereas deletions removing part of the rod domain and hinge 3 have a later-onset DCM (mid-40s).Further, we modeled the effects of the most common mutations occurring in the rod domain on the overall structure of the dystrophin protein. By combining genetic and protein information, this analysis revealed a strong correlation between specific protein structural modifications and DCM age of onset. Conclusions-We identified specific regions of the dystrophin gene that when mutated predispose BMD patients to early-onset DCM. In addition, we propose that some mutations lead to early-onset DCM by specific alterations in protein folding. These findings have potential implications for early intervention in the cardiac care of BMD patients and for therapeutic approaches that target the heart in dystrophinopathies. (Circ Cardiovasc Genet. 2009;2:544-551.)Key Words: cardiomyopathy Ⅲ genetics Ⅲ risk factors Ⅲ muscular dystrophy Ⅲ dystrophin T he dystrophin gene, located on the X-chromosome, is the largest known human gene (2.4 Mb, 79 exons), resulting in a high rate of spontaneous disease-causing mutations (30% of cases) with deletions forming the majority (Ϸ60%). Dystrophin plays an essential structural role in both cardiac and skeletal muscle, protecting the sarcolemma from mechanical stresses of muscle contraction. Complete loss of dystrophin leads to Duchenne muscular dystrophy (DMD), the most common severe form of childhood muscular dystrophy, complicated by skeletal muscle degeneration and dilated cardiomyopathy (DCM). Clinical Perspective on p 551In contrast to the well-defined clinical course of DMD, mutations that do not disrupt the reading frame can result in expression of an altered dystrophin protein, leading to a more variable clinical presentation. This includes Becker muscular dystrophy (BMD) that presents primarily with progressive skeletal muscle degeneration with variable age of onset and severity, and X-linked DCM (XLDCM) that typically has no detectable skeletal muscle signs accompanyin...

Heterotopic ossification (HO) is defined as the formation of bone inside soft tissue. Symptoms include joint stiffness, swelling, and pain. Apart from the inherited form, the common traumatic form generally occurs at sites of injury in damaged muscles and is often associated with brain injury. We investigated bone morphogenetic protein 9 (BMP-9), which possesses a strong osteoinductive capacity, for its involvement in muscle HO physiopathology. We found that BMP-9 had an osteoinductive influence on mouse muscle resident stromal cells by increasing their alkaline phosphatase activity and bone-specific marker expression. Interestingly, BMP-9 induced HO only in damaged muscle, whereas BMP-2 promoted HO in skeletal muscle regardless of its state. The addition of the soluble form of the ALK1 protein (the BMP-9 receptor) significantly inhibited the osteoinductive potential of BMP-9 in cells and HO in damaged muscles. BMP-9 thus should be considered a candidate for involvement in HO physiopathology, with its activity depending on the skeletal muscle microenvironment. ß

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.