Federica Montanaro

Expression profiling and identification of novel genes involved in myogenic differentiation

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, Vc1, 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.

NF-κB-mediated Pax7 dysregulation in the muscle microenvironment promotes cancer cachexia

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.

Skeletal muscle engraftment potential of adult mouse skin side population cells

Adult bone marrow and skeletal muscle have been shown to contain a subpopulation of cells, called side population (SP) cells, that can be isolated with the fluorescence-activated cell sorter. We used a similar method to identify SP cells in the skin of adult mice. These cells express surface markers similar to SP cells isolated from skeletal muscle, but differ from bone marrow SP cells and do not express hematopoietic markers. When transplanted into nonirradiated mdx mice, nuclei from donor skin SP cells are found within myofibers that express dystrophin. Thus, adult skin SP cells can engraft in dystrophic skeletal muscle even in the absence of total body irradiation.

Analysis of Dystrophin Deletion Mutations Predicts Age of Cardiomyopathy Onset in Becker Muscular Dystrophy

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 ≈70% 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.

BMP‐9‐induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

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.

Current understanding and management of dilated cardiomyopathy in Duchenne and Becker muscular dystrophy

Purpose: To review the current understanding of the pathophysiology of dilated cardiomyopathy (DCM) in patients with Duchenne and Becker muscular dystrophies, assessment of cardiac dysfunction for these patients, and the recommended pharmacological treatment options and ongoing research directions. Data sources: Reviews and original research articles from scholarly journals and books. Conclusions: Duchenne and Becker muscular dystrophies are debilitating neuromuscular disorders, both caused by mutations in the dystrophin gene. Most patients develop DCM as part of the disease course; in fact, DCM is the leading cause of death among these patients. Cardiac surveillance, including routine monitoring of electrocardiograms, echocardiograms, and appropriate blood biomarkers, may detect early DCM development. Although previous studies have shown that early administration of cardiac medications may delay the development of DCM, current standard of care does not emphasize cardiac surveillance and timely treatment. This, in turn, limits clinicians, including advanced practice nurses, to be optimally engaged in providing the most aggressive and proactive patient care. Implications for practice: Implementing a routine cardiac assessment and timely pharmacological treatment in primary or specialty care settings is highlighted as an important step to optimize cardiac health among patients with Duchenne and Becker muscular dystrophies.

Proteomic Analysis Reveals New Cardiac-Specific Dystrophin-Associated Proteins

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.

mdx5cv Mice Manifest More Severe Muscle Dysfunction and Diaphragm Force Deficits than Do mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx(5cv) mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx(⁵cv) mice. Our study revealed that mdx(⁵cv) mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx(⁵cv) mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx(⁵cv) mouse models of DMD are not interchangeable and identify the mdx(⁵cv) mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.

Comparative proteomic profiling of dystroglycan-associated proteins in wild type, mdx and Galgt2 transgenic mouse skeletal muscle

Dystroglycan is a major cell surface glycoprotein receptor for the extracellular matrix in skeletal muscle. Defects in dystroglycan glycosylation cause muscular dystrophy and alterations in dystroglycan glycosylation can impact extracellular matrix binding. Here we describe an immunoprecipitation technique that allows isolation of beta dystroglycan with members of the dystrophin-associated protein complex (DAPC) from detergent-solubilized skeletal muscle. Immunoprecipitation, coupled with shotgun proteomics, has allowed us to identify new dystroglycan-associated proteins and define changed associations that occur within the DAPC in dystrophic skeletal muscles. In addition, we describe changes that result from overexpression of Galgt2, a normally synaptic muscle glycosyltransferase that can modify alpha dystroglycan and inhibit the development of muscular dystrophy when it is overexpressed. These studies identify new dystroglycan-associated proteins that may participate in dystroglycans roles, both positive and negative, in muscular dystrophy.

Muscle side population cells from dystrophic or injured muscle adopt a fibro-adipogenic fate.

Muscle side population (SP) cells are rare multipotent stem cells that can participate in myogenesis and muscle regeneration upon transplantation. While they have been primarily studied for the development of cell-based therapies for Duchenne muscular dystrophy, little is known regarding their non-muscle lineage choices or whether the dystrophic muscle environment affects their ability to repair muscle. Unfortunately, the study of muscle SP cells has been challenged by their low abundance and the absence of specific SP cell markers. To address these issues, we developed culture conditions for the propagation and spontaneous multi-lineage differentiation of muscle SP cells. Using this approach, we show that SP cells from wild type muscle robustly differentiate into satellite cells and form myotubes without requiring co-culture with myogenic cells. Furthermore, this myogenic activity is associated with SP cells negative for immune (CD45) and vascular (CD31) markers but positive for Pax7, Sca1, and the mesenchymal progenitor marker PDGFRα. Additionally, our studies revealed that SP cells isolated from dystrophic or cardiotoxin-injured muscle fail to undergo myogenesis. Instead, these SP cells rapidly expand giving rise to fibroblast and adipocyte progenitors (FAPs) and to their differentiated progeny, fibroblasts and adipocytes. Our findings indicate that muscle damage affects the lineage choices of muscle SP cells, promoting their differentiation along fibro-adipogenic lineages while inhibiting myogenesis. These results have implications for a possible role of muscle SP cells in fibrosis and fat deposition in muscular dystrophy. In addition, our studies provide a useful in vitro system to analyze SP cell biology in both normal and pathological conditions.