Antioxidants Reduce Muscular Dystrophy in the dy2J/dy2J Mouse Model of Laminin α2 Chain-Deficient Muscular Dystrophy.

Congenital muscular dystrophy with laminin α2 chain-deficiency (LAMA2-CMD) is a severe neuromuscular disorder without a cure. Using transcriptome and proteome profiling as well as functional assays, we previously demonstrated significant metabolic impairment in skeletal muscle from LAMA2-CMD patients and mouse models. Reactive oxygen species (ROS) increase when oxygen homeostasis is not maintained and, here, we investigate whether oxidative stress indeed is involved in the pathogenesis of LAMA2-CMD. We also analyze the effects of two antioxidant molecules, N-acetyl-L-cysteine (NAC) and vitamin E, on disease progression in the dy2J/dy2J mouse model of LAMA2-CMD. We demonstrate increased ROS levels in LAMA2-CMD mouse and patient skeletal muscle. Furthermore, NAC treatment (150 mg/kg IP for 6 days/week for 3 weeks) led to muscle force loss prevention, reduced central nucleation and decreased the occurrence of apoptosis, inflammation, fibrosis and oxidative stress in LAMA2-CMD muscle. In addition, vitamin E (40 mg/kg oral gavage for 6 days/week for 2 weeks) improved morphological features and reduced inflammation and ROS levels in dy2J/dy2J skeletal muscle. We suggest that NAC and to some extent vitamin E might be potential future supportive treatments for LAMA2-CMD as they improve numerous pathological hallmarks of LAMA2-CMD.

Effects of metformin on congenital muscular dystrophy type 1A disease progression in mice: a gender impact study.

Congenital muscular dystrophy with laminin α2 chain-deficiency (LAMA2-CMD) is a severe muscle disorder with complex underlying pathogenesis. We have previously employed profiling techniques to elucidate molecular patterns and demonstrated significant metabolic impairment in skeletal muscle from LAMA2-CMD patients and mouse models. Thus, we hypothesize that skeletal muscle metabolism may be a promising pharmacological target to improve muscle function in LAMA2-CMD. Here, we have investigated whether the multifunctional medication metformin could be used to reduce disease in the dy2J/dy2J mouse model of LAMA2-CMD. First, we show gender disparity for several pathological hallmarks of LAMA2-CMD. Second, we demonstrate that metformin treatment significantly increases weight gain and energy efficiency, enhances muscle function and improves skeletal muscle histology in female dy2J/dy2J mice (and to a lesser extent in dy2J/dy2J males). Thus, our current data suggest that metformin may be a potential future supportive treatment that improves many of the pathological characteristics of LAMA2-CMD.

Bioenergetic Impairment in Congenital Muscular Dystrophy Type 1A and Leigh Syndrome Muscle Cells.

Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients.

Skeletal muscle wasting and renewal: a pivotal role of myokine IL-6.

Adult skeletal tissue is composed of heterogeneous population of cells that constantly self-renew by means of a controlled process of activation and proliferation of tissue-resident stem cells named satellite cells. Many growth factors, cytokines and myokines produced by skeletal muscle cells play critical roles in local regulation of the inflammatory process and skeletal muscle regeneration during different pathological conditions. IL-6 is a pleiotropic cytokine released in large amount during infection, autoimmunity and cancer. Low levels of IL-6 can promote activation of satellite cells and myotube regeneration while chronically elevated production promote skeletal muscle wasting. These distinct effects may be explained by a crosstalk of the IL-6/IL-6 receptor and gp130 trans-signaling pathway that oppose to regenerative and anti-inflammatory of the classical IL-6 receptor signaling pathway. Here we discuss on potential therapeutic strategies using monoclonal antibodies to IL-6R for the treatment of skeletal muscle wasting and cachexia. We also highlight on the IL-6/JAK/STAT and FGF/p38αß MAPK signaling pathways in satellite cell activation and the use of protein kinase inhibitors for tailoring and optimizing satellite cell proliferation during the skeletal muscle renewal. Future investigations on the roles of the IL-6 classical and trans-signaling pathways in both immune and non-immune cells in skeletal muscle tissue will provide new basis for therapeutic approaches to reverse atrophy and degeneration of skeletal muscles in cancer and inflammatory diseases.

Dermcidin exerts its oncogenic effects in breast cancer via modulation of ERBB signaling.

BACKGROUND: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines. METHODS: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform. RESULTS: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands. CONCLUSIONS: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.

Quantitative proteomic analysis reveals metabolic alterations, calcium dysregulation, and increased expression of extracellular matrix proteins in laminin α2 chain-deficient muscle.

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).

Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.