A guide to the composition and functions of the extracellular matrix.

Extracellular matrix (ECM) is a dynamic 3-dimensional network of macromolecules that provides structural support for the cells and tissues. Accumulated knowledge clearly demonstrated over the last decade that ECM plays key regulatory roles since it orchestrates cell signaling, functions, properties and morphology. Extracellularly secreted as well as cell-bound factors are among the major members of the ECM family. Proteins/glycoproteins, such as collagens, elastin, laminins and tenascins, proteoglycans and glycosaminoglycans, hyaluronan, and their cell receptors such as CD44 and integrins, responsible for cell adhesion, comprise a well-organized functional network with significant roles in health and disease. On the other hand, enzymes such as matrix metalloproteinases and specific glycosidases including heparanase and hyaluronidases contribute to matrix remodeling and affect human health. Several cell processes and functions, among them cell proliferation and survival, migration, differentiation, autophagy, angiogenesis, and immunity regulation are affected by certain matrix components. Structural alterations have been also well associated with disease progression. This guide on the composition and functions of the ECM gives a broad overview of the matrisome, the major ECM macromolecules, and their interaction networks within the ECM and with the cell surface, summarizes their main structural features and their roles in tissue organization and cell functions, and emphasizes the importance of specific ECM constituents in disease development and progression as well as the advances in molecular targeting of ECM to design new therapeutic strategies.

A Family of Laminin α2 Chain-Deficient Mouse Mutants: Advancing the Research on LAMA2-CMD.

The research on laminin α2 chain-deficient congenital muscular dystrophy (LAMA2-CMD) advanced rapidly in the last few decades, largely due to availability of good mouse models for the disease and a strong interest in preclinical studies from scientists all over the world. These mouse models continue to provide a solid platform for understanding the LAMA2-CMD pathology. In addition, they enable researchers to test laborious, necessary routines, but also the most creative scientific approaches in order to design therapy for this devastating disorder. In this review we present animals belonging to the laminin α2 chain-deficient "dy/dy" mouse family (dy/dy, dy 2J/dy 2J, dy 3K/dy 3K, dy W/dy W, et al.) and a summary of the scientific progress they facilitated. We also raise a few questions that need to be addressed in order to maximize the usefulness of laminin α2 murine mutants and to further advance the LAMA2-CMD studies. We believe that research opportunities offered by the mouse models for LAMA2-CMD will continuously support our efforts to find a treatment for the disease.

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.

Early skeletal muscle pathology and disease progress in the dy3K/dy3K mouse model of congenital muscular dystrophy with laminin α2 chain-deficiency.

Deficiency of laminin α2 chain leads to a severe form of congenital muscular dystrophy (LAMA2-CMD), and dystrophic symptoms progress rapidly in early childhood. Currently, there is no treatment for this detrimental disorder. Development of therapies is largely hindered by lack of understanding of mechanisms involved in the disease initiation and progress, both in patients but also in mouse models that are commonly used in the preclinical setup. Here, we unveil the first pathogenic events and characterise the disease development in a mouse model for LAMA2-CMD (dy3K/dy3K), by analysing muscles at perinatal, neonatal and postnatal stages. We found that apoptotic muscle fibres were present as early as postnatal day 1. Other typical dystrophic hallmarks (muscle degeneration, inflammation, and extensive production of the extracellular matrix proteins) were clearly evident already at postnatal day 4, and the highest degree of muscle deterioration was reached by day 7. Interestingly, the severe phenotype of limb muscles partially recovered on days 14 and 21, despite worsening of the general condition of the dy3K/dy3K mouse by that age. We found that masticatory muscles were severely affected in dy3K/dy3K mice and this may be an underlying cause of their malnutrition, which contributes to death around day 21. We also showed that several signalling pathways were affected already in 1-day-old dy3K/dy3K muscle. Therapeutic tests in the dy3K/dy3K mouse model should therefore be initiated shortly after birth, but should also take into account timing and correlation between regenerative and pathogenic events.

A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene.

Neuromuscular disorders are often caused by heterogeneous mutations in large, structurally complex genes. Targeting compensatory modifier genes could be beneficial to improve disease phenotypes. Here we report a mutation-independent strategy to upregulate the expression of a disease-modifying gene associated with congenital muscular dystrophy type 1A (MDC1A) using the CRISPR activation system in mice. MDC1A is caused by mutations in LAMA2 that lead to nonfunctional laminin-α2, which compromises the stability of muscle fibres and the myelination of peripheral nerves. Transgenic overexpression of Lama1, which encodes a structurally similar protein called laminin-α1, ameliorates muscle wasting and paralysis in mouse models of MDC1A, demonstrating its importance as a compensatory modifier of the disease1. However, postnatal upregulation of Lama1 is hampered by its large size, which exceeds the packaging capacity of vehicles that are clinically relevant for gene therapy. We modulate expression of Lama1 in the dy2j/dy2j mouse model of MDC1A using an adeno-associated virus (AAV9) carrying a catalytically inactive Cas9 (dCas9), VP64 transactivators and single-guide RNAs that target the Lama1 promoter. When pre-symptomatic mice were treated, Lama1 was upregulated in skeletal muscles and peripheral nerves, which prevented muscle fibrosis and paralysis. However, for many disorders it is important to investigate the therapeutic window and reversibility of symptoms. In muscular dystrophies, it has been hypothesized that fibrotic changes in skeletal muscle are irreversible. However, we show that dystrophic features and disease progression were improved and reversed when the treatment was initiated in symptomatic dy2j/dy2j mice with apparent hindlimb paralysis and muscle fibrosis. Collectively, our data demonstrate the feasibility and therapeutic benefit of CRISPR-dCas9-mediated upregulation of Lama1, which may enable mutation-independent treatment for all patients with MDC1A. This approach has a broad applicability to a variety of disease-modifying genes and could serve as a therapeutic strategy for many inherited and acquired diseases.

Exploratory Profiling of Urine MicroRNAs in the dy2J/dy2J Mouse Model of LAMA2-CMD: Relation to Disease Progression.

Circulating microRNAs (miRNAs) are being considered as non-invasive biomarkers for disease progression and clinical trials. Congenital muscular dystrophy with deficiency of laminin α2 chain (LAMA2-CMD) is a very severe form of muscular dystrophy, for which no treatment is available. In order to identify LAMA2-CMD biomarkers we have profiled miRNAs in urine from the dy2J /dy2J mouse model of LAMA2-CMD at three distinct time points (representing asymptomatic, initial and established disease). We demonstrate that unique groups of miRNAs are differentially expressed at each time point. We suggest that urine miRNAs can be sensitive biomarkers for different stages 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.

Laminin α1 reduces muscular dystrophy in dy2J mice.

Muscular dystrophies, including laminin α2 chain-deficient muscular dystrophy (LAMA2-CMD), are associated with immense personal, social and economic burdens. Thus, effective treatments are urgently needed. LAMA2-CMD is either a severe, early-onset condition with complete laminin α2 chain-deficiency or a milder, late-onset form with partial laminin α2 chain-deficiency. Mouse models dy3K/dy3K and dy2J/dy2J, respectively, recapitulate these two forms of LAMA2-CMD very well. We have previously demonstrated that laminin α1 chain significantly reduces muscular dystrophy in laminin α2 chain-deficient dy3K/dy3K mice. Among all the different pre-clinical approaches that have been evaluated in mice, laminin α1 chain-mediated therapy has been shown to be one of the most effective lines of attack. However, it has remained unclear if laminin α1 chain-mediated treatment is also applicable for partial laminin α2 chain-deficiency. Hence, we have generated dy2J/dy2J mice (that express a substantial amount of an N-terminal truncated laminin α2 chain) overexpressing laminin α1 chain in the neuromuscular system. The laminin α1 chain transgene ameliorated the dystrophic phenotype, restored muscle strength and reduced peripheral neuropathy. Thus, these findings provide additional support for the development of laminin α1 chain-based therapy for LAMA2-CMD.

Absence of microRNA-21 does not reduce muscular dystrophy in mouse models of LAMA2-CMD.

MicroRNAs (miRNAs) are short non-coding RNAs that modulate gene expression post-transcriptionally. Current evidence suggests that miR-21 plays a significant role in the progression of fibrosis in muscle diseases. Laminin-deficient congenital muscular dystrophy (LAMA2-CMD) is a severe form of congenital muscular dystrophy caused by mutations in the gene encoding laminin α2 chain. Mouse models dy3K/dy3K and dy2J/dy2J, respectively, adequately mirror severe and milder forms of LAMA2-CMD. Both human and mouse LAMA2-CMD muscles are characterized by extensive fibrosis and considering that fibrosis is the final step that destroys muscle during the disease course, anti-fibrotic therapies may be effective strategies for prevention of LAMA2-CMD. We have previously demonstrated a significant up-regulation of the pro-fibrotic miR-21 in dy3K/dy3K and dy2J/dy2J skeletal muscle. Hence, the objective of this study was to explore if absence of miR-21 reduces fibrogenesis and improves the phenotype of LAMA2-CMD mice. Thus, we generated dy3K/dy3K and dy2J/dy2J mice devoid of miR-21 (dy3K/miR-21 and dy2J/miR-21 mice, respectively). However, the muscular dystrophy phenotype of dy3K/miR-21 and dy2J/miR-21 double knock-out mice was not improved compared to dy3K/dy3K or dy2J/dy2J mice, respectively. Mice displayed the same body weight, dystrophic muscles (with fibrosis) and impaired muscle function. These data indicate that miR-21 may not be involved in the development of fibrosis in 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.

Potent pro-inflammatory and pro-fibrotic molecules, osteopontin and galectin-3, are not major disease modulators of laminin α2 chain-deficient muscular dystrophy.

A large number of human diseases are caused by chronic tissue injury with fibrosis potentially leading to organ failure. There is a need for more effective anti-fibrotic therapies. Congenital muscular dystrophy type 1A (MDC1A) is a devastating form of muscular dystrophy caused by laminin α2 chain-deficiency. It is characterized with early inflammation and build-up of fibrotic lesions, both in patients and MDC1A mouse models (e.g. dy3K/dy3K). Despite the enormous impact of inflammation on tissue remodelling in disease, the inflammatory response in MDC1A has been poorly described. Consequently, a comprehensive understanding of secondary mechanisms (impaired regeneration, enhanced fibrosis) leading to deterioration of muscle phenotype in MDC1A is missing. We have monitored inflammatory processes in dy3K/dy3K muscle and created mice deficient in laminin α2 chain and osteopontin or galectin-3, two pro-inflammatory and pro-fibrotic molecules drastically increased in dystrophic muscle. Surprisingly, deletion of osteopontin worsened the phenotype of dy3K/dy3K mice and loss of galectin-3 did not reduce muscle pathology. Our results indicate that osteopontin could even be a beneficial immunomodulator in MDC1A. This knowledge is essential for the design of future therapeutic interventions for muscular dystrophies that aim at targeting inflammation, especially that osteopontin inhibition has been suggested for Duchenne muscular dystrophy therapy.

Bortezomib Does Not Reduce Muscular Dystrophy in the dy2J/dy2J Mouse Model of Laminin α2 Chain-Deficient Muscular Dystrophy.

Congenital muscular dystrophy with laminin α2 chain-deficiency, also known as MDC1A, is a severe neuromuscular disorder for which there is no cure. Patients with complete laminin α2 chain-deficiency typically have an early onset disease with a more severe muscle phenotype while patients with residual laminin α2 chain expression usually have a milder disease course. Similar genotype-phenotype correlations can be seen in the dy3K/dy3K and dy2J/dy2J mouse models of MDC1A, respectively, with dy3K/dy3K mice presenting the more severe phenotype. Recently, we demonstrated that the proteasome inhibitor bortezomib partially improves muscle morphology and increases lifespan in dy3K/dy3K mice. Here, we explore the use of bortezomib in dy2J/dy2J animals. However, bortezomib neither improved histological hallmarks of disease nor increased muscle strength and locomotive activity in dy2J/dy2J mice. Altogether our data suggest that proteasome inhibition does not mitigate muscle dysfunction caused by partial laminin α2 chain-deficiency. Still, it is possible that proteasome inhibition could be useful as a supportive therapy in patients with complete absence of laminin α2 chain.

Laminin-α2 Chain-Deficient Congenital Muscular Dystrophy: Pathophysiology and Development of Treatment.

Laminin-211 is a major constituent of the skeletal muscle basement membrane. It stabilizes skeletal muscle and influences signal transduction events from the myomatrix to the muscle cell. Mutations in the gene encoding the α2 chain of laminin-211 lead to congenital muscular dystrophy type 1A (MDC1A), a life-threatening disease characterized by severe hypotonia, progressive muscle weakness, and joint contractures. Common complications include severely impaired motor ability, respiratory failure, and feeding difficulties. Several adequate animal models for laminin-α2 chain deficiency exist and analyses of different MDC1A mouse models have led to a significant improvement in our understanding of MDC1A pathogenesis. Importantly, the animal models have been indispensable tools for the preclinical development of new therapeutic approaches for laminin-α2 chain deficiency, highlighting a number of important disease driving mechanisms that can be targeted by pharmacological approaches. In this chapter, I will describe laminin-211 and discuss the cellular and molecular pathophysiology of MDC1A as well as progression toward development of treatment.

Dystrophin deficiency reduces atherosclerotic plaque development in ApoE-null mice.

Dystrophin of the dystrophin-glycoprotein complex connects the actin cytoskeleton to basement membranes and loss of dystrophin results in Duchenne muscular dystrophy. We have previously shown injury-induced neointima formation of the carotid artery in mice with the mdx mutation (causing dystrophin deficiency) to be increased. To investigate the role of dystrophin in intimal recruitment of smooth muscle cells (SMCs) that maintains plaque stability in atherosclerosis we applied a shear stress-modifying cast around the carotid artery of apolipoprotein E (ApoE)-null mice with and without the mdx mutation. The cast induces formation of atherosclerotic plaques of inflammatory and SMC-rich/fibrous phenotypes in regions of low and oscillatory shear stress, respectively. Unexpectedly, presence of the mdx mutation markedly reduced the development of the inflammatory low shear stress plaques. Further characterization of the low shear stress plaques in ApoE-null mdx mice demonstrated reduced infiltration of CD3(+) T cells, less laminin and a higher SMC content. ApoE-null mdx mice were also found to have a reduced fraction of CD3(+) T cells in the spleen and lower levels of cytokines and monocytes in the circulation. The present study is the first to demonstrate a role for dystrophin in atherosclerosis and unexpectedly shows that this primarily involves immune cells.

Deletion of integrin α7 subunit does not aggravate the phenotype of laminin α2 chain-deficient mice.

Laminin-211 is a major constituent of the skeletal muscle basement membrane, exerting its biological functions by binding to cell surface receptors integrin α7ß1 and dystroglycan (the latter is part of the dystrophin-glycoprotein complex). The importance of these molecules for normal muscle function is underscored by the fact that their respective deficiency leads to different forms of muscular dystrophy with different severity in humans and animal models. We recently demonstrated that laminin α2 chain and members of the dystrophin-glycoprotein complex have overlapping but non-redundant roles despite being part of the same adhesion complex. To analyse whether laminin-211 and integrin α7 subunit have non-redundant functions we generated mice deficient in laminin α2 chain and integrin α7 subunit (dy(3K)/itga7). We show that lack of both molecules did not exacerbate the severe phenotype of laminin α2-chain deficient animals. They displayed the same weight, survival and dystrophic pattern of muscle biopsy, with similar degree of inflammation and fibrosis. These data suggest that laminin-211 and integrin α7ß1 have intersecting roles in skeletal muscle.

Laminin α2 Chain-Deficiency is Associated with microRNA Deregulation in Skeletal Muscle and Plasma.

microRNAs (miRNAs) are widespread regulators of gene expression, but little is known of their potential roles in congenital muscular dystrophy type 1A (MDC1A). MDC1A is a severe form of muscular dystrophy caused by mutations in the gene encoding laminin α2 chain. To gain insight into the pathophysiological roles of miRNAs associated with MDC1A pathology, laminin α2 chain-deficient mice were evaluated by quantitative PCR. We demonstrate that expression of muscle-specific miR-1, miR-133a, and miR-206 is deregulated in laminin α2 chain-deficient muscle. Furthermore, expression of miR-223 and miR-21, associated with immune cell infiltration and fibrosis, respectively, is altered. Finally, we show that plasma levels of muscle-specific miRNAs are markedly elevated in laminin α2 chain-deficient mice and partially normalized in response to proteasome inhibition therapy. Altogether, our data suggest important roles for miRNAs in MDC1A pathology and we propose plasma levels of muscle-specific miRNAs as promising biomarkers for the progression of MDC1A.

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.

Loss of dystrophin and ß-sarcoglycan significantly exacerbates the phenotype of laminin α2 chain-deficient animals.

The adhesion molecule laminin α2 chain interacts with the dystrophin-glycoprotein complex, contributes to normal muscle function, and protects skeletal muscles from damage. Complete loss of the laminin α2 chain in mice results in a severe muscular dystrophy phenotype and death at approximately 3 weeks of age. However, it is not clear if the remaining members of the dystrophin-glycoprotein complex further protect laminin α2 chain-deficient skeletal muscle fibers from degeneration. Hence, we generated mice deficient in laminin α2 chain and dystrophin (dy(3K)/mdx) and mice devoid of laminin α2 chain and ß-sarcoglycan (dy(3K)/Sgcb). Severe muscular dystrophy and a lack of nourishment inevitably led to massive muscle wasting and death in double-knockout animals. The dy(3K)/Sgcb mice were generally more severely affected than dy(3K)/mdx mice. However, both double-knockout strains displayed exacerbated muscle degeneration, inflammation, fibrosis, and reduced life span (5 to 13 days) compared with single-knockout animals. However, neither extraocular nor cardiac muscle was affected in double-knockout animals. Our results suggest that, although laminin α2 chain, dystrophin, and ß-sarcoglycan are all part of the same adhesion complex, they have complementary, but nonredundant, roles in maintaining sarcolemmal integrity and protecting skeletal muscle fibers from damage. Moreover, the double-knockout mice could potentially serve as models in which to study extremely aggressive muscle-wasting conditions.

Isobaric Tagging-Based Quantification for Proteomic Analysis: A Comparative Study of Spared and Affected Muscles from mdx Mice at the Early Phase of Dystrophy.

Duchenne muscular dystrophy (DMD) is the most common childhood myopathy, characterized by muscle loss and cardiorespiratory failure. While the genetic basis of DMD is well established, secondary mechanisms associated with dystrophic pathophysiology are not fully clarified yet. In order to obtain new insights into the molecular mechanisms of muscle dystrophy during earlier stages of the disease, we performed a comparative proteomic profile of the spared extraocular muscles (EOM) vs. affected diaphragm from the mdx mice, using a label based shotgun proteomic approach. Out of the 857 identified proteins, 42 to 62 proteins had differential abundance of peptide ions. The calcium-handling proteins sarcalumenin and calsequestrin-1 were increased in control EOM compared with control DIA, reinforcing the view that constitutional properties of EOM are important for their protection against myonecrosis. The finding that galectin-1 (muscle regeneration), annexin A1 (anti-inflammatory) and HSP 47 (fibrosis) were increased in dystrophic diaphragm provides novel insights into the mechanisms through which mdx affected muscles are able to counteract dystrophy, during the early stage of the disease. Overall, the shotgun technique proved to be suitable to perform quantitative comparisons between distinct dystrophic muscles and allowed the suggestion of new potential biomarkers and drug targets for dystrophinopaties.