Proteomic analysis of the sarcolemma-enriched fraction from dystrophic mdx-4cv skeletal muscle.

The highly progressive neuromuscular disorder dystrophinopathy is triggered by primary abnormalities in the Dmd gene, which causes cytoskeletal instability and loss of sarcolemmal integrity. Comparative organellar proteomics was employed to identify sarcolemma-associated proteins with an altered concentration in dystrophic muscle tissue from the mdx-4cv mouse model of dystrophinopathy. A lectin agglutination method was used to prepare a sarcolemma-enriched fraction and resulted in the identification of 190 significantly changed protein species. Proteomics established differential expression patterns for key components of the muscle plasma membrane, cytoskeletal network, extracellular matrix, metabolic pathways, cellular stress response, protein synthesis, immune response and neuromuscular junction. The deficiency in dystrophin and drastic reduction in dystrophin-associated proteins appears to trigger (i) enhanced membrane repair involving myoferlin, dysferlin and annexins, (ii) increased protein synthesis and the compensatory up-regulation of cytoskeletal proteins, (iii) the decrease in the scaffolding protein periaxin and myelin PO involved in myelination of motor neurons, (iv) complex changes in bioenergetic pathways, (v) elevated levels of molecular chaperones to prevent proteotoxic effects, (vi) increased collagen deposition causing reactive myofibrosis, (vii) disturbed ion homeostasis at the sarcolemma and associated membrane systems, and (viii) a robust inflammatory response by the innate immune system in response to chronic muscle damage. SIGNIFICANCE: Duchenne muscular dystrophy is a devastating muscle wasting disease and represents the most frequently inherited neuromuscular disorder in humans. Genetic abnormalities in the Dmd gene cause a loss of sarcolemmal integrity and highly progressive muscle fibre degeneration. Changes in the neuromuscular system are associated with necrosis, fibrosis and inflammation. In order to evaluate secondary changes in the sarcolemma membrane system due to the lack of the membrane cytoskeletal protein dystrophin, comparative organellar proteomics was used to study the mdx-4cv mouse model of dystrophinopathy. Mass spectrometric analyses identified a variety of altered components of the extracellular matrix-sarcolemma-cytoskeleton axis in dystrophic muscles. This included proteins involved in membrane repair, cytoskeletal restoration, calcium homeostasis, cellular signalling, stress response, neuromuscular transmission and reactive myofibrosis, as well as immune cell infiltration. These pathobiochemical alterations agree with the idea of highly complex secondary changes in X-linked muscular dystrophy and support the concept that micro-rupturing of the dystrophin-deficient plasma membrane is at the core of muscle wasting pathology.

Murphy S ，Zweyer M ，Henry M ，Meleady P ，Mundegar RR ，Swandulla D ，Ohlendieck K ... -  《-》

Proteomic analysis of dystrophin deficiency and associated changes in the aged mdx-4cv heart model of dystrophinopathy-related cardiomyopathy.

Cardiomyopathy is a serious complication in Duchenne muscular dystrophy, an X-linked neuromuscular disease of childhood that is triggered by primary abnormalities in the dystrophin gene. In order to directly correlate the deficiency in the membrane cytoskeletal protein dystrophin to secondary abnormalities in the dystrophic heart, this study has used label-free mass spectrometry to compare protein expression patterns in the aged mdx-4cv heart model of dystrophinopathy versus wild type heart. This report is the first successful identification of members of the cardiac dystrophin-glycoprotein complex by comparative whole tissue proteomics. The mass spectrometric analysis confirmed the loss of dystrophin and concomitant reduction of syntrophin and sarcoglycans in the dystrophin-deficient heart. Proteomic profiling of secondary changes identified distinct alterations in the basal lamina component laminin, the Ca(2+)-binding protein sarcalumenin, the matricellular protein periostin, the proteoglycans asporin and lumican, the cardiac-specific myosin light chain kinase, heat shock proteins and a large number of mitochondrial and glycolytic enzymes. The proteomic findings indicate that the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy is highly complex and involves impairments, modulations and/or adaptations of mitochondrial metabolism, glycolysis, protein chaperoning and ion homeostasis, as well as the maintenance of the contractile apparatus, the intracellular cytoskeleton and the extracellular matrisome. The X-linked inherited disorder Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood. Primary abnormalities in the dystrophin gene trigger progressive skeletal muscle wasting and impaired cardiorespiratory functions. In order to improve our general understanding of the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy and to identify new marker candidates of cardiac changes in dystrophinopathy, we have carried out a comparative proteomic study of the mdx-4cv mouse model of Duchenne muscular dystrophy. The mass spectrometric profiling of whole heart preparations has identified the reduction in the dystrophin-glycoprotein complex and a large variety of secondary changes in the dystrophic heart. Cardiac proteins with a changed abundance were shown to be involved in fibre contraction, energy metabolism, cellular signalling, the cytoskeletal network, the extracellular matrix and the stress response. In the future, the newly identified cardiac proteins may be useful to improve predictive, diagnostic, prognostic or therapy-monitoring approaches in the field of muscular dystrophy and cardiomyopathy.

Murphy S ，Dowling P ，Zweyer M ，Mundegar RR ，Henry M ，Meleady P ，Swandulla D ，Ohlendieck K ... -  《-》

Proteomic profiling of the dystrophin complex and membrane fraction from dystrophic mdx muscle reveals decreases in the cytolinker desmoglein and increases in the extracellular matrix stabilizers biglycan and fibronectin.

Murphy S ，Brinkmeier H ，Krautwald M ，Henry M ，Meleady P ，Ohlendieck K ... -  《-》

Comparative Label-Free Mass Spectrometric Analysis of Mildly versus Severely Affected mdx Mouse Skeletal Muscles Identifies Annexin, Lamin, and Vimentin as Universal Dystrophic Markers.

Holland A ，Henry M ，Meleady P ，Winkler CK ，Krautwald M ，Brinkmeier H ，Ohlendieck K ... -  《MOLECULES》

Comparative proteomic profiling of soleus, extensor digitorum longus, flexor digitorum brevis and interosseus muscles from the mdx mouse model of Duchenne muscular dystrophy.

Carberry S ，Brinkmeier H ，Zhang Y ，Winkler CK ，Ohlendieck K ... -  《-》