Elastase levels and activity are increased in dystrophic muscle and impair myoblast cell survival, proliferation and differentiation

Abstract

In Duchenne muscular dystrophy, progressive loss of muscle tissue is accompanied by fibrosis, chronic inflammation and reduced muscle regenerative capacity. Although much is known about the development of fibrosis and chronic inflammation in muscular dystrophy, less is known about how they are mechanistically linked to loss of muscle regenerative capacity. We have developed a proteomics method to discover dystrophy-associated changes in the muscle progenitor cell niche, which identified serine proteases, and especially neutrophil elastase, as candidates. We show that elastase activity is increased in dystrophic (mdx4cv) muscle and impairs myoblast survival in culture. While the effect of elastase on C2C12 cell survival correlates with the kinetics of elastase-mediated degradation of the substrate to which the cells adhere, the effect of elastase on satellite cell-derived primary myoblast growth and differentiation is substrate-independent and even more dramatic than the effect on C2C12 cells, suggesting a detrimental role for elastase on myogenesis in vivo. Additionally, elastase impairs differentiation of both primary and C2C12 myoblasts into myotubes. Our findings evidence the importance of neutrophil-mediated inflammation in muscular dystrophy and indicate elastase-mediated regulation of myoblast behaviour as a potential mechanism underlying loss of regenerative capacity in dystrophic muscle.

Neutrophil dysregulation is pathogenic in idiopathic inflammatory myopathies

“…It is known that neutrophils can cause muscle fiber damage in vitro and in vivo (25) and that oxidants released by these cells can cause severe cellular damage. Recent studies also implicate a role for HNE in muscular dystrophies by impairing myoblast cell survival, proliferation, and differentiation (26). We found that, while NETs do not impair myoblast proliferation, they significantly decrease skeletal myotube viability.…”

“…This is relevant, as biopsies from IIM subjects, but not healthy muscle, displayed evidence of infiltrating netting neutrophils and NET structures. Given the high levels of HNE present in NETs, it is possible that it and other proteases play prominent roles in the induction of myotube cell death (26). However, blocking the effects of HNE did not improve myotube viability, while citrullinated histones recapitulated the toxic effect of NETs on myotube viability.…”

Emerging proteomic biomarkers of X-linked muscular dystrophy

“…Altered CA3 levels and its leakage into the circulatory system may be related to fibre type-specific susceptibilities and accompanying changes in the dystrophic phenotype [3]. Consistent changes were also reported for annexin isoforms ANX1, ANX2 and ANX5, which may be related to disturbed Ca 2+ -homeostasis and repair mechanisms within dystrophic fibres [42,45,50,61,64,67,[71][72][73][74]. Potential compensatory mechanisms that are based on the up-regulation of cytoskeletal elements were confirmed by mass spectrometry.…”

“…These types of muscle proteins are more appropriately analyzed by one-dimensional gel electrophoretic techniques that can be combined with on-membrane digestion and mass spectrometry [34,43,72,77] or affinity purification and immunoprecipitation approaches [39,41,44,77]. Alternatively, skeletal muscle proteins that are present in crude tissue extracts or subcellular fractions can be conveniently analyzed by bottom-up proteomics, which employs trypsination of total protein complements in combination with liquid chromatography and mass spectrometry [30,45,56,60,62,64,[66][67][68][69]71,[73][74][75][76]78].…”

“…Reactive myofibrosis is a histopathological hallmark of X-linked muscular dystrophy and closely related to loss in motor functions [17,[19][20][21]. Proteomics established an increased density of a variety of markers of the extracellular matrix, such as collagens, dermatopontin, the matricellular protein periostin, fibronectin, biglycan, lumican and asporin [42,58,61,66,69,71,73]. Additional biomarker candidates of dystrophinopathy are represented by albumin, myosin light chains, the actin binding-protein profilin, the lipid dropletassociated protein perilipin and various glycolytic and mitochondrial enzymes [30,[39][40][41][42]44,45,.…”