Aberrant repair and fibrosis development in skeletal muscle

“A nomenclature for polarized macrophages has been proposed [24,25] and they are now referred to as classically and alternatively activated macrophages, or M1 and M2 macrophages, respectively (Figure 2, Figure 3).”

“Activation requires proteolytic cleavage of the inactive precursor by either membrane-type (MT)1MMP [105] or plasmin, and cleavage of their corresponding inhibitors [106].”

“These cells are associated with tissue repair and fibrosis in many tissues and organs, including muscle, skin, liver, lung, bone and cartilage [56].”

“An important development in our understanding of muscle repair and fibrosis was the demonstration that a heterogeneous population of macrophages exists in regenerating muscle after injury, exhibiting opposing activities (either pro-inflammatory or anti-inflammatory) and different kinetics [23].”

“Whether FAP conversion also occurs during aging, and whether Basal lamina ECM deposition Satellite cell Fibro/adipogenic progenitor Resident macrophage Fibroblast Increased number of fibroblasts Fibrogenic-converted myogenic cells Myofiber Atrophic myofiber Young Old Figure 5 Myofiber growth and extracellular matrix (…”

“An earlier study by the same group had already highlighted the role of the canonical Wnt pathway in ageassociated fibrosis, linking increased collagen deposition in aged regenerating muscles to a greater percentage of fibrogenic cells arising from the conversion of myogenic into non-myogenic cells [65].”

“outcome of muscle repair, or alternatively, fibrosis [12,14,17,18].”

“A nomenclature for polarized macrophages has been proposed [24,25] and they are now referred to as classically and alternatively activated macrophages, or M1 and M2 macrophages, respectively (Figure 2, Figure 3).”

“Whether FAP conversion also occurs during aging, and whether Basal lamina ECM deposition Satellite cell Fibro/adipogenic progenitor Resident macrophage Fibroblast Increased number of fibroblasts Fibrogenic-converted myogenic cells Myofiber Atrophic myofiber Young Old Figure 5 Myofiber growth and extracellular matrix (…”

“The earliest phases of tissue repair are generally characterized by local activation of the innate immune system, even though the original immunogenic stimuli are not always known (see below) [20].”

“Immediately after skeletal-muscle injury, cytokines and growth factors are released from both the injured blood vessels and from infiltrating inflammatory cells [13,14].”

“In vitro, PDGF production can be induced by TGFb, which also stimulates fibroblasts to contract collagen matrices and differentiate into myofibroblasts [88].”

“This fibrotic response is specific to TGFb and CTGF, and is not mimicked by EGF, PDGF or FGF2 [87].”

“Mutant mice were able to remove necrotic tissue from injured muscle, but they exhibited severe defects in myofiber regeneration [43].”

“interleukin (IL)-4 was shown to regulate fusion of myoblasts in vitro and in vivo [129].”

“Several studies of macrophage depletion or impaired macrophage recruitment have revealed crucial functions of macrophages in the regulation of fibrogenesis in dystrophic muscle, and the potential for therapeutic intervention [19,35,37,38].”

“Indeed, similar shifts in macrophage polarization and macrophage competition for arginine metabolism were seen to influence the severity of the muscle pathology in mdx dystrophic mice [28].”

“For example, analysis of the microniche of aged murine muscle stem cells found high levels of TGFb and its activated effector Smad3 in both differentiated muscle fibers and satellite cells, which was reciprocal to the levels of active Notch, which is more abundant in the young microniche [62].”

“However, in chronic human diseases such as DMD and many other dystrophies, newly generated fibers are also prone to degeneration because they retain the underlying molecular defect, producing constant cycles of fiber degeneration associated with chronic inflammation (Figure 1) [4].”

“Furthermore, the ECM sequesters and presents heparinbinding growth factors to the fibers, such as hepatocyte growth factor (HGF) and fibroblast growth factor (FGF), and signals to the differentiated fibers through dystroglycan and sarcoglycan complexes [102,103].”

“In addition, PDGF can induce c-abl kinase activity, similar to TGFb, in a non-canonical manner [89].”

“In addition, there is infiltration of fat and other non-contractile material, which causes a reduction in muscle ‘quality’ [8].”

“Similarly, mdx mice lacking myostatin displayed less fibrosis in the diaphragm, and were also stronger and more muscular than their normal mdx counterparts [97].”

“Interestingly, there is the suggestion that the type of injury in acute models may drive the repair response towards more fibrotic or more adipogenic repair, for example after glycerol injury and acute ischemia, respectively [68,69], although the mechanisms have not been extensively investigated.”

“Deficiency of myostatin increases skeletal muscle mass, myofiber diameter and strength [93,94].”

“Perhaps more importantly, the skeletal muscle of adult mdx mice expresses high levels of latent and active MMP-2 and MMP-9, and inhibiting MMP-9 activity was shown to significantly improve regeneration and contractile functions by reducing the structural deterioration of muscle, necrosis, inflammation and fibrosis [16…”

“PGs have been shown to play various roles in many stages of myogenesis, and are secreted from regenerating muscle [48,49].”

“One example of this was the deletion of IL-10 in mdx mice, which increased muscle damage and reduced muscle strength, due to an imbalance between M1 and M2 macrophages [29].”

“These results correlated well with improvements in muscle strength and reduced diaphragmatic and cardiac fibrosis [53].”

“Direct injection of recombinant TGFb into skeletal muscle in vivo stimulates TGFb expression in myogenic cells in an autocrine fashion and induces connective-tissue formation in the area of the injection [73,74].”

“Immediately after skeletal-muscle injury, cytokines and growth factors are released from both the injured blood vessels and from infiltrating inflammatory cells [13,14].”

“when followed by anti-CD4 and/or antiCD8 antibody treatment, failed to improve diaphragm fibrosis at 6 months of age in mdx mice [51,54,55].”

“Local expression of a muscle-restricted IGF-1 transgene accelerates the regenerative process in injured skeletal muscle, modulating the inflammatory response and limiting fibrosis [39].”

“However, myostatin not only regulates the growth of muscle cells, but also regulates muscle fibroblast activation and hence the progression of fibrosis [96].”

“Fibrin(ogen) deposits may serve as important triggers for the inflammatory responses, because of their capacity to bind to the integrin receptor Mac-1 on classically activated M1 macrophages [27] (see above).”

“The net consequence of these alterations firstly involves progressive atrophy and loss of individual muscle fibers, associated with concomitant loss of motor units [7].”

“Because Let-7 family members been related to cell-cycle control, they may function to downregulate other genes involved in muscle-cell proliferation, which in turn might negatively affect the reduced regenerative capacity of muscle seen with aging [126].”

“The large family of MMPs includes various collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-7, MMP-10 and MMP-11), membrane-type metalloproteinases (MMP-14, MMP-15, MMP-16, MMP-17, MMP24 and MMP-25), and the metalloelastase MMP-12 [16].”

“Recent studies have shown that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to injury, which is linked to adaptive immunity through inflammatory dendritic cells [21].”

“Several studies of macrophage depletion or impaired macrophage recruitment have revealed crucial functions of macrophages in the regulation of fibrogenesis in dystrophic muscle, and the potential for therapeutic intervention [19,35,37,38].”

“As a potential therapeutic approach, immunological neutralization of myostatin in mdx mice was shown to attenuate dystrophy by improving muscle regeneration and reducing ECM accumulation [98].”

“Finally, latent TGFb-binding protein (LTBP)4 regulates the release and bioavailability of TGFb from the ECM, where it has been shown to modulate fibrosis in the context of muscular dystrophy in mice [77] Taken together, these studies highlight the crucial role of TGFb in the initiation of fibrotic processes, and in th…”

“Reduced tissue fibrosis after injury was also detected in mice treated with both decorin- and angiotensin receptor antagonists, with decorin also preventing TGFb-induced differentiation of myogenic cells into fibrotic cells [80].”

“In patients with DMD, fibrosis has also been associated with increased numbers of alternatively activated macrophages [42].”

“Normal myostatin signaling can be counter-regulated by the extracellular protein follistatin [95].”

“A more recent follow-up study [118] related the increased fibrosis of Sca-1-deficient mice to a reduced recruitment of IgM and C3 to injured muscle, due to a specific reduction in peritoneal IgM-producing B-1a cells.”

“In normal muscle, the external lamina is composed of collagen IV, laminin, and heparan sulfate proteoglycans, while the interstitial matrix that surrounds the basal lamina contains collagens I, III and V, fibronectin and perlecan [10,11,102].”

“Although fibroblasts are the major collagen-producing cells, myofiber-associated satellite cells and C2C12 myoblasts have also been shown to express significant levels of interstitial collagens I and III, which diminish during the process of differentiation [58].”

“Perhaps more importantly, the skeletal muscle of adult mdx mice expresses high levels of latent and active MMP-2 and MMP-9, and inhibiting MMP-9 activity was shown to significantly improve regeneration and contractile functions by reducing the structural deterioration of muscle, necrosis, inflammation and fibrosis [16…”

“Because Sca-1 also plays a role in the maintenance of progenitor cells [119], it was hypothesized that the reduction in B-1a cells was due to lineage-specific defects in self-renewal.”

“Conversely, BM transplantation experiments have revealed an important role for the CC chemokine receptor 2/monocyte chemotactic protein-1 (CCR2/MCP-1) ligand, and for the proteolytic activity of urokinase plasminogen activator (uPA)/plasmin in skeletal-muscle repair by regulating the recruitment of BMderived macrophag…”

“Rho/ Rho kinase signaling also appeared to be implicated in the TGFb-mediated transition of myoblasts into myofibroblasts downstream of SK1 activation [60].”

“whereas loss of biglycan in transgenic mice does not affect regenerative capacity, despite inducing delays in new myofiber growth [100], decorin delivery to regenerating muscle via adenoassociated viral (AAV) vectors prevents TGFb activation and fibrosis [76].”

“In fibroblasts, myostatin induces activation of the Smad, p38 MAPK and AKT pathways to promote ECM synthesis [99].”

“Similar results were reported in animals treated with suramin, which acts by competing with TGFb for receptor binding [82].”

“drugs has been their ability to promote fiber atrophy [47], in addition to their negative effect on prostaglandin (PG) synthesis via interference with cyclooxygenase (COX) gene functions.”

“For example, direct immunomodulation of TGFb inhibited connective-tissue accumulation and the progression of fibrosis in the diaphragm of mdx mice, although inflammation increased [79].”

“These cells restored a vascular network and diminished collagen deposition, raising the exciting possibility of using similar cell-based or gene therapies to improve muscle function in patients with currently untreatable advanced-stage dystrophies [114].”

“In nu/nu/ mdx mice, (immunodeficient nude mice in the mdx background) the lack of functional T cells alone was associated with less diaphragm fibrosis at 3 months, supporting the pathogenic role for T cells in mdx muscle, and revealing this lymphocyte subclass to be an important source of TGFb1 [52].”

“For example, MT1-MMP is presumed to help maintain myofiber integrity, as transgenic mice deficient in MT1-MMP develop centrally nucleated myofibers that are smaller than normal [105].”

“These results suggest a role for both TGFb and CTGF in skeletal-muscle remodeling by inducing fibrosis, inhibiting myogenesis and promoting dedifferentiation of myoblasts into myofibroblast-like cells [86].”

“Conversely, BM transplantation experiments have revealed an important role for the CC chemokine receptor 2/monocyte chemotactic protein-1 (CCR2/MCP-1) ligand, and for the proteolytic activity of urokinase plasminogen activator (uPA)/plasmin in skeletal-muscle repair by regulating the recruitment of BMderived macrophag…”

“Increased levels of CTGF have been reported in skeletal muscle from patients with DMD, dystrophic dogs and mdx mice [85,86].”

“Whereas it has been shown that both uPA and plasmin activity are necessary for skeletal-muscle regeneration, tPA activity is dispensable, indicating that no redundancy exists in muscle [33,35].”

“Several studies of macrophage depletion or impaired macrophage recruitment have revealed crucial functions of macrophages in the regulation of fibrogenesis in dystrophic muscle, and the potential for therapeutic intervention [19,35,37,38].”

“In additional studies of aged muscle, the fate of muscle stem-cell progeny was reported to be controlled by an interaction between the Wnt and Notch pathways in which glycogen synthase kinase (GSK)3b plays an important role [64].”

“Another recent study identified an increase in a muscle-resident stromal cell (mrSC) population in the muscles of mdx mice [122].”

“when followed by anti-CD4 and/or antiCD8 antibody treatment, failed to improve diaphragm fibrosis at 6 months of age in mdx mice [51,54,55].”

“Consequently, administering imatinib to mdx mice reduced fibrosis in the diaphragm, and attenuated skeletal-muscle necrosis and inflammation, with concomitant improvements in muscle function [91].”

“PGs have been shown to play various roles in many stages of myogenesis, and are secreted from regenerating muscle [48,49].”

“whereas loss of biglycan in transgenic mice does not affect regenerative capacity, despite inducing delays in new myofiber growth [100], decorin delivery to regenerating muscle via adenoassociated viral (AAV) vectors prevents TGFb activation and fibrosis [76].”

“In normal muscle, the external lamina is composed of collagen IV, laminin, and heparan sulfate proteoglycans, while the interstitial matrix that surrounds the basal lamina contains collagens I, III and V, fibronectin and perlecan [10,11,102].”

“Aged human muscle also behaves differently from young muscle after a similar modest level of damage, with dysregulation of a particular set of transcripts including transcripts related to fibrosis and connectivetissue function [127].”

“TGFb is expressed in normal muscle after injury and in the dystrophic muscle of patients with DMD and mdx mice [70], where it has the potential to induce fibrosis around myofibers, probably by stimulating fibroblasts to produce ECM proteins such as collagen and fibronectin.”

“Indeed, halofuginone treatment was also shown to enhance myogenesis, a feature that may be relevant to improve muscle regeneration and function in muscular dystrophy [84].”

“Direct injection of recombinant TGFb into skeletal muscle in vivo stimulates TGFb expression in myogenic cells in an autocrine fashion and induces connective-tissue formation in the area of the injection [73,74].”

“For example, a major role for myofibers themselves in controlling the homeostasis of the ECM during age-related sarcopenia has been proposed [125].”

“In addition, NO, which is released by local and invading cells during injury, can act as a pro-inflammatory molecule, and is able to activate MMP-2, which in turn releases HGF. Importantly, blockers of NOS function, such as nitro- Larginine methyl ester (L-NAME), have been shown to decrease the acute inflammatory reac…”

“Based on all of the emerging data, it is tempting to speculate that the pronounced fibrosis in seen in patients with DMD is related to altered net proteolytic activity in the dystrophic muscles, due to imbalances in expression and activity of PA/MMP system components [101,116].”

“Perhaps more importantly, the skeletal muscle of adult mdx mice expresses high levels of latent and active MMP-2 and MMP-9, and inhibiting MMP-9 activity was shown to significantly improve regeneration and contractile functions by reducing the structural deterioration of muscle, necrosis, inflammation and fibrosis [16…”

“CCL17 in particular has been shown to enhance fibrosis in several mouse models of pulmonary disease by binding to CC chemokine receptor (CCR)4 [30].”

“Similar benefits on the dystrophic phenotype in mdx mice were reported after using a variety of anti-inflammatory agents acting on cytokines such as TNFa and on their cellular receptors, or on other major pro-inflammatory mediators such as nuclear factor (NF)- B [39-41].”

“The main function of the plasminogen activation system main function is to degrade fibrin [104], but it also fulfills an important role in muscle ECM remodeling by cleaving ECM-associated molecules, liberating and/or activating latent forms of bioactive molecules such as growth factors, angiogenic factors and cytokine…”

“In aged mice, a Sca-1+, non-myogenic (MyoD-) and non-immunohematopoietic (CD45-) cell population has been proposed to favor fibrosis [121].”

“At the ultrastructural level, aging has also been associated with myofibril disarrangements in a dystrophic animal model, and drops in force without alterations in motor protein function as measured by in vitro motility assays [9].”

“Similar benefits on the dystrophic phenotype in mdx mice were reported after using a variety of anti-inflammatory agents acting on cytokines such as TNFa and on their cellular receptors, or on other major pro-inflammatory mediators such as nuclear factor (NF)- B [39-41].”

“intervention, or impairs the efficiency of these therapies [5].”

“Microarray analyses of myogenic cells from aged animals also revealed major alterations in the expression of many genes dependent upon the activation of TGFb signaling [78].”

“Collagen modifications, such as non-enzymatically regulated crosslinking to produce advanced glycation end (AGE) products, also increase the stiffness of muscle connective tissue, thereby contributing to impaired muscle function in the older person [59].”

“MMP-13 and MMP-1 also participate in ECM remodeling during muscle repair [16,107,108], while gelatinase MMP-2 affects new fiber formation by promoting collagen IV degradation in the basement membrane during myoblast proliferation, migration and fusion.”

“Finally, Notch2 can inhibit the differentiation of myoblasts into myofibroblasts by directly counter-regulating Notch3 and limiting its expression [61].”

“Based on all of the emerging data, it is tempting to speculate that the pronounced fibrosis in seen in patients with DMD is related to altered net proteolytic activity in the dystrophic muscles, due to imbalances in expression and activity of PA/MMP system components [101,116].”

“MMP-13 and MMP-1 also participate in ECM remodeling during muscle repair [16,107,108], while gelatinase MMP-2 affects new fiber formation by promoting collagen IV degradation in the basement membrane during myoblast proliferation, migration and fusion.”

“Increased STAT3-dependent responses in aged versus young muscles have also been reported in humans after resistance exercise, which has being hypothesized to be related to the efficiency of muscle repair and regeneration [128].”

“when followed by anti-CD4 and/or antiCD8 antibody treatment, failed to improve diaphragm fibrosis at 6 months of age in mdx mice [51,54,55].”

“Similar effects were also seen in aged muscle [124].”

“Although more detailed studies of the role of IgM and C3 in muscle regeneration and fibrosis are necessary, C3 has already been shown to influence the development of liver fibrosis in other animal models [120].”

“repair seen after injury in PAI-1-deficient animals [36].”

“Basement membranes and temporary ECM components are also crucial for guiding the formation of neuromuscular junctions (NMJs) [15].”

“Similar scenarios of reduced Notch activation and increased TGFb/pSmad3 signaling have been reported recently in aged human muscles [63].”

“The protein ADAM12 (a disintegrin and metalloproteinase 12) has been shown to produce benefits in the context of muscle dystrophy and aging by upregulating utrophin and stabilizing the plasma membrane [115].”

“Complementary studies showed that imatinib ameliorated dystrophy in mdx mice after exercise, although treated mice also exhibited significant weight loss [92].”

“For example, the anti-neoplastic agent imatinib selectively and competitively blocks the ATP binding site of c-abl and several other tyrosine kinases, including PDGF receptors and c-kit, and it diminishes tissue fibrosis in many experimental mouse models of fibrotic disorders [90].”

“One other limitation of the drugs used to treat IIMs is that they do not produce complete recovery, nor do they address the underlying defects, which in most cases are multifactorial and not fully defined [50].”

“More importantly from the clinical perspective, long-term dietary supplementation with arginine increased skeletal and cardiac muscle fibrosis in dystrophic mice, in contrast to the reported benefits from short-term supplementation, thus suggesting caution is needed regarding dietary arginine supplementation for patie…”

“It is still not clear if Sca-1 acts directly or indirectly to upregulate MMP activity, which could in turn increase matrix breakdown and efficient muscle regeneration, while halting fibrosis [117].”

“84 There is no direct evidence of muscle fi brosis with age in humans due to the dif fi culty in assessing fi brosis in population studies, but animal work indicated > 17% increase in fi brotic tissue in older rats as compared with younger rats.”

“This uncontrolled and irreversible ECM expansion accompanied by an accumulation of ECM due to inhibited degradation (turnover), results in a fibrotic phenotype within muscle (Mann et al., 2011), especially in chronic diseases such as dystrophinopathies (Serrano & Muñoz-Cánoves, 2017).”