Luc E. Gosselin

Localization and early time course of TGF-β1 mRNA expression in dystrophic muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy (DMD). In the dystrophic (mdx) mouse model of DMD, the diaphragm is more severely affected than other skeletal muscles. The level of transforming growth factor‐beta1 (TGF‐β1), an inflammatory cytokine, is significantly elevated in mdx diaphragm. However, little is known about the onset of TGF‐β1 messenger ribonucleic acid (mRNA) expression, or which cells express the mRNA. In this study, we characterized the location and time course of expression of TGF‐β1 mRNA in diaphragm from mdx mice. TGF‐β1 mRNA was significantly elevated in mdx diaphragm at 6 and 9 but not 12 weeks of age, and these changes corresponded with changes in type I collagen mRNA and hydroxyproline concentration. Mononucleated cells localized to areas of fiber necrosis highly expressed the TGF‐β1 transcript in mdx diaphragm. Neutralization of TGF‐β1 by decorin administration resulted in a 40% reduction in the level of diaphragm muscle type I collagen mRNA. These findings support a role for TGF‐β1 during the early stages of fibrogenesis in dystrophic diaphragm muscle. Therapeutic interventions aimed at neutralizing this cytokine may be beneficial in slowing the development of fibrosis in DMD. Muscle Nerve, 2004

Impact of prednisone on TGF‐β1 and collagen in diaphragm muscle from mdx mice

The purpose of this study was to assess the impact of prednisone treatment for 8 weeks on the level of transforming growth factor‐beta 1 (TGF‐β1), hydroxyproline (HYP) concentrations, and level of the mature, nonreducible collagen cross‐link hydroxylysylpyridinoline (HP) in diaphragm muscle from 12‐week‐old mdx mice. Diaphragm muscle from untreated mdx mice had a significantly higher level of TGF‐β1, HYP, and HP cross‐link compared with normal C57BL/10J (control) mice. Prednisone treatment significantly reduced the level of TGF‐β1 and HYP in diaphragm from mdx mice to values similar to control mice, but resulted in a higher level of the HP cross‐link compared with untreated mdx mice. These findings indicate that short‐term treatment of mdx mice with prednisone can attenuate the fibrotic response in diaphragm muscle, possibly by mediating the level of TGF‐β. Although prednisone was beneficial in preventing collagen accumulation, it resulted in a higher level of the HP cross‐link, presumably by decreasing collagen turnover

A comparison of factors associated with collagen metabolism in different skeletal muscles from dystrophic (mdx) mice : Impact of pirfenidone

Skeletal muscles in mdx mice exhibit differential degrees of pathological changes and fibrosis. The purpose of this study was to examine differences in various indices of collagen metabolism in skeletal muscles with widely different functions and activity profiles in mdx mice, and to determine whether pirfenidone would attenuate the development of fibrosis. Mice in the pirfenidone group were orally fed pirfenidone (500 mg/kg) daily for 4 weeks. Marked differences were noted in hydroxyproline concentration between muscles, which could not be explained solely by the level of type I collagen and transforming growth factor‐β1 (TGF‐β1) mRNA. In normal mice, matrix metalloproteinase (MMP)‐2 mRNA was significantly higher in the gastrocnemius than in the diaphragm or genioglossus muscles, suggesting that collagen degradation plays an important role in regulating collagen accretion in skeletal muscle. In mdx mice, the levels of both MMP‐2 and MMP‐9 mRNA were significantly elevated relative to control, although the response was muscle specific. Pirfenidone treatment resulted in a significant reduction in the level of hydroxyproline concentration across all muscles, although the effect was small. Results from this study reveal intrinsic dissimilarities in collagen metabolism between functionally different skeletal muscles. Moreover, the pharmacological use of pirfenidone may be beneficial in preventing fibrosis in muscular dystrophy. Muscle Nerve, 2006

Impact of TNF‐α Blockade on TGF‐β1 and type I collagen mRNA expression in dystrophic muscle

Dystrophin-deficient diaphragm muscle generally follows a pathological cascade of muscle damage, necrosis, and fibrosis,13 leading to increased weakness and stiffness.13 The precise mechanisms leading to fibrosis in dystrophic muscle are not known but likely involve the action of several inflammatory cytokines such as tumor necrosis factor– (TNF) and transforming growth factor– 1 (TGF1). TGF1 is upregulated in dystrophic skeletal muscle2 and is known to influence collagen metabolism.7 However, it is unknown whether TGF1 is solely responsible for development of muscle fibrosis or whether it works in concert with other cytokines. TNFis a proinflammatory cytokine produced by activated macrophages3 and T cells, and its level is increased in dystrophic muscle.10 By magnifying the cellular and mediator responses both locally and systemically, TNFmay play a critical role during inflammation.1 Although TNFstimulates collagen metabolism in vitro, little is known about its role in the pathogenesis of fibrosis in dystrophic diaphragm muscle. We hypothesized that TNFblockade via Enbrel administration would significantly attenuate the expression of both TGF1 and type I collagen messenger RNA (mRNA) expression in diaphragm muscle from young mdx mice. Male control C57BL/10ScSn and mdx mice (Jackson Laboratories, Bar Harbor, Maine) were used in this study. Mice were provided with water and chow ad libitum and were housed in the Laboratory Animal Facility at the University at Buffalo, where all procedures were approved by the local Animal Care and Use Committee. Type I collagen mRNA and TGF1 were assessed in diaphragm muscle from 6-week-old control, untreated mdx, and Enbrel-treated mdx mice (n 5 per group). The Enbrel-treated mdx mice were given a daily dose of Enbrel (10 g/kg body weight i.p.) dissolved in phosphate-buffered saline (pH 7.4) for 10 consecutive days. Enbrel (Amgen, Thousand Oaks, CA) is a soluble receptor fusion protein that acts by binding TNF, thereby reducing the bioavailability of TNFmolecules to TNFreceptors. Real-time quantitative polymerase chain reaction (PCR) was used to assess changes in TGF1, collagen type I (Col 1A2) mRNA. Total RNA was isolated using the Tri-spin method according to Reno et al.11 Total RNA was quantified by optical density at 260 nm. Total RNA was reversetranscribed using SuperScript II RNase H (Invitrogen Corp., Carlsbad, CA) according to instructions. The 5 -Taqman quantitative PCR assays were performed using an MX-4000 Real Time Q-PCR instrument (Stratagene Inc., La Jolla, CA). The sequences for forward and reverse primers and fluorescent reporter probes (5 FAM and 3 -BHQ-1 Black HoleTM quenchers) (BioSearch Technologies, Novato, CA) were designed using Beacon Designer 2 software (Premier Biosoft, Palo Alto, CA) spanning at least one exon–exon junction to eliminate genomic contamination. Standards were generated using synthetic-DNA amplicon oligonucleotides (BioSource Int., Camarillo, CA) spanning the entire amplification region. Standard curves [ R 0.995; efficiencies (slope), 3.2 to 3.3] were generated to determine copy number of gene targets using synthetic DNA aliquots (10 to 10 copies). Quantitative PCR reactions were performed under the following conditions: 12 min at 95°C followed by 40 cycles of two-step thermocycling (45 s at 94°C and 12 s at 60°C) using Hotstar Taq polymerase (Qiagen, Inc., Valencia, CA). Each sample was run in triplicate. Changes in mRNA levels were analyzed using a one-way analysis of variance with post hoc (Student–Newman–Keuls) analysis. Statistical significance was placed at P 0.05. Figure 1 illustrates the mRNA levels for TGF1 (Fig. 1A) and 2(I) collagen (Fig. 1B) in diaphragm muscle from the three groups of mice. Compared with controls, the mean TGF1 mRNA level was increased over threefold in untreated mdx mice (P 0.002). Diaphragm muscle from Enbrel-treated mdx mice had a significantly lower TGF1 mRNA level (P 0.001) than untreated mdx mice, whereas the Enbrel-treated group did not significantly differ from control. Type I collagen gene expression was elevated over sevenfold in mdx diaphragm relative to control (P 0.001). In contrast, treating mdx mice with Enbrel significantly reduced diaphragm muscle type I collagen mRNA expression (P 0.001) compared with untreated mdx mice—no difference existed between control mice and Enbrel-treated mdx mice.

Episodic hypoxia exacerbates respiratory muscle dysfunction in DMDmdx mice

Many patients with Duchenne muscular dystrophy (DMD) are eventually diagnosed with sleep‐disordered breathing (SDB). SDB is associated with reduced ventilation, decreased arterial oxygen tension, and increased respiratory muscle recruitment during sleep, factors that could be especially detrimental to respiratory muscles in DMD. To assess whether SDB impacts dystrophin‐deficient respiratory muscle function and fibrosis, diaphragm strength, and collagen content were evaluated in dystrophic mice (Dmdmdx) exposed to experimental SDB. Diurnal exposure to episodic hypoxia resulted in a 30% reduction in diaphragm strength without affecting collagen content. Episodic hypoxia secondary to SDB can exacerbate respiratory muscle dysfunction in DMD. Muscle Nerve, 2007

Pentoxifylline fails to attenuate fibrosis in dystrophic (mdx) diaphragm muscle

Fibrosis is a common pathological feature observed in muscle from patients with Duchenne muscular dystrophy and in mdx diaphragm. The purpose of this study was to determine whether pentoxifylline (PTX) treatment for 4 weeks (16 mg/kg/day) could significantly attenuate the process of fibrosis in diaphragm muscle from mdx mice. PTX treatment had no impact on in vitro diaphragm muscle contractile function. In addition, diaphragm muscle hydroxyproline concentration and the level of type I and III collagen and TGF‐β1 mRNA were unaffected by PTX treatment. These findings do not support the use of PTX as an antifibrotic drug for the treatment of muscular dystrophy. Muscle Nerve, 2006

Skeletal Muscle Collagen: Age, Injury and Disease

Collagen is the most common protein of the extracellular matrix and has several important functions in skeletal muscle, including the provision of both tensile strength and elasticity, the transmission of muscular forces to the bones, the regulation of cell attachment and differentiation, and mechanical and ionic filtration by the basal lamina. Aging is associated with significant changes in the connective tissue compartment of skeletal muscle. This chapter describes the effect of aging on skeletal muscle collagen, how injury affects collagen metabolism, how collagen is remodeled with advancing age and in severe muscle diseases like Duchenne muscular dystrophy. The regulation of collagen metabolism in normal and damaged skeletal muscle is complex and likely involves the interaction of several cell types and growth factors. Muscles with different activation patterns exhibit marked differences in collagen mRNA levels as well as collagen characteristics, indicating that mechanical load mediates collagen biosynthesis. Injured skeletal muscle contains elevated levels of inflammatory cells, which are known to secrete pro- and anti-inflammatory cytokines. Chronic inflammation plays a key role in the development of fibrosis in dystrophic muscle, although the mechanisms that regulate this process are not well understood. Both neutrophils and macrophages play important roles in the regulation of collagen remodeling post-injury by releasing various cytokines that mediate the behavior of inflammatory cells, fibroblasts and satellite cells. The behavior of these cells can be affected by extrinsic factors such as basal levels of growth hormone, which also changes with advancing age.