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G.S Lynch (ed.), Sarcopenia – Age-Related Muscle Wasting and Weakness,

DOI 10.1007/978-90-481-9713-2_8, © Springer Science+Business Media B.V 2011

Abstract 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

Keywords Aging • Collagen • Fibrosis • Force transmission • Inflammation

• Growth factors • Mechanical loading • Muscle architecture • Muscular dystrophy • Tissue remodeling

L.E Gosselin (*)

Department of Exercise and Nutrition Sciences, University at Buffalo,

211 Kimball Tower, Buffalo, NY 14214-8028, USA

e-mail: gosselin@buffalo.edu

Skeletal Muscle Collagen: Age, Injury

and Disease

Luc E Gosselin

160 L.E Gosselin

1 Overview of Collagen in Skeletal Muscle

Collagen is the most common protein of the extracellular matrix (ECM) (Laurent

1987) and has several important functions in skeletal muscle, including: (1) provi-sion of both tensile strength and elasticity; (2) transmisprovi-sion of muscular forces to the bones; (3) regulation of cell attachment and differentiation; and (4) mechanical and ionic filtration by the basal lamina (Minor 1980; Nimni and Harkness 1988; Hay

1991) From the collagen family of proteins, fibrillar collagen type I and type III, the basement membrane collagen type IV, and some of the minor types (e.g V, VI, VII,

XV, XVIII) have been characterized in skeletal muscle (Duance et al 1977; Light and Champion 1984; Kovanen et al 1988; Hurme et al 1991) The epimysium is composed primarily of type I collagen whereas the perimysium contains both type I and III (with type I predominating) (Light and Champion 1984) On the basis of their structural properties type I collagen is suggested to confer tensile strength and rigid-ity (Mays et al 1988) whereas type III collagen confers compliance (Burgeson

1987) to intramuscular connective tissue Fibroblasts synthesize the fibrillar colla-gen types in muscle (Hurme et al 1991), although skeletal muscle cells are known

to produce mRNA for types I and III collagen (Takala and Virtanen 2000)

Collagen is unique because the protein undergoes extensive post-translational modification both in the intra- and extracellular space Prolyl-4-hydroxylase (P4H)

is an intracellular posttranslational enzyme involved in the hydroxylation of prolyl residues necessary for the formation of the stable collagen triple-helix (Kovanen

2002) Molecular maturation of collagen (i.e., formation of reducible and nonreducible cross-links) is an essential extracellular post-translational process that affords tensile strength to the protein (Viidik 1968; Eyre et al 1984) The rate-limiting step involves the extracellular oxidation of lysine and hydroxylysine resi-dues by the enzyme lysyl oxidase, thus forming semialdehydes that can undergo further chemical transformations throughout the life of the protein (Eyre et al 1984; Reiser et al 1992) The maturation of collagen alters its mechanical and biochemical properties, leading to increased tensile strength (Viidik 1968; Eyre et al 1984), decreased solubility (Ricard-Blum and Ville 1989) and enhanced resistance to some proteases (Cheung and Nimni 1982)

Collagen concentration in the extracellular space can be controlled either intracellularly prior to secretion or extracellularly following secretion Intracellular procollagen turnover may be influenced by altering synthesis and/or degradation rate (Bienkowski et al 1978; Laurent et al 1985; Laurent 1987; McAnulty and Laurent 1987) As much as 90% of procollagen may be degraded intracellularly within minutes of synthesis (Laurent 1987) Two pathways for this intracellular degradation are proposed: Golgi apparatus and the lysosomes (Laurent 1987) In the extracellular space, the newly synthesized forms of collagen are degraded more quickly than the mature, cross-linked collagen (Laurent 1987) Matrix metallopro-teinases (MMPs), also known as collagenases, are the enzymes responsible for the initiation of the extracellular degradation of the collagen triple-helix (Stetler-Stevenson 1996) Fibrillar collagens (I, II, III) are degraded by MMP-1, MMP-8,

161 Skeletal Muscle Collagen: Age, Injury and Disease

and MMP-13, whereas the gelatinases MMP-2 and MMP-9 degrade type IV collagen and gelatin (Birkedal-Hansen et al 1993) Tissue inhibitors of matrix metalloproteinases (TIMP-1,-2,-3, and -4) regulate the activity of MMPs by binding either the active or latent forms of MMPs (Edwards et al 1996) In skeletal muscle, MMP-2 is constitutively expressed, whereas MMP-9 appears following acute skeletal muscle damage (Kherif et al 1999) In vivo, fibroblasts, polymor-phonuclear leukocytes, neutrophils, and macrophages are responsible for the secre-tion of MMPs as well as the growth factors involved in the regulasecre-tion of the expression of the MMPs and TIMPs (Birkedal-Hansen et al 1993)

2 Effect of Aging on Skeletal Muscle Collagen

Aging is associated with significant changes in the connective tissue compartment

of skeletal muscle The relative distribution of type I collagen increases from birth

to senescence, whereas the relative distribution of type III collagen decreases dur-ing the same period (Kovanen and Suominen 1989) The concentration of type IV collagen also increases in skeletal muscle with age (Kovanen et al 1988) In addi-tion to these changes, both concentraaddi-tion of collagen and extent of nonreducible cross-linking significantly increase in senescent skeletal muscle (Zimmerman

et al 1993; Gosselin et al 1994, 1998) and cardiac tissue (Thomas et al 1992) The age-related increase in skeletal muscle collagen content occurs without any changes in the activities of P4H or galactosylhydroxylysysl glucosyltransferase (Kovanen and Suominen 1989), two post-translational modification enzymes whose activities reflect collagen synthesis rate Moreover, Mays et al (1988) reported that the fractional synthesis rate of collagen in rat skeletal muscle decreases approximately tenfold from 1- to 24-months of age These results sug-gest that increases in collagen concentration in senescent skeletal muscle are a result of a decreased rate of resorption out of proportion to the reduced biosyn-thetic activity Biopsies from the vastus lateralis muscles of young and old seden-etary men and women revealed that intramuscular endomysial collagen and collagen cross-linking (hydroxylsylpyridoline) were unchanged with aging but that the advanced glycation end product, pentosidine, was increased by ~200% (Haus et al 2007) These data suggested that the synthesis and degradation of contractile proteins (actin and myosin) and proteins involved in the transfer of muscle forces (collagen and pyridinoline cross-links), were tightly regulated during aging and that changes in the glycation-related cross-linking of intramuscular con-nective tissue possibly contributes to the age-related changes in force transmission and overall muscle function (Haus et al 2007)

Endurance exercise training can lower the extent of collagen cross-linking in senescent cardiac (Thomas et al 1992) and skeletal (Zimmerman et al 1993; Gosselin et al 1998) muscle, suggestive that collagen turnover is increased during periods of altered use The impact of increased collagen concentration and cross-linking on repair of injured senescent skeletal muscle is unknown Increased

162 L.E Gosselin cross-linking increases collagen’s resistance to proteolytic degradation (Cheung and Nimni 1982), allowing slower collagen degradation in senescent skeletal muscle Whether or not this affects muscle repair is unknown It is also possible that increased collagen concentration may impair the migration of satellite cells in cases where the basement membrane is destroyed in the damaged area, though this remains speculative

3 Effect of Injury on Skeletal Muscle Collagen Metabolism

Despite positive benefits achieved from exercise training, some studies have indi-cated that skeletal muscles of older adults are more susceptible to injury during exercise than muscles of younger adults (Zerba et al 1990; Brooks and Faulkner

1994; Faulkner et al 1995) Senescent skeletal muscles can be further compro-mised since repair occurs more slowly compared to young muscle (Brooks and Faulkner 1990), and because of a limited potential for satellite cell activation (Schultz and Lipton 1982) The slowed response time for repair may be partially attributed to decreases in protein synthesis observed with aging (Welle et al 1993) Thus, any beneficial gains from exercise may be lost during a prolonged period of muscle repair due to inactivity

Although exercises involving lengthening or ‘eccentric’ contractions, appear to cause more injury (Armstrong et al 1983; McCully and Faulkner 1986) than short-ening contractions, muscle injury has also been reported to occur with the latter (McCormick and Thomas 1992) Muscle injury is typically manifested by a decre-ment in maximal specific force (force/cross sectional area), and morphologically by alterations in Z-line pattern (i.e., Z-line streaming) (Friden et al 1983) and infiltration by inflammatory cells (Tidball; see Chapter 16) Catabolism of dam-aged intra- and extracellular proteins is a necessary step in the injury/repair process and involves the activity of calpains (Tidball and Spencer 2000) Additionally, satellite cells and muscle fibroblasts are activated (Tidball 1995), presumably from local growth factors such as fibroblast growth factor (FGF) and insulin-like growth factor I (IGF-I) Participation by these cells as well as inflammatory cells is essen-tial for the repair of the damaged muscle fibers Thus, repair of the muscle involves the coordinated processes from several cell types, each of which having separate and distinct roles Successful repair of skeletal muscle depends not only on remod-eling the damaged intracellular (contractile, cytoskeletal) proteins, but also the surrounding extracellular matrix, including collagen

Extensive evidence indicates that the extracellular matrix is remodeled during muscle repair Following acute exercise-induced muscle damage, the mRNA level

of type IV collagen increases within 6 h after inducement of damage (Han et al

1999) The level of mRNA for types I and III collagen subsequently increase coor-dinately with mRNA of P4H a- and b- subunits and lysyl oxidase, in addition to

163 Skeletal Muscle Collagen: Age, Injury and Disease

the P4H activity As determined by immunohistochemistry, a qualitative transitory increase in the expression of type III collagen has been noted in mouse skeletal muscle following exercise-induced injury (Myllyla et al 1986) It is known that collagen metabolism is down-regulated with aging (Mays et al 1988), and that accumulation of intramuscular connective tissue occurs (Kovanen and Suominen

1989; Zimmerman et al 1993; Gosselin et al 1994, 1998) together with altered functional properties (Kovanen et al 1984; Gosselin et al 1994, 1998) However, there is a dearth of information regarding how collagen expression is regulated in aged skeletal muscle following muscle injury

4 Do Extrinsic Factors Affect Collagen Remodeling

in Aged Damaged Muscle?

Growth hormone (GH) has pronounced effects on organ and tissue growth Body growth of hypophysectomized rats and Lewis dwarf rats deficient in GH is mark-edly reduced but can be reversed by GH supplementation (Guler et al 1988; Gosteli-Peter et al 1994; Martinez et al 1996) During aging, myofibrillar protein synthesis decreases (Welle et al 1993) as do the circulating levels of serum GH (Florini et al 1985) However, when old rats are supplemented with GH, protein synthesis is increased to levels similar to that observed in young rats (Sonntag et al

1985) It was reported recently that increased GH availability stimulates matrix collagen synthesis in skeletal muscle and tendon, but with no effect on myofibrillar protein synthesis, indicating that GH might be more important in strengthening the matrix tissue than for skeletal muscle hypertrophy in adult human musculotendi-nous tissue (Doessing et al 2010)

GH is thought to function indirectly on skeletal muscle via the action of insulin-like growth factor I (IGF-I), a growth promoting peptide factor (Schwander et al

1983) When physiological concentrations of IGF-I are applied to myoblasts grown

in tissue culture, cell mitotic activity and protein synthesis significantly increases (Florini 1987; Johnson and Allen 1990) The target of IGF-I not only includes myoblasts but other cell types as well For example, cultured fibroblasts exposed to physiological concentrations of IGF-I increase collagen synthesis (Goldstein et al

1989; Gillery et al 1992), whereas addition of an antibody specific to the IGF-I receptor (aIR-3) inhibits fibroblast collagen synthesis (Goldstein et al 1989) Although the liver produces the majority of IGF-I (Sonntag et al 1985), other tissues, including skeletal muscle, can also produce IGF-I (Sonntag et al 1985; Jennische and Hansson 1987; Jennische and Olivecrona 1987; Yan et al 1993) The action of IGF-I on muscle is dependent not only upon the local concentration of IGF-I, but also on the pattern of growth factor receptor expression (Rubin and Baserga 1995) Whether or not aging alters IGF-I receptor density in skeletal muscle, and what impact this may have during muscle repair is unclear

164 L.E Gosselin

5 Duchenne Muscular Dystrophy: Collagen Metabolism Run Amok

DMD is an X-chromosome linked disorder resulting in the loss of the muscle protein dystrophin (Hoffman et al 1987), a large protein localized to the inner surface of the muscle cell membrane (Watkins et al 1988) Dystrophin-deficient muscle is damaged

to a greater degree given the same recruitment history due to its innate membrane fragility (Petrof et al 1993; Petrof 1998) Consequently, the muscles undergo cycles

of injury and repair that result in progressive muscle fiber loss, weakness, and exten-sive fibrosis The diaphragm is particularly affected, and humans typically suffer from respiratory failure early in life (Inkley et al 1974)

The mdx mouse shares a genetic and biochemical homology with human

muscular dystrophy and is commonly used to study DMD Although limb skeletal

muscles from mdx mice are capable of significant regeneration, the diaphragm

muscle exhibits progressive degeneration similar to that observed in skeletal mus-cle from patients with DMD (Stedman et al 1991) The mechanisms responsible for this divergent response are not known, but may be due to differences in inflam-mation secondary to muscle activation pattern

Data indicates that the process of diaphragm fibrosis has commenced by 6

weeks of age in mdx mice (Gosselin et al 2004), and that the extent of diaphragm fibrosis increases progressively thereafter such that by 16 months of age,

hydroxy-proline concentration in mdx diaphragm is elevated ~sevenfold (Stedman et al

1991) These biochemical changes are associated with a significant increase in diaphragm stiffness (Stedman et al 1991) Collagen is also involved in the regulation of cell attachment and differentiation, and mechanical and ionic filtration

by the basal lamina (Minor 1980; Nimni and Harkness 1988; Hay 1991) Hence, excessive collagen may therefore serve as a barrier for targeted drug or gene therapy In spite of these important physiological functions, there is a dearth of information regarding the mechanisms that regulate collagen metabolism in damaged and dystrophic skeletal muscle

Collagen accretion in the extracellular space is a function of both synthesis and degradation Significant increases in type I collagen mRNA (Goldspink et al 1994; Gosselin and Martinez 2004; Gosselin et al 2004; Gosselin and Williams 2006)

have been observed in mdx diaphragm Interestingly, the level of type I collagen

mRNA, expressed per mg RNA, is similar in diaphragm and gastrocnemius muscle

from 9-week-old mdx mice, despite the fact that the diaphragm accumulates

signifi-cantly more collagen (Gosselin and Williams 2006) RNA concentration in mdx diaphragm is ~80% higher than in mdx gastrocnemius (Gosselin and Williams

2006), suggestive that a hypercellular environment exists in mdx diaphragm

Assuming a constant mRNA to RNA ratio in both muscles, the diaphragm muscle contains approximately 80% more type I collagen mRNA per unit weight This difference could theoretically result in significantly greater collagen synthesis and accretion in the diaphragm Whether or not fibroblast proliferation occurs in vivo

in dystrophic diaphragm muscle and contributes to the hypercellularity remains to be

165 Skeletal Muscle Collagen: Age, Injury and Disease

determined Such a finding however would be of significant biological consequence, even in the absence of elevated levels of pro-fibrotic cytokines

Matrix metalloproteinases (MMPs) are a group of zinc-dependent enzymes that initiate the extracellular degradation of collagen (Hay 1991; Nagase et al 2006) Of the 20 or so different MMPs (Nagase et al 2006), MMP-9 and MMP-2 have been the most studied in mammalian skeletal muscle MMP-2 is constitutively expressed

in normal skeletal muscle whereas MMP-9 is absent (Kherif et al 1999) However,

in response to various forms of injury, such as that induced by cardiotoxin (Kherif

et al 1999) or ischemia-reperfusion (Muhs et al 2003), MMP-9 mRNA and activity significantly increase within 24 h post-injury and appears to be expressed primarily by neutrophils (Kherif et al 1999; Muhs et al 2003) In contrast, the active form of MMP-2 does not begin to increase until ~72 h post-injury, and increases further at 7 days, suggestive that these two MMPs have unique roles in the remodeling of the ECM Interestingly, MMP-9 and MMP-2 are elevated in

skeletal muscle from 3-month-old mdx mice (Kherif et al 1999), findings that are paradoxical to the development of fibrosis in dystrophic skeletal muscle

MMP-9 has been shown to be involved in the recruitment of inflammatory cells

in the post-ischemic liver model (Khandoga et al 2006) In other models of injury and fibrosis, MMP-9 blockade significantly decreases the extent of inflammation and fibrosis (Corbel et al 2001a, b; Tan et al 2006), suggestive that MMP-9 may either directly or indirectly mediate the behavior of inflammatory cells or fibro-blasts The basal lamina, which contains type IV collagen, is known to bind a number of growth factors, including bFGF (DiMario et al 1989; Yamada et al

1989) Given the rapidity of MMP-9 up-regulation following muscle damage and

of its action on type IV collagen, MMP-9 may play a crucial role in the

pathogen-esis of fibrosis in mdx muscle, either through stimulating the inflammatory response

or through its action on the basal lamina (i.e growth factor release/activation)

Indeed, when mdx mice were administered with Batimastat, an inhibitor of MMP’s,

resulted in reduced muscle necrosis and infiltration with inflammatory cells (Kumar

et al 2010) Additionally, MMP-9 gene deletion in mdx mice significantly reduced

the extent of skeletal muscle injury and inflammation (Li et al 2009)

An interesting feature of dystrophin-deficiency across species is the expression

of grouped and segmental necrosis (Cazzato 1968; Anderson et al 1988; Cox et al

1993; D’Amore et al 1994) Grouped fiber necrosis is more typical of extracellular rather than intracellular events (Bridges 1986) As a consequence of muscle activation, the sarcolemma accumulates transient breaks, which allow the release of factors that initiate wound healing (McNeil and Khakee 1992) DNA microarray

analysis of adult mdx limb muscle revealed that approximately 30% of all

differen-tially regulated genes were associated with inflammation (Porter et al 2002), and

that several of the inflammatory genes identified in the muscle from mdx mouse

were also found to be upregulated in muscle from DMD patients (Chen et al 2000) The leakage of material from dystrophin-deficient muscle results in the accumula-tion of inflammatory cells in both endomysial and perimysial connective tissue (Tanabe et al 1986; Carnwath and Shotton 1987; McDouall et al 1990; Spencer

et al 2000) Dystrophin-deficient muscle is damaged to a greater degree given the