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159
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:
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) transmission 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,
161Skeletal 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 regulation 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 concentration 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
163Skeletal 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
165Skeletal 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