Enter the Matrix: Shape, Signal, and Superhighway

Abstract

Mammalian skeletal muscle is notable for both its highly ordered biophysical structure and its regenerative capacity following trauma. Critical to both of these features is the specialized muscle extracellular matrix (ECM), comprising both the multiple concentric sheaths of connective tissue surrounding structural units from single myofibers to whole muscles and the dense interstitial matrix that occupies the space between them. ECM-dependent interactions affect all activities of the resident muscle stem cell population, the satellite cell, from the maintenance of quiescence and stem cell potential to the regulation of proliferation and differentiation. This review will focus on the role of the extracellular matrix in muscle regeneration, with a particular emphasis on regulation of satellite cell activity.

Introduction

Our approximately six hundred and forty individual skeletal muscles, comprising roughly 40% of an adult human’s total body mass, are collectively responsible for maintaining posture and balance, for respiration, and for nearly all movements from the most delicate microsurgeries and brushstrokes to marathon running and power-lifting. Possibly due to the potential for damage implicit in such diverse and critical functions, skeletal muscle is one of the most highly regenerative tissues in the body; such regeneration requires the activity of a population of tissue-specific adult stem cells referred to as satellite cells.

Briefly, muscle satellite cells are the obligate tissue-specific stem cells of skeletal muscle: if the satellite cell population (defined by expression of the satellite cell marker Pax7 [1]) is genetically ablated after maturity in the mouse, muscle regeneration fails [2–4]. Satellite cells are derived from a somitic lineage during development, and are thought to disperse throughout the developing musculature concomitantly with the myoblasts that will contribute to embryonic and fetal myogenesis [5–7]. They are maintained in a quiescent state in the absence of physiological signals of damage, overuse, or disease in a minimal niche consisting of the cell membrane of the multinucleate, differentiated myofiber they are associated with and its overlying basal lamina (described further below.) When activated from quiescence by stress or damage, satellite cells will enter the cell cycle and proliferate extensively to form a population of replacement myocytes, which will fuse with each other or existing myofibers to reconstitute the muscle (reviewed in [8, 9].) Due in large part to their potential as either vectors or targets for cell therapy of human myopathies, particular Duchenne muscular dystrophy, satellite cells have been the focus of intensive research to try to unravel the molecular mechanisms governing their maintenance and activity in vivo, in both healthy and pathological tissue. We will describe briefly the role the extracellular matrix plays in the structure and function of healthy skeletal muscle, then address key interactions with the ECM during satellite cell-mediated muscle regeneration, with particular emphasis on potential new and emerging areas of research.

Structure of the skeletal muscle extracellular matrix

The architecture of each individual skeletal muscle is maintained in part by concentric arrangements of connective tissue layers, which give the muscles both structure and strength [10, 11]; a recent review [12] describes the muscle ECM in detail with respect to its composition, ultrastructure, and interconnectivity. The myofiber plasma membrane is referred to as the sarcolemma; it encases each myofiber and forms the most proximal layer of the myofiber basal lamina. The sarcolemma and its associated basal lamina are a key feature of muscle fibers: first described in Bowman’s classic monograph “On the Minute Structure and Movements of Voluntary Muscle” in 1840 [13] as a thin, tough, transparent membrane that remained after hypercontracture of the contents of a myofiber, Bowman’s tubes “would seem not improbably to consist of a very close and intricate interweaving of threads, far too minute for separate recognition.” The basal lamina and its associated interstitial matrix are of particular interest for the purposes of this review due to their physical proximity and intimate interactions with quiescent and activated satellite cells and the sarcolemma of myofibers. In fact, the “gold standard” definition of a quiescent satellite cell is as originally described by Mauro in 1961 [14], and elaborated on in further electron microscopy-based studies [15]: small, bipolar, cells with a heterochromatic nucleus, scanty cytoplasm, and cytoplasmic processes of up to 25 μm, located beneath the basal lamina but outside the sarcolemma.

The basal lamina is also referred to as the endomysium, and is the smallest unit of the ECM in muscle. It overlays the sarcolemma and includes a tight sheath of laminin α2β1γ1 (also known as laminin-2 or merosin) connected to a mesh like layer of collagen IV (reviewed in [12, 16].) Deposition of these components of the basal lamina requires the interactions of myoblasts with muscle fibroblasts [17, 18], consistent with the recent observation that muscle connective tissue fibroblasts are absolutely essential for muscle development [19] and regeneration [2]: laminin is secreted by myofibers [20] while collagen IV is secreted by muscle fibroblasts [18]. This dense, tough lamina surrounds and protects the myofiber, transduces contractile force to the muscle unit, and forms the apical side of the quiescent satellite cell niche [11]. Electron microscopy studies [21, 22] of myofibers and their associated endomysium highlight the intimate associations of each muscle fiber with the three-dimensional network of the endomysium; the endomysium also harbors capillaries and axons serving each individual muscle fiber [23].

The cytoskeleton of each myofiber is physically attached to the basal lamina, mainly through integrins and dystrophin-glycoprotein complexes [24, 25]; these connections are continued via additional crosslinking molecules such as entactin, nidogen, and agrin to the interstitial matrix between myofibers [26] (Figure 1.) It is disruptions in this linkage that are most often responsible for human myopathies, including Duchenne’s muscular dystrophy [27]. The interstitial matrix that surrounds myofibers accounts for 1–10% of muscle tissue and fills all of the space between muscle fibers while maintaining mechanical continuity with tendons. The major roles of the interstitial ECM are to transduce mechanical force from the muscles and to serve as a structural support for the muscle and its associated blood vessels and nerves. It is more porous than the basal lamina, and is composed of fibrous components providing tensile strength (primarily collagens) and proteoglycans (including chondroitin, heparan, and dermatan sulfates), which comprise 10% of the weight but 90% of the volume of the matrix and provide both a labile environment for other cells and components to move within and a sink for other matrix-associated molecules (proteases, chemokines, cytokines, mitogens, and growth factors) (reviewed in [16].) The most significant fibrous components of the interstitial matrix are the collagens: collagen comprises roughly 90% of the protein mass of the ECM. Local fibroblasts secrete collagen VI into the interstitium [28] where the triple-helical subunits form double-beaded collagen VI microfibrils by end-to-end association. Collagen VI in the interstitial matrix acts to bind the muscle ECM into functional units and provide structure and stability.

Figure 1.

A quiescent satellite cell (identified by Pax7 staining, red) in a frozen section of mouse skeletal muscle; myofiber basal lamina is stained for laminin (green.) Schematic representations of the local matrix near the satellite cell and in an adjacent area where neighboring myofibers appose one another are above and to the right, respectively.

Bundles of muscle fibers bounded by epimysia are then formed into fascicles, which are surrounded by the perimysium; this layer contains and organizes larger blood vessels and nerves as well as connecting to the interstitial matrix. The whole muscle is then surrounded by the epimysium, which will broaden and flatten at the extremities of each muscle to form the myotendinous junction. The perimysium and epimysium differ from the endomysium in their composition (primarily collagen I and III instead of IV and VI, and different complements of proteoglycans) and function; their specific role in preservation of muscle architecture and function is also somewhat less well defined (reviewed in [12].) The epimysium, perimysium, and endomysium are linked by interconnecting collagen fibers to evenly and effectively distribute contractile forces without damaging the muscle structure during movement [29].

As would be expected, loss or mutation of basal lamina or interstitial matrix components frequently leads to muscle pathologies [30]: approximately 50% of congenital muscular dystrophies are the result of deficiencies in laminin-2 (congenital muscular dystrophy type 1A, or MDC1A), mutations in collagen IV have pleiotropic effects which include some forms of myopathy, and defects in collagen VI lead to Ullrich (the second most frequent form of congenital muscular dystrophy) and Bethlem myopathies [31]. While myopathies caused by defects in the structure of the endomysium itself are the most common in patients, disruptions in the connectivity between the myofiber cytoskeleton and the endomysium, including Duchenne’s muscular dystrophy, collectively represent a larger class of mutations. In all of these cases, a direct molecular connection to satellite cell biology is unclear: structural proteins of mature muscle such as these are not expressed by undifferentiated satellite cells, but as the cells responsible for muscle repair and homeostasis they are necessarily affected by the chronic over-activation, inflammation, and alteration of the muscle environment itself that attend muscle disease. As described below, there remain significant gaps in our understanding of what signals satellite cells receive from the muscle ECM and how they respond, even in healthy tissue; it is important to bear in mind that parallel studies on the signals prevalent in pathological muscle are also being conducted, although not discussed further here.

Signaling to satellite cells by the extracellular matrix

It has been argued that ECM signals are at least as important as soluble signals in regulating cellular determination, differentiation, proliferation, survival, polarity, and migration [32]. In the case of satellite cell-ECM signaling interactions, there are published examples of regulation and presentation of ‘soluble’ factors by the ECM, specific adhesion signaling, and biophysical stress or stiffness-induced signals that are each critical for satellite cell activity and function. It is likely that not only are all three signaling modalities directly affecting satellite cell activity, but that they are cooperative and interactive.

The niche of the quiescent satellite cell is composed of the sarcolemma of the host muscle fiber and the interior side of the basal lamina, thus any niche factors involved in maintenance of quiescence must derive from these two matrix sources: during quiescence, satellite cells are isolated electrically and chemically from both the myofiber cytoplasm and the extracellular environment [33]. Because of the difficulties in studying quiescent satellite cells in situ, comparatively little is known regarding the input of the niche to satellite cell activity, however it has been established that disruption of the matrix components of the quiescent niche will negatively affect satellite cell activity [34] while preservation of the niche in cell transplant will dramatically enhance engraftment and function [35].

In contrast to the limited understanding of ECM signaling during quiescence, the critical role of the matrix in the earliest events of the satellite cell response has been well-established. Initial activation of the satellite cell occurs downstream of physical stretch [36, 37] or rupture of the myofiber membrane and basal lamina (reviewed in [38].) Nitric oxide synthase-1 (NOS1 or N-NOS) is anchored to the myofiber sarcolemma by association with the dystrophin-glycoprotein complex and has mechanosensory as well as enzymatic functions: following myofiber stretch, a bolus of nitric oxide (NO) is released locally [39]. This leads to release of the active form of hepatocyte growth factor/scatter factor (HGF) (which is itself sequestered within the extracellular matrix [40]), potentially through the activity of the matrix metalloprotease MMP-2 [41, 42]. HGF remains the only protein factor with the ability to directly activate satellite cells from quiescence [43, 44], thus these matrix-mediated events are critical to satellite cell activity.

Once activated, satellite cell proliferation and differentiation are modulated by multiple extracellular signaling pathways, the majority of which include a matrix component; however, this aspect of ‘soluble’ factor signaling is rarely taken into account in in vitro studies. For example, the binding of FGFs to their receptors requires specific sequences of heparan sulfates as components of the ternary complex [45]; transforming growth factor-β (TGF-β) ligands must bind to proteoglycans in the matrix to be ‘presented’ to their cellular receptors [46]; and HGF complexes with its receptor c-met can include fibronectin or vitronectin as well as integrins [47]. In particular, a requirement for ligand binding and presentation by proteoglycans, in cis or in trans, introduces an additional level of specificity to the organism, and analytical difficulty to the experimenter: carbohydrate chains are added and modified post-translationally via multiple different steps at different locations within the cell. The length, sequence, epimerization, and sulfation pattern of the sugars comprising the carbohydrate chains of matrix proteoglycans are extremely dynamic (reviewed in [48]) and are also highly specific in their capacity for interaction with growth factors such as FGF [49]. Thus, the colocalization of growth factor, receptor, and proteoglycan does not necessarily, or even usually, imply either ligand-receptor binding or downstream signaling activity [50]! Proteoglycan side chains are by far the most complex biopolymer, however current technology for analyzing even the sequence of carbohydrates lags dramatically behind that for DNA, RNA or protein (reviewed in [51]). Unfortunately, realistic integration of this aspect of satellite cell-ECM signaling in vivo is therefore beyond the current capacity of the field. However, progress made primarily in vitro has identified roles for ECM molecules in directing the satellite cell response to soluble growth factors. Work in the Brandan lab has identified roles for the ECM proteoglycans decorin, biglycan, and dermatan sulfate in modulating the bioavailability and signaling potential of key growth factors including FGF-2 and HGF [52], and TGF-β [53]; other groups have identified similar effects on myostatin [54]. It is important to note that these interactions are with extracellular proteoglycans that would presumably act in trans, unlike the interactions in cis that characterize cell-surface proteoglycans expressed by satellite cells themselves such as syndecans and glypican [55–58].

In addition to ECM molecules produced by either muscle fibroblasts or differentiated myofibers, quiescent and activated satellite cells have themselves been shown to be a source of ECM components that impinge on multiple aspects of satellite cell activity. Most recently, an increase in fibronectin expression by satellite cells immediately following activation has been implicated in active remodeling of the local ECM to promote self-renewal by facilitating Wnt7a binding to a complex of its receptor and the transmembrane heparan sulfate proteoglycan syndecan-4 [59].

Another modality of signaling from the ECM is via integrins, with or without coordinated signaling of a growth factor receptor. Adhesion signaling to myoblasts/myocytes has been shown to be necessary for myogenesis, particularly in the case of differentiation. Hauschka and Konigsberg were the first to demonstrate a requirement for a specific ECM factor (collagen I) for differentiation of chick myoblasts [60], and many studies since have highlighted the importance of both complete ECM and its components for differentiation of myogenic cell lines and primary myoblasts from developing chick and mouse [61–65]. Integrins are heterodimeric transmembrane adhesion receptors with specificity for different ECM components based on the identity of the α and β chains they are comprised of (reviewed in [66].) While satellite cells appear transcriptionally competent to express nearly all known integrin chains [67], only a limited number of functional dimers have shown a biological activity or phenotype. Of these, the laminin receptor integrin α7β1 is the most prominent (reviewed in [11].) Integrin α7β1 is localized throughout the sarcolemma, myotendinous junctions, and neuromuscular junctions of myofibers [68] where it mediates adhesion to laminin as well as interacting with the dystroglycan-glycoprotein complex, syndecans, and sulfatides (reviewed in [11, 30, 69].) It is also considered a molecular marker for the satellite cell population even in quiescence [70], where its primary role appears to be in migration [67, 71, 72], discussed further below. Of the other integrins expressed by satellite cells and their progeny, most have not yet been correlated with a molecular function, although activity in proliferation, differentiation, and fusion have been suggested. Making analysis more complicated is the key role many of these same integrins play in the maintenance and function of differentiated muscle: not only do integrin α7β1 deficiencies result in myopathy [69], but changes in integrin α7β1 expression are associated with muscle pathologies caused by many other different mutations as well [73]. Unraveling the roles of specific integrins in vivo within a tissue in which myofibers and satellite cells both coexist and coexpress the same integrins will most likely require cell type-specific genetic deletions and careful downstream analysis, as affected satellite cells will give rise to affected and/or chimeric myofibers.

An aspect of satellite cell/ECM interactions that has only recently been closely examined is the biophysical influence of substrate stiffness and organization on satellite cell behavior. While physical stress has been widely understood to influence muscle differentiation and organization and the role of transient stretch in activation of satellite cells is well-established (see discussion of NOS above), the effects of two-dimensional versus three-dimensional culture [74] and varying substrate rigidity on satellite cell physiology are an emerging area of inquiry. Of particular interest, the Blau lab has recently described the effects of gradually altering substrate rigidity on multiple satellite cell activities including maintenance of stem cell potential and capacity for differentiation using single satellite cells cultured on a wide array of PEG hydrogels [75, 76]. Since the great majority of in vitro studies are still done in two dimensions on rigid surfaces which are either uncoated or coated with purified recombinant matrix factors, these new systems may serve to highlight areas where our current modles of potential in vivo signaling events may be incomplete or incorrect. This will be particularly important in understanding the differences in satellite cell activity associated with either pathological or aged muscle tissue [77, 78], since fibrosis and changes in matrix stiffness are characteristic of both of these conditions.

Experimental paradigms that should be of increasing interest in establishing physiologically-relevant biophysical influences on satellite cell activity include simple three-dimensional culture systems using hydrogel or collagen, three-dimensional culture in reconstituted native ECM, and decellularized tissue that can be seeded with live cells. While all three of these methods are currently in use, there are significant drawbacks to each: inert or single-component hydrogels are easily created and manipulated, but are necessarily lacking the majority of ECM-derived signals discussed above; purified and reaggregated muscle matrix lacks the ordered arrangement of matrix proteins as well as their carbohydrate side chains; and even decellularized muscle tissue implanted in vivo fails to provide an enhanced environment for myogenesis [79]. In this respect, the muscle field lags behind studies of other tissues in the ability to produce bioactive, biomimetic scaffolds for either research or clinical use, such as have been described for cartilage and bone [80–82]. For many of the reasons outlined here, further development of three-dimensional systems replicating both the biochemical and biomechanical properties of native muscle matrix will be a key step in developing both physiologically relevant in vitro systems and therapeutically useful clinical tools.

Role of the extracellular matrix in satellite cell motility

As both the surface that motile cells adhere to and travel on and the substance that they must travel through, the different components of the ECM are critical influences on cell migration. Myogenic precursor cell motility is required for muscle development, during which emigration form the somites to the presumptive muscle fields is dependent on signaling of HGF via c-met [83]. Members of the FGF and PDGF families have also been implicated in myoblast motility during development, and as discussed above all of these growth factors have requirements for matrix presentation and/or interaction to signal, however the role of the matrix in embryonic muscle development has not been widely addressed. Thus, this area represents the increasingly rare case in which cellular and molecular mechanisms are better understood in the context of regeneration than development.

Once activated, satellite cells leave their quiescent position beneath the basal lamina then proliferate and migrate in the interstitial space before differentiating and fusing with a new or damaged myofiber (Figure 2.) This exit from the sublaminar space appears to rely on physical force rather than protease activity to create a tear in the basal lamina, as it can be blocked by preventing adhesion to laminin in ex vivo fiber culture using blocking antibodies but not by exposure to the broad-spectrum matrix metalloprotease inhibitor GM6001 (Siegel and Cornelison, unpublished results.) This results in both a change in the aspect of the basal lamina the satellite cells are in contact with, and exposure to the interstitial matrix with the accompanying potential for signaling. Laminin is the preferred substrate for satellite cell motility in vitro [67, 84], and engagement of laminin by α7β1 integrin is necessary for satellite cell motility on the surface of the myofiber [67]. Once satellite cells have exited the niche and adhered to the exterior of the myofiber of the basal lamina, they are capable of extensive motility under ex vivo conditions: timelapse observations have recorded cell velocities of up to 250 μm/hour. However, it is important to note that this does not completely or accurately mimic an in vivo situation, because the single myofiber system does not include the interstitial matrix through which cells are required to navigate in vivo, additional cells present in a regenerating muscle, or any matrix or soluble factors not derived from the host myofiber.

Figure 2.

An activated satellite cell (identified by CD34 staining, red) on an isolated mouse myofiber 18 hours postisolation; myofiber basal lamina is stained for laminin (green.) Left, schematic representation of the local matrix interactions during multiple phases of satellite cell activity in vivo.

The fact that satellite cells can and do move both along and between myofibers during regeneration in vivo is well-established [85, 86], however the question of whether muscle precursor cell migration is necessary for regeneration in vivo (as it is in development) remains open. While there are theoretically adequate local populations of satellite cells available to restore lost myonuclei without recruiting cells from more distal areas [87], it is possible that the regeneration response could be both accelerated and enhanced by mobilizing additional cells. A role for long-range motility of satellite cells, including recruitment of cells from distal uninjured muscle [88], was suggested based on early in vivo analyses of muscle regeneration. Evidence for long-distance activation and motility of satellite cells includes: detection of BrdU positive satellite cells in uninjured areas of the TA distal to a needle injury [89] and movement of satellite cells from distal uninjured areas towards a focal crush site [90]. In addition, after free grafting of large muscles satellite cells will migrate from the central necrotic areas toward the periphery [91] then back again after revascularization [92], and have been observed to migrate from the viable half of a longitudinally split autograft into the killed half [93].

Several groups have described soluble factors that promote satellite cell motility and migration in vitro (reviewed in [67]). Of these, the small chemokine SDF-1 is particularly intriguing: a key mediator of stem cell homing in several other tissues (reviewed in [94–96]) it is expressed following injury by muscle-derived fibroblasts and is chemotactic to satellite cells [97]. The transmembrane heparan sulfate proteoglycan syndecan-4, which is expressed by satellite cells during both quiescence and activation [58], is the obligate coreceptor for SDF-1 along with CXCR4 [98], which is also a marker for satellite cells [70]: all three components of the ternary complex are implicated in satellite cell motility [99], donor cell engraftment [100] and enhancement of muscle regeneration in vivo [101]. HGF may also have a role in addition to initial activation: it has been characterized as a potent motogen in many systems including both developing and adult muscle [83, 102], once having been named ‘scatter factor’ for this activity [103]. It stimulates cell motility in developmental and physiologically normal contexts as well as being one of the primary signaling pathways hijacked during tumorigenesis and metastasis (reviewed in [104, 105]). This makes the HGF/c-Met axis a frequent target for therapeutic downregulation or inhibition [106], and many tools have been developed in the context of tumor therapy that may also be useful for evaluating the role and requirement for HGF in satellite cell biology and motility.

The requirement for digestion and remodeling of the interstitial matrix to facilitate satellite cell migration in vivo is suggested by experiments in which matrix-modifying enzymes such as urokinase plasminogen activator (uPA) (reviewed in [107]), MMPs (matrix metalloproteases) [108–113] or their inhibitors (TIMPS) [114, 115] are experimentally manipulated to yield a positive effect on satellite cell spread and/or muscle regeneration. This would be consistent with the idea that mobilization and relocalization of satellite cells is either beneficial or necessary for successful muscle regeneration. However, while these results have been promising, the overall mechanism(s) by which matrix-modifying enzymes act to enhance satellite cell motility or activity remain elusive: indeed, the question of whether MMP activity should be enhanced or diminished is still highly dependent on the context of the in vivo experiment and the particular enzyme being studied. Significantly more work focusing on both endogenously-released soluble motogens and chemattractants and matrix remodeling enzymes therefore remains to be done before a definitive role for satellite cell migration can be assigned. However, even in the absence of such a role during normal muscle regeneration, the difficulty in achieving adequate spreading of therapeutically-engrafted satellite cells or their progeny [116] makes a better understanding of local factors affecting satellite cell movement through the muscle tissue an important goal.

Signposts?

While it was not the authors’ intent at the outset, a primary theme that emerged in writing this review is the emphasis of areas in which understanding the matrix’s place in muscle regeneration is incomplete, potentially inaccurate, or currently impossible. Although the ECM clearly must play multiple critical roles in satellite cell-mediated myogenesis, its influence is rarely taken into account in in vitro studies, and is somewhat cryptic in many in vivo situations. While mimicking the properties of the muscle ECM is difficult, it should not be impossible: advances in decellularized organs [81, 117], purification of native muscle matrix for seeding myoblasts [61, 118], and three-dimensional biomaterials-based culture systems [74, 75] are promising developments that will ideally soon resolve many of our unanswered questions (and suggest new ones!)

Abbreviations used

ECM

extracellular matrix