Research and Reviews: Poultry and Swine

Special Circular 164-99

Skeletal Muscle Collagen Organization May Be Associated with Proteoglycan Expression

Sandra G. Velleman1
The Ohio State University
Department of Animal Sciences

1 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 213 Gerlaugh Hall, 1680 Madison Ave., Wooster, OH 44691; 330-263-3905; Fax: 330-263-3949; e-mail: velleman.1@osu.edu.

Abstract

Myogenesis is a highly regulated process involving specific gene regulation and cell migration events. It should not be overlooked that the material outside the cell, extracellular matrix, plays a key regulatory role in the myogenic process. The extracellular matrix has been described as an instructive component in the formation of complex tissue structures. This organizational property of the extracellular matrix involves, in part, the interaction of cells with proteoglycans. Certain proteoglycans interact with the cell and both collagenous and noncollagenous extracellular matrix molecules. The proteoglycan decorin is a regulator of collagen fibrillogenesis and cell growth. In the chicken genetic muscle weakness Low Score Normal, decorin levels are elevated prior to hatching, followed by subsequent increases in collagen cross-linking. These extracellular matrix modifications affect the function of the muscle tissue by decreasing tissue elasticity. In terms of meat quality, shear force values are increased which would result in a less desirable product.

Introduction

Myogenesis involves the precise regulation of a number of developmental events which include cell adhesion and cell-cell recognition (Miller, 1992; Buckingham, 1994). It is well recognized that the process of myoblast differentiation is primarily regulated by the expression of muscle-specific transcription factors (Weintraub et al., 1991; Olson, 1992; Edmondson and Olson, 1993; Weintraub, 1993; Lassar et al., 1994). However, it is also known that other macromolecules such as growth factors and the extracellular matrix components play a critical role in regulating muscle differentiation (Fernandez et al., 1991; Florini et al., 1991; McLennan, 1993).

The extracellular matrix contains a network of macromolecules secreted by the cell. The extracellular matrix has been classically described as a structural scaffold containing a proteinaceous fiber component (collagen) and an "amorphous ground substance" (proteoglycan). This definition implies that the extracellular matrix is a static structure with limited ability to influence tissue structure, function, development, or gene expression. It is now known that the extracellular matrix is a dynamic structure which regulates cell behavior through the interaction of extracellular matrix molecules with each other, interaction with growth factors, and through cell-extracellular matrix signal transduction pathways. Furthermore, different tissues have unique extracellular matrices that change as an animal ages.

Collagen Biosynthesis

Collagen biosynthesis is an extremely complex process. There are at least 19 different vertebrate collagens with tissue-specific distributions and unique functional properties. These unique collagen types can be subdivided into the following classes based on function or size: fibrillar, fibril-associated, network forming, filamentous, short chain, and long chain (van der Rest and Garrone, 1991). Bone contains collagen Type I, cartilage contains Type II, and skeletal muscle contains collagens Type I and III. These collagens are fibrillar in nature. The fibrillar collagens like Types I through III contain a single triple-helical domain consisting of three separate peptide chains. The three chains wrap around each other forming an alpha helix and are linked together by interchain disulfide bonds. After the collagen molecules are synthesized, they are secreted from the cell into the extracellular space and align into a quarter-stagger array; cross-linking between the microfibrils is initiated and larger diameter fibrils form.

The cross-linking of collagen will alter tissue structure and function. For example, the toughening of meat is related to the collagen cross-link type and concentration. With age and maturation, there is an increase in cross-link formation. These changes contribute to meat toughening (Light, 1987; McCormick, 1994) and reduce tissue elasticity. Although collagen is a dynamic component of the extracellular matrix, it does not influence tissue properties independently. Collagen is intimately associated with proteoglycans.

The Extracellular Matrix

Proteoglycan Component

Extracellular matrix proteoglycans are proteins that contain carbohydrates called glycosamino-glycans covalently attached to a central core protein. Figure 1 is a schematic illustration of a proteoglycan structure. The glycosaminoglycans are polymers of disaccharide repeats that are highly sulfated, except for hyaluronic acid, and are negatively charged. Typical glycosaminoglycans attached to the proteoglycan central core protein are chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. Proteoglycans interact with cells, collagen, growth factors, and water. Due to the diversity of the extracellular matrix proteoglycan family, proteoglycans may be divided into two major classes: aggregating and nonaggregating interstitial proteoglycans. Aggregating proteoglycans generally possess core proteins greater than 200 KDa and ionically interact with hyaluronic acid. The nonaggregating proteoglycans consist of proteoglycans involved in both the control of cell proliferation and differentiation, and in the regulation of extracellular matrix architecture.

Figure 1: Generalized structure of a proteoglycan illustrating the central core
protein and attached glycosaminoglycan chains.

The large aggregating proteoglycan of cartilage, aggrecan, is the most well characterized of all the proteoglycans. Aggrecan has a high negative charge density due to the attachment of chondroitin and keratan sulfate chains to the core protein. The glycosaminoglycan chains allow the proteoglycan to exhibit ionic interactions with other molecules. The negative charge attracts counter ions and as a result water is drawn into the matrix. The proteoglycan molecule, therefore, creates a water compartment. In cartilage, the negative charge of aggrecan draws water into the cartilage matrix which is crucial in distributing load in weight-bearing joints. The importance of aggrecan is well illustrated by the avian mutation, nanomelia (Argraves et al., 1981). In nanomelia, the aggrecan proteoglycan is absent from the cartilage extracellular matrix. As a result of this aberration in extracellular matrix organization, the cartilage contains less water, is unable to support load-bearing stress, and collapses.

Although the identification and characterization of skeletal muscle proteoglycans has lagged relative to that of cartilage proteoglycans, it is now thought that proteoglycans play a key role in muscle formation and function (Young et al., 1990; Fernandez et al., 1991). Like cartilage, avian skeletal muscle contains a large chondroitin sulfate proteoglycan, versican (Carrino and Caplan, 1989). It is possible that versican functions in a manner similar to aggrecan by ionically interacting with water. Early in myogenesis, the skeletal muscle extracellular is rich with this proteoglycan which may space developing myofibrils. The spacing of myofibrils would result in an extracellular matrix area between fibrils. If versican draws water into the skeletal muscle extracellular matrix, this would have a direct impact on the "juiciness" of meat and the ability to retain a hydrated state.

In addition to the aggregating proteoglycan, nonaggregating proteoglycan also exists. One class of nonaggregating proteoglycans, the small leucine-rich proteoglycans (SLRPs), is characterized by their functional roles in the regulation of collagen fibrillogenesis, modulation of growth factor activity, and regulation of cell growth properties (Iozzo and Murdock, 1998). The SLRPs include five distinct but structurally related proteoglycans: decorin, biglycan, fibromodulin, lumican, and epiphycan. These proteoglycans all have central core protein composed of leucine-rich repeats that confer most of the biological functions. Unlike the aggregating proteoglycans which possess up to 100 attached glycosaminoglycan chains, decorin, biglycan, and epiphycan contain only one to two chondroitin or dermatan sulfated side chains.

Proteoglycan-Collagen Interactions

Considerable in vitro data has suggested that a specific functional interaction occurs between decorin and fibrillar collagen (Figure 2) (Vogel et al., 1984; Vogel and Trotter, 1987; Scott, 1988; Uldbjerg and Danielsen, 1988; Brown and Vogel, 1989; Weber et al., 1996). Understanding collagen maturation into a fibrillar structure has significance in terms of tissue function. A collagen molecule consists of a triple helical arrangement containing three collagen alpha chains. Collagen molecules aggregate in parallel to form a fibril. The collagen molecules arrange themselves into a structure where the individual molecules overlap each other by three-quarters of their length (quarter stagger arrangement). This overlap or aggregation of collagen molecules gives rise to a D periodicity or D-staggered array (1D staggered array = 67 nm) of fibrils in which each D period is divisible into a collagen overlap zone or a gap zone. The gap zone is approximately 40 nm between the collagen molecules. Through molecular modeling studies (Weber et al., 1996), it has been proposed that decorin binds to collagen in the gap zone between collagen molecules. The correct positioning of decorin will properly position the collagen molecules. The precise spatial arrangement of collagen molecules is critical in the formation of collagen cross-links. If decorin binds to other potential binding sites on the collagen molecule, the inaccurate spacing of the collagen molecules will result in a lateral fusion or increased cross-linking between collagen molecules.

Figure 2: Collagen fibril composed of four collagen molecules in a quarter
stagger arrangement. Decorin is the shaded rectangle interacting with collagen in
the gap zone. Other potential decorin binding sites are indicated by the solid
rectangle interacting with collagen in the gap zone. Other potential decorin
binding sites are indicated by the solid rectangles on the collagen molecule
(adapted from Weber et al., 1996).

Extracellular Matrix Regulation of Skeletal Muscle Formation

Pectoral muscle structural and extracellular matrix characteristics have been examined in the chicken Low Score Normal (LSN) genetic muscle disorder. The LSN condition is characterized by subnormal skeletal muscle development and function. The LSN defect appears to be localized to the skeletal muscle extracellular matrix component and does not affect muscle fiber type or myosin expression (Velleman et al., 1993). Therefore, the LSN chicken is an excellent model to determine how the extracellular matrix influences skeletal muscle formation and function.

Prior to 20 days of embryonic development, LSN glycosaminoglycan and proteoglycan concentrations do not vary significantly from levels expressed in normal muscle (Velleman et al., 1996). However, at 20 days of embryonic development there is a dramatic increase in decorin proteoglycan levels. Subsequent to the increase in decorin levels, LSN collagen cross-link levels are elevated nearly 200% by six-week- posthatch. Although collagen cross-linking is modified, collagen concentration is unaffected.

By transmission electron microscopy, collagen fibril organization has been examined in six-week-posthatch normal and LSN pectoral muscle (Figure 3). Coinciding with the biochemical observation of increased LSN collagen cross-linking, collagen fibrils in the LSN pectoral muscle exhibit a lateral fusion or parallel alignment. This arrangement of collagen fibrils is not observed in normal muscle and would result in a high proportion of cross-linking. When collagen fibrils form a laterally fused structure like that observed in the LSN muscle, tissue elasticity will most likely decrease. These data are suggestive of a decorin-induced remodeling of collagen fibril organization.

Figure 3: Representative transmission electron micrographs of A: Normal and B:
LSN six-week- posthatch skeletal muscle collagen fibers. A box has been placed
around the collagen fibers.

Does Decorin Mediated Collagen Remodeling Affect Meat Quality?

Tenderness is one of the most important characteristics by which consumers judge meat quality. The connective tissue component of muscle through its mechanical strength plays a significant role in regulating meat tenderness. Much of the research on the influence of connective tissue meat textural properties has focused on collagen because of its tensile strength (Bailey et al., 1974; Duance et al., 1977; Bailey, 1984). The toughening of meat is related to collagen cross-link type and concentration (McCormick, 1994). Collagen fibril organization and cross-linking are, in part, under the regulation of the extracellular matrix proteoglycan decorin. Altered expression of decorin as observed in LSN pectoral muscle modifies collagen fibril organization. The effect of changes in decorin expression as it relates to meat tenderness has not been well investigated. Six-week-posthatch LSN and normal pectoral muscle was examined for shear force properties (Table 1). The results showed a significant increase in LSN shear press values (Nn) compared to normal muscle (28.46 vs. 16.50 Nn, respectively). These data suggest that modified collagen fibril organization, which may be mediated by altered proteoglycan expression, does play a role in meat quality.

Summary

To fully understand tissue development and function requires the consideration of the extracellular matrix. Much research attention, in terms of skeletal muscle development, has been devoted to the study of muscle specific transcription factors, myosin isoform transitions, and sarcomere formation. The role of the extracellular matrix in this process is relatively unknown. The proteoglycan decorin, due to its role as a regulator of collagen fibril organization and cell proliferation, could play a significant role in myogenesis. Studies on skeletal muscle formation in the LSN chicken support the potential significance of decorin and extracellular matrix organization in myogenesis.

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