Figure 1. Twenty-day embryonic decorin transcript levels. Decorin pectoral muscle mRNA levels were quantitated in 20-day embryonic control and LSN animals.
S.G. Velleman
Department of Animal Sciences
Extracellular matrix development of chicken pectoral muscle was examined in the Low Score Normal (LSN) genetic muscle weakness and compared to normal pectoral muscle development. At 20 days of embryonic development, significant elevations were noted in LSN decorin transcript levels. At 6 weeks posthatch, decorin transcript levels were indistinguishable from the normal control. These data suggest that the alteration in decorin transcription is limited to a late embryonic stage in development.
Myogenesis involves the precise regulation of a number of developmental events which includes cell adhesion and cell-cell recognition (Miller, 1992; Buckingham, 1994). These processes, in part, involve the interaction of the cell with extracellular glycosaminoglycans or proteoglycans. Proteoglycans are defined as any molecule with a core protein and at least one attached glycosaminoglycan chain. Skeletal muscle contains dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans. These proteoglycans are capable of interacting with the cell and both collagenous and non-collagenous extracellular matrix molecules (Vogel et al., 1984; Vogel and Trotter, 1987; Scott, 1988). Based on these properties, proteoglycans have a significant potential to influence muscle growth and development.
Although extracellular matrix macromolecules have been identified in skeletal muscle, their role in muscle development and function is relatively unknown. Decorin is a chondroitin/dermatan sulfate proteoglycan which interacts with both collagen types I and II and has been identified in a wide range of tissues including skeletal muscle. Investigations have shown decorin to bind to fibrillar collagen and inhibit fibrillogenesis (Vogel et al., 1984; Vogel and Trotter, 1987; Scott, 1988; Brown and Vogel, 1989; Birk et al., 1995; Scott, 1995). Birk et al. (1995) have suggested that decorin may regulate the maturation of collagen fibrils into larger fibrils and fiber networks.
In the avian Low Score Normal (LSN) genetic muscle weakness, pectoral muscle at 20 days of embryonic development had a significant increase in decorin proteoglycan protein levels but not in total collagen concentration (Velleman et al., 1996). At 6 weeks posthatch, collagen concentration was not significantly different from normal muscle, but collagen crosslinking exhibited close to a 200% increase (Velleman et al., 1996).
The interaction of decorin proteoglycans with collagen fibers may play a significant role in regulating muscle growth and development. Additionally, the regulation of collagen crosslinking has been shown to influence meat tenderness (McCormick, 1994). Collagen crosslinking is a progressive process: the toughening of meat is related to collagen crosslink concentration. With age and maturation, there is a general increase in collagen crosslinking.
The avian LSN genetic muscle weakness is a unique tool to investigate the influence of the extracellular matrix on muscle development. In the present study, we have examined decorin mRNA levels to determine if the increase in decorin protein levels is due to an increase in gene expression or translation of existing message.
Animals and Husbandry. Control White Leghorn and LSN birds used in this study were from flocks maintained by the Department of Animal Genetics at the University of Connecticut. The LSN phenotype was detected among F2 progeny in an out cross between chickens with hereditary muscular dystrophy and a commercial White Leghorn stock. The LSN nomenclature was selected to distinguish two separate classes of birds which showed impaired ability to right themselves when repeatedly placed on their backs (exhaustion score test) at 2 to 3 months posthatch. Exhaustion scores of 0 to 3 characterized the homozygotes; scores of 9 to 12, the LSN phenotype. Pectoral muscle mass in LSN birds 1 week posthatch was 1.9% of total bodyweight compared to 3.2% for control birds. The LSN phenotype has been reproduced for at least 20 generations.
Slot Blot Protocol. Total RNA was extracted by the method of Chomczyncski and Sacchi (1987) and stored at -70oC in 100 g aliquots in 90% EtOH. RNA samples were prepared for use by pelleting and then drying. The dried pellet was resuspended in 50 µ-l TE (10 mM Tris pH 8.0/1 mM EDTA). After resuspension, 30 µ-l 20X SSC and 20 µ-l 37% formaldehyde were added to denature the RNA. The samples were then incubated for 15 minutes in 10X SSC and then placed in a slot blot manifold (Schleicher and Schuell, Keene, NH). RNA diluted to the appropriate concentration was then applied in a volume of 100 micro-l and pulled through the membrane with a slight vacuum suction. The membrane was baked at 800C for 1 hour and then prehybridized and hybridized as described below.
Prehybridization and Hybridization. Filters were prehybridized at 40oC for 2 hours in a 50% formamide, 4X SSPE, 0.1% SDS, 1X Denhardt's, and 10 g/ml salmon sperm DNA solution. Hybridizations were carried for 48 hours at 40oC. Randomly labeled (Feinberg and Vogelstein, 1983) decorin cDNA or 18S RNA (Ambion, Austin, TX) were used in the hybridizations. Blots were washed to final conditions of 2X SSPE.
Analysis of Results. The resulting hybridized blots were exposed to Fuji RX film for 3 to 5 days at -70oC in cassettes containing a Fisher Biotech L-Plus intensifying screen. The films were scanned and analyzed using a PDI Discovery Series Quantity One Densitometer. Band intensity is reported as the average optical density.
Total cellular RNA was extracted from 20-day embryonic and 6-week posthatch normal control and LSN pectoral muscle. Total RNAs ranging in concentration from 50 micro-g to 100 g were hybridized to a decorin cDNA probe and message optical density levels were determined by quantitative densitometric scanning. At 20 days of embryonic development, decorin transcript expression was significantly increased compared to control levels (Figure 1). By 6 weeks posthatch, decorin mRNA levels did not significantly differ from normal control values (Figure 2).
Figure 1. Twenty-day embryonic decorin
transcript levels. Decorin pectoral muscle mRNA levels were quantitated
in 20-day embryonic control and LSN animals.
Figure 2. Six-week posthatch decorin transcript
levels. Decorin pectoral muscle mRNA levels were quantitated in 6-week
posthatch control and LSN animals.
Existing biochemical data describing the LSN defect (Velleman et al., 1996) did not indicate whether the increase in decorin protein levels was due to altered transcriptional regulation or a modification in translation rate. In the present study, we have shown that the late embryonic elevation in decorin is associated with an increase in decorin transcription. These data suggest that the alteration in embryonic decorin expression was due to a modification in a signal transduction pathway regulating the transcription of the decorin gene. The upregulation in decorin gene expression at embryonic day 20 was a temporary increase, and by 6 weeks posthatch, decorin transcript levels did not differ significantly from normal control values.
What causes the aberration in LSN decorin transcription is currently under investigation. Increases in the amount of transforming growth factor beta-1 and -2 (TGF-beta) have been shown to upregulate the biosynthetic rate of the decorin core protein and to increase the molecular mass of the glycosaminoglycan chain attached to the core protein (Bassols and Massagué, 1988). It is possible that the LSN defect results from an alteration in a signal transduction involving TGF-beta. In skeletal muscle, TGF-beta is a potent inhibitor of myoblast differentiation and proliferation.
The alteration in decorin has significant biological implications in terms of muscle growth, function, and muscle meat quality. Decorin is a regulator of collagen fibrillogenesis. We have already shown a significant elevation in collagen crosslinking (Velleman et al., 1996). The increase in collagen crosslinking will decrease muscle elasticity in response to contraction. In terms of muscle meat quality, the increase in collagen crosslinking will toughen the meat, making it less desirable. The results described above demonstrate that appropriate levels of extracellular matrix molecules may be required for normal skeletal muscle development, structure, and function.
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