Contribution of extracellular matrix components to the stiffness of skeletal muscle contractures in patients with cerebral palsy

Abstract

Purpose: Joint contractures in children with cerebral palsy contain muscle tissue that is mechanically stiffer with higher collagen content than typically developing children. Interestingly, the correlation between collagen content and stiffness is weak. To date, no data are available on collagen types or other extracellular matrix proteins in these muscles, nor any information regarding their function. Thus, our purpose was to measure specific extracellular protein composition in cerebral palsy and typically developing human muscles along with structural aspects of extracellular matrix architecture to determine the extent to which these explain mechanical properties. Materials and Methods: Biopsies were collected from children with cerebral palsy undergoing muscle lengthening procedures and typically developing children undergoing anterior cruciate ligament reconstruction. Tissue was prepared for the determination of collagen types I, III, IV, and VI, proteoglycan, biglycan, decorin, hyaluronic acid/uronic acid and collagen crosslinking. Results: All collagen types increased in cerebral palsy along with pyridinoline crosslinks, total proteoglycan, and uronic acid. In all cases, type I or total collagen and total proteoglycan were positive predictors, while biglycan was a negative predictor of stiffness. Together these parameters accounted for a greater degree of variance within groups than across groups, demonstrating an altered relationship between extracellular matrix and stiffness with cerebral palsy. Further, stereological analysis revealed a significant increase in collagen fibrils organized in cables and an increased volume fraction of fibroblasts in CP muscle. Conclusions: These data demonstrate a novel adaptation of muscle extracellular matrix in children with cerebral palsy that includes alterations in extracellular matrix protein composition and structure related to mechanical function.

Keywords: Cerebral palsy; collagen; extracellular matrix; muscle; stiffness.

Figures

Figure 1:

Differences in bundle stiffness and hydroxyproline between TD and CP muscles. A) CP patients showed significantly higher muscle bundle stiffness compared to TD (TD: 24.11± 2.73; CP: 37.37± 3.70 (kPa); p < 0.05). B) Hydroxyproline content, representing overall collagen, was markedly higher in patients with CP compared to TD (CP: 5681± 407; TD: 753± 93; p < 0.0001). N = 13 for both CP and TD samples.

Figure 2:

Increased levels of individual collagen types in CP. A) Collagen I, III, IV and VI levels measured by ELISA were increased significantly in children with CP compared with TD children. All collagens measured showed a 3–5 fold increase. B) Because collagen I had the lowest increase and collagen III had the highest fold increase, the I/III ratio was lower in CP samples, although this difference was not significant (p=0.10). C) Collagen I made up the largest proportion of collagen types measured (~70%) followed by collagen IV, III, and VI. Overall collagen proportions were not significantly changed with CP.

Figure 3:

Differences in GAGs and Proteoglycans in CP. A) Uronic acid present in all GAGs had an ~2-fold increase while hyaluronic acid was not significantly altered in CP compared to TD. B) Overall proteoglycan content was not significantly altered in CP. SLRPs were differentially regulated with decorin demonstrating a 2.7 fold significant increase and biglycan a 2.7 fold significant decrease.

Figure 4:

Stepwise linear regression of bundle stiffness on ECM biochemistry. A) Multiple stepwise linear regression performed on bundle stiffness for all 26 samples with 14 parameters of extracellular matrix components generated a model accounting for 65% of variance that included positive predictors collagen I (COL1), III (COL3), and total proteoglycan (PG) with biglycan (BG) as a negative predictor. B) Multiple stepwise linear regression on TD and CP samples separately resulted in better fitting models (TD: R2 = 84; CP: R2 = 0.89). Both included total proteoglycan and biglycan together with additional inclusion of positive predictors of hydroxylysyl pyridinoline (HP), collagen I and IV in TD and collagen I/III ratio and hydroxyproline (HPL) in CP.

Figure 5:

Stereological quantification of subcellular components of muscle tissue in CP and TD. A) Low magnification transmission electron micrograph of CP muscle shown with a 400-point stereological grid. Low and high magnification micrographs (n = 845) were analyzed for volume fraction of myofibrils, basal lamina, mitochondria, myonuclei, satellite cells, collagen fibrils in cables, single collagen fibrils, fibroblasts, and blood vessels, each shown in a different color (note that some components were only measured at high magnification and are identified here as examples). B) Stereological results comparing TD and CP muscle. Note the significantly greater volume fraction of fibrils in cables and fibroblasts in CP muscle compared to TD.