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, 589 (Pt 10), 2625-39

Hamstring Contractures in Children With Spastic Cerebral Palsy Result From a Stiffer Extracellular Matrix and Increased in Vivo Sarcomere Length

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Hamstring Contractures in Children With Spastic Cerebral Palsy Result From a Stiffer Extracellular Matrix and Increased in Vivo Sarcomere Length

Lucas R Smith et al. J Physiol.

Abstract

Cerebral palsy (CP) results from an upper motoneuron (UMN)lesion in the developing brain. Secondary to the UMNl esion,which causes spasticity, is a pathological response by muscle - namely, contracture. However, the elements within muscle that increase passive mechanical stiffness, and therefore result in contracture, are unknown. Using hamstring muscle biopsies from pediatric patients with CP (n =33) and control (n =19) patients we investigated passive mechanical properties at the protein, cellular, tissue and architectural levels to identify the elements responsible for contracture. Titin isoform, the major load-bearing protein within muscle cells, was unaltered in CP. Correspondingly, the passive mechanics of individual muscle fibres were not altered. However, CP muscle bundles, which include fibres in their constituent ECM, were stiffer than control bundles. This corresponded to an increase in collagen content of CP muscles measured by hydroxyproline assay and observed using immunohistochemistry. In vivo sarcomere length of CP muscle measured during surgery was significantly longer than that predicted for control muscle. The combination of increased tissue stiffness and increased sarcomere length interact to increase stiffness greatly of the contracture tissue in vivo. These findings provide evidence that contracture formation is not the result of stiffening at the cellular level, but stiffening of the ECM with increased collagen and an increase of in vivo sarcomere length leading to higher passive stresses.

Figures

Figure 1
Figure 1. Images of biopsy collection method using clamps for in vivo sarcomere length determination
A, hamstring muscle is exposed. B, clamp is secured around gracilis muscle with joint position at 90 deg of hip and knee flexion. C, close-up view of muscle clamps around the biopsy tissue. D, biopsy is dissected from muscle while clamped and fixed in Formalin for subsequent sarcomere length measurement.
Figure 2
Figure 2. Passive tension as a function of sarcomere length for fibres and bundles, after stress relaxation
Plots represent the average of the fits from each individual sample ± SEM. The stress vs. sarcomere length fit was linear for fibres with a R2 value of 0.962 ± 0.003 (A and B) and quadratic for bundles with a R2 value of 0.985 ± 0.002 (C and D). A, gracilis fibres show no difference between CP and control. B, semitendinosus fibres show no difference between CP and control. C, CP gracilis bundles show a significant increase in stress at high sarcomere lengths compared to control. D, CP semitendinosus bundles show a significant increase in stress at high sarcomere lengths compared to control.* inside symbol designates the approximate sarcomere length at 90 deg of hip and knee flexion.
Figure 3
Figure 3. Tangent stiffness of fibres and bundles
Samples are represented with either a linear fit for fibres or a quadratic fit for bundles. A, tangent stiffness values at 4.0 μm for single fibres are not changed with CP for either gracilis or semitendinosus muscles. B, tangent stiffness values at 4.0 μm for fibre bundles are significantly greater in CP compared to control bundles in both gracilis and semitendinosus (P < 0.05). C, tangent stiffness values at measured average in vivo sarcomere length for CP bundles or the predicted in vivo sarcomere length for control bundles show highly elevated values in CP for a joint configuration of 90 deg hip and knee flexion.
Figure 4
Figure 4. In vivo sarcomere length of gracilis and semitendinosus
A, measured in vivo sarcomere length with 90 deg of hip and knee flexion ± SEM for CP subjects in gracilis and semitindenosus (P < 0.05). Solid white line represents predicted sarcomere length for control children. B, correlation between in vivo sarcomere length measured for CP subjects and their Gross Motor Function Classification System (GMFCS) shows a positive significant correlation (P < 0.05), meaning subjects with longer in vivo sarcomeres are more severely affected patients. C, correlation between in vivo sarcomere length and popliteal angle is negative and significant (P < 0.05), meaning subjects with less knee extension have longer sarcomere lengths.
Figure 5
Figure 5. Molecular mass of titin isoforms of CP and control subjects in gracilis and semitendinosus muscles
Two-way ANOVA shows no significant effect of pathology on molecular mass (P > 0.05). Although not significant, the trend for molecular mass of titin in CP muscles is larger than control suggesting, if anything, more compliant fibres due to titin alterations.
Figure 6
Figure 6. Collagen content of muscle biopsies shows significantly higher collagen content in CP biopsies
The results of this assay are consistent with the increased stiffness observed in fibre bundles (Fig. 4). *indicates a significant post hoc difference between control and CP for the semintendinosus (p < 001).
Figure 7
Figure 7. Immunohistochemistry of muscle biopsies show qualitatively increased levels of ECM in CP (B, D, F and H) compared to control (A, C, E and G) children
Representative images with primary antibody to fibrillar collagen type I in cross section (A and B) and longitudinal section (C and D). Representative images with primary antibody to laminin of the basal lamina, in cross section (E and F) and longitudinal section (G and H). Note that muscle fibers from children with CP are slightly smaller with a great amount of Collagen I and laminin, two of the major components of the extracellular matrix. Scale bars represent 100 μm.
Figure 8
Figure 8. Myosin heavy chain isoforms
There was a significant increase in myosin heavy chain I in CP muscles compared to control suggesting contractured fibres have a slower phenotype. There was no significant difference between muscles. *represents significant difference in MyHC 1 percentage between control and CP muscles (p < 0.001)

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