Synthesis and degradation of muscle collagen during immobilization, glucocorticoid treatment and in neuromuscular diseases

Anne Ahtikoski

Department of Physiology, University of Oulu
Neuromuscular Research Center, Department of Biology of Physical Activity, University of Jyväskylä
Department of Sports Medicine, Oulu Deaconess Institute

Abstract

To investigate the turnover of type IV collagen in skeletal muscle in conditions where muscle function is impaired, type IV collagen and proteins regulating its degradation were studied during 1, 3 and 7 days of immobilization, 3- and 10-day glucocorticoid treatment and in neuromuscular diseases. In addition, fibrillar type I and III collagens were studied during immobilization and in neuromuscular diseases. The mRNA levels of type I, III and IV collagens were decreased during immobilization and during 10-day dexamethasone treatment. Gene expression and quantity of (pro)MMP-2 was increased during immobilization but decreased during dexamethasone treatment. The expression of TIMP-2 was decreased both during immobilization and dexamethasone treatment. Decreased gene expression and increased degradation caused decreased concentration of type IV collagen, suggesting net degradation of type IV collagen during immobilization. While the gene expression and degradation were decreased during dexamethasone treatment, the amount of type IV collagen was not changed. Dexamethasone thus seemed to slow down the turnover of type IV collagen. Decreased mRNA levels of collagens and prolyl 4-hydroxylase suggest decreased biosynthesis of collagens during immobilization. The mRNA levels of collagens I, III and IV were increased in polyneuropathy and polymyositis. The concentration and staining intensity of type IV collagen was increased in polyneuropathy, as was also the quantity and staining intensity of (pro)MMP-9. The results suggest accumulation of type IV collagen in the basement membranes of muscle cells and capillaries in polyneuropathy muscles. Lengthened position during immobilization partly prevented the atrophy and changes in collagen metabolism in plantarflexors. Endurance running was effective in preventing muscle atrophy during dexamethasone treatment, but exercise did however fail to prevent the changes observed in type IV collagen synthesis and degradation.

Dedication

To Anssi, Roope and Veeti

Table of Contents

Acknowledgements

Abbreviations

List of original articles

1. Introduction

2. Review of the literature

2.1. Extracellular matrix in skeletal muscle

2.2. Fibril forming and associated collagen types in skeletal muscle

2.3. Collagens in the basement membrane of skeletal muscle

2.4. Collagen synthesis in skeletal muscle

2.4.1. Intracellular events
2.4.2. Extracellular events

2.5. Degradation of collagens

2.5.1. MMPs
2.5.2. Other forms of collagen degradation

2.6. Adaptation of connective tissue in skeletal muscle

2.6.1. Effects of physical activity
2.6.2. Effects of disuse
2.6.3. Effects of glucocorticoids
2.6.4. Effects of neuromuscular diseases

3. Purpose of the study

4. Material and methods

4.1. Animals (I, II, III)

4.2. Human subjects (IV)

4.3. Immobilization (I, II)

4.4. Glucocorticoid treatment and exercise (III)

4.5. Muscle samples

4.6. mRNA analyses

4.7. Biochemical assays

4.7.1. Prolyl 4-hydroxylase activity (I)
4.7.2. Proportions of type I and III collagens (I)
4.7.3. Total collagen concentration (I, II)
4.7.4. Radioimmunoassay for 7-S (II, III, IV)
4.7.5. Zymography (II, III, IV)
4.7.6. Reverse zymography (II, III)

4.8. Immunohistochemistry (IV)

4.9. Statistical analyses

5.1. General responses to immobilization, glucocorticoids and exercise (I, II, III)

5.2. Effects of immobilization on collagens in skeletal muscle (I, II)

5.2.1. Total collagen biosynthesis
5.2.2. Fibrillar collagens
5.2.3. Type IV collagen
5.2.4. MMP-2
5.2.5. TIMP-2

5.3. Effects of dexamethasone and exercise on type IV collagen synthesis and degradation in skeletal muscle (III)

5.3.1. Type IV collagen
5.3.2. MMP-2
5.3.3. TIMP-1 and TIMP-2

5.4. Effects of neuromuscular diseases on collagen and MMPs (IV)

5.4.1. Fibrillar collagens
5.4.2. Type IV collagen
5.4.3. MMPs

6. Discussion

6.1. General responses to immobilization, glucocorticoids and exercise
6.2. Synthesis of fibrillar collagens
6.3. Expression of MMPs
6.4. Inhibition of MMPs
6.5. Type IV collagen turnover
6.6. Compensatory effects of stretch and exercise

7. Conclusions

List of Tables
1. Family of matrix metalloproteinases (MMPs) with their major substrates and modulators.
2. The effects of immobilization, exercise, neuromuscular diseases and glucocorticoids on collagen synthesis and the activities of MMPs and TIMPs
3. Main analyses used in the studies.
4. Summary of the main findings.

List of Figures
1. Scanning electron micrographs of epimysium (EP, top right), perimysium (P, bottom) and endomysium (E, top left and bottom) of bovine semitendinosus muscle. Reprinted from Nishimura et al. (1994). Used with permission from S. Karger AG.
2. Collagens in skeletal muscle. (Kuo et al.1997, Aumailley & Gayraud 1998 and Tu et al. 2002)
3. Synthesis of fibrillar collagens.
4. Dexamethasone treatment and exercise in study III. The number of animals in each group is presented inside the boxes. All groups have two subgroups with 6 animals in each for the 3- and 10-day treatments. D = dexamethasone; S = sedentary; E = endurance running; U = uphill running.
5. Activity of TIMP-1 in TA and EDL during dexamethasone treatment and exercise. Dex = dexamethasone.

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