Relationship between extracellular matrix, contractile apparatus, muscle mass and strength in case of glucocorticoid myopathy

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Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 117–120

Relationship between extracellular matrix, contractile apparatus, muscle mass and strength in case of glucocorticoid myopathy Eva-Maria Riso a,∗ , Anne Ahtikoski b , Karin Alev a , Priit Kaasik a , Ando Pehme a , Teet Seene a a

Department of Functional Morphology, University of Tartu, 18 Ylikooli, 50090 Tartu, Estonia b Department of Physiology, University of Oulu, Finland Received 16 February 2007; accepted 20 June 2007

Abstract The purpose of this study was to evaluate the effect of dexamethasone on the contractile apparatus and extracellular matrix (ECM) components of slow-twitch (ST) soleus (Sol) and fast-twitch (FT) extensor digitorum longus (EDL) muscle. The specific aim was to assess the development of glucocorticoid-induced myopathy on the level of contractile apparatus and ECM, paying attention to the expression of fibrillar forming collagen types I and III and nonfibrillar type IV collagen expression in extracellular compartment of muscle. Degradation of myofibrillar proteins increased from 2.62 ± 0.28 to 5.58 ± 0.49% per day during glucocorticoids excess. Both fibril- and network-forming collagen-specific mRNA levels decreased at the same time in both types of skeletal muscle. Specific mRNA level for MMP-2 did not change significantly during dexamethasone administration. Hindlimb grip strength simultaneously decreased. The effect of excessive glucocorticoids on the extracellular compartment did not differ significantly in skeletal muscles with different twitch characteristics. © 2007 Elsevier Ltd. All rights reserved. Keywords: Excess of glucocorticoids; Contractile proteins; Extracellular matrix

1. Introduction It is well known that the glucocorticoids excess significantly decreases muscle mass, mainly at the expense of myofibrillar proteins, diminishes muscle strength and motor activity of laboratory animals [1] and humans [2], but changes in collagen metabolism in atrophic muscle have not been studied so well. It has been shown that excessive glucocorticoids inhibit the expression of types I and III collagen on mRNA level [3], but increase the concentration of type IV collagen in skeletal muscle [4]. As the function of contractile proteins in single muscle fibers occurs on the level of muscle in cooperation with extracellular matrix (ECM) components, it is important to clarify, how densely these two systems are linked in atrophic muscle. Knowledge about the influence of glucocorticoid-induced myopathy on the coherence and mechanical strength of musculoskeletal system helps to

∗

Corresponding author. Tel.: +372 7 375 364; fax: +372 7 375 362. E-mail address: [email protected] (E.-M. Riso).

0960-0760/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2007.06.002

understand the mechanism of development of atrophy via contractile apparatus and ECM. The purpose of the present study was to evaluate the effect of dexamethasone on the contractile apparatus and ECM of slow-twitch (ST) soleus (Sol) and fast-twitch (FT) extensor digitorum longus (EDL) muscle. The specific aim was to assess the development of glucocorticoid myopathy on the level of ECM, paying attention to fibril-forming collagen types I and III and network-forming type IV collagen expression in extracellular compartment of muscle. 2. Materials and methods Laboratory animals were used in accordance with the European convention for the protection of vertebrate animals used for experimental and other scientific purposes and their use was monitored by the Committee of Laboratory Animal Science of the University of Tartu and by the Committee of Laboratory Animal Experiments, University of Jyv¨askyl¨a.

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2.1. Animals, dexamethasone treatment, labelled amino acid infusion and muscle removal The used animals (National Laboratory Animal Centre, Kuopio, Finland) were Sprague–Dawley rats, 16 weeks old at the beginning of the experiment, 10 rats per group. Animals were housed in identical environmental conditions in polycarbonate type III cages, at 21 ◦ C, two per cage at 12/12 h light/dark period. They received diet (SDS-RM1(C) 3/8, Witham, Essex, England) and water ad libitum. The rats were assigned to control and dexamethasone-treated groups. Dexamethasone (Glucocortin-20, Interchemie, Holland) was diluted to 200 ␮g/ml with 0.15 M NaCl and administered intraperitoneally 100 ␮g/100 g bw during 10 days. The control animals received appropriate amounts of 0.15 M NaCl. l-[4.5-3 H] leucine (170 Ci/mmol) was infused intraperitoneally for 6 h, 250 ␮Ci/100 g bw. Rats were anesthetized 24 h after the last infusion by the intraperitoneal injection of Ketamin (Calycol, Gedeon Richter A.O., Budapest, Hungary) and diazepam (Lab Renaudin, France) and sacrificed. The EDL, Sol and Gastrocnemius muscles were removed, weighed, frozen and stored in liquid nitrogen until further processing. Due to the small size of soleus and extensor digitorum longus these muscles were pooled for mRNA analyses, so that one sample consisted of right and left muscle of the same animal. 2.2. Grip strength measurement The hindlimb grip strength was used as to make reference to development of muscle myopathy and measured with grip strength meter 0167-004L (Columbus Instruments) and expressed as N/100 g bw. 2.3. Estimation of 3-methylhistidine (3-MeHis) in skeletal muscle and urine The 3-MeHis in skeletal muscle and urine was used as an indicator for contractile protein degradation. The determination was performed as described previously [1,5]. Briefly, total muscle protein from Gastrocnemius muscle was hydrolyzed in 6 M HCl for 20 h at 110 ◦ C in vacuum-sealed flasks. HCl was removed by evaporation, and the hydrolysate was dissolved in 0.2 M pyridine to achive a concentration of 10–20 mg/ml. 3-MeHis pool excreted was expressed as percentage per day. 2.4. Protein assay Total muscle protein and myofibrillar protein was assayed by using the technique described by Bradford [6].

Technologies, Paisley, Scotland, UK). Other steps were performed as described in the manufacturer’s protocol. The purity and concentration of total RNA were assessed spectrofotometrically. 2.6. mRNA analyses For Northern blotting, 30 ␮g of total RNA was denatured in loading buffer, electrophoresed in a 1% agarose/formaldehyde gel, and transferred to a nylon membrane (GeneScreen Plus, Biotechnology Systems, Boston, MA, USA) with a standard procedure [7]. For slot blotting, 20 ␮g of total RNA was spotted on a nylon membrane using a vacuum filtration manifold (Minifold II; Schleider and Schuell, Dassel, Germany) [8]. All the membranes were incubated in 0.05 N NaOH for 5 min to bind the RNA to the membrane. Pre-hybridization was carried out in a solution containing 5× saline sodium citrate (SSC), 5× Denhart’s solution, 50% formamide, ssDNA 100 ␮g ml−1 , 50 mM sodium phosphate pH 6.8, 10% dextran sulphate and 1% sodium dodecyl sulphate (SDS) for 2 h at 42 ◦ C. The RNA–cDNA hybridization was performed for 24 h at 42 ◦ C using the solution containing the same components as the pre-hybridization solution and [32 P]-labelled cDNA probe, labelled with a Ready-To-Go-DNA Labelling Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The cDNA probes were 321 bp long mouse cDNA for the pro␣1(I)chain mRNA, 500 bp long rat cDNA for the pro␣1(III)-chain mRNA, 1800 bp cDNA for mouse pro␣1(IV)-chain mRNA and 1668 bp cDNA for rat MMP-2 mRNA. After hybridization, the membranes were washed at 65 ◦ C with 2× SSC + 2% SDS solution. The membranes were exposed to Kodak X-Omat film at −70 ◦ C. Bands were analysed using densitometry (Personal Densitometer SI, Molecular Dynamics, Sunnyvale, CA, USA). The signal obtained by hybridization with a 24-mer oligonucleotide for 18S ribosomal RNA was used to normalize RNA loading/transfer amount. Results are presented as specific mRNA level (mRNA OD 18S OD−1 ). Because absolute OD values were not used, the results are presented as percentage of changes in comparison with control group mean values. 2.7. Statistics Means and standard errors of means were calculated from individual values by standard procedures of Excel. The data were analysed by SAS, using the analysis of variance (ANOVA). Differences were considered significant at p < 0.05.

2.5. Total RNA analyses

3. Results

For total RNA isolation, muscle samples were homogenized with an Ultra-Turrax homogenizer in Trizol (Life

Excess of glucocorticoids decreased the body mass and FT muscle mass significantly (Table 1). Increased degradation

E.-M. Riso et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 117–120

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Table 1 Changes in body and muscle mass during 10-day dexamethasone treatment Group

Control (n = 10) dex (n = 10)

Body weight g

275 ± 8 220 ± 7*

EDL

Sol

mg

mg/g bw

mg

mg/g bw

132 ± 3.9 100 ± 4.1*

0.48 ± 0.05 0.45 ± 0.04

106 ± 3.1 104 ± 4.5

0.38 ± 0.04 0.47 ± 0.05

EDL, extensor digitorum longus muscle; Sol, soleus muscle; control, control group; dex, dexamethasone-treated group and *p < 0.001 in comparison with control group.

Fig. 1. Changes daily proportion of 3-MeHis pool excreted (B) during 10-day dexamethasone treatment. 3-MeHis, 3-methylhistidine. 3-MeHis pool excreted was used for characterization of contractile proteins degradation rate. contr, control group; dex, dexamethasone-treated group and ***p < 0.001 in comparison with control group.

Fig. 3. Changes in specific mRNA for MMP-2 in comparison with control group (100%) in fast- and slow-twitch skeletal muscles after 10-day dexamethasone treatment. EDL, extensor digitorum longus muscle and Sol, soleus muscle.

4. Discussion of myofibrillar proteins is a typical effect of glucocorticoids’ action in skeletal muscle (Fig. 1). It was previously shown that MyHC IIb isoform is more sensitive to the activity of proteinases [1]. Both fibril- and network-forming collagen-specific mRNA levels decreased during dexamethasone treatment in both FT and ST skeletal muscles (Fig. 2). Specific mRNA level for MMP-2 did not change significantly during dexamethasone treatment (Fig. 3). Hindlimb grip strength decreased about from 4.6 ± 0.4 to 3.4 ± 0.3 N/100 g bw (p < 0.05) during 10 days of dexamethasone treatment.

Fig. 2. Changes in specific mRNA for types I, III and IV collagen in comparison with control group (100%) in fast- and slow-twitch skeletal muscles after 10-day dexamethasone administration. EDL, extensor digitorum longus muscle; Sol, soleus muscle; col I, type I collagen mRNA (fibril-forming); col III, type III collagen mRNA (fibril-forming); col IV, type IV collagen mRNA (network-forming); *p < 0.05 in comparison with control group and **p < 0.01 in comparison with control group.

Skeletal muscle weakness in case of glucocorticoid myopathy was caused by lesions of myofibrillar apparatus [1,9] and by changes in neuromuscular synapses in FT muscles fibers [10]. The extracellular matrix outside of muscle fibers forms dynamic meshwork, which gives coherence and mechanical strength and functions as stress-tolerant [11] system. This system distributes the forces of muscle contractions both in muscle and tendon. Extracellular matrix is also influenced by glucocorticoids [12,13]. Excess of glucocorticoids has some similarities in action on the intracellular and extracellular compartments of skeletal muscle, like decreased synthesis of proteins [4,9]. Down regulation of collagen synthesis during dexamethasone administration shows that ECM components decrease [4]. As shown by us previously [1], MyHC synthesis rate decreased only in FT muscles. In the present study, the expression of collagen I, III and IV mRNA decreased in both FT and ST muscles. It seems that 10 days of dexamethasone treatment influenced similarly fibril- and network-forming collagen expression in ST and FT muscles and differed at this point from contractile protein myosin synthesis, which was depressed in FT muscles only. The second principle difference between contractile proteins and ECM during excess of glucocorticoids revealed in this study was the degradation rate of proteins. Dexamethasone increased the degradation of contractile proteins in skeletal muscle about 2.1 times but the expression of MMP-2

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mRNA did not simultaneously change significantly although the degradation of collagens occurs mainly through matrix metalloproteinase (MMP) activity [14]. This is surprising, as it was shown earlier that ST muscles contain about 50% more collagen than FT muscles [15]. The concentration of endomysial collagen is also higher around FT fibers [16], but as the down regulation of synthesis and unchanged degradation of type IV collagen are very similar with fibrillar collagen, it gives us reason to conclude that the mechanical stability in skeletal muscle fibers, ensured by collagen IV, does not differ between ST and FT fibers. The effect of excessive glucocorticoids on muscle weakness is applied through damaged contractile machinery of FT muscle fibers’ intracellular compartment [1]. In conclusion, excess of glucocorticoids has some similarities in its action on the intracellular and extracellular compartments of skeletal muscle [1], like decrease in protein synthesis and collagen expression. Difference between intracellular and extracellular compartments appears in FT and ST fibers. Intracellular compartment of muscle reacts to the excess of glucocorticoids with decrease of synthesis only in FT muscles. In extracellular matrix the decrease of protein synthesis appears in both, FT and ST muscles. The second difference between intracellular and extracellular compartments is the reaction to the protein degradation. In intracellular compartment of skeletal muscle, significant degradation of contractile proteins is a typical effect of excess of glucocorticoids, while in extracellular compartment the expression of matrix metalloproteinases did not change significantly.

Acknowledgements This study was supported by the funds of Ministry of Education and Research of Estonia, research project number 1787, by Estonian Scientific Foundation, Grant number 6501, and by the Ministry of Education, Finland. We are grateful to Ms. Mervi Koistinen and Ms. Aila Ollikainen for their skilful technical assistance.

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