Collagen and muscle pathology in fibromyalgia patients

S. T. Gronemann, S. Ribel-Madsen, E. M. Bartels1, B. Danneskiold-Samsøe and H. Bliddal

The Parker Institute, Department of Rheumatology, Frederiksberg Hospital, H:S University Hospital and 1The Danish National Library of Science and Medicine, Copenhagen, Denmark.

Correspondence to: S. Ribel-Madsen, The Parker Institute, Department of Rheumatology, Frederiksberg Hospital, H:S University Hospital, DK-2000 Frederiksberg, Denmark. E-mail: soren.ribel.madsen{at}fh.hosp.dk

Abstract

Objective. To measure collagen concentration and search for muscle pathology in muscle non-tender-point areas from fibromyalgia (FM) patients.

Methods. Muscle biopsies were obtained from m. vastus lateralis of 27 carefully selected, female fibromyalgia patients, and from eight age-matched female control subjects. Amino acids were determined by HPLC and electron microscopy was performed.

Results. The FM patients had lower hydroxyproline and lower total concentration of the major amino acids of collagen than the controls. No significant difference was seen in the concentration of the major amino acids of myosin or of total protein. Electron microscopy showed no significant differences between FM patients and controls although atrophied muscle fibrils occurred in FM patients only, but frequencies were not significantly different.

Conclusion. Fibromyalgia patients had a significantly lower amount of intramuscular collagen. This may lower the threshold for muscle micro-injury and thereby result in non-specific signs of muscle pathology.

KEY WORDS: Fibromyalgia, Collagen, Muscle, Electron microscopy.

Fibromyalgia (FM) is characterized by chronic musculoskeletal pain. The criteria for the classification, defined by the American College of Rheumatology (ACR) [1], are based on case history and clinical findings, while no histological or biochemical analyses to substantiate the diagnosis have yet been presented.

A relationship between disorganization of myofibrils and muscle soreness, pain and stiffness after exercise has been described [2, 3]. A balance between repeated injury and subsequent repair will establish itself, but signs of previous injury remain as deposits of collagen in the muscle interstitium [4]. A change in collagen metabolism, which may reflect a similar development in FM muscle as described here, has been indicated by a decreased ratio between pyridinoline and deoxypyridinoline in urine and in serum, and a lower concentration of hydroxyproline in urine, when compared with control subjects [5]. In the same study, highly ordered collagen cuffs were found around terminal nerves in the tender-point areas in skin, and were suggested to be a result of neurogenic inflammation.

It may be speculated that the collagen defect is a general feature of FM, and not only related to tender points or to skin. This is supported by the finding of a low concentration of procollagen type III amino-terminal peptide in serum (S-PIIINP) in FM patients, and the known correlation between low S-PIIINP and high intensity of muscle pain [6]. The presence of a neurogenic inflammation in FM is supported by the finding of a high amount of substance P stored in nerve endings in the trapezius muscle [7]. Accordingly, collagen cuffs might be expected around nerve endings both in skin and in muscle, but the alterations in FM muscle described in several electron microscopy (EM) studies have seemed to be non-specific, with no characteristic collagen structures, and no suggested relation to collagen [812].

In muscle, collagen is the major fraction of the non-contractile constituents. The organization and co-operation between muscle fibres and intramuscular connective tissue determine the properties of the whole muscle. Hydroxyproline mainly occurs in collagen and is routinely used as a collagen marker.

The aim of this study was to quantify collagen-related amino acids in muscle biopsies taken from FM patients' non-tender-point areas, representing the perimysium and endomysium, and to search for histological signs of connective tissue and general muscle pathology.

Patients and methods

The Copenhagen Ethics Committee accepted the protocol, and each subject gave her informed consent according to the declaration of Helsinki before entering the study.

Patient group
Eighty-one FM patients were identified from medical records, and 27 participated in the final study. The inclusion criteria were: female who fulfilled the ACR criteria of FM and in addition had no depression, no concurrent muscle or joint disease, no motor trauma, nor a history of monotonous physical work, and no daily sports activity. Before any biopsies were taken, blood samples were subjected to haematological examination, determination of plasma electrolytes and determination of substances reflecting metabolic status and rheumatological status (IgM rheumatoid factor and antinuclear antibodies).

Assessments of tender points and four negative control points were carried out by two independent examiners as defined [1]. Patients indicated their pain on a visual analogue scale (VAS) and reported their daily activity. A record of patient's age, height, body weight, time of onset of FM symptoms and current medication was made.

Control group
The control group consisted of eight women undergoing knee arthroscopy who matched the FM patients in age and physical activity. Apart from exercise-related pain from a knee during a period ranging from 3 weeks to several years, they had no daily universal muscle or joint pain, and were otherwise healthy. They had no daily sports activity [13].

Muscle biopsies
The biopsies were taken with a Bergström's needle from vastus lateralis m. quadriceps femoris, 10 cm proximally of the upper margin of patella. The site was outside the defined FM tender-point areas. There was uncertainty on the validity of the sample taken for biochemistry from one patient. Data referring to this patient were therefore excluded from the amino acid results.

Electron microscopy
A part of each biopsy was fixed immediately after extraction in cold 0.1 M cacodylate buffer at pH 7.35 with 3% glutaraldehyde and 7.5% sucrose. Post-fixation was in phosphate-buffered 1% osmic acid. The specimens were then dehydrated through a series of increasing concentrations of ethanol, and finally embedded in Epon epoxy resin (Merck for EM). Sections were cut to a thickness of 60 nm, post-stained with uranyl acetate and lead citrate, and finally studied in a Jeol 1010 electron microscope at 80 kV.

Investigators, blinded to the clinical data, evaluated the parameters mentioned below. The scoring system chosen was: ‘normal’ and ‘altered’ to make the allocation more certain and statistical analysis possible. The parameters were: the overall number and distribution of lipid droplets in the muscle cell and on the outside of sarcolemma; regularity of Z lines; atrophy of muscle fibrils seen as spaces more than 1 µm wide in longitudinal sections and in cross-sections; collagen appearance in general, and occurrence of collagen cuffs around terminal nerves, if any.

Amino acid determination
Another part of each biopsy, resembling a cylinder with length 2 mm and diameter 1 mm, was delipidized, dried and hydrolysed in hydrogen chloride in an oxygen-free atmosphere. The hydrolysates were evaporated to dryness, redissolved and duplicate aliquots were taken for determination of amino acids by a routine method [14] including derivatization with phenylisothiocyanate followed by high-performance liquid chromatography (HPLC), identifying and quantifying the collagen-related amino acids hydroxyproline, hydroxylysine and proline, and 14 other, generally occurring, amino acids. The amount of total protein per milligram of dry, delipidized tissue was calculated by summarizing the products of the determined molar amount of each amino acid and the residual weight of this amino acid, expressing the resulting amount as a proportion of the dry tissue weight. The coefficient of variation of the entire analytical procedure was 7.3%.

Statistics
Statistical analysis was performed with the SPSS 9.0 software. Means were compared with Mann–Whitney U-test since data on muscle hydroxyproline and data on body mass index deviated significantly from a normal distribution and variance homogeneity, as judged from Kolmogorov–Smirnov and Levene test. The distribution of current medication was compared by calculation of the Pearson {chi}2-test. The distribution of scores from evaluation of electron micrographs, and the proportions between users and non-users of medicine, were compared by means of Fisher's exact test. The relationship between muscle hydroxyproline and the individual's current medication, or the most important medication if more than one drug was taken, was analysed with Kruskal–Wallis test. All tests were two-sided and the 0.05 level of significance was chosen.

Results

Patients and control group data
The age of FM patients and controls, given as mean (S.E.M.), was 38.3 (1.0) and 40.5 (3.0) yr, and body mass index was 24.1 (0.7) and 25.2 (2.4), respectively. In the FM group, the duration of FM was 81 (17) months, the number of positive tender points 14.2 (0.5), and VAS of pain score 68.6 (4.2). None of the subjects included had any clinical biochemical values outside the reference interval.

FM patients had a much higher intake of analgesics, antidepressants and tranquillizing agents than the controls. The difference was significant when considering the entire distribution (P = 0.008), or the proportion between number of subjects using one or more of these drugs and the number who used none of them (P = 0.001).

Amino acid analysis
The amount of hydroxyproline per milligram of dry muscle was lower in the FM group than in the control group (P = 0.006). The sum of the three most abundant amino acids in collagen—hydroxyproline, proline, and glycine—was also significantly lower in the FM group (P = 0.020). No difference in the sum of the amounts of the major amino acids of myosin, lysine, leucine and isoleucine or in total protein was found (Table 1).


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TABLE 1. Results of amino acid analysis

 
No significant difference in the respective amino acids was seen between FM patients with or without daily medication. No relationship was found between amount of hydroxyproline and the number of positive tender points or VAS of pain.

Electron microscopy study
Figure 1 shows micrographs of FM and control muscle. The percentage distribution of scores assigned to the micrographs are listed in Table 2. Lipid droplets between muscle fibrils tended to be more numerous in biopsies from FM patients, whereas the proportion of biopsies with remarkable clustering of lipid droplets around mitochondria was slightly higher in the control group than in the FM patients. Only in the FM group was disorganization of the Z lines observed, appearing as irregularity of their alignment across a fibre in an area where the plane of section was exactly longitudinal, and atrophy of muscle fibrils, appearing as fibrils spaced more than 1 µm apart, most clearly seen in longitudinal sections. The differences between the proportions of occurrence to non-occurrence in the FM patient group and the corresponding proportions in the control group were not statistically significant.



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FIG. 1. Electron micrographs of muscle tissue (x3000) from a fibromyalgia patient (left panel) and a control (right panel). Lipid droplets, areas with distorted Z line registration and, in the left panel, atrophy of fibrils occur.

 

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TABLE 2. Distribution of percentage of scores from evaluation of electron micrographs of muscle biopsies

 

Discussion

In the present study, we found a significantly lower concentration of hydroxyproline in tissue from vastus lateralis m. quadriceps femoris of FM patients than of control subjects. The relatively low concentration of hydroxyproline in the perimysium and endomysium in FM muscle is in agreement with an earlier study based on measurements from urine samples, where low urine hydroxyproline concentrations in FM patients were described [5]. We also found a lower sum of the amounts per milligram of muscle tissue of the three major amino acids of collagen, and in contrast, no difference in total muscle protein or in the amino acids related to myosin.

An animal study has shown a biphasic response with an increase in hydroxyproline content during the first week of immobilization followed by a decrease with a reduction of the total muscle hydroxyproline after 3 weeks of immobilization [15]. The control group in the present study had been chosen with a view of being as similar to the FM patients as possible with regard to activity level, without adding the complication of other pain-creating diseases. Inactivity is therefore not the sole cause of decreased hydroxyproline in FM patients.

Analysis of data referring to the group of FM patients revealed no correlation between muscle hydroxyproline and the clinical parameters ‘VAS of pain’ and ‘number of positive tender points’, again indicating that other genesis than pain-related inactivity is relevant.

Pathological changes of FM muscle have been described in several studies [812]. In our study, the comparison of ultrastructure of muscle from the FM patients with that of control subjects showed a tendency towards a pathological muscle, but no outstanding signs of abnormality, and some signs of repair. The control subjects were reported to be as sedentary as the FM patients, excluding the differences being due to activity level. Larger biopsies might have been preferable, but we adhered to the technique that is locally routine and considered ethically acceptable.

The reduced collagen in FM muscle, compared with control, may result in a weakness of the supporting connective tissue, and normal increase in interstitial muscle collagen in response to strenuous exercise may be reduced. This may in turn lower the threshold for micro-injury in muscle, known from excessive exercise performance [3, 16, 17], or another possible mechanism could be focal remodelling activity, as seen with delayed-onset muscle soreness, with visible disorganization [18]. Both conditions would result in pain. Neurogenic inflammation as a factor in FM pain experience cannot be excluded either.

Injuries or remodelling processes will normally cause a rise in the hydroxyproline concentration [19]. If FM patients have a lower collagen concentration in muscle tissue than normal, and the muscles constantly show smaller injuries, which the electron microscopy finding might indicate, the healing processes may slow down due to low hydroxyproline concentration. Less supportive connective tissue may also increase the number of micro-injuries. In healthy people, muscle collagen increases 3 weeks after heavy exercise that has caused micro-injuries [20]. If this is not so in FM patients, their muscles may be prone to injuries during exercise. The micro-injury hypothesis could explain the characteristic pain in FM as being partly due to a peripheral component, while a central mechanism determining the collagen content in muscle may also be present [21, 22].

Acknowledgments

The authors wish to thank D. Hansen (Department of Pathology, Hvidovre Hospital, Denmark) for her skilful technical assistance, J. Nielsen and S. Hirschorn (Department of Rheumatology, Frederiksberg Hospital, Denmark) for their clinical assistance with the FM patients, and T. Riis Johannessen for her careful preparation of specimens. In addition, we wish to thank Dr T. Sandberg Sørensen (Department of Orthopaedic Surgery, Frederiksberg Hospital) for providing the control biopsies, and to Dr T. Kobayashi (Department of Dermatology, Research Unit, Bispebjerg Hospital, Denmark) for instructions and advice on preparation for electron microscopy. This study was supported by grants from The IMK Foundation, The OAK Foundation, The Danish Health Foundation and H:S University Hospital.

The authors have declared no conflicts of interest.

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Submitted 28 January 2003; Accepted 28 May 2003





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