PSACH is a skeletal dysplasia that in the most severe form is debilitating for the patient's well-being and lifestyle owing to the dramatic skeletal phenotype, which includes short-limbed dwarfism and early onset degenerative joint disease (44
). In contrast, the related, but milder MED is sometimes misdiagnosed or not reported until later in life (17
). In this study, we showed that mutations in Comp
that cause PSACH-MED result in altered collagen fibril diameters in force-loaded tendons and ligaments and is associated with a mild muscle myopathy in the absence of a detectable muscle pathology.
One of the clinical complications recently recognized as a part of the PSACH-MED phenotype is a mild myopathy, which in some cases may manifest earlier than the skeletal phenotype (17
). In these instances, the patients are reported as having difficulties with standing up or tire easily during exercise and are often referred to the clinic with an unclassified ‘neuromuscular disorder’ prior to the diagnosis of a underlying skeletal dysplasia. On the basis of these and other reported cases, it has been suggested that if a child presents with a ‘difficult to explain’ myopathy (i.e. waddling gait, increasing muscle weakness, but with none or only mild changes in a muscle biopsy), the child should be referred for a skeletal survey with a view to identifying an underlying skeletal dysplasia (46
). Interestingly, in many PSACH-MED patients with a reported myopathy, a causative mutation was identified in the CTD of COMP. In these patients, the myopathy was not comprehensively documented, but included features such as mildly elevated PCK levels (25
), basophilic and/or atrophic muscle fibres (17
). Furthermore, a mild myopathy has also been reported in several MED families with type IX collagen mutations, a protein which is not expressed in skeletal muscle, but is present in tendons at the insertion of the tendon into the bone (i.e. the enthesis) (47
). In these families, the proband was also referred to a neuromuscular clinic for the evaluation of proximal muscle weakness prior to the diagnosis of MED (27
) (M.D.B., submitted for publication).
The musculoskeletal complications of the PSACH-MED phenotype have not been studied in detail, primarily due to the difficulty in obtaining suitable pathological samples and aged/site-matched controls. A detailed analysis of the myopathy associated with COMP
mutations would therefore enable earlier diagnosis of mild PSACH-MED, improve our understanding of the disease mechanisms and ultimately improve patient care (48
). In this study, we have clinically demonstrated muscle weakness in mutant mice and analysed the gastrocnemius and soleus muscles and Achilles tendon to gain insights into the key pathological features and disease mechanisms of this mild myopathy.
T585M mutant mice, which we have previously characterized as a relevant model of mild PSACH (20
), also suffer from a progressive myopathy as demonstrated by grip strength testing. Furthermore, when we analysed skeletal muscle from the mutant mice, we found a progressive increase in the number of myofibres with central nuclei, specifically around the perimysium and the MTJ. This observation is indicative of skeletal muscle stress and the subsequent remodelling of the gastrocnemius and soleus muscles in these specific areas (28
). To test that the myopathy in mutant mice was not a generalized muscle pathology, we analysed the skeletal muscle tissue of wild-type and mutant mice in detail. We used immunohistochemistry to determine the localization of several important ECM proteins in murine skeletal muscle and did not detect any differences in the distribution of wild-type and mutant COMP, or in the distribution of types I, IV and VI collagen, desmin or vimentin. We have also confirmed that COMP was present in the ECM of skeletal muscle, specifically in the endo- and perimysium, and that there were no differences in its localization between wild-type and mutant tissues. Interestingly, these observations are in direct contrast to changes in the localization of mutant COMP seen in the cartilage growth plates of Comp
T585M mutant mice (20
) and suggests that the ECM of muscle and cartilage may assemble and respond differently to the presence of mutant COMP. We have previously shown that mild ER stress was detected in chondrocytes from mutant mice expressing Comp
T585M. However, the analysis of an ER stress marker (BiP) and an apoptosis marker (Bcl-2) showed no differences between the wild-type and mutant cells. Overall, these data suggest that although secreted by myocytes and present in the ECM, mutant COMP did not have an effect on general skeletal muscle architecture and did not elicit an ER stress response in the mutant cells.
It has been shown that the transmission of forces between the skeletal muscle and tendon depends on the interaction of the muscle fibres with the surrounding ECM and also on the collagen fibrillar organization of the tendon (37
). Perimysium has been shown to form a connective tissue with a ‘lattice-like’ structure and is important for conveying forces between the tendon and skeletal muscle (37
). These observations led us to hypothesize that myopathy in the mutant mice may, in fact, be the result of an underlying tendinopathy. Therefore, to gain insight into the mechanisms underlying the restricted localization of the myopathic changes, we studied the ultrastructure of the Achilles tendon from wild-type and mutant mice at 3 and 9 weeks of age. Tendons are an important skeletal tissue since they act as buffers for muscle stretch during locomotion (50
). We have shown that Comp
is expressed in murine tendon and ligament by a variety of techniques, and that the localization of mutant COMP was not altered in mutant tissues. To determine the effect of mutant COMP on collagen organization, we measured the diameters of collagen fibrils in the Achilles tendon of wild-type and Comp
T585M mutant mice. Interestingly, we found that the distribution of collagen fibril diameters in the mutant tendon was dramatically altered, with a higher proportion of larger diameter fibrils. There was a similar difference in the distribution of collagen fibrils in wild-type and mutant spinal ligament, further supporting a role for COMP in collagen fibrillogenesis. The total area occupied by the collagen fibrils was not altered between the wild-type and mutant tendons, but the number of fibrils per unit area of tendon was decreased in the mutant mice. Furthermore, the overall ‘interfibrillar area’ was not altered in the mutant tendons, suggesting that the amount of inter-territorial matrix in the wild-type and mutant tendons was similar. PGs comprise ~0.5% of tendon dry weight and play a role in tendon fibril spacing (37
). We therefore analysed the PG content of wild-type and mutant Achilles tendons from 3 week-old mice and found that the PG content was similar for both genotypes.
The abnormal changes seen in collagen fibril diameters in the mutant tendon might be due directly to the mutation in the CTD of COMP. For example, this region of COMP contains a potential collagen-binding site (15
), and a role for COMP as a catalyst in collagen fibrillogenesis has been proposed (16
). It is therefore possible that this C-terminal COMP mutation (T585M), which is close to the potential collagen-binding site (15
), has a detrimental effect on the ‘catalyst’ function of COMP that could alter collagen fibril diameter in the mutant tissue and ultimately its biomechanical properties. The detrimental effect of COMP mutations on collagen fibrillogenesis has been previously demonstrated in vitro
). In addition, we also observed an increase in the number of fused/bifurcating collagen fibrils in mutant tendon and ligament compared with the wild-type tissues. Similar observations have previously been seen in a ligament sample from a PSACH. In wild-type animals, fibril bifurcation is abundant in mouse fetal tendon tissue, but decreases with age (52
). Furthermore, increased fibril bifurcations have also been found in tendon scar tissue and at the scar to tissue junctions (53
), and it has been suggested that an increase in fibril bifurcations may be indicative of wound healing and that they may be required to connect neighbouring fibrils to transform the force properly from the scar to the residual tissue (53
). It has been proposed that the tendon biomechanical function depends on the precise collagen fibre alignment in the tissue (54
). It is therefore interesting to speculate that owing to the altered biomechanical properties of the mutant tendons, some microdamage could occur in the mutant tendon, and the increased number of fused fibrils may be indicative of repair mechanisms in the mutant tissue, which could in turn affect the biomechanical properties of the tissue.
Finally, the cross-sectional area of whole mutant tendons was also significantly less than that of the wild-type tendons. This may be due to tendon remodelling and/or disuse (55
). It could also potentially be a compensatory mechanism for the thicker collagen fibrils in the mutant tendons, which would make the mutant tissue stiffer. The dimensions of tendons directly influence their ability to stretch, store and release kinetic energy (56
). In physiological conditions, the cross-sectional area of the tendon, relative to that of the fascicles of the attached muscle, dictates the maximum tensile stress to which a tendon can be subjected (57
). The smaller cross-sectional area of mutant tendons, and the abnormal structural similarities between mutant tendons and ligaments, may therefore explain the joint laxity seen in PSACH-MED patients (2
). In addition, the variability in the diameters of individual collagen fibrils can have a dramatic impact on the tissue's biomechanical properties. The biomechanical properties of tendons are directly related to fibril length, diameter and modulus and inversely to interfibrillar spacing (58
), and thick fibrils are predicted to withstand higher tensile forces owing to the higher number of intrafibrillar crosslinks (59
To assess the biomechanical properties of the wild-type and mutant tendons, we performed a series of tensile strength experiments. In a stretch-to-failure experiment, mutant tendons were able to withstand higher stresses and were stretched more before failure. However, when the mutant tissues were tested in a cyclic strain experiment, they became more lax with an increasing number of cycles, which is analogous to the progressive joint laxity observed in PSACH-MED patients (2
). Lax tendons would also be less suitable for conveying the appropriate forces from muscle to bone, which might help explain the neuromuscular symptoms seen in PSACH-MED patients. By analogy to the structural changes seen in mouse mutant tendon and ligament, we can hypothesize that joint laxity in PSACH-MED patients might also stem from an altered distribution of collagen fibrils.
Tendons are known to adapt to mechanical load requirements, and inactivity has been shown to decrease collagen turnover in tendon (37
). It was also shown that training increased the cross-sectional area of tendons in pigs (62
) and that an increase in cross-sectional area of tendons correlates with an increase in tendon stiffness (37
). Collagen fibril diameters also shift towards thicker fibrils with age (63
), resulting in less compliant tissues (50
). Therefore, it is likely that the tendon and ligament pathology seen in this mouse model of mild PSACH, and in PSACH-MED patients, becomes exacerbated with age as short-limbed dwarfism and increasing joint pain greatly reduce the patients' mobility. The increased joint laxity may in turn have an effect on the joint stability and the progression of degenerative joint disease in PSACH patients (44
In summary, we have shown that COMP is expressed in the Achilles tendon and skeletal muscle of adult mice. We have demonstrated that Comp
T585M mutant mice suffer from a progressive myopathy that is specifically localized to the perimysium and the MTJ. The distribution of collagen fibril diameters is altered in the mutant tendon and ligament, whereas the PG content is comparable between the wild-type and mutant tissues. The biomechanical properties of mutant tendon are dramatically altered, making the tissue more resilient to failure stresses, but ultimately more lax following cyclic strain. We therefore hypothesize that the mild myopathy reported in PSACH-MED results from altered forces transmitted by the mutant tendon, and that the joint laxity may be directly due to the structural abnormalities in the ligament. Furthermore, our study suggests that the difficulties in walking and easy tiredness experienced by some PSACH-MED patients might be a combination of the skeletal phenotype and an underlying tendinopathy. Since tendons are able to adapt to mechanical load and environmental requirements (36
), certain physiotherapeutic treatments may help alleviate some of clinical symptoms of PSACH-MED, such as muscle weakness and joint instability. Our detailed characterization of the mild myopathy and tendinopathy in the mouse model of mild PSACH may also help in the management and early diagnosis of some forms of PSACH-MED, specifically in those children that present with tiredness and muscle weakness prior to the diagnosis of an underlying skeletal dysplasia.