Background
Duchenne muscular dystrophy (DMD) is an X-linked, lethal disorder of skeletal muscle caused by mutations in the dystrophin gene, which encodes a large sub-sarcolemmal cytoskeletal protein, dystrophin. DMD is characterized by a high incidence (1 in 3,500 boys) and a high frequency of
de novo mutation [
1]. The absence of dystrophin is accompanied by the loss of dystrophin-associated glycoprotein complex from the sarcolemma, leading to reduce membrane stability of myofibers. This dysfunction results in progressive muscle weakness, cardiomyopathy, and subsequent early death by respiratory or heart failure in DMD patients.
For basic and therapeutic studies of DMD, it is very important to perform analysis and evaluation using dystrophin-deficient animal models, such as the
mdx mouse and dystrophic dog. The
mdx mouse has been well utilized in many DMD studies, but the murine model shows moderate dystrophic changes unlike severe human DMD [
2]. In contrast, golden retriever muscular dystrophy (GRMD) shows similar dystrophic phenotypes to those of human patients: elevated serum CK level, gross muscle atrophy with joint contracture, cardiomyopathy, prominent muscle necrosis, degeneration with mineralization and concurrent regeneration, and endomysial and perimysial fibrosis [
3]. Therefore, the dystrophic dog is more suitable than the
mdx mouse for studies to gain insight into the pathogenic and molecular biological mechanisms of human DMD, as well as for pre-clinical trials [
4]. Therefore, we have recently established a colony of beagle-based canine X-linked muscular dystrophy in Japan (CXMD
J) [
5], and have demonstrated that CXMD
J also exhibited severe symptoms similar to GRMD. To date, we have utilized the littermates of the CXMD
J colony for pathological [
6,
7], molecular biological [
8], and therapeutic examinations [
9] of DMD.
Skeletal muscles are composed of heterogeneous populations of muscle fiber types, which contribute to a variety of functional capabilities. In addition, muscle fibers can adapt to diverse situations, such as aging, exercise, and muscular diseases, by changing fiber size or fiber type composition. Therefore, it is important to analyze fiber types to evaluate the condition of skeletal muscle with disease. Fiber types can be distinguished by biochemical, metabolic, morphological, and physiological properties. One of the most informative methods for identification of fiber types is detection of myosin heavy chain (MHC) [
10,
11]. Myofibers express various MHC isoforms containing slow (type I), fast (types IIA, IIX, IIB), embryonic, and neonatal forms. MHC expression, however, seems to differ between animal species and muscle types. Three MHC isoforms (types I, IIA, and IIX) have been identified in limb skeletal muscles of human and dog, while the fourth isoform, MHC IIB, is abundantly present in small mammals including mouse [
10,
11]. In addition, expression profiles of MHCs in dystrophin-deficient muscles have been widely examined in limb skeletal muscles of DMD patients [
12] and animal models, such as the
mdx mouse [
13] and GRMD [
14], but it has not been fully analyzed in skeletal muscles of a canine model. Furthermore, expanded studies of the diaphragm were restricted to that of the
mdx mouse [
13,
15]. Therefore, it is important to perform detailed evaluation of fiber types and fiber sizes in limb skeletal muscles and the diaphragm of CXMD
J to understand adaptations toward disease by changes in fiber type composition in the skeletal muscles of human DMD.
In this study, to investigate fiber types of myofibers in dystrophin-deficient skeletal muscles of dystrophic dogs, we evaluated the expression profiles of MHCs in tibialis cranialis (TC) muscles and diaphragms of CXMDJ at various ages, by immunohistochemical and electrophoretic techniques. Briefly, we detected myofibers expressing fast type, slow type, and/or developmental MHCs. In addition, the numbers of fast or slow MHC fibers and the size distribution of these myofibers were analyzed among populations of muscle fibers with or without developmental MHC. The composition of MHC isoforms was also examined in pairs of normal and affected dogs at various ages. This is the first report of evaluation of the detailed distribution of fiber types in TC muscles and diaphragms of dystrophic dogs.
Discussion
To investigate the alterations in fiber types in skeletal muscles of a canine DMD model, we examined MHC expression in the TC muscle and diaphragm of CXMDJ at various ages. Our results indicated that the influences of dystrophin deficiency on fiber type composition were significantly different between TC muscle and diaphragm.
To analyze MHC expression in details, we compared fiber type composition and fiber size distribution of MHC-expressing fibers between a normal dog (10 months old) and an affected dog (11 months old). In normal and affected dogs, body weight rapidly increased to approximately 9 kg at 4 months old, and then slightly increased to approximately 14 and 11 kg at 12 months old, respectively [
5]. As body weight reflects muscle weight, muscle mass and fiber size would not extremely change in 1 month after 4 months old, especially in normal dogs. In fact, in TC muscles or diaphragms of normal dogs, there were no significant differences among compositions of fiber types and MHC isoforms after 4 months old (Fig
6 and
7). In addition, we examined normal dogs at 11, 12 and 14 months old, and affected dogs at 10, 12, 13 and 15 months old. Normal muscles of adult dogs showed similar expression of fast type, slow type, or developmental MHC at all adult ages, and affected muscles also showed similar MHC expression at examined ages (data not shown). These observations implied that there would be no significant difference in MHC expression between at 10 and 11 months old, in both of normal and affected dogs.
Common features between TC muscle and diaphragm of CXMDJ
TC muscle and diaphragm of CXMD
J shared the features that slow MHC fibers increased and enlarged selectively in non-regenerating populations, while fast type IIX or IIA MHC isoform decreased. Similar observations have been reported in skeletal muscles of the
mdx mouse [
13], GRMD [
14], and human DMD [
12,
21]. In general, increasing and enlarging of slow fibers may be a consequence of adaptive responses by metabolic enzyme systems and energy consumption, because slow fibers have lower capacity for power output and consume less energy than fast fibers [
22]. Our results also supported the hypothesis that slow fibers would be more adaptable to dystrophic stress than fast fibers, to compensate for the reduced abilities of muscle function.
Two mechanisms were considered to explain the selective increase in slow fibers during progressive muscle degeneration. One possibility is that slow fibers may be more resistant to dystrophic stress than fast fibers, leading to selective survival of slow fibers. This was supported by the observation that slower muscle fibers contained significantly more utrophin, a homolog of dystrophin, in comparison to faster counterparts [
23,
24]. Another is transition of MHC isoforms, where type IIA or IIX MHC isoforms could be transited to type I, as seen in hypertrophy and exercise [
25]. MHC I, IIa, IIx, and IIb gene expression are known to be regulated by the calcineurin pathway [
26,
27]. Dystrophin deficiency may accelerate MHC transition to slower types
via calcineurin/NFAT signaling in skeletal muscles of CXMD
J, because calcineurin and activated NFATc1 protein content were higher in muscles from
mdx than wild-type mice [
28]. However, it remains possible that both mechanisms may be active at the same time, because the calcineurin/NFAT cascade can regulate not only the MHC promoters but also the utrophin A promoter [
24,
29,
30].
Differences between TC muscle and diaphragm of CXMDJ
The CXMD
J diaphragm developed severe degenerative lesions from earlier stages than TC muscle, which corresponded to previous reports [
3,
5,
31]. In addition, dystrophic changes in the CXMD
J diaphragm not only markedly altered the expression of fast and slow type MHCs but also decreased the amount of the developmental (embryonic and/or neonatal) MHC with growth, unlike affected TC muscle. Especially, fast MHC fibers disappeared and slow MHC fibers enlarged in the adult CXMD
J diaphragm. The greater cross-sectional area of slow fibers in affected diaphragms might be due to hypertrophy in compensation for loss of fast fibers, relating to plasticity of muscle fibers, as mentioned above. The diaphragm keeps continuous contraction of muscle fibers without resting, while limb skeletal muscle regularly rests its movement. Therefore, replacement with slow fibers may be particularly enhanced in the diaphragm rather than TC muscle, depending on pathological severity and contractile activity of skeletal muscles.
Fiber type determination and fiber type-specific gene expression are regulated by multiple signaling pathways and transcription factors. As partially described above, a key mediator, calcineurin, plays an important role in acquisition of fiber phenotype [
29,
30] and may induce not only transition of MHC isoforms from faster to slower types but also transformation of myofiber phenotypes in mouse or rat muscles [
26,
27,
32]. In addition, calcineurin signaling activity was greater in the diaphragm than in the tibialis anterior muscle of the
mdx mouse [
28]. Therefore, replacement with slow fibers may be up-regulated to a greater extent in the diaphragm than in the TC muscle of CXMD
J.
We also showed age-related changes of MHC expression in affected diaphragms after 6 months old, in contrast to TC muscles (Fig
4,
5 and
7). In addition, fiber type compositions in non-regenerating or regenerating fibers were also different between the TC muscle and the diaphragm, depending on age. In non-regenerating fibers of affected TC muscles, fast MHC fibers at 4 months old was higher than those at 2 and 11 months old (Fig.
6B). It might be partially involved in pathological changes that degenerative lesions appeared obviously in affected TC muscles after 4 months old, as described previously [
3,
5,
31]. In regenerating fibers of the CXMD
J diaphragm, the proportion of myofibers expressing slow type MHC increased markedly after 4 months old (Fig.
6C). These results suggested that MHC expression in TC muscle and the diaphragm of CXMD
J would be influenced by different mechanisms after 4 months old. These age-dependent MHC expression might be related to body growth, particularly increasing of muscle mass. One possibility is participitation of insulin-like growth factor (IGF)-1, which is important for postnatal growth of skeletal muscles [
33] and can activate multiple Ca
2+-dependent signaling pathways, including the calcineurin/NFAT pathway [
30]. When growth rate of body weight decreases after 4 months old [
5], signaling activity of IGF-1 might reduce and MHC expression might be regulated predominantly by alternative signaling pathways.
Comparison among mdx, CXMDJ, and DMD diaphragms
MHC expression in normal skeletal muscle has been well studied in mice [
15,
34], dogs [
11], and humans [
35]. In normal dogs, the proportions of fiber types in TC muscle were relatively similar to those in the representative tibialis anterior muscles of mice and humans. In the diaphragm, however, the proportion of fiber types differed markedly among these species. The murine diaphragm is composed mainly of fast type IIA and IIX isoforms [
15,
34], but the canine diaphragm consists of equal populations of slow type MHC I and fast type MHC IIA [
11], as also shown in our study. In normal human diaphragm, the distribution of myosin isoforms has been estimated that types I, IIA, and IIX account for approximately 45%, 40%, and 15%, respectively [
35]. Thus, the proportions of MHC isoforms in the diaphragm of healthy dogs are much closer to those of humans than those of mice.
Some groups have studied expression profiles of MHC isoforms in the diaphragm of the
mdx mouse. The
mdx diaphragm shows increases in MHC type I fibers and elimination of type IIX population at 2 years old, but not at young ages (3 to 6 months old) [
13,
15,
34]. In contrast to the
mdx diaphragm, that in CXMD
J exhibited drastic changes even in younger animals (6 months old). On the other hand, there is no direct information available regarding the changes in fiber type composition in the diaphragm in human DMD. In addition, there is an important difference of MHC expression even in limb skeletal muscles between large mammals (including dogs and humans) and mammals with smaller body mass, especially rodents. The former do not express the fastest MHC IIB isoform in limb muscles [
10,
11,
36], while it is abundantly expressed in the latter [
34]. Therefore, changes/adaptations in skeletal muscles of dogs with muscular dystrophy are likely to be more relevant to human DMD, than that in the
mdx mouse. As it is difficult to examine the diaphragms of DMD patients, it would be important to investigate the differences between murine and canine models for understanding the mechanisms of respiratory failure in human DMD.
Conclusion
Based on fiber type classification using MHC expression, we demonstrated the predominant replacement with slow fibers and reduced muscle regeneration with progression of muscular dystrophy in the diaphragm of a canine DMD model, but these phenomena were much less strict in affected TC muscle. In addition, the expression profiles of MHC isoforms in the CXMDJ diaphragm were evidently different from those of the mdx mouse. Our results indicated that dystrophic dog is a more appropriate model than a murine one for human DMD, and would be useful for investigation of the mechanisms of respiratory failure in DMD, as well as pathological and molecular biological backgrounds, and therapeutic effects in clinical trials.
Acknowledgements
We are grateful to Dr. Madoka Yoshimura, Dr. Nobuyuki Urasawa, Dr. Naoko Yugeta, Ms. Ryoko Nakagawa, and Dr. Masayuki Tomohiro for technical assistance. We also thank Mr. Hideki Kita and Mr. Shinichi Ichikawa for care and management of experimental animals. This work was supported by Grants-in-Aid for Center of Excellence (COE), Research on Nervous and Mental Disorders (13B-1, 16B-2, 17A-10, 19A-7), Health Science Research Grants for Research on Psychiatry and Neurological Disease and Mental Health (H12-kokoro-025, H15-kokoro-021, H18-kokoro-019), and the Human Genome and Gene Therapy (H13-genome-001, H16-genome-003) from the Ministry of Health, Labor, and Welfare of Japan, and Grants-in-Aid for Scientific Research to KY and High-Tech Research Center Project for Private Universities (matching fund subsidy, 2004-2008) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
KY designed the study, carried out the pathological and immunohistological examinations, and drafted the manuscript. AN participated in interpretation of data, and helped to draft the manuscript. TH participated in coordination of the study. ST participated in the design, planning, and coordination of the study, and helped to draft the manuscript. All authors read and approved the final manuscript.