Initial Pulmonary Respiration Causes Massive Diaphragm Damage and Hyper-CKemia in Duchenne Muscular Dystrophy Dog

The molecular mechanism of muscle degeneration in a lethal muscle disorder Duchene muscular dystrophy (DMD) has not been fully elucidated. The dystrophic dog, a model of DMD, shows a high mortality rate with a marked increase in serum creatine kinase (CK) levels in the neonatal period. By measuring serum CK levels in cord and venous blood, we found initial pulmonary respiration resulted in massive diaphragm damage in the neonates and thereby lead to the high serum CK levels. Furthermore, molecular biological techniques revealed that osteopontin was prominently upregulated in the dystrophic diaphragm prior to the respiration, and that immediate-early genes (c-fos and egr-1) and inflammation/immune response genes (IL-6, IL-8, COX-2, and selectin E) were distinctly overexpressed after the damage by the respiration. Hence, we segregated dystrophic phases at the molecular level before and after mechanical damage. These molecules could be biomarkers of muscle damage and potential targets in pharmaceutical therapies.ArticleSCIENTIFIC REPORTS. 3:2183 (2013)journal articl

α1-Syntrophin–deficient skeletal muscle exhibits hypertrophy and aberrant formation of neuromuscular junctions during regeneration

α1-Syntrophin is a member of the family of dystrophin-associated proteins; it has been shown to recruit neuronal nitric oxide synthase and the water channel aquaporin-4 to the sarcolemma by its PSD-95/SAP-90, Discs-large, ZO-1 homologous domain. To examine the role of α1-syntrophin in muscle regeneration, we injected cardiotoxin into the tibialis anterior muscles of α1-syntrophin–null (α1syn−/−) mice. After the treatment, α1syn−/− muscles displayed remarkable hypertrophy and extensive fiber splitting compared with wild-type regenerating muscles, although the untreated muscles of the mutant mice showed no gross histological change. In the hypertrophied muscles of the mutant mice, the level of insulin-like growth factor-1 transcripts was highly elevated. Interestingly, in an early stage of the regeneration process, α1syn−/− mice showed remarkably deranged neuromuscular junctions (NMJs), accompanied by impaired ability to exercise. The contractile forces were reduced in α1syn−/− regenerating muscles. Our results suggest that the lack of α1-syntrophin might be responsible in part for the muscle hypertrophy, abnormal synapse formation at NMJs, and reduced force generation during regeneration of dystrophin-deficient muscle, all of which are typically observed in the early stages of Duchenne muscular dystrophy patients

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMDJ) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle

<p>Abstract</p> <p>Background</p> <p>Skeletal muscles are composed of heterogeneous collections of muscle fiber types, the arrangement of which contributes to a variety of functional capabilities in many muscle types. Furthermore, skeletal muscles can adapt individual myofibers under various circumstances, such as disease and exercise, by changing fiber types. This study was performed to examine the influence of dystrophin deficiency on fiber type composition of skeletal muscles in canine X-linked muscular dystrophy in Japan (CXMD_J), a large animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used tibialis cranialis (TC) muscles and diaphragms of normal dogs and those with CXMD_Jat various ages from 1 month to 3 years old. For classification of fiber types, muscle sections were immunostained with antibodies against fast, slow, or developmental myosin heavy chain (MHC), and the number and size of these fibers were analyzed. In addition, MHC isoforms were detected by gel electrophoresis.</p> <p>Results</p> <p>In comparison with TC muscles of CXMD_J, the number of fibers expressing slow MHC increased markedly and the number of fibers expressing fast MHC decreased with growth in the affected diaphragm. In populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of CXMD_Jmuscles, slow MHC fibers were predominant in number and showed selective enlargement. Especially, in CXMD_Jdiaphragms, the proportions of slow MHC fibers were significantly larger in populations of myofibers with non-expression of developmental MHC. Analyses of MHC isoforms also indicated a marked increase of type I and decrease of type IIA isoforms in the affected diaphragm at ages over 6 months. In addition, expression of developmental (embryonic and/or neonatal) MHC decreased in the CXMD_Jdiaphragm in adults, in contrast to continuous high-level expression in affected TC muscle.</p> <p>Conclusion</p> <p>The CXMD_Jdiaphragm showed marked changes in fiber type composition unlike TC muscles, suggesting that the affected diaphragm may be effectively adapted toward dystrophic stress by switching to predominantly slow fibers. Furthermore, the MHC expression profile in the CXMD_Jdiaphragm was markedly different from that in <it>mdx </it>mice, indicating that the dystrophic dog is a more appropriate model than a murine one, to investigate the mechanisms of respiratory failure in DMD.</p

Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMD(J)): electrocardiographic, echocardiographic, and morphologic studies

BACKGROUND: Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMD(J)) and examined the cardiac involvement. METHODS: The cardiac phenotypes of eight CXMD(J )and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations. RESULTS: Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMD(J )dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6–7 months of age in all CXMD(J )dogs. In the echocardiogram, one of eight of CXMD(J )dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6–7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMD(J )dogs by 6–7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMD(J )dogs by 21 months of age. CONCLUSION: Cardiac involvement in CXMD(J )dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMD(J )may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart

Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ)

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMD_J) model, a suitable animal model for Duchenne muscular dystrophy.</p> <p>Methods</p> <p>We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMD_Jdogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and <it>in situ </it>zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.</p> <p>Results</p> <p>In CXMD_Jskeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMD_Jmuscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMD_Jmuscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMD_Jdid not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMD_Jdogs compared to the levels observed in normal controls.</p> <p>Conclusion</p> <p>MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.</p

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle-8

<p><b>Copyright information:</b></p><p>Taken from "Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle"</p><p>http://www.biomedcentral.com/1471-2474/9/1</p><p>BMC Musculoskeletal Disorders 2008;9():1-1.</p><p>Published online 9 Jan 2008</p><p>PMCID:PMC2257929.</p><p></p>sitive fiber. Furthermore, fast (white), hybrid (gray), or slow MHC myofibers (black) were analyzed among populations of muscle fibers with non-expression of developmental MHC (A, B) or with expression of developmental MHC (C) in terms of fiber numbers (see Table 1) and fiber sizes (A-C). Note that larger sizes of slow MHC fibers were noticeable in populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of affected muscles (B)

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle-4

<p><b>Copyright information:</b></p><p>Taken from "Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle"</p><p>http://www.biomedcentral.com/1471-2474/9/1</p><p>BMC Musculoskeletal Disorders 2008;9():1-1.</p><p>Published online 9 Jan 2008</p><p>PMCID:PMC2257929.</p><p></p

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle-5

<p><b>Copyright information:</b></p><p>Taken from "Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle"</p><p>http://www.biomedcentral.com/1471-2474/9/1</p><p>BMC Musculoskeletal Disorders 2008;9():1-1.</p><p>Published online 9 Jan 2008</p><p>PMCID:PMC2257929.</p><p></p>scle and diaphragm of a normal (A) or an affected dog (B, C) at adolescent (2 or 4 months old) or adult stages (10 or 11 months old). The numbers under the ages show total fibers examined. MHC expression between two groups (normal, dMHC (-) affected, dMHC (-); affected, dMHC (-) affected, dMHC (+)), between muscles (TC muscle diaphragm), or among ages (2, 4, and 10 or 11 months) was analyzed by Yates's chi-square test. Significant differences (< 0.05) were detected in all tests, except for no significant differences between 4 and 10 months old in normal TC muscles or diaphragms. Note that slow MHC fibers were consistently larger than other fibers, in populations of muscle fibers without developmental MHC of affected diaphragms. In populations of muscle fibers co-expressing developmental MHC and other MHC isoform(s), slow MHC and hybrid fibers were increased markedly in the affected diaphragm at 4 and 11 months old, unlike TC muscles

Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle-1

<p><b>Copyright information:</b></p><p>Taken from "Dystrophin deficiency in canine X-linked muscular dystrophy in Japan (CXMD) alters myosin heavy chain expression profiles in the diaphragm more markedly than in the tibialis cranialis muscle"</p><p>http://www.biomedcentral.com/1471-2474/9/1</p><p>BMC Musculoskeletal Disorders 2008;9():1-1.</p><p>Published online 9 Jan 2008</p><p>PMCID:PMC2257929.</p><p></p>sitive fiber. Furthermore, fast (white), hybrid (gray), or slow MHC myofibers (black) were analyzed among populations of muscle fibers with non-expression of developmental MHC (A, B) or with expression of developmental MHC (C) in terms of fiber numbers (see Table 1) and fiber sizes (A-C). Note that larger sizes of slow MHC fibers were noticeable in populations of muscle fibers expressing fast and/or slow MHC(s) but not developmental MHC of affected muscles (B)