Kowashi Yoshioka

Immunohistochemical dystrophin reaction in synaptic regions

To investigate the functional significance of dystrophin, we studied various tissues of control and mdx mice, and rats immunohistochemically, using anti-dystrophin antibodies. In control animals, we observed the immunohistochemical localization of dystrophin in synaptic regions such as neuromuscular junctions, the cornea and outer plexiform layer of the retina, the taste buds and neurons in the brain, as well as on the surface membrane of skeletal, cardiac and smooth muscle fibers. In mdx mice, dystrophin was absent from the surface membrane of muscle fibers and the outer plexiform layer of the retina. These results suggest that dystrophin plays an important physiological and/or structural role in the conduction system.

Possible systemic smooth muscle layer dysfunction due to a deficiency of dystrophin in Duchenne muscular dystrophy

The localization of dystrophin was examined immunohistochemically in various tissues from mice and rats as well as from biopsied human muscle specimens, using polyclonal antibodies against dystrophin. Although dystrophin was completely absent in biopsied muscle specimens from 3 male DMD patients, it was faintly observed in the surface membrane of almost all muscle fibers examined in a female DMD patient. In all controls, human and animal, a strong dystrophin reaction was observed in the surface membrane of intrafusal muscle fibers and at the neuromuscular junctions, rather than in the surface membrane of skeletal and cardiac muscle fibers. In addition, dystrophin was clearly localized in the surface membrane of smooth muscle fibers in the viscera, bronchial system, ureter, and tunica media of blood vessels, including arteries and veins, in all examined animal tissues. In mdx mice, dystrophin was absent in almost all muscle and smooth muscle fibers in various tissues and blood vessels. These results suggested a possible systemic dysfunction of smooth muscle layers, especially those of blood vessels, as well as skeletal muscle fibers, due to a deficiency of dystrophin in Duchenne muscular dystrophy.

Immunohistochemical study of calpain and its endogenous inhibitor in the skeletal muscle of muscular dystrophy.

A calcium-dependent proteinase (calpain) has been suggested to play an important role in muscle degradation in Duchenne muscular dystrophy (DMD). In immunohistochemical studies, calpain and its endogenous inhibitor (calpastatin) were located exclusively in the cytoplasm in normal human muscles. The intensity of the staining was stronger in type 1 than in type 2 fibers. Quantitative immunohistochemical study showed an increase of calpain in biopsied muscles from the patients with DMD and Becker muscular dystrophy. Abnormal increases in calpain and calpastatin were demonstrated mainly in atrophic fibers, whereas necrotic fibers showed moderate or weak immunoreactions for the enzymes. Opaque fibers and hypertrophic fibers were negative. Not all dystrophin-deficient muscle fibers necessarily showed a strong reaction for calpain. We suggest that calpain may play an important role in muscle fiber degradation, especially in the early stage of muscle degradation in muscular dystrophy.

Vascular endothelial cell injury and platelet embolism in Duchenne muscular dystrophy at the preclinical stage

Blood vessels in muscle biopsy specimens from 4 Duchenne muscular dystrophy (DMD) patients (including 3 at the preclinical stage) were examined by electron microscopy and compared with those in non-diagnostic biopsy specimens from age-matched controls and cases of other childhood neuromuscular disorders. The most striking feature was the blister-like swelling of vascular endothelial cells in the biopsied muscle specimens from the 3 preclinical stage DMD patients, which was observed in 23-39% of the small blood vessels examined. Other noticeable features in the preclinical DMD patients were: (1) replication of the basement membrane, there being more than 3 layers in 30% of the capillaries; (2) many degenerating and regenerating capillaries; and (3) platelet adhesion and aggregation in small blood vessels including small arteries and veins. Morphometric analysis showed that the capillary and endothelial cell areas were much greater in the preclinical DMD patients than in the controls or the cases of the other neuromuscular disorders. These phenomena strongly suggest an as yet undetermined process in blood vessels in preclinical DMD.

Dystrophin and dystrophin-related protein in the central nervous system of normal controls and Duchenne muscular dystrophy

To clarify the localization and characterization of dystrophin and dystrophin-related protein (DRP) in the human central nervous system (CNS), we carried out immunoblotting and immunostaining studies using three region-specific anti-dystrophin and one anti-DRP antibodies. With immunostaining, punctate immuno-reactivity of dystrophin was seen along the cell bodies and dendrites of the cerebral cortical neurons and cerebellar Purkinje cells in the normal controls autopsied. By contrast, dystrophin was not detected at all in the CNS of Duchenne muscular dystrophy (DMD) patients with intellectual disturbance. Immunoreactivity of DRP was observed in the vascular walls of both normal and DMD brains, but not in the neuronal cells. Compensatory increase of DRP was not noted in DMD brains. This study suggests that in DMD the brain-type dystrophin originally present in neurons is absent and may be related to the intellectual disturbance.

Dystrophin in control and mdx retina

To determine whether or not dystrophin really exists in the outer plexiform layer (OPL) of the retina, we studied control and mdx mice, using four kinds of polyclonal antibodies (DMDP-II, 60 kd, 30 kd and DMDP-IV) against dystrophin. Although control OPL showed a positive immunohistochemical reaction with all four antibodies, mdx OPL showed a positive reaction with DMDP-II and DMDP-IV, a negative reaction with 60 kd and 30 kd antibodies. Immunoblot analysis showed the positive band compatible with the immunohistochemical reaction.

A 900 bp genomic region from the mouse dystrophin promoter directs lacZ reporter expression only to the right heart of transgenic mice

In order to study the regulatory mechanism of developmental and tissue‐specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZgene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum, and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.

Dystrophin and a dystrophin-related protein in intrafusal muscle fibers, and neuromuscular and myotendinous junctions

SummaryTo determine whether or not and how dystrophin exists in neuromuscular junctions (NMJs) and myotendinous junctions (MTJs), we studied the mid-belly and peripheral portions of control and mdx muscles, immunohistochemically and immunoelectrophoretically, using six kinds of polyclonal antibodies, and an antibody against a dystrophin-related protein (DRP). In controls these regions and the polar region of intrafusal muscle fibers showed a rather clearer immunohistochemical dystrophin reaction than those of extrafusal muscle fibers with all antibodies used. In the muscles of mdx mice NMJs only showed a positive dystrophin reaction with the c-terminal antibody, that is, no reaction with the other five antibodies, and MTJs in mdx showed a positive reaction with the c-terminal antibody and a faint to negative reaction with the other five antibodies. In biopsied human muscles NMJs and MTJs also showed a clear reaction with all ten antibodies, i.e., six polyclonal and four monoclonal ones. Although an immunohistochemical DRP reaction was clearly seen at NMJs, only a faint or no reaction was seen on MTJs and on intrafusal muscle fibers in both mouse and human materials. Western blot analysis of control mouse muscle for dystrophin showed a clearer band for the peripheral portion, which contains many MTJs, than for the mid-belly portion. These data suggest that dystrophin really exists on MTJs, and that dystrophin and DRP exist on NMJs in mouse and human muscles.

Developmental studies of dystrophin-positive fibers in mdx, and DRP localization

Dystrophin positive fibers (DPFs) were observed in about 1% of the total muscle fibers in 1-year-old mice. Some of these fibers were found to have positive staining with all six antibodies, while others showed a negative reaction with specific antibodies. These results suggest that the most likely mechanism giving rise to these DPFs is a second site mutation which prepares in-frame deletion. A study of the frequency of DPF during development showed single and scattered DPFs in younger mice, which gradually increased in number and began to form small groups with age. DRP was observed constantly on the neuromuscular junctions in both control and mdx muscle, and surface membrane of immature muscle fibers such as regenerating fibers in mdx and newborn muscle during 2 weeks of age in control and mdx.

Novel mutation in splicing donor of dystrophin gene first exon in a patient with dilated cardiomyopathy but no clinical signs of skeletal myopathy

One cause of X-linked dilated cardiomyopathies is mutation of the dystrophin gene. We report the case of a young boy who suffered from dilated cardiomyopathy caused only by dystrophin-deficient cardiac muscle, but who did not present with any clinical signs of skeletal myopathy. Sequence analysis of the patients dystrophin gene revealed the presence of a novel single point mutation at the first exon—intron boundary, inactivating the 5′ splice site consensus sequence of the first intron. The lack of muscle weakness observed clinically can be explained by expression of the brain and Purkinje dystrophin isoforms in skeletal muscle.