Hidetsugu Ueyama

Proteasome expression in the skeletal muscles of patients with muscular dystrophy.

Abstract Previous investigators have suggested that proteolysis by calpain, a Ca2+-dependent protease, causes muscle fiber degradation in Duchenne and Becker muscular dystrophies (DMD/BMD). Recent evidence indicates that the nonlysosomal ATP-ubiquitin-dependent proteolytic complex (proteasomes) participates in muscle wasting during various catabolic states and in muscle fiber degradation in physiological or pathological conditions. To elucidate the possible role of proteasomes in dystrophic muscles, routine histochemistry and immunohistochemistry of 26S proteasomes were performed on muscle biopsy specimens obtained from patients with various neuromuscular disorders including DMD/BMD, polymyositis (PM), amyotrophic lateral sclerosis, and peripheral neuropathies, and on normal human muscle specimens. Immunohistochemically, proteasomes were located in the cytoplasm in normal human muscle, but their staining intensity was faint. Compared to control muscles, abnormal increases in both proteasomes and ubiquitin were demonstrated mainly in the cytoplasm of necrotic fibers and to a lesser extent in regenerative fibers in DMD/BMD and PM. Non-necrotic, atrophic fibers in all diseased muscles showed moderate or weak immunoreactions for the proteins; their staining intensities were stronger than those of control muscle fibers. Both proteins often colocalized well. Not all dystrophin-deficient muscle fibers showed a strong reaction for proteasomes. Our results showed increased proteasomes in necrotic and regenerative muscle fibers in DMD/ PMD, although this may not be disease-specific up-regulation. We suggest that the ATP-ubiquitin-dependent proteolytic pathway as well as the nonlysosomal calpain pathway may participate in muscle fiber degradation in muscular dystrophy.

Expression of three calpain isoform genes in human skeletal muscles

Calpain is thought to be involved in muscular degradation in progressive muscular dystrophy (PMD), especially Duchenne and Becker muscular dystrophies. To assess the expression of calpain genes in skeletal muscles of patients with myopathies, we examined mRNA levels of three calpain isoforms by the quantitative reverse transcriptase-polymerase chain reaction method in biopsied muscles from control, PMD and amyotrophic lateral sclerosis (ALS) patients. There was a statistically significant increase in calpain 1 and calpain 2 mRNA levels in PMD and ALS patients as compared to controls. In contrast, there was a decrease in expression of calpain 3 mRNA in PMD, but it was not statistically significant. Expression of calpain 1 and calpain 2 positively correlated with each other, but not with calpain 3. These results indicate that expression of calpain 1 and calpain 2, but not calpain 3, are upregulated in diseased human muscles, likely playing a regulatory role in the process of myofibrillar degradation at the transcriptional as well as posttranslational level.

Calpain and Cathepsins in the Skeletal Muscle of Inflammatory Myopathies

To clarify the significance of intracellular lysosomal (cathepsins B, L and H) and extralysosomal (calpain) proteolytic systems in the process of muscle fiber degradation in inflammatory myopathies, biopsied muscle specimens were examined from patients with polymyositis (PM) and dermatomyositis (DM). Generally, in specimens from patients with PM and DM, but not in those from normal controls, muscle fibers surrounding inflammatory infiltrates or in the perifascicular regions, and occasionally mononuclear cell infiltrates demonstrated positive immunostaining for calpain and cathepsins B, L, and H. In addition, enzyme activities of cathepsins B and L increased in specimens with inflammatory myopathy. These results suggest that calpain and cathepsins play a significant role in the process of muscle fiber destruction in inflammatory myopathy.

Expression of lysosome-related proteins and genes in the skeletal muscles of inclusion body myositis

Despite the unknown etiology and pathogenesis of sporadic inclusion body myositis (s-IBM), investigators have speculated that the lysosome system in muscle fiber plays a central role in rimmed vacuole formation, a hallmark of s-IBM. We explored the role of receptor-mediated intracellular transport and autophagy in the lysosomal system in the abnormal accumulation of rimmed vacuoles in s-IBM. Expressions of mannose 6-phosphate receptor (M6PR), clathrin and hApg5 and hApg12 were analyzed in muscle biopsy specimens from patients with s-IBM, amyotrophic lateral sclerosis (ALS) or peripheral neuropathy and in normal human muscle specimens by means of immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Most muscle fibers in control specimens showed little or no immunoreactivity for clathrin and M6PR, which are involved in the receptor-mediated intracellular transport. Abnormal increases in both proteins were observed mainly in the sarcoplasm of atrophic fibers in all diseased specimens. In s-IBM muscles in particular, clathrin and M6PR were often observed inside rimmed vacuoles and in the sarcoplasm of vacuolated or non-vacuolated fibers. mRNA levels of hApg5 and hApg12, which are involved in autophagic vacuole formation, as well as of M6PR and clathrin were significantly increased in s-IBM muscles in comparison to levels in normal and ALS/peripheral neuropathy muscles. Our results suggest that the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus and autophagic vacuole formation (i.e., autophagy) in the lysosome system are activated in s-IBM muscles. Remarkable accumulation of rimmed vacuoles is thought to occur because of abnormal lysosome function, especially the formation or turnover of autolysosomes after the fusion of autophagic vacuoles with the early endosomes or because of the increase in the rate of muscle fiber breakdown.

Neurogenic pulmonary edema induced by primary medullary hemorrhage: a case report

We report a case of neurogenic pulmonary edema occurring in association with primary medullary hemorrhage. A pervious healthy 28-year-old man suddenly developed severe dyspnea without cardiac failure. Radiographs and computed tomography of the chest showed pulmonary edema. A diagnosis of primary medullary hemorrhage was made some weeks later by cranial magnetic resonance imaging showing an area of low signal intensity in both T1- and T2-weighted images in the right ventrolateral, medial, and dorsal medulla, extending from low to mid levels. We suspect that edema surrounding the lesion had superimposed an element of left dorsal medullary dysfunction and that bilateral dorsal medullary involvement had induced neurogenic pulmonary edema.

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.

A new dysferlin gene mutation in two Japanese families with limb-girdle muscular dystrophy 2B and Miyoshi myopathy

We found a new dysferlin gene mutation in two Japanese families, one with limb-girdle muscular dystrophy 2B and the other with Miyoshi myopathy. All patients in the limb-girdle muscular dystrophy 2B family showed apparent proximal dominant muscle atrophy and weakness, whereas a patient with Miyoshi myopathy in the second family showed distal muscle involvement at an early stage. The common clinical feature of all patients in both families was preferential involvement of calf muscles rather than the tibialis anterior muscle, which was confirmed by muscle computed tomography scan. All patients in both families shared the same homozygous alleles for chromosome 2p13 markers, and dysferlin gene analysis revealed a novel missense mutation, a G to A transition at nt 5882, which changed aspartic acid to asparagine at codon 1837. Allele-specific polymerase chain reaction analysis was used for confirmation of the mutation and for genotype analysis of the family members.

Myosin Loss in Denervated Rat Soleus Muscle after Dexamethasone Treatment

Objective: Critical illness myopathy (CIM) is an acute myopathy that appears in the setting of critical illness or during exposure to corticosteroids and neuromuscular blocking agents. Its pathological feature is selective loss of thick myosin filaments. Our aim is to gain further insight into the pathomechanism of myosin loss in this myopathy. Methods: To clarify the expression of myosin heavy chain (MHC) and ubiquitin ligase atrogin-1 in this myopathy, histological, immunohistochemical, SDS-PAGE, and semiquantitative reverse transcriptase-polymerase chain reaction studies were performed on innervated and denervated rat soleus muscles after saline and dexamethasone treatments. Results: Denervated muscles from dexamethasone-treated rats showed marked MHC loss. The mRNA expression of ubiquitin ligase atrogin-1 was significantly increased in denervated dexamethasone-treated muscles, suggesting that the ubiquitin-proteasome pathway plays an important role in muscular wasting in CIM. Furthermore, mRNA levels of MHC I, a myosin isoform, were decreased in the denervated dexamethasone-treated muscles. Conclusion: Our findings suggest that an altered transcription rate of myosin, as well as the upregulation of multiple ubiquitin ligases, may be responsible for selective myosin loss in this myopathy.

Expression of autophagy-associated genes in skeletal muscle: an experimental model of chloroquine-induced myopathy.

Objective: Chloroquine modulates autophagocytic protein degradation in the lysosome system, thereby inducing the formation of rimmed vacuoles consisting of autophagosomes and autolysosomes in skeletal muscle. The goal of this study was to investigate the contribution of the lysosomal system, particularly autophagosome formation (an autophagic process) at the molecular level, to the abnormal accumulation of vacuoles in an experimental model of chloroquine-induced myopathy. Methods: Histological, immunohistochemical and semiquantitative reverse transcriptase-polymerase chain reaction studies were performed on innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Results: Accumulation of rimmed vacuoles was observed only in chloroquine-treated denervated muscles. Microtubule-associated protein-1 light chain-3 (LC3) protein and mRNA levels were significantly increased exclusively in denervated muscles from chloroquine-treated rats, whereas Apg5 and Apg12 mRNA levels did not change significantly. Further, the mRNA levels of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), which are associated with distal myopathy with rimmed vacuoles showing numerous rimmed vacuoles in its skeletal muscle, were not decreased in denervatedmuscles treated with chloroquine. Conclusions: LC3 mRNA may increase in association with rimmed vacuole formation in denervated muscles from chloroquine-treated rats, suggesting an increase in autophagy at the molecular level. Abnormal accumulation of rimmed vacuoles in this myopathy does not appear to be mediated by inhibition of autophagosome-related genes or GNE gene.

Elevated soluble intercellular adhesion molecules‐1 in inflammatory myopathy

Introduction – We evaluated the serum level of soluble intercellular adhesion molecule‐1 (sICAM‐1) in patients with polymyositis (PM) and dermatomyositis (DM) and investigated the correlation between the serum level of sICAM‐1 and clinical findings. Material and methods – We measured the serum level of sICAM‐1 using an enzyme‐linked immunosorbent assay in 19 untreated patients with inflammatory myopathy (14 patients with PM and 5 patients with DM), 20 patients with other neuromuscular disorders in which immunological mechanisms are unlikely to be involved, and 14 normal healthy controls. Results – The serum level of sICAM‐1 was significantly higher in patients with PM/DM compared with patients with other neurological disorders and control subjects. The sICAM‐1 level was, however, not correlated with the clinical characteristic including disease severity, the duration of illness, and the serum level of CK. Conclusion – These findings suggest that sICAM‐1 is involved in the inflammatory process of PM and DM.