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Immunohistochemical analysis of the inflammatory cells and complement C3 in the rat skeletal muscle was performed chronologically in bupivacaine-induced myonecrosis. At 30 minutes after injection, polymorphonuclear leukocytes appeared and increased in number, with a peak value at 12 hours, while macrophages reached the highest level at 2 days. In contrast, T cells comprised only a small population. Two weeks after the injection, all types of the inflammatory cells returned to the normal level. Deposition of complement C3 was recognized at 60 minutes at the surface membrane of degenerating muscle fiber. Our observation suggests the importance of both polymorphonuclear leukocytes and complement C3 in the early stage, and macrophages in the later stage of bupivacaine-induced myonecrosis. In addition, our findings cast doubt on the pathological significance of T cells in this model.

We have examined the effect of cycloheximide on bupivacaine-induced, macrophage-mediated muscle degeneration in rats and obtained the following results. Direct intramuscular injection of bupivacaine into soleus muscle caused uniform muscle degeneration (Ishiura, S. et al. (1983) J. Biochem. 94, 311-314). The degeneration was most prominent 48 h after injection, with many infiltrating macrophages. Intraperitoneal administration of cycloheximide prevented the bupivacaine-induced macrophage invasion and decrease of structural proteins 48 h after injection. Cycloheximide inhibited the increase in lysosomal enzymes in bupivacaine-treated muscle. The peritoneal macrophages increased in number immediately after bupivacaine injection, while cycloheximide inhibited their accumulation. These results indicated that cycloheximide inhibited muscle degradation by preventing macrophage proliferation.

Three patients including a father and his daughter as well as a sporadic case who had a history of delayed developmental milestones showed symptoms of generalized muscle weakness predominantly in the neck flexors, high-arched palate, slender stature, myopathic face and nasal tone vocalisation. Histological and histochemical examinations on the biopsied muscles demonstrated minimal nonspecific changes; mild variation in fiber size, slight abnormality in fiber type distribution and an increased number of undifferentiated type 2C fibers. The abnormal muscle pathology was assumed to have resulted from delayed muscle fiber growth and differentiation due to a probable defective neural influence upon the developing muscles.

Experimental acute degenerative changes in skeletal muscle accompanied by a drastic increase in cathepsins B&L were induced in rats by intramuscular injection of a local anesthetic, bupivacaine. Cathepsins B&L have been implicated in the rapid disappearance of muscle fibers. Degenerating muscle showed a spotty fluorescence when stained with antibodies against cathepsin B, indicating that the increased cathepsin B did not originate from the muscle itself, but from invading phagocytes. We report here results showing that cathepsin B of nonmuscle cell origin is involved in the breakdown of myofibrillar proteins in acute bupivacaine-induced muscle degeneration.

High AMP deaminase reactivity was detected in the rimmed vacuoles in skeletal muscles in adult onset acid maltase deficiency and distal myopathy with rimmed vacuole formation histochemically as well as immunohistochemically. Acid phosphatase activity was positive but myosin ATPase activity was negative in the vacuoles. AMP deaminase found in rimmed vacuoles does not seem to be associated with myosin but is possibly bound to lysosomes or other related organelles in accordance with the proliferation of autophagic vacuoles.

Localized high AMP deaminase activity was found in the rimmed vacuoles of skeletal muscles in acid maltase deficiency, distal myopathy with rimmed vacuole formation, and experimental chloroquine myopathy on histochemical staining. Acid phosphatase activity was also increased in and around these vacuoles, but the vacuoles were negative for other histochemical stainings such as with NADH-tetrazolium reductase, ATPase and phosphorylase. These findings suggest that AMP deaminase is bound to membranous components in addition to myosin in skeletal muscle.

The administration of 50 mg/kg/day of chloroquine to rats for 8 weeks produced the chloroquine myopathy characterized by autophagic vacuole formation and increases in lysosomal enzymes, especially cathepsins B & L. Coadministration of 10 mg/kg/day of a potent cysteine proteinase inhibitor, EST, and chloroquine prevented the induction of the chloroquine myopathy. Rats already suffering from the chloroquine myopathy were treated with 10 mg/kg/day of EST together with chloroquine injections for 5 weeks and also recovered remarkably from the myopathy. Thus, EST may be beneficial for myopathies associated with autophagic vacuoles.

Skeletal muscle obtained from a patient with distal myopathy with rimmed vacuole formation (DMRV) was examined by a new direct labeled antibody enzyme method of immunohistochemistry. Abnormal increases of cathepsins B and H, capable of degrading the myofibrillar proteins, were demonstrated to be localized at the site of vacuoles and other intramyofibral portions and not to be associated with concomitant increases of their endogenous inhibitors. Autodigestion by these intramyofibral lysosomal proteinases may be of major importance in focal destruction of myofibers and formation of vacuoles, because no myofiber necrosis with invasion of macrophages, overt myositis, and neuropathy was seen in the muscle. These findings provide the first reasonable explanation of myofibral breakdown and atrophy in DMRV, and should be helpful in further studies on the mechanism of myofibral breakdown in various vacuolar myopathies and other myopathies of so far unknown pathogenesis.