Endocytosis in chronically denervated mouse skeletal muscle. A biochemical and ultrastructural study with horseradish peroxidase

Abstract

Endocytosis of horseradish peroxidase was studied in vivo in innervated mouse extensor digitorum longus and soleus muscles and in the same muscles 6 days after surgical denervation, by a combination of biochemical and morphological techniques. Biochemical determination of peroxidase activity in extensor digitorum longus and soleus muscles at different times after a single intravenous injection of horseradish peroxidase followed by extensive washing of the muscles, showed that peak levels of horseradish peroxidase in muscle tissue were obtained 2 h after injection. The uptake of horseradish peroxidase in denervated extensor digitorum longus and soleus muscles was approximately three times greater than in innervated control muscles. Light and electron microscopic examination of denervated and innervated extensor digitorum longus muscles, demonstrated that horseradish peroxidase was present in the extracellular space, i.e. between muscle fibers and in transverse tubules already 30 min after an intravenous horseradish peroxidase injection. Light-microscopic examination, 2–4 h after an intravenous injection of horseradish peroxidase, revealed that a population of denervated muscle fibers contained horseradish peroxidase in bodies or vacuoles restricted to segments of the fibers. Such bodies, containing horseradish peroxidase, were not observed in innervated muscle fibers. Ultrastructural examination showed that these horseradish peroxidase-bodies were limited by a single membrane and located adjacent to transverse tubules between myofibrils showing early signs of degeneration. Coated or uncoated vesicles of about 50–100 nm appeared to originate from horseradish peroxidase-labelled transverse tubules in denervated muscle fibers. These findings suggest that endocytosis (micropinocytosis) occurs from transverse tubules in segments of denervated muscle fibers where signs of degeneration can be observed. Following the entry of horseradish peroxidase, the tracer accumulates in heterophagosomes and secondary lysosomes of diverse appearance.