Exocytotic "constipation" is a mechanism of tubulin/lysosomal interaction in colchicine myopathy

Colchicine, a known microtubule disrupting agent, produces a human myopathy, characterized by accumulation of lysosomes. We have created a reliable animal model of colchicine myopathy that replicates the subacute myopathy seen in humans, reproducing the chronic proximal weakness and vacuolar changes in nonnecrotic myofibers. If a microtubule network plays a role in lysosomal function in muscle, disturbance of it could alter degradation of intrinsic membrane receptors, presumably at some intracellular processing site or at exocytosis. Thus, we examined, as a possible cellular pathogenesis of colchicine myopathy, how the muscle cytoskeleton affects the degradation of membrane proteins, which are processed through the endosomal/lysosomal pathway. We used the acetylcholine receptor as a model membrane component in cultured myotubes allowed to preincubate with colchicine. We tested at which step colchicine interferes with receptor trafficking by accounting for internalization, delivery to lysosomes, hydrolysis, or exocytotic release of debris. We report that colchicine significantly decreases the exocytosis of AChRs but does not affect receptor internalization, lysosomal hydrolysis, or the number of surface membrane receptors. Further, our immunofluorescence observations revealed a morphologic tubulin network in rat skeletal muscle that is more densely distributed in white (mitochondria-poor) muscle fibers than in red (mitochondria-rich) fibers but is present in both. Ultrastructurally, immunogold labeling localized tubulin in the intermyofibrillar region in a long and linear fashion, unassociated with myofibers or mitochondria. Taken together, our findings suggest the following: (1) Microtubules likely play a functional role in the pathway of lysosomal degradation in normal adult skeletal muscle; (2) The observed decrease in overall apparent degradation of membrane receptors by colchicine must be due primarily to inhibition of exocytosis. These data indicate that lysosomal "constipation" underlies colchicine myopathy. (3) An animal model faithful to the human disorder will allow further pathogenetic studies.