The overall goals of this project are to understand the regulation and pathways of intracellular proteolysis in cardiac and skeletal muscle. Our preliminary results indicate that myotubes actively take up several labelled proteins by pinocytosis; these proteins are subsequently degraded. In addition, methods for selective osmotic lysis of pinosomes permitting microinjection of proteins into the cytoplasm have been adapted for muscle; such microinjected proteins also undergo degradation. These methods, as well as red cell-mediated microinjection, offer the possibility of directing proteins and inhibitors into different cellular compartments e.g. lysosomes and cytosol. Using these procedures, information will be obtained on the sites of protein degradation, responsible proteases, degradative intermediates and aspects of protein structure important in determining rates of proteolysis under different physiological conditions. Such conditions will include the presence and absence of serum, multiplication stimulating activity, calcium and stretch. Degradation of myofibrillar vs. soluble enzymes, native vs. denatured proteins, and methylated vs. native proteins will be compared to identify features of proteins which influence rates as well as sites of degradation. Proteins initially microinjected into the cytoplasm or allowed to remain in pinocytic vacuoles will be followed as a function of time by use of light and E.M. autoradiography and histochemistry to determine whether particular proteins are routed to lysosomal or cytoplasmic locations. Degradation and accumulation of proteins in different compartments will be examined in the presence and absence of known lysosomal protease inhibitors, and inhibitors of non-lysosomal ATP- and calcium-dependent proteolysis. Attempts will also be made to detect intermediates in proteolysis which are either fragments of labelled proteins or possibly higher molecular weight covalent complexes formed by conjugation of ubiquitin to proteins. These studies should clarify our understanding of intracellular proteolysis in muscle. In addition, optimization and characterization of mass microinjection methodology in cardiac and skeletal muscle cells should be of broad interest to workers in many areas of muscle biology.
