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Heat-shock protein 72 (HSP72), the inducible isoform of the HSP70 family, is constitutively expressed in rat hindlimb muscles in proportion to the content of type I muscle fibers. To determine whether this relationship was maintained after fiber transformation, male Sprague-Dawley rats were treated with 3,5,3'-triiodo-DL-thyronine (T3) for 40 days or underwent surgical removal of the left gastrocnemius muscle, after which the left plantaris muscle was allowed to hypertrophy for 30 days. Hypertrophied plantaris muscles exhibited an increased number of type I fibers, type I myosin heavy-chain (MHC) protein, and HSP72 content compared with contralateral muscles. Soleus muscles from rats administered T3 exhibited an increased number of type II fibers, citrate synthase activity, and decreased HSP72 content compared with soleus muscles from controls. These results indicate that the relationship between HSP72 content and type I muscle fiber-MHC composition is maintained when muscles undergo fiber transformation and substantiate that HSP72 content in rat skeletal muscle is not directly linked to a muscle's oxidative capacity.

The most prominent group of stress or heat-shock proteins (HSPs) has an Mr of approximately 70,000 and is collectively referred to as the HSP70 family. The extent of stress inducibility and subcellular location of the various HSP70 isoforms differ, but all appear to be involved with ATP-dependent stabilization or solubilization of proteins. One isoform, termed the inducible isoform of HSP70 (HSP72i), is normally absent in unstressed cells. In a previous study, we detected a protein corresponding in Mr and pI to HSP72i in unstressed rat muscle. Therefore, it was of interest to determine if this expression in unstressed muscle cells is general or confined to specific muscle fiber types. To answer this question we have employed various rat hindlimb muscles that differ in fiber type proportion from predominantly type I (soleus) to predominantly type IIB (white gastrocnemius). Proteins from muscle homogenates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to a nylon membrane, probed with a monoclonal antibody for HSP72i, and visualized using an alkaline phosphatase-conjugated secondary antibody. Immunoblot analyses demonstrate the constitutive expression of HSP72i in rat muscles comprised primarily of type I muscle fibers (soleus), but not in muscles comprised primarily of type IIB fibers (white gastrocnemius). In muscles of mixed fiber type, HSP72i content is roughly proportional to the percentage of type I fibers. These results substantiate that unstressed rat muscles express the inducible HSP72 isoform and demonstrate that its constitutive expression is proportional to the type I muscle fiber composition.

Subjecting 24-h-old cultures of quail myoblasts to incubation at an elevated temperature causes the pattern of protein synthesis to shift from the production of a broad spectrum of different proteins to the enhanced synthesis of a small number of heat-shock proteins. The synthesis of four major heat-induced polypeptides with Mrs of 88,000, 82,000, 64,000 and 25,000 achieve levels comparable to that of the major structural protein, actin. Two-dimensional electrophoretic separation and fluorographic analysis of these polypeptides establish that those with Mrs of 94,000, 88,000, 82,000, and 64,000 and pIs of 5.1, 5.2, 5.2, and 5.4, respectively, are synthesized by heat-shocked as well as by control (albeit not as intense) cultures. However, the synthesis of polypeptides with Mrs of 94,000, 64,000, and 25,000 and pI's of 5.2, 5.8, and 5.4, respectively, is detectable only in myoblasts shifted to a higher temperature. Recovery of heat-shocked myoblasts, to a normal preinduction pattern of polypeptide synthesis, takes approximately 8 h. Similar studies, completed in older, more differentiated myogenic cells, demonstrated that as cells progress through myogenesis their ability to respond to a similar temperature shift is diminished. The synthesis of some myoblastlike heat-shock proteins by fusing of cells or by myotubes requires that they be maintained at an elevated temperature at least twice as long as myoblasts. This observation and the demonstration that heat-shocked myotubes do not synthesize detectable levels of the 25,000-dalton polypeptide found in heat-shocked myoblasts, suggest that the synthetic response of myogenic cells to heat shock is dependent on the differentiative state of these cells.

Cultured quail myoblasts were labelled with 32Pi and nuclear proteins extracted before and after myoblast fusion. Histones H1, H4 and the H1-H2B-H2A complex were all phosphorylated in proliferating prefusion cultures, while histone phosphorylation was absent in B1-arrested postfusion cultures except for minor phosphorylation of the H3-H2B-H2A complex. Postfused cultures were distinguished by the appearance of the histone-like protein whch migrated slightly faster than H1. Histone phosphorylation is therefore correlated with cell proliferation, while the appearance of the new histone-like protein is associated with G1 arrest and the absence of cell division.

Electrophoretic separation on polyacrylamide gels of polypeptides extracted from skeletal and cardiac muscle of BIO 14.6 dystrophic, carrier, and random-bred normal hamsters demonstrates similar quantities and electrophoretic mobility of a protein having a relative mass of 52 000 daltons (apparent isoelectric point 6.2) from all sources examined; we have tentatively identified this protein as the intermediate filament protein desmin. Reaction of such separated polypeptides transferred to nitrocellulose blots with antibodies raised against this protein fails to show immunological differences in this 52 000 dalton protein in cardiac and skeletal muscle from normal and dystrophic animals. Indirect immunofluorescence analysis of skeletal myofibrils from 30-to 60-day normal and dystrophic animals shows no differences in Z-line staining when immunoglobulins from anti-alpha-actinin serum are used as primary antibodies. Immunoglobulins from the putative anti-desmin serum also produce Z-line staining of skeletal myofibrils from normal animals, but fail to bind to the Z-lines of some skeletal myofibrils from dystrophic hamsters.

Subjecting 9-day-old quail embryos to an elevated temperature in ovo causes limb, breast, and brain tissues to shift their patterns of protein synthesis from the production of a broad spectrum of different proteins to the new and (or) enhanced synthesis of a small number of heat-shock proteins (HSPs). The HSPs synthesized by undifferentiated breast tissue in ovo (relative masses (Mrs) 88 000, 82 000, 64 000, and 25 000) are similar to those synthesized by explanted breast tissue or by primary cultures of breast myoblasts heat-shocked in culture. Heat-shocked, 120-hour-old myotube cultures synthesize HSPs similar to those detected in heat-shocked myoblasts except that myotubes also exhibit enhanced synthesis of a 55 000 dalton polypeptide and little or no synthesis of a 25 000 dalton HSP; the failure to thermally induce a 25 000 dalton polypeptide in myotubes is related to the fused nature of these cells rather than to their state of differentiation. Myoblasts, as well as myotubes, cultured in the presence of elevated amounts of arsenite, copper, or zinc also synthesize new and (or) enhanced amounts of polypeptides with isoelectric points and immunochemical properties similar to the 25 000 and 64 000 dalton HSPs. However, elevated levels of these metal ions fail to stimulate new and (or) enhanced synthesis of other HSP-like proteins. These results demonstrate that, although the protein synthetic response of myogenic cells to chemical and thermal stress may be similar in some respects, a number of the synthetic responses are clearly different.

Recent in vivo and in vitro studies with polyribosomes from the tail muscle of T3-treated tadpoles establish that this hormone initiates a regulating effect on tadpole tail muscle which operates at the translational level and results in an overall decreased rate of protein synthesis (Saleem, M. & Atkinson, B. G. (1978) J. Biol. Chem. 253, 1378-1384). This hormone-induced decrease in the rate of protein synthesis is partially, if not wholly, due to the presence of a sarcoplasmic factor(s) inhibiting ribosomal translational efficiency. This research employs the use of a reconstituted, cell-free polypeptide synthesizing system as a means to substantiate the presence of an inhibitor and further elucidate the mechanism by which this inhibitory factor(s) depresses protein synthesis. The results of this study further demonstrate the presence of an inhibitor of protein synthesis in the tail muscle sarcoplasm of T3-treated tadpoles and suggest that this depressed synthetic activity results from an interaction of the inhibitor with ribosomal or polyribosomal constituents.

Skeletal muscle cells of genetically dystrophic mice (dy/dy) of the REJ-129 Bar Harbor strain exhibit reduced cytoplasmic levels of the enzyme creatine phosphokinase (CPK) when compared with normal (+/+) mice following SDS-gel electrophoresis of sarcoplasmic proteins. This observation has been thought to reflect "leakage" of CPK from dystrophic muscle cells through lesions in the sarcolemma. The present study has employed the freeze-fracture method to examine vast expanses of sarcolemma fracture face for determination of whether lesions do exist in the membrane or an alternate route is present for extravasation of CPK from dystrophic muscle cells. Most of the dystrophic cells examined in this study appeared intact and were therefore presumed viable. The intramembrane lipoprotein particles characteristic of PF-fracture face membrane were reduced in dystrophic as compared with normal murine skeletal muscle, and the plasmalemma possessed a greatly amplified population of caveolae as compared with nondiseased sarcolemma. No abnormal structural feature of these dystrophic muscle plasma membranes could be interpreted as a perforating focal "delta" lesion, such as the structures seen in thin plastic sections by other investigators. However, a second group of cells, generally few in number, that exhibited features indicative of necrosis (and loss of viability), were seen in both thin sections and platinum replicas. These moribund cells were usually embedded in dense sheaves of connective tissue along with other dystrophic cells that lacked signs of necrosis. The cytoplasm of the necrotic muscle cells was disorganized, as was the contractile machinery. The sarcolemma showed numerous perforations, through which CPK could escape into the tissue extracellular compartment. We conclude on the basis of our observations that the "focal lesions" reported by other investigators are not a structural feature of viable dystrophic muscle cell plasma membranes and are found only in necrotic or dying cells, and that the elevated serum levels of CPK associated with muscular dystrophy may result either from escape of the enzyme through lesions present in necrotic or dying cells or by extravasation along avenues provided by the hyperplastic mass of membrane caveolae present in dystrophic sarcolemma.