Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch-hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher levels of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fibers revealed a large cytoplasmic space devoid of myofibrils, but containing polysomes, sarcoplasmic reticulum and T-membranes, mitochondria, Golgi complexes, and nascent filament assemblies. Tallies from electron micrographs indicate that myofibril assembly in stretched fibers followed a set sequence of events. (a) In stretched fiber ends almost the entire sarcolemmal membrane was electron dense but only a portion had attached myofibrils. Vinculin, detected by immunofluorescence, was greatly increased at the MTJ membrane of stretched muscles. (b) Thin filaments were anchored to the sarcolemma at the electron dense sites. (c) Thick filaments associated with these thin filaments in an unregistered manner. (d) Z-bodies splice into thin filaments and subsequently thin and thick filaments fall into sarcomeric register. Thus, the MTJ is a site of mRNA accumulation which sets up regional protein synthesis and myofibril assembly. Stretched muscles also lengthen by the addition of myotubes at their ends. After 6 d of stretch these myotubes make up the majority of fibers at the muscle ends. Essentially all these myotubes repeat the developmental program of primary myotubes and express slow MHC. MHC mRNA distribution in myotubes is disorganized as is the distribution of their myofibrils.

A monoclonal antibody (mAb 37) specific for alpha-myosin heavy chain (alpha-MHC) is used to follow the spatial and temporal incorporation of alpha-MHC into rabbit left ventricular myocytes. The expression of the two adult cardiac MHC genes, alpha and beta, is regulated by manipulating the thyroid hormone level of the animal. 10 wk on a propylthiouracil diet down-regulates expression of alpha-MHC to near 0%. alpha-MHC gene expression is up-regulated by injecting L-triiodothyronine (100 micrograms/kg per d) for 1-4 d. This protocol provides a means by which to follow the redistribution pattern of alpha-MHC within the myocyte in vivo. A uniform distribution of immunofluorescent signal is seen within every myocyte throughout the left ventricle. Ultracryomicrotomy without fixation is used to obtain sections for immunogold-electron microscopy. To quantify the immunogold method the density of gold-labeled antibody per unit of area tissue is determined for various regions of the sarcomere. Tissue from normal and 2-wk baby has a uniform distribution of gold density along the length of the A band. The average gold density of the A band increases with days of thyroid injection from 38 +/- 4 grains/micron 2 (n = 2 animals) (mean +/- SE) at day 1 to 182 +/- 59 grains (n = 2 animals) at day 4. There is a nonuniform incorporation of the newly synthesized alpha-MHC within the A band of thyroid-treated animals since 50% more of the alpha-MHC is found at the end of the A band while the center of the A band has 40% less than the average alpha-MHC content (grains/micron 2, n = 7 animals). These results support a thick filament assembly model that allows every myosin in a thick filament to be exchanged with new myosin. However, in the intact functioning myocyte, there is greater exchange of new myosin at the ends than in the central region of the thick filament.