After 2 or 4 mo of bed rest (6 degrees head-down tilt) and 1 mo of ambulation, there was a tendency toward a higher percentage of fibers expressing fast myosin heavy chain (MHC) isoforms and a de novo appearance of fibers coexpressing type I+IIa+IIx and IIa+IIx MHC in human soleus fibers. After 2 and 4 mo of bed rest, the mean size of type I fibers decreased by 12 (P > 0.05) and 39%, respectively. Because myonuclear number/mm of fiber length was unchanged, myonuclear domain was smaller after bed rest than before. The mean size and myonuclear domain of type I fibers were largest after 1 mo of recovery. The effects of wearing an antigravity device (Penguin suit), which had a modest but continuous resistance at the knee and ankle (Penguin-1) or knee resistance without loading on the ankle (Penguin-2), for 10 consecutive h/day were determined during 2 mo of bed rest. Mean fiber sizes in Penguin-1, but not Penguin-2, group were maintained at or above pre-bed-rest levels, whereas neither group showed phenotype changes. Myonuclear domain in type I fibers was larger in Penguin-1 and smaller in Penguin-2 group post- compared with pre-bed rest, indicating that a single daily 10-h bout of modest muscle loading can prevent bed-rest-induced soleus fiber atrophy but has minimal effect on myosin phenotype. The specific adaptive cellular strategies involved may be a function of the duration and magnitude of the adaptive stimulus as well as the immediate activity history of the fiber before the newly changed functional demands.

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Abstract. To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic illaments. Further, striations of •4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterallyassociated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.

In an earlier study connectin, an elastic protein of striated muscle, was found to be associated with "gap filaments" originating from the thick filaments in the myofibril, but it was not clear whether it extends to Z lines or not (Maruyama, K., H. Sawada, S. Kimura, K. Ohashi, H. Higuchi, and Y. Umazume, 1984, J. Cell Biol., 99:1391-1397. In the present immunoelectron microscopic study using polyclonal antibodies against native connectin, we have concluded that the connectin structures are directly linked to Z lines from the thick (myosin) filaments in myofibrils of skinned fibers of frog skeletal muscle. There were five distinct antibody-binding stripes in each half of the A band and two stripes in the A-I junction region. Deposits of antibodies were recognized in I bands and Z lines. We suggest that connectin filaments run alongside the thick filaments, starting from a region ~0.15/~m from the center of the A band.Connectin (also called titin) is a very long flexible protein of striated muscle (1-3) that is assumed to be responsible for passive tension generated upon stretch (4,5).Immunofluorescent studies have shown that connectin is mainly located in the A-I junction area in a sarcomere (6-9). In a recent paper (8) we showed that the connectin filaments are associated with the "gap filaments" that Sj6strand described in 1962 (10). Trinick and associates (3) have demonstrated that "end filaments" extruding from the isolated native thick (myosin) filaments are morphologically indistinguishable from connectin filaments (11).On the other hand, it was not clear whether the connectin filaments run through the entire I band to Z lines or not. Wang has presented the view that connectin filaments are linked to "nebulin meshwork" that is assumed to be connected with Z lines (12). Our previous study using immunoelectron microscopic observations was inconclusive with respect to this (8). Present reinvestigation has clearly shown that connectin structures are longitudinally located through the I band reaching the Z lines from the both sides of the thick filaments. MATERIALS AND METHODS Preparation of Skinned Fibers:Single muscle fibers were dissected from semitendinous muscle of the bullfrog (Rana castesbeiana), and mechanically skinned fibers were prepared in a relaxing solution (90 mM KCI, 5.2 mM MgCI2, 4.3 mM ATP, 4.0 mM EGTA, and 10 mM PIPES, pH 7.0). Partial dissociation of thick filaments in skinned fibers was performed by the extraction with a solution of ionic strength of 0.35 (relaxing solution containing 290 mM KCI) for 10 rain at 20°C at appropriate sarcomere lengths. Skinned fibers were fixed in situ for 10 rain at 20"C with the relaxing solution or the solution of ionic strength of 0.35 containing 10% formalin.Another type of skinned fiber was prepared at low ionic strength. Skinned fibers in the relaxing solution were incubated for 10 min at 5"C in a rigor solution (110 mM KCI, 1.2 mM MgC12, 4.0 mM EGTA, and 10 mM PIPES, pH 7.0). They were then immersed for 5 min at 20"C in 0.5 mM PIPES buffer, p...