Cody, Mark J.

It has been found that maximum specific force (Fmax; force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in Fmax reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHCslow and MHCneo, respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHCslow and coexpressing MHCneoproduced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHCslow and MHCneo contributes to the lower Fmax seen in early postnatal development.

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Diam) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Diamfibers. Maximum specific force was determined by activation of single Diamfibers in the presence of a high-calcium solution (pCa, −log Ca2+concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.

We hypothesize that 1) the effect of denervation (DNV) is more pronounced in fibers expressing fast myosin heavy chain (MHC) isoforms and 2) the effect of DNV on maximum specific force reflects a reduction in MHC content per half sarcomere or the number of cross bridges in parallel. Studies were performed on single Triton X-100-permeabilized fibers activated at a pCa (−log Ca 2+ concentration) of 4.0. MHC content per half sarcomere was determined by densitometric analysis of SDS-PAGE gels and comparison to a standard curve of known MHC concentrations. After 2 of wk DNV, the maximum specific force of fibers expressing MHC 2X was reduced by ∼40% (MHC 2B expression was absent), whereas the maximum specific force of fibers expressing MHC 2A and MHC slow decreased by only ∼20%. DNV also reduced the MHC content in fibers expressing MHC 2X, with no effect on fibers expressing MHC 2A and MHC slow . When normalized for MHC content per half sarcomere, force generated by DNV fibers expressing MHC 2X and MHC 2A was decreased compared with control fibers. These results suggest the force per cross bridge is also affected by DNV.

The effects of the nitric oxide (NO) donor spermine NONOate (Sp-NO, 1.0 mM) on cross-bridge recruitment and cross-bridge cycling kinetics were studied in permeabilized rabbit psoas muscle fibers. Fibers were activated at various Ca2+concentrations (pCa, negative logarithm of Ca2+concentration), and the pCa at which force was maximal (pCa 4.0) and ∼50% of maximal (pCa505.6) were determined. Fiber stiffness was determined using 1-kHz sinusoidal length perturbations, and the fraction of cross bridges in the force-generating state was estimated by the ratio of stiffness during maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+activation to stiffness during rigor (at pCa 4.0). Cross-bridge cycling kinetics were evaluated by measuring the rate constant for force redevelopment after quick release (by 15% of optimal fiber length, Lo) and restretch of the fiber to Lo. Exposing fibers to Sp-NO for 10 min reduced force and the fraction of cross bridges in the force-generating state at maximal and submaximal (pCa50) Ca2+activation. However, the effects of Sp-NO were more pronounced during submaximal Ca2+activation. Sp-NO also reduced the rate constant for force redevelopment but only during submaximal Ca2+activation. We conclude that Sp-NO reduces Ca2+sensitivity by decreasing the number of cross bridges in the strongly bound state and also impairs cross-bridge cycling kinetics during submaximal activation.

The present study examined Ca2+sensitivity of diaphragm muscle (Diam) fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that Diamfibers expressing the MHCslowisoform have greater Ca2+sensitivity than fibers expressing fast MHC isoforms and that this fiber-type difference in Ca2+sensitivity reflects the isoform composition of the troponin (Tn) complex (TnC, TnT, and TnI). Studies were performed in single Triton-X-permeabilized Diamfibers. The Ca2+concentration at which 50% maximal force was generated (pCa50) was determined for each fiber. SDS-PAGE and Western analyses were used to determine the MHC and Tn isoform composition of single fibers. The pCa50for Diamfibers expressing MHCslowwas significantly greater than that of fibers expressing fast MHC isoforms, and this greater Ca2+sensitivity was associated with expression of slow isoforms of the Tn complex. However, some Diamfibers expressing MHCslowcontained the fast TnC isoform. These results suggest that the combination of TnT, TnI, and TnC isoforms may determine Ca2+sensitivity in Diamfibers.

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Diam) fibers, 2) Hyp decreases the maximum specific force (Fmax) of Diamfibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and Fmaxto a greater extent in fibers expressing MHC type 2X (MHC2X) and/or MHC type 2B (MHC2B). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC2Xand/or MHC2B. On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased Fmaxacross all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (∼40 vs. ∼20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC2Awas reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC2Xand/or MHC2Bin the Hyp Diamthan for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC2Xand/or MHC2Band that the reduction of Fmaxwith Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.