No description available.

Appendix 2: Movie Clips is supplied as a Zip Archive and will need to be unzipped before viewing. The mechanism of force transfer from contracting sarcomeres to the membrane and endomysium of striated muscle fibres is unclear. The caveolar complex array in striated muscle membranes is a local concentration of cholesterol, sphingomyelin, signalling molecules and the protein caveolin-3. Immunofluorescence microscopy of caveolin-3 in the membrane reveals a regular pattern of fluorescent nodes arranged in longitudinal and transverse rows. The primary aim of this study was to analyse this pattern and how caveolin-3 behaves during contraction. Dynamic imaging and Fast Fourier Transforms (FFTs) were used to study force transmission across the fibre membrane. This pattern was studied in frozen sections of both shortened and rest-length striated muscle fibres. Direct and FFT measurements of spacings between these nodes demonstrated significant reductions in longitudinal measurements in shortened muscle when compared to rest-length muscle. Caveolin-3 nodes lay in register with underlying actin bands in both muscle states, and co-localised with dystrophin. Caveolin-3 was not detectable in C2C12 myoblasts. During differentiation expression became detectable at 2 days. Caveolin-3 was present during myoblast fusion, before forming the regular pattern on the membrane from days 4-5. Fibres became contractile after 5-6 days of development. By 12 days, muscle fibres are 1-2 mm long, multinucleated myotubes with evidence of the caveolin-3 immunofluorescence pattern seen in mature fibres. Knockdown of caveolin-3 expression greatly reduced the number of differentiated myotubes at 12 days. This pattern was not demonstrated in contracting myotubes, possibly owing to lack of permeability to the antibody. The results are consistent with the hypothesis that the force of contraction is transferred across the whole membrane rather than at fibre distal ends.

The biology of tendons has been extensively investigated previously. Many studies have looked into the development and healing of tendons. An overall picture of the events in tendon development and healing, in terms of recently recognised functional molecules is missing, particularly in a single model. The recent understanding of the importance of metalloproteinases and other enzymes in ECM degradation suggests they may be of importance in tendon healing and development. Another unexplored area with relevance to wound healing and development is the functional integrin molecules. This thesis looked at the spatial and temporal localisation of a wide range of functional molecules and at cell proliferation. The subject groups were tendon development, from the limb bud to juvenile animal, and a defined reproducible adult healing model, with time points to six months. The study was then extended to fetal tendon healing. The results were analysed qualitatively using immunohistology and the findings were supported by histological and ultrastructural analysis, which reproduced the findings of previous studies and allowed novel observations. Adult tendon healing was accessible to quantitative analysis, which added further weight to the results. Tendon wounds in the adult model were noted to be asymmetrical in terms of tissue fibrillation, and immunohistology on opposite sides of the wound. Observations of integrin and cytoskeletal patterning in the wound, suggested mechanical load may have modulated this effect. A further study looked at a defined set of wound models. These modified the mechanical load and tissue perfusion of the wound to reproduce the effects in symmetrical wounds. The results showed that the spatial and temporal localisation of collagens, growth factors, enzymes and integrins and cell proliferation were modulated over a six month healing period. This was supported by quantitative analysis. Similar, but different effects were noted in development and fetal healing. Collagen types had a similar pattern in the three models, although the results demonstrated profound differences in timing and events surrounding new matrix production. The results of the wound modulation study using a cell proliferation marker, immunohistology, Western blots and zymograms of these wounds, were analysed and found to show marked differences in morphology, enzyme production and cell proliferation. The results excluded growth factors and tissue perfusion as the cause when these factors were controlled for. Significant (p<O.05) differences were observed between wounds. The novel results are discussed, showing modulation of several collagens, MMPs, TIMPs, cathepsins, growth factors and integrins during tendon healing, development and fetal healing with reference to other studies and proposals for the modulation of tendon healing principally by mechanical forces on the wound are made. The observation is made that tendon fetal and adult healing are profoundly different. This is consistent with other models of fetal healing. A novel proposal from the results based on matrix organisation is made for the dissimilar nature of adult and fetal wound healing.

No description available.

No description available.

Muscle contraction is brought about by the interaction of the proteins actin and myosin. Xray diffraction is a useful tool for gaining information about this process. This thesis reports on the effect of sarcomere length on the X-ray diffraction patterns from active and rigor bony fish muscle. During contraction, sarcomere I.ength changes are known to slow the development of tension (Cecchi et al., 1991) and effect X-ray reflection intensities, (Elliott et al., 1963). Up until now the size of the contractile sarcomere length change in these muscles was unknown and its effect on the tension and X-ray intensities had been neglected (Harford and Squire, 1992). Previously, a time lag was observed between the intensity changes of . the first two major equatorial X-ray reflections (A(10) and A(11�». This led to a hypothesis that the low and high force attached crossbridge states are structurally distinct (Harford and Squire, 1992). In this project, a sarcomere length measurement and control system was developed. The contractile performance of the bony fish muscles was also improved. For the first time, the sarcomere length change during contraction of whole Plaice fin muscle was measured (a reduction of (3.10�±0.06)% per sarcomere) and the system could halve this change. This sarcomere length control was found to significantly increase the rate of tension development. X-ray data also showed a reduction of over half in the lag between the two intensity changes (A(10) and A(11�», providing less clear evidence that the two crossbridge states are structurally different, a finding closer to that seen in frog muscle (Cecchi et al., 1991). Using two pre-existing X-ray datasets, the effect of initial sarcomere length on the state induced in rigor bony fish muscle was also investigated. Evidence from the intensity distributions on the actin layer-lines and from electron density maps of the muscle crosssection, (Harford et al., 1994), suggested that in rigor muscles with a longer initial sarcomere length than the conventional 2.2lJm, a different state was induced in the specimens. This was characterised by a smaller tropomyosin shift, a different crossbridge labelling pattern and a different average head shape, possibly closer to that seen in active muscle.

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Inspiratory muscle fatigue (IMF) is knovm to attenuate exercise performance. One way in which this may arise is via a reduction of blood flow to other working skeletal muscle. Previous reports have shovm that IMF causes a reflexive reduction in resting limb blood flow, secondary to an h'1spiratory muscle metaboreflex, which reduces leg vascular conductance (LVC). '''hether this reflexive decline in leg blood flow is overridden in the presence of functional hyperaemic stimuli was unknown. Therefore, the purpose of the thesis was to assess the impact of inspiratory muscle work history upon limb exercise. Results: In the presence of pre-existing IMF, time to calf fatigue (calf Tlim) was significantly curtailed (9.93 ± 1.95 and 6.28 ± 2.24 min, control and IMP + PF, respectively; p < 0.01). As LVC declined during IMP, we propose that the exacerbated time course ofcalf fatigue was due to a decrease in Lve, secondary to the inspiratory muscle metaboreflex, which was elicited before calf exercise. Conversely, when the inspiratory muscles were allowed to recover prior to calf exercise, or the relative inspiratory muscle load was reduced following 4-weeks of inspiratory muscle training (IMT), calf THm was restored to its control value (calf THm = 9.55 ± 1.88 min and 9.52 ± 1.88 min, respectively; p > 0.05). However, when the inspiratory muscle load was increased after IMT to reflect the improvements in inspiratory muscle strength (19%; P < 0.01), calfTlim was attenuated (6.33 ± 1.67 min; p < 0.01). Importantly, the metaboreflex was not elicited when a significantly lower level of lMF was induced. This suggests that fatigue per se is not the trigger, rather, that a critical level of fatigue is required. These results demonstrate that; 1) calf THm is curtailed with pre-existing IMF, probably because of increased sympathetic outflow to the limbs; 2) IMT abolishes this response when an identical inspiratory muscle load is used; and 3) a critical level ofIMF is required to evoke an inspiratory muscle metaboreflex. Thus, our data suggests that the metaboreflex is potent enough to overwhelm the functional hyperaemia.

No description available.

No description available.