| Cardiac muscle fibers consist of bundles of myofibrils in which runs a series of sarcomeres where the contractile forces are produced by cyclic interactions between actin (thin filament) and myosin (thick filament) in overlap, and then transmitted to extracellular matrix and adjacent fibers and to the heart wall to squeeze out the blood filled in the heart cavity through a series of sarcomeres. Since the elastic properties influence the response of fibers to applied forces and may cause cardiac dysfunction, it is of importance to study the elastic behavior of fibers in response to forces in both directions. As the main component of endomysium, collagen fiber should be studied to make sure the function of cardiac muscle. The ultrastructural and biomechnical studies on collagen fiber would help us to understand how collagen affect on the conmunication pathway between cardiac muscle cells and extracellular matrix, and how collagen play a key role on tissue growth, development, disease, repaire and regeneration. Both of them will provide a scientific basis for clinical treatment.In this study, surface ultrastructure and biomechanics of bovine cardiac muscle fibers, insect flight muscle and rat tail tendon collagen I fibers in different physiological conditions were examined with high resolution atomic force microscopy and nanoindentation in order to find the relation among fiber ultrastructure, biomechanics and physiological function. Through the studies above, several important results were achieved as follows:(1) Sarcomere length of 1.22±0.02μm (n=5) in rigor with a significant 9% increase in sarcomere length in relaxing state (1.33±0.03μm, n=5), indicating that overlap move with the changing physiological conditions. Compression elasticity curves along with sarcomere locations have been taken by AFM compression processing. Coefficient of Z-line, I-band, Overlap, and M-line are 25±2pN/nm, 8±1pN/nm, 10±1pN/nm, and 17±1.5pN/nm respectively in rigor state, and 18±2.5pN/nm, 4±0.5pN/nm, 6±1pN/nm, and 11±0.5pN/nm respectively in relaxing state. Young's Modulus in Z-line, I-band, Overlap, and M-line are 115±12kPa, 48±9kPa, 52±8kPa, and 90±12kPa respectively in rigor, and 98±10kPa, 23±4kPa, 42±4kPa, and 65±7kPa respectively in relaxing state. The elasticity curves has shown a similar appearance to the section analysis profile of AFM height images of sarcomere and the distance between adjacent largest coefficient and Young's Modulus is equal to the sarcomere length measured from the AFM height images using section analysis, indicating that mechanic properties of fibers have a similar periodicity to the topography of fibers.(2) Sarcomere length of insect flight muscle fiber in rigor, relaxed and 3 activated states were 2.10±0.05μm(rigor), 3.10±0.10μm(relaxed), 2.50±0.15μm(activated, 2mM Ca2+), 2.60±0.25μm(activated, 5mM Ca2+) and 2.55±0.15μm(activated, 10mM Ca2+) respectively. A-band kept 1.50μm in length whatever the physiological state the flight muscle fibers be at when A-band had changed from 0.7μm to 1.6μm,which was consistent with the slip model of the mechanical and space structure of actin and myosin filaments (i.e. thin and thick filaments) in overlap zone. The spacing between the adjacent thick filaments were 53nm(relaxed),58.5nm(rigor),56.7nm(activated, 2mM Ca2+),54.8nm(activated, 5mM Ca2+) and 55.6nm(activated, 10mM Ca2+), then a equal volume contractile model was introduced according to the change of sarcomere length. And the distance of 37.5±0.5nm between adjacent crossbridges on myosin filament was achieved. And the Young's Modulus of the same muscle fiber in the same physiological state in different sarcomere locations were in the order of Z-line>M-line>overlap>I-band;the value in the same loaction were in the order of activated(5mM Ca2+)>activated(2mM Ca2+)> activated (10mM Ca2+)。(3) D-spacing length of air-dried collagen fiber kept around 66.67nm in different drying time, when the gap depth increased from 4.4nm to 4.7nm (~7%) and the diameter decreased from 201nm to 187nm (~7%). 1h antibody binded collagen fibers were chosed to analysis the binding position of antibody according to the image quality, and the overlap was determined to be the binding location of anti-collagen I antibody on collagen I fiber through the amplifier(gold nanoparticles) and high resolution atomic force microscopy.(4) Friction and hieght images of labeled samples using unfunctionized tips showed us the overlaps were the binding location of antibody on collagen fibers. And the adhesive force on overlap increased by 17.8%,48.9% and 82.2% in 20min, 1h and 2h labeling time respectively,which demonstrated that the antibody were fixed to overlap zone. And single interaction between antibody and collagen fiber was ~ 250pN acording to the results from the force-distance curves recorded with the tip functionized by anti-collagen I antibody.
|
PDF Full Text Request