Filament overlap affects TnC extraction from skinned muscle fibres.

Recent studies on calcium regulation of muscle contraction selectively extract troponin C (TnC) from skinned skeletal muscle fibres with a low ionic strength rigor solution containing a Ca2+/Mg2+ chelator. As previous results from this laboratory and others demonstrate a crossbridge effect, especially rigor, on many of the properties of TnC, the effects of filament overlap on TnC extraction from skinned rabbit psoas muscle fibres were investigated. Tension-pCa relationships at a sarcomere length of 2.7 microns were determined before and after a 5 min TnC extraction at sarcomere lengths of 2.3, 2.5, 2.7, 3.1, 3.3 or 3.5 microns with 20 mM Tris, pH 7.8, 5 mM EDTA. The decrease in the post-extraction maximum Ca2+ activated tension, an indicator of the amount of TnC extracted, was linearly related to the overlap of the thick and thin filaments with decreases in tension being associated with a decrease in filament overlap. The smaller fibre diameter at the longer sarcomere length could facilitate diffusion of TnC from fibre segments. However, the wide range of measured diameters, 40-120 microns, accounted for only 14% of the observed tension decrement and shrinking the fibre with polyvinylpyrrolidone did not increase the tension decrement. Increasing the sarcomere length before extraction was also found to decrease the TnC content of fibre segments along with the post-extraction maximum tension. Thus, TnC appears to be preferentially extracted from non-overlap than overlap regions of the sarcomere. These results further indicate that rigor crossbridges affect TnC other than through increased Ca2+ binding and that under the conditions used here, they retard its extraction.

Force-calcium relations in skinned twitch and slow-tonic frog muscle fibres have similar sarcomere length dependencies.

The sarcomere length (SL) dependence of the calcium sensitivity of force was measured in skinned single twitch and slow-tonic muscle fibres from frog and toad. Twitch and slow-tonic fibres were characterized by location, appearance, physiological response to calcium and by protein band patterns from sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Force-calcium relations were determined for each fibre type at two sarcomere lengths, 2.4 and 3.1 microns. Bathing solution ionic strength (IS) was 200 mM and solution pH was 7.0, 6.0 or 5.5; experiments were also done at IS = 120 mM and pH 7.0. At all pHs and ionic strengths tested, slow-tonic fibres exhibited a slower time course of force development and were more sensitive to calcium than were twitch fibres. Lowering IS increased calcium sensitivity and lowering pH decreased calcium sensitivity in both fibre types. Increasing SL increased the calcium sensitivity of force in both twitch and slow-tonic fibres at pH 7.0 and at both 200 and 120 mM IS. Lowering pH caused a decrease in the length dependence of calcium sensitivity of both fibre types; at pH 5.5 the calcium sensitivity of force in slow-tonic fibres exhibited a slight decrease with increasing SL.

Ca2+ and Sr2+ activation: comparison of cardiac and skeletal muscle contraction models.

The mechanism of contraction in rabbit fast-twich, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponin-tropomyosin regulated actin heavy meromyosin. The Ca2+ and Sr2+ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca2+ or Sr2+. By contrast, rabbit adductor magnus (fast-twich) skinned fibers required substantially higher [Sr2+] than [Ca2+] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr2+ than Ca2+ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca2+ and Sr2+ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca2+- and Sr2+-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.