Extra calcium on shortening in barnacle muscle. Is the decrease in calcium binding related to decreased cross-bridge attachment, force, or length?

Abstract

Barnacle single muscle fibers were microinjected with the calcium-specific photoprotein aequorin. We have previously shown (Ridgway, E.B., and A. M. Gordon, 1984, Journal of General Physiology, 83:75-104) that when barnacle fibers are stimulated under voltage clamp and length control and allowed to shorten during the declining phase of the calcium transient, extra myoplasmic calcium is observed. The time course of the extra calcium for shortening steps at different times during the calcium transient is intermediate between those of free calcium and muscle force. Furthermore, the amplitude increases with an increased stimulus, calcium transient, and force. Therefore, the extra calcium probably comes from the activating sites on the myofilaments, possibly as a result of changes in calcium binding by the activating sites. The change in calcium binding may be due, in turn, to the change in muscle length and/or muscle force and/or cross-bridge attachment per se. In the present article, we show that the amount of the extra calcium depends on the initial muscle length, declining at shorter lengths. This suggests lengthdependent calcium binding. The relation between initial length and extra calcium, however, parallels that between initial length and peak active force. The ratio of extra calcium to active force is therefore virtually independent of initial length. These data do not distinguish between a direct effect of length on calcium binding and an indirect effect owing to changes in cross-bridge attachment and force through some geometrical factor. The amount of extra calcium increases with the size of the shortening step, tending toward saturation for steps of >_10%. This experiment suggests that calcium binding depends on muscle force or cross-bridge attachment, not just length (if at all). There is much less extra calcium seen with shortening steps at high force when the high force results from stretch of the active muscle than when it results from on May 11, 2018 jgp.rupress.org Downloaded from http://doi.org/10.1085/jgp.90.3.321 Published Online: 1 September, 1987 | Supp Info: 322THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 90 9 1987 increased stimulation of muscle. Thus, the number of attached cross-bridges appears to be more important than force itself. After a conditioning step decrease in length, force redevelops. A second test shortening step during force redevelopment produces less extra calcium than is seen in the absence of the conditioning step. Delaying the test step until more force has redeveloped increases the extra calcium seen with the test step, the increase paralleling the force redevelopment. This implies that calcium binding is increased by crossbridge reattachment. These experiments do not exclude the existence of lengthdependent calcium binding, but strongly suggest that there is increased calcium binding to the activating sites when cross-bridges attach.

Actin‐filament motion in the in vitro motility assay has a periodic component

“…The possibility of actin-based cooperativity comes from the observations of Gordon and Ridgeway [1987], who provided evidence that the presence of an active cross-bridge enhances further cross-bridge formation. A similar indication of cooperativity can be drawn from tension-noise measurements: In skinned fibers [Iwazumi, 1987] and in the motility assay [Ishijima et al, 1991], tension noise was found to be too low in magnitude to be explained if cross-bridges were acting randomly and independently.…”

The role of troponin C in the length dependence of Ca(2+)‐sensitive force of mammalian skeletal and cardiac muscles.

“…Similarly, there are recent indications that both rigor and active cross-bridges modify the fluorescent properties of skeletal TnC, indicating some functional modification of this moiety (Guth & Potter, 1987;Gordon, Ridgeway, Yates & Allen, 1988). Such a mechanism appears of major importance in explaining the hysteresis loop of contraction (Gordon & Ridgeway, 1987;Harrison, Lamont & Miller, 1988;, as well as the related phenomenon of Ca2' release from binding sites on myofilaments Sys & Brutsaert, 1989) in skinned fibres. However, it seems unlikely that variation in the cross-bridge population provides a primary mechanism with which to explain Starling's law because: (1) the decrease in the number of cycling cross-bridges at the short length is relatively small, if any (5-15 %; Kentish, ter Keurs & Allen, 1988;0% in Stephenson, Stewart & Wilson, 1989), (2) stretching the length beyond 24,um continues to increase Ca2+ sensitivity (Endo, 1972;Fabiato & Fabiato, 1978), even as cross-bridge number decreases with partial filament overlap.…”

Myosin Crossbridge Activation of Cardiac Thin Filaments

“…19 -21 Considerable support for the physiological relevance of crossbridge-induced increases in Ca 2ϩ binding affinity of TnC has emerged from experiments in which intracellular [Ca 2ϩ ] is measured with Ca 2ϩ indicators. For example, in invertebrate striated muscles, shortening of the muscle during the relaxation phase was found to give rise to an extra Ca 2ϩ transient, which the authors interpreted as dissociation of Ca 2ϩ from TnC subsequent to the shortening-induced reduction in numbers of strongly bound crossbridges (Gordon and Ridgway 22 and references therein). Similarly, extra Ca 2ϩ appeared in the myoplasm of ferret ventricular muscle when the muscle was allowed to shorten, thereby reducing force and the number of crossbridges bound to the thin filament.…”