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

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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 length-dependent 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 .gtoreq. 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 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 cross-bridge reattachment. These experiments do not exclude the existence of length-dependent calcium binding, but strongly suggest that there is increased calcium binding to the activating sites when cross-bridges attach.