UNLOADED SHORTENING OF SKINNED MUSCLE-FIBERS FROM RABBIT ACTIVATED WITH AND WITHOUT CA2+

Unloaded shortening velocity (V-US) was determined by the slack method and measured at both maximal and submaximal levels of activation in g lycerinated fibers from rabbit psoas muscle. Graded activation was ach ieved by two methods. First, [Ca2+] was varied in fibers with endogeno us skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified f orm of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (V-E) followed by a slower late phase (V-L). In fib ers with endogenous sTnC, both V-E and V-L varied with [Ca2+], but V-E was less affected than V-L. Similar results were obtained after extra ction of TnC and reconstitution with either sTnC or cine, except for a small increase in the apparent activation dependence of V-E. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, V-E and V-L were similar to those obta ined in fibers reconstituted with sTnC or cTnC at equivalent force lev els. In these fibers, which contained aTnC and cTnC, V-E and V-L incre ased with isometric force when [Ca2+] was increased from pCa 9.2 to 4. 0. Fibers that contained a mixture of aTnC and cTnC were then extracte d a second time to selectively remove cTnC. In fibers containing aTnC only, V-E and V-L were proportional to the resulting submaximal isomet ric force compared with maximum Ca2+-activated control. With aTnC alon e, force, V-E and V-L were not affected by changes in [Ca2+]. The simi larity of activation dependence of V-US whether fibers were activated in a Ca2+-sensitive or -insensitive manner implies that V-US is determ ined by the average level of thin filament activation and that, with s TnC or cTnC, V-US is affected by Ca2+ binding to TnC only.