Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle.
Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.
Martyn
D A
DA
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA. dmartyn@boieng.washington.edu
Gordon
A M
AM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012235:Rhodamines / Q000378:metabolism; D012518:Sarcomeres / Q000737:chemistry / Q000378:metabolism; D019209:Troponin C / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D012235:Rhodamines; D019209:Troponin C; C005358:tetramethylrhodamine; D002118:Calcium