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 ISL) 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 acid dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa(50) 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.