Effects of Cycling and Rigor Crossbridges on the Conformation of Cardiac Troponin C

The results of work by several investigators indicate that crossbridge attachment serves as a positive feedback mechanism that transiently increases the Ca affinity of troponin C (TnC) during each normal heartbeat. To monitor structural changes in the cardiac isoform of TnC (cTnC) associated with Ca binding and crossbridge attachment in muscle, we labeled cTnC with the sulfhydryl-specific fluorescent probe 2-(4ʼ-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS). When IAANS-labeled cTnC (cTnCIAANS) was substituted for endogenous TnC, the fluorescence intensity of cardiac and skeletal muscle preparations increased substantially during rigor crossbridge attachment in the absence of Ca (pCa 9.2). In cardiac muscle, the fluorescence signal increased the same amount in rigor and maximal activation, whereas in skeletal muscle, it was higher in rigor (rigorcardiac and skeletal=1; pCa 4.0cardiac=0.98±0.13, skeletal=0.59±0.05). This indicates that crossbridge attachment alone is capable of influencing the structure of cTnCIAANS. Because the relative fluorescence intensity of cTnCIAANS was more sensitive to Ca than was force in both preparations (cardiacpCaso fluorescence=6.05±0.05, pCa50 force=5.51±0.11; skeletalpCa50 fluorescence=5.94±0.13, pCaso force=5.65±0.14), we measured the Ca sensitivity of the strong crossbridge attachment (sinusoidal stiffness was measured by imposing 1 kHz at 0.1–0.2% muscle length) in rat trabeculae. Like fluorescence, stiffness was also more sensitive to Ca than force was (pCa50 stiffness=5.49±0.05, pCaso force=5.41±0.05, p