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 Ca2+ affinity of troponin C (TnC) during each normal heartbeat. To monitor structural changes in the cardiac isoform of TnC (cTnC) associated with Ca2+ binding and crossbridge attachment in muscle, we labeled cTnC with the sulfhydryl-specific fluorescent probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS). When IAANS-labeled cTnC (cTnC(IAANS)) was substituted for endogenous TnC, the fluorescence intensity of cardiac and skeletal muscle preparations increased substantially during rigor crossbridge attachment in the absence of Ca2+ (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 (rigor. cardiac and skeletal=1; pCa 4.0: cardiac=0.98+/-0.13, skeletal=0.59+/-0.05). This indicates that crossbridge attachment alone is capable of influencing the structure of cTnC(IAANS). Because the relative fluorescence intensity of cTnC(IAANS) was more sensitive to Ca2+ than was force in both preparations (cardiac: PCa50 fluorescence=6.05+/-0.05, pCa50 force=5.51+/-0.11; skeletal: pCa50 fluorescence=5.94+/-0.13, PCa50 force=5.65+/-0.14), we measured the Ca2+ 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 Ca2+ than force was (pCa50- stiffness=5.49+/-0.05, pCa50 force=5.41+/-0.05, p<0.05), and this suggests that strong attachment of cycling crossbridges in zero- or low-force states may also influence the conformation of cTnC. The results from a computer model of crossbridge attachment also support this interpretation.