Evaluation of the cross-bridge-dependent change in the Ca2+ affinity of troponin C in aequorin-injected ferret ventricular muscles

Ca2+ affinity of cardiac troponin C (TnC) is regulated by the active cross-bridges (downstream-dependent mechanism). In the present study, we showed one of the methods to evaluate the downstream-dependent change in the Ca2+ affinity of TnC during contraction using the aequorin-injected ferret papillary muscle. For this purpose, the tension-dependent change in the extra-Ca2+ (a transient increase in the intracellular Ca2+ concentration (Ca2+]i) in response to a quick length reduction) was measured under various conditions. We examined whether the regression line between the magnitude of tension reduction and the magnitude of the normalized extra-Ca2+ (the extra-Ca2+ was divided by Ca2+]i immediately before length change) (the normalized extra-Ca2+-tension relation) in twitch contraction can be used for the estimation of the downstream-dependent change in the Ca2+ affinity of TnC. The normalized extra-Ca2+-tension relation became shallow by EMD 57033 (EMD) (one of the Ca2+ sensitizers) and by an increase in Ca2+ concentration in the solution (Ca2+]o) in a concentration-dependent manner. However, 2,3-butanedione monoxime (BDM) (one of the desensitizers) antagonized the effects of EMD and higher Ca2+]o in a concentration-dependent manner. These effects of EMD and BDM were also observed in the normalized extra-Ca2+-tension relation in tetanic contraction. The normalized extra-Ca2+-tension relation became steep by shortening the initial muscle length before contraction in tetanic contraction. Length-tension relation in twitch contraction was significantly shifted upward by higher Ca2+]o and EMD, but BDM showed the opposite effects on them in a concentration-dependent manner. Thus, the downstream-dependent change in the Ca2+ affinity of TnC which physiologically functions in intact cardiac muscle can be evaluated using the normalized extra-Ca2+-tension relation.