Spectrofluorometric analysis of length-dependent conformational changes in cardiac troponin C

Abstract

Length modulation of cardiac muscle is manifested in the Frank–Starling relation of the heart. Recently, it has been shown that length-dependent changes in SH reactivity of cardiac troponin C (cTnC) occurred in association with cross-bridge attachment and Ca²⁺. However, the presence of two SH groups (Cys-35 and Cys-84) in the regulatory region of cTnC complicates efforts to detect conformational changes. In this study skinned porcine cardiac fibers were reacted with 7-diethylamino-3-[4′maleimidylphenyl]-4-methylcoumarin (CPM). Alkaline urea gel electrophoresis, along with protein elution, was used to isolate filament bound cTnC. Analysis of fluorescence measurement showed that there is a Ca²⁺-increased fluorescence for CPM-labeled cTnC in long fibers (sarcomere length = 2.2 ∼ 2.5 μm) but not in short fibers (sarcomere length = 1.6 ∼ 1.8 μm). In addition, the labeled cTnC was measured for the fluorescence decrease over time by adding a non-fluorescence energy acceptor, 4-dimethylaminophenylazophenyl-4′maleimide (DABMI), in the presence and absence of Ca²⁺. Fluorescence quenching by DABMI is not affected by Ca²⁺ in long fibers but it is significantly increased in short fibers. However, the fibers maintained in the relaxed state with 5 mM MgATP and 1 mM Vanadate showed no length effect on the CPM-labeled cTnC in terms of the Ca²⁺-mediated changes in fluorescence spectrum and in fluorescence quenching by DABMI. All together, our results suggest that the relative reactivities of Cys-35 and Cys-84 vary with sarcomere length.