Kinetics of Thin Filament Activation Probed by Fluorescence of N-((2-(Iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-Labeled Troponin I Incorporated into Skinned Fibers of Rabbit Psoas Muscle: Implications for Regulation of Muscle Contraction

Abstract

Making use of troponin with fluorescently labeled troponin I subunit ( N-((2-(iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (Trybus and Taylor, 1980. Proc. Natl. Acad. Sci. 77:7209–7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (Brenner et al., 1999. Biophys. J. 77:000–000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca 2+ concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca 2+ concentrations. At all Ca 2+ concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca 2+ is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca 2+-dependent equilibration among different states of the thin filament can fully account for the Ca 2+ dependence of force redevelopment and the fluorescence changes described in this study.