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We investigated how strong cross-bridge number affects sliding speed of regulated Ca2+-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca2+. Second, we varied the number of Ca2+-activatable troponin complexes at maximal Ca2+using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca2+binding at both N-terminal, low affinity Ca2+-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa50decreased by 0.12–0.18 when either heavy meromyosin density was reduced to ∼60% or the fraction of Ca2+-activatable regulatory units was reduced to ∼33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca2+. These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.

In the past decade the otolaryngologist has become interested in the problem of muscle reinnervation as it relates to laryngeal and facial paralysis. Although reinnervation by neuromuscular pedicle transfer has shown promising results in the laboratory and clinic, some investigators have had difficulty in achieving reliable results with this procedure. To further assess the technique's validity, we investigated the neuromuscular pedicle. This study utilized a strap muscle neuromuscular pedicle transfer to a contralateral strap muscle in the rabbit. The results were analyzed by the use of a number of independent measures, including electrical stimulation of the nerve, muscle contractibility, electromyography, enzyme histochemistry, reduced-silver staining for normal fibers, and the retrograde transport of the enzyme marker horseradish peroxidase. The physiologic and anatomic results demonstrated that morphologic and functional reinnervation of the experimentally isolated muscle by the transferred neuromuscular pedicle occurred. The most convincing data were produced by gross electrical stimulation, twitch and tetanic contraction, and horseradish peroxidase labeling. Electromyographic activity and other histologic findings supported the above conclusions.

The kinetics of the binding of Ca2+to the single regulatory site of cardiac muscle troponin was investigated by using troponin reconstituted from the three subunits, using a monocysteine mutant of troponin C (cTnC) labeled with the fluorescent probe 2-[(4′-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS) at Cys-35. The kinetic tracings of binding experiments for troponin determined at free [Ca2+] > 1 μmwere resolved into two phases. The rate of the fast phase increased with increasing [Ca2+], reaching a maximum of about 35 s−1at 4 °C, and the rate of the slow phase was approximately 5 s−1and did not depend on [Ca2+]. Dissociation of bound Ca2+occurred in two phases, with rates of about 23 and 4 s−1. The binding and dissociation results obtained with the binary complex formed between cardiac troponin I and the IAANS-labeled cTnC mutant were very similar to those obtained from reconstituted troponin. The kinetic data are consistent with a three-step sequential model similar to the previously reported mechanism for the binding of Ca2+to a cTnC mutant labeled with the same probe at Cys-84 (Dong et al.(1996) J. Biol. Chem.271, 688–694). In this model, the initial binding in the bimolecular step to form the Ca2+-troponin complex is assumed to be a rapid equilibrium, followed by two sequential first-order transitions. The apparent bimolecular rate constant is 5.1 × 107m−1s−1, a factor of 3 smaller than that for cTnC. The rates of the first-order transitions are an order of magnitude smaller for troponin than for cTnC. These kinetic differences form a basis for the enhanced Ca2+affinity of troponin relative to the Ca2+affinity of isolated cTnC. Phosphorylation of the monocysteine mutant of troponin I by protein kinase A resulted in a 3-fold decrease in the bimolecular rate constant but a 2-fold increase in the two observed Ca2+dissociation rates. These changes in the kinetic parameters are responsible for a 5-fold reduction in Ca2+affinity of phosphorylated troponin for the specific site.