The goal of this project is to understand myofibrillar factors that control striated muscle relaxation. There is controversy regarding the relative contribution of the Ca2+ dissociation rate from the regulatory site(s) of troponin C (TnC), the number of force generating cross-bridges and the kinetics of cross-bridge detachment to the process of relaxation. The effect of altering these factors on the relaxation rate in skinned rabbit skeletal muscle and rat cardiac trabeculae will be determined. Aim #1: Develop a biochemical model that mimics Ca2+ exchange with TnC during muscle relaxation. Working hypotheses: A) For skeletal muscle, TnC-Tnl, but not TnC alone, is an adequate model system. B) For cardiac muscle, the effects of crossbridges on Ca2+ dissociation from TnC must be considered and thus the minimally sufficient model is the reconstituted thin filament or isolated myofibril. Aim #2: Elucidate the molecular communication pathway between TnC and Tnl. Working hypothesis: The hydrophobic interactions between the regulatory domain of TnC and Tnl are critical for binding to Tnl, transferring the high Ca2+ affinity and slow Ca2+ dissociation rates to the complex and sustaining maximal force in muscle. Aim #3: Determine the effects of modifying Ca2+ exchange with TnC, cross-bridge number and cross-bridge kinetics on the rates of muscle contraction and relaxation. Working hypotheses for skeletal muscle: A) Ca2+ dissociation from TnC and cross-bridge detachment rates are similar, such that decreasing either rate slows relaxation but accelerating either rate has limited effect on relaxation. B) Relaxation rate is independent of the number of force generating crossbridges. C) The rates of Ca2+ exchange with TnC do not affect the kinetics of contraction or Vmax. Working hypothesis for cardiac muscle: Relaxation rate is modulated by the Ca2+ dissociation rate from TnC, cross-bridge number and cross-bridge kinetics. Fluorescently labeled mutants of TnC and Tnl will be utilized to alter Ca2+ dissociation rate from TnC. Cross-bridge number will be altered by [Ca2+], crossbridge inhibitors and NEM myosin S1. Cross-bridge kinetics will be modified with inorganic phosphate. Contraction and relaxation will be induced by flash photolysis of NP-EGTA and diazo-2, respectively. Elucidating the fundamental factors that modulate relaxation will improve understanding of muscle diseases that exhibit defects in relaxation.