increased the calcium sensitivity. 2D gel electrophoresis, indicated myosin as a critical target for glutahionylation under these conditions. The RMLC was phosphorylated in the psoas ﬁbers and the level remained constant during ox- idant - glutathione treatment. In summary, our data suggest that glutathionyla- tion of myoﬁlament proteins can modulate calcium sensitivity, and may play an important role in maintaining muscle function during oxidative stress. 778-Pos The Small Molecule Skeletal Sarcomere Activator, CK-2017357, is a Calcium Sensitizer that Binds Selectively to the Fast Skeletal Troponin Complex Raja Kawas, Alan Russell, Alex Muci, Bradley Morgan, Fady Malik, Jim Hartman. Cytokinetics, Inc., South San Francisco, CA, USA. Striated muscle contraction is governed by the release of Ca 2þ from the sarco- plasmic reticulum via the sarcomeric calcium sensor, the troponin complex. A trimer consisting of troponins T, I, and C, the complex undergoes calcium-de- pendent conformational changes that regulate the accessibility of myosin bind- ing sites along actin ﬁlaments. We used a high throughput screen to identify compounds that activate the ATPase activity of skinned fast skeletal myoﬁbrils; optimization of the initial hit compounds has resulted in compounds with im- proved potency and medicinal chemical properties. The most advanced exem- plar of this chemical series, CK-2017357, shifts the calcium sensitivity of de- tergent-skinned fast skeletal myoﬁbrils by >10-fold in a concentration dependent manner. This compound speciﬁcally activates fast skeletal myoﬁ- brils, with no effect on either slow skeletal or cardiac myoﬁbrils. A reconsti- tuted sarcomere assay using combinations of fast skeletal, slow skeletal, and cardiac components demonstrates that the activity of CK-2017357 requires the presence of fast skeletal troponin. Isothermal titration calorimetry indicates the compound binds directly to fast skeletal troponin with a sub-micromolar dissociation constant, while experiments with the ﬂuorescent calcium chelator Quin-2 demonstrate that CK-2017357 slows calcium dissociation from tropo- nin. Consistent with this ability to stabilize the calcium-troponin complex, CK-2017357 increases sub-maximal force development in vitro and in vivo, suggesting this mechanism may increase power or strength in diseases where muscle function is compromised due to injury, disease or age. 779-Pos Effect of Temperature on The Rates of Calcium Dissociation and Cross- Bridge Detachment in Cardiac Myoﬁbrils Reported by Troponin C Sean C. Little, Kristopher Kline, Bin Liu, Jonathan P. Davis. The Ohio State University, Columbus, OH, USA. It has been proposed that the rate limiting step of cardiac muscle relaxation re- sides in the myoﬁlaments. The primary mechanism is thought to be the rate of cross-bridge detachment (strongly governed by ADP) since it is generally as- sumed to be substantially slower than the rate at which the thin ﬁlament inac- tivates (governed by Ca 2þ dissociation from TnC). This stems from the fact that the rate of Ca 2þ dissociation from isolated TnC is orders of magnitude faster than the rate of relaxation. However, TnC does not function in isolation but as an integral component of the myoﬁlament contractile system. Furthermore, the Ca 2þ binding properties of TnC can be drastically affected by the other thin ﬁlament proteins and by myosin binding to actin. Thus, we wanted to determine the Ca 2þ dissociation rate from TnC in cardiac myoﬁbrils during different cross-bridge states. To achieve this goal, rabbit ventricular myoﬁbrils were ex- changed with human cardiac troponin containing a TnC (C35S, C84S, T53C) ﬂuorescently labeled with IANBD. Unexpectedly, via the change in TnC ﬂuo- rescence, not only could we observe the rate of Ca 2þ dissociation from TnC in the myoﬁbrils, but also what we think is the rate of cross-bridge detachment. At 15 o C and in the presence of ADP, the cross-bridge detachment rate was ~7/s, three times slower than the rate of Ca 2þ dissociation from TnC (~21/s). How- ever, at near physiological temperature (35 o C) the two rates were very similar (~60/s). Based on the temperature dependence of the rates, at temperatures be- low 25 o C, cross-bridge detachment may very well be rate limiting for relaxa- tion, but at higher temperatures both rates may be able to modulate the rate of relaxation. 780-Pos The Effect of Rigor Myosin Upon the pCa of Calcium Binding to Native Cardiac Thin Filaments Ahmed Houmedia 1 , David Heeley 2 , Howard D. White 3 . 1 University of Noachott, Noachott, Mauritania, 2 Memorial University, Saint Johns, NL, Canada, 3 Eastern Virginia Medical School, Norfolk, VA, USA. We have used double mixing stopped-ﬂow ﬂuorescence to measure the effect of calcium on the kinetics of the dissociation of the hydrolysis product deoxy- mantADP (mdADP) from cardiac myosin-mdADP and cardiac myosin- mdADP-Pi by native cardiac thin ﬁlaments. Increasing the calcium concentra- tion from pCa > 7 to pCa < 4 increased the rate of dissociation of mdADP from cardiac myosin-S1-mdADP-Pi ~100 fold from 0.5 s 1 to 50 s 1 while the rate of dissociation of mdADP from cardiac myosin-S1-ADP increased only ~10 fold from 15 s 1 to 150 s 1 . Rigor myosin-S1 bound to the thin ﬁlaments in- creased the apparent pCa of mdADP dissociation myosin-S1-mdADP-Pi from 0.12 to 0.79 uM. The change in pCa is similar to the increase in the rate of ADP dissociation but is much less than the acceleration in the rate of rate of product dissociation from myosin-ADP-Pi, ~ 100 fold. These results in- dicate that slow dissociation of phosphate limits the rate of ADP dissociation from acto(thinﬁlaments)myosin-ADP-Pi and that there are different mecha- nisms for the calcium regulation of dissociation of the two products of myosin ATP hydrolysis, ADP and phosphate. These results support a mechanism in which phosphate dissociation from actomyosin-ADP-Pi is the step of the hy- drolysis cycle that is principally regulated by calcium and do not support a mechanism such as the three state mechanism in which the regulation is a re- sult different distributions of thin ﬁlament states in presence and absence of bound calcium that occur prior to myosin binding. This work is supported by a NIH HL84604. 781-Pos Model for Transient Activation of Isometric Force by Calcium Henry G. Zot, Javier E. Hasbun, Nguyen V. Minh. University of West Georgia, Carrollton, GA, USA. The purpose of this study is to model force transients generated by vertebrate striated muscle in response to calcium pulses. We have developed an equilib- rium model for calcium activation of isometric force based on three positions of tropomyosin, i.e., troponin-dependent (B), central (C), and myosin-depen- dent (M). From the equilibrium model, we derived a complete set of ordinary differential equations that can be solved simultaneously given arbitrary cal- cium. By setting the differential equations equal to zero, steady-state activa- tion was found to reproduce the equilibrium results of the parent model. A time-dependent solution resulted by providing a pulse of calcium using a Gaussian function to control the duration and amplitude of the calcium tran- sient. The results report the fraction of tropomyosin in Position M (activation transient) as function of calcium changes over time. For a given calcium pulse, several characteristics of the activation transient varied with the rate constants used, including the amplitude of peak activation, the time lag in the peak activation, and the duration of the activation transient. If a train of calcium pulses were sufﬁciently separated in time, identical activation tran- sients returned to baseline before each pulse. However, as the time between the pulses was shortened, the activation transients became progressively fused and the amplitude increased. Using a train of submaximum calcium pulses, the activation transients were seen to rise in amplitude with each pulse and approach plateau amplitude similar in appearance to the staircase phenome- non observed for tetanic muscle stimulation. Thus, we describe a model con- sistent with the known positions of tropomyosin that reproduces the transient behavior of force development of vertebrate striated muscle. A derivation of differential equations and application to muscle activation may be found on- line (www.westga.edu/STEMresearch). This work was supported by NSF grant MCB-0508203 (HGZ). 782-Pos Equilibrium Model for Cooperative Activation of Muscle by Calcium Henry G. Zot, Javier E. Hasbun, Nguyen V. Minh. University of West Georgia, Carrollton, GA, USA. The purpose of this study is to model the cooperative activation of muscle. Current models for calcium activation are based on three positions of tropo- myosin, i.e., troponin-dependent (B), central (C), and myosin-dependent (M). Regulation of molluscan muscle, which lacks troponin, may be the basis for cooperative activation of all ﬁlamentous myosin systems. However, ﬁtting actual calcium-dependent isometric force (F-Ca) data has been difﬁcult to achieve for all muscle and, among existing models, none has been shown to be compatible with muscle that lacks troponin. We describe a mass action mechanism for cooperative activation that involves only Positions C and M. We show that our model ﬁts F-Ca of scallop striated adductor muscle (RM Simmons and AG Szent-Gyorgyi, 1985, J. Physiol. 358: 47-64). Furthermore, given troponin that regulates simply by binding actin in Position B, we show that this same model will ﬁt F-Ca of vertebrate striated muscle regulated by both native and mutant forms of troponin (MA Regnier et al., 2002, J. Physiol. 15: 485-497). Our model also ﬁts paired myosin binding and thin ﬁlament ac- tivation data (KM Trybus and EW Taylor, 1980, Proc. Natl. Acad. Sci 77: 7209-7213). The results suggest that myosin binding couples energetically to a conformational change in tropomyosin that propagates in position M. Ex- pansion of segments of tropomyosin in position M promotes the association of uncoupled myosin, which stabilizes one coupled myosin for each segment. Sunday, February 21, 2010 151a