mutations affect the rate of nucleotide exchange from the nucleotide-binding pocket and show altered ATP-binding and ADP release rates. Homology models of myosin S1, either D45 or Mhc5, suggest a possible mechanism by which the single point mutation can alter the kinetic properties of the myosin head. 2562-Pos Board B532 Comparison Of Mechanical Properties Of Single Intact Fibres From Wild- type And Mlc/migf-1 Mouse Muscle Barbara Colombini 1 , Giulia Benelli 2 , Marta Nocella 2 , Antonio Musaro ` 3 , Giovanni Cecchi 1 , Maria Angela Bagni 1 . 1 Interuniversity Institute of Myology, Dept. Scienze Fisiologiche, University of Florence, Firenze, Italy, 2 University of Florence, Firenze, Italy, 3 Interuniversity Institute of Myology, Dept. Istologia ed Embriologia Medica, University of Rome ‘‘La Sapienza’’, Rome, Italy. In this study we compared the mechanical properties of single intact muscle ﬁbres of wild-type (WT) and MLC/mIgf-1 (TG) mice, in which the localized Igf-1 transgene expression sustains hypertrophy (Musaro ` et al., Nat. Genet. 27, 2001). The study has been focussed on ‘‘static stiffness’’ (SS), a non cross- bridge calcium-dependent stiffness previously identiﬁed in activated frog muscle ﬁbres (Bagni et al., Biophys. J. 82, 2002). Single intact ﬁbres, dissected from the ﬂexor digitorum brevis muscle, were mounted in an experimental chamber (~23  C) between the lever arms of a force transducer and of an electromagnetic motor to apply fast stretches. Sarcomere length was measured by means of a videocamera and with laser diffraction. Te- tanic tension and force-velocity relation in WT and TG mice were not signiﬁ- cantly different, however, the maximum shortening velocity (Vmax) was faster than previously reported and comparable with frog muscle. Compared to frog ﬁbres, the plateau of length-tension relation shifted according to the different myoﬁlament lengths. TG ﬁbres exhibited an increase in diameter and maximum force, but speciﬁc force was the same as for WT ﬁbres. SS was present either in WT or in TG ﬁbres and its time course, independent from isometric tension, was faster than in frog. A preliminary analysis suggests that the only signiﬁcant mechanical difference between WT and TG ﬁbres is in the SS properties. This may be related to a dif- ferent compliance of the structure responsible of the SS that we speculated could be titin. 2563-Pos Board B533 Reactive Oxygen Species Alter Activation Of Cardiac Myoﬁlaments And Modify Sarcomeric Proteins Benjamin S. Avner, R. John Solaro. University of Illinois at Chicago, Chicago, IL, USA. The generation of reactive oxygen species [ROS] such as hydrogen peroxide [H 2 O 2 ] is elevated in acute and chronic cardiac pathophysiology. Post-trans- lational oxidative modiﬁcation of sarcomeric proteins important to cardiac function, such as actin and tropomyosin [Tm], represents a possible mech- anism by which ROS may induce changes in cardiac function. Here we present data that test the hypothesis that ROS modify the function of ven- tricular muscle through oxidation of sarcomeric components. We directly exposed cardiac muscle to oxidation by treatment of detergent-extracted myoﬁbril bundles from murine papillary muscles with 2.5 mM H 2 O 2 . From the same hearts, we prepared homogenates of the ventricular sarco- meric proteins; each sample was divided and processed with and without ex- posure to H 2 O 2 . We compared the oxidation state of the proteins, employing electrophoresis to analyze the formation of reduction-sensitive disulﬁdes by oxidized cysteine residues. Compared to untreated ﬁber bundles, those treated with H 2 O 2 showed signiﬁcantly blunted cooperative activation in re- sponse to strong actin-myosin cross-bridge binding, as measured by addition of N-ethylmaleimide modiﬁed myosin sub-fragment 1 [NEM-S1]. Cross- bridge dependent effects are important for full activation of the cardiac thin ﬁlament and are believed to control the kinetics of ejection and relax- ation. Results from ‘‘diagonal’’ gels (SDS-PAGE run successively under non-reducing and reducing conditions) revealed reduction-sensitive products which were more abundant in peroxide-treated compared to untreated tissue samples. Western immuno-blot analysis conﬁrmed that these products con- tained actin and Tm. Overall, our ﬁndings represent evidence for the ROS-induced oxidation of myoﬁbrillar proteins along with impairment in cardiac muscle function. Investigation of whether the endogenously gener- ated ROS observed in pathological settings have similar effects in vivo will aid in assessing the signiﬁcance of these modiﬁcations, and may suggest a therapeutic target. 2564-Pos Board B534 Effects Of Blebbistatin And BDM (2,3-Butanedione Monoxime) On The Short-range Mechanical Properties Of Murine Diaphragm Muscle Fibers Mihail I. Mitov, Kenneth S. Campbell. Department of Physiology, University of Kentucky, Lexington, KY, USA. Blebbistatin (BLEB) and 2,3-butanedione monoxime (BDM) are well-know inhibitors of myoﬁlament force production and useful tools in structural and functional studies of cell motility and muscle contraction. In this study, we investigated the effects of BLEB and BDM on the short-range mechanical properties of single chemically permeabilized murine diaphragm ﬁbers. BLEB and BDM were used in separate sets of experiments to reduce isomet- ric force in saturating Ca2þ solution to approximately 50% of the control value. Muscle ﬁbers were subjected to repeated triangular length changes (paired ramp stretches/releases, 0.04 l 0 , 0.33 l 0 s 1 ) imposed under ﬁber length control in solutions with free Ca 2þ concentrations ranging from pCa 9.0 to pCa 4.5. Short-range stiffness values were calculated from the slopes of regression lines ﬁtted to the ﬁrst 15 ms of XY plots of force against muscle length for each stretch response and expressed as Young’s Moduli. Analysis of results obtained in control Ca2þ solutions (without BLEB or BDM) showed that short-range stiffness increased proportionately with the level of isometric force. Experiments performed with BLEB showed that short-range stiffness declined in proportion with the reduction in isometric force (no change in the stiffness/force ratio). In contrast, BDM produced a dispropor- tionately large decrease in isometric force (that is, the stiffness/force ratio in- creased in the presence of BDM, ANACOVA test, p<0.001). These results support the hypothesis that BLEB and BDM reduce isometric force in skeletal muscles by different mechanisms. BLEB seems to prevent myosin heads from attaching to the thin ﬁlament while BDM probably reduces force by decreas- ing the rate at which myosin heads undergo tension-generating biochemical transitions. Cardiac Muscle I 2565-Pos Board B535 Stretching Cardiac Trabeculae Increases the Force by Decreasing the Cross-bridge Weakening Rate in a Velocity Dependent Manner Moran Yadid, Amir Landesberg. Technion - Israel Institute of Technology, Haifa, Israel. Background: Stretch increases the force and decreases energy consumption in skeletal muscle. However, the underlying mechanisms and the effects of stretching cardiac muscle remain elusive. We hypothesized that stretch in- creases the force by modulating the cross-bridge (XB) cycling rates. Methods: Trabeculae (n¼6) were isolated from rat right ventricles. Sarco- mere length was measured by laser diffraction and controlled by a fast servo- motor. The number of strong XBs (N XB ) was quantiﬁed by measuring the dy- namic stiffness. Ramp stretches (n¼42) at different velocities and onset times were imposed on sarcomere isometric twitches. Normalized stress (stiffness) enhancement, s E (K E ), was deﬁned as the increase in the stress (stiffness) dur- ing stretch normalized by the instantaneous isometric stress (stiffness). Re- sults: Stretches yielded identical increases in s E and K E , implying that the stretch increases force by increasing N XB . A unique linear relationship was ob- served between the instantaneous normalized stress and stiffness, for all the stretch velocities (1.0350.078, R 2 ¼0.9950.026), suggesting that the unitary force per XB is constant for all stretch velocities (in contrast, a velocity de- pendent decrease in the force per XB was obtained during sarcomere shorten- ing, in congruent with previous publication). The rate of s E development de- pended linearly on the stretch velocity (7.3551.07 [1/mm]). Interestingly, the rate of s E development was independent of the stretch onset time, indicating that it is not dominated by changes in XB recruitment, but is an inherent prop- erty of the strong XB, since the population of available XB varies during the twitch. Conclusions: Constant force per XB, independence on the recruitment rate, and the linear dependence of s E on the stretch velocity, strongly suggest that stretch decreases the rate of XB turnover from strong to weak conforma- tion in a velocity dependent manner. 2566-Pos Board B536 Microfabricated Post Array Detectors to assess cardiomyocyte forces in- duced on their environment via focal adhesions Anthony G. Rodriguez, Sangyoon Han, Michael Regnier, Nathan Sniadecki. U. of Washington, Seattle, WA, USA. There is vast potential in regenerative medicine to improve cardiac muscular dysfunction, but difﬁculties arise in functionally integrating implanted cells Tuesday, March 3, 2009 497a