suggesting regularity in the domains interactions. In the constructs from the first two sub-periods, side-by-side dimers were observed and oligomers were seen only rarely. Co-sedimentation and solid phase assays showed binding of the constructs to the light meromyosin part of myosin but did not reveal any significant differences in the interactions. Modelling of the proteins folds and electrostatics was consistent with the observed interactions being mainly electrostatic. 815-Pos Board B570 Titin Visco-Elasticity Modulated by Limiting Ig Domain Unfolding and Refolding Jens Herzog 1 , Timothy R. Leonard 2 , Azim Jinha 2 , Walter Herzog 2 . 1 Mount Allison University, Sackville, NB, Canada, 2 University of Calgary, Calgary, AB, Canada. Titin is a molecule with spring-like domains that have specific visco-elastic properties which come into play in an orderly fashion. When lengthened within the physiologically relevant range of sarcomere excursion, Ig domains align first, followed by stretching of the PEVK region. While these spring elements behave elastically, Ig domain un/refolding is thought to be highly visco-elastic and occurs beyond physiological stretching. Using rabbit psoas myofibrils, we investigated whether titin behaves elastically at long sarcomere lengths when un/refolding of Ig domains is prevented. The first protocol consisted of stretching myofibrils to 4.0-6.0 mm/sarcomere and then superposing ten small stretch-shortening cycles. The second protocol was identical to the first, except that the ten stretch-shortening cycles followed a two minute stress relaxation hold at 4.5mm/sarcomere. Protocol 1 was associated with a loss of peak force in the ten stretch-shortening cycles indicating persistent Ig domain unfolding (Figure 1a). Protocol 2 gave steady peak cycling forces suggesting no un/refolding of Ig domains, but there was a persistent hyster- esis (Figure 1b). This result suggests that there is visco-elasticity in myofibrillar titin even when Ig domain un/refolding is prevented which contradicts observations made in isolated titin experiments. 816-Pos Board B571 An Active Role for Titin in Skeletal Muscle Krysta Powers, Azim Jinha, Walter Herzog. University of Calgary, Calgary, AB, Canada. The sliding filament theory of muscle contraction is widely accepted as the means by which muscles generate active force. Recent studies have observed enhanced titin-based force in activated rabbit psoas myofibrils that were stretched to lengths which exceed filament overlap. These forces cannot be ac- counted for in the current theory, thus, alternative mechanisms of force produc- tion should be considered. Titin has been speculated to play a role in active force by binding to the thin filament, shortening and stiffening its spring length during skeletal muscle activation. To further investigate this, the present study uses a model (mdm) in which the titin protein is mutated. This study aims to test the hypothesis that the region of titin that binds to the thin filament during acti- vation is contained within the mdm deletion in titin. If the deleted region of mdm titin modulates titin-based stiffness via activation-dependent binding to the thin filament, mdm would be deficient in this binding, resulting in a more compliant titin spring. To test this hypothesis, mouse psoas myofibrils were passively and actively stretched to ~6.0 mm/sarcomere. When wild-type myo- fibrils were actively stretched to average sarcomere lengths that exceed fila- ment overlap, force continued to increase and remained greater than the passive force at all lengths. This trend was not observed in mdm myofibrils. Actively stretched mdm myofibrils were more compliant than wild-type myo- fibrils and did not differ from passive wild-type or mdm myofibrils. An enhanced state of titin-based force has now been demonstrated in rabbit and mouse myofibrils, suggesting that modulation of titin force during activation may be an inherent property of skeletal muscle. This property is disrupted in mdm, suggesting that a critical function in the enhancement of titin-based force is lost in mdm titin. 817-Pos Board B572 Titin-Based Modulation of Sarcomere Structure as Revealed by Equato- rial X-Ray Diffraction Karen H. Hsu 1 , Younss Ait-Mou 2 , Pieter P. de Tombe 2 , Thomas C. Irving 1 . 1 BCPS, Illinois Institute of Technology, Chicago, IL, USA, 2 Moelcular Physiology, Loyola University, Maywood, IL, USA. Myofilament Length Dependent Activation (LDA) is the cellular mechanism underlying the Frank-Starling Law of the Heart. LDA is defined as an increase in force for a given amount of calcium when the muscle is stretched to a longer sarcomere length. Despite its physiological importance, the "length sensor that underlies LDA is not known. To address this issue we use X-ray diffraction of intact twitching rat papillary muscle where muscle initially at maximum sarcomere length (Lmax) is quickly released to slack length (Slack). X-ray patterns are taken is a 10 ms window just prior to force generation. Previous analyses of the data did not support the hypothesis that there was a radially outward movement of myosin heads at Lmax requiring some other explanation. The equatorial portion of our X-ray patterns show 5 pairs of well-resolved diffraction spots from the 1,0 to the 3, 0 equatorial reflection. This allowed calculation of two dimensional electron density maps (ED maps) comparing Lmax and Slack from both wild type rats and mutant rats with exceptionally long titin (J Mol Cell Cardiol. 44:983-91 2008) where the passive tension generated at Lmax, as well as LDA, is much reduced. Differ- ence ED maps (Lmax-Slack) from WT rats indicate a better localization of both thick and thin filaments at Lmax plus the existence of bridging density joining the thick and thin filaments at Lmax and not at slack. In difference ED maps from the titin mutant rats, the bridging structures are absent and only the thick filaments appeared better localized. These results support a model where a small number of crossbridges transmit titin-based strain from the thick filament to the thin filament as part of the mechanism of LDA. Supported by NIH R01HL075494 and 9 P41 GM103622. 818-Pos Board B573 Cardiac Thin Filament Structural Modulation by Sarcomere Length Younss Aitmou 1 , Karen H. Hsu 2 , Mohit Kumar 1 , Danuta Szczesna-Cordary 3 , Marion L. Greaser 4 , Tom C. Irving 2 , Pieter P. deTombe 1 . 1 Cell and molecular physiology, Loyola University Chicago, Maywood, IL, USA, 2 Biological Chemical Physical Science, Illinois Institute of Technology, Chicago, IL, USA, 3 Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, FL, USA, 4 Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI, USA. Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. LDA has been studied intensively and appears to be modulated through various mechanisms, such as contractile proteins composition and their phosphorylation status. However, the cellular molecular mechanisms that underlie this phenomenon are still not well characterized. The aim of our study is to determine whether LDA is regulated through cTnC structural changes upon stretch. Accordingly, we used a single attached skinned cardiac myocyte in combination with confocal fluorescent measurement. Using this technique, we found that stretch of a relaxed cell, in the absence of Ca 2þ , resulted in marked alterations of cTnC structure as reported by cTnC-T53C- IAF confocal fluorescence. Moreover, titin mutant cells show a drastic alter- ation of both passive tension and myofilament sensitivity to calcium (pCa50) upon stretch. Consistent with this finding, by employing time-resolved x-ray diffraction of intact, electrically stimulated rat myocardium, we found marked changes in troponin and myosin structure upon stretch in the diastolic phase (i.e. when cross-bridges are not active). Moreover, we repeated these x-ray ex- periments using rat myocardium that expresses an unusually long titin molecule and found that, when compared to WT, diastolic stretch in these muscles did not induce the structural changes in troponin and myosin observed in WT. These results strongly suggest a direct effect of sarcomere length on thin and thick filament and implicate titin to be the molecule underling the length signal trans- duction for LDA. 819-Pos Board B574 Atomic-Level Visualization of Smooth Muscle Activation by Phosphoryla- tion of the Myosin Regulatory Light Chain Brett A. Colson, Matthew A. Mauseth, David J. Kast, L. Michel Espinoza- Fonseca, Osha Roopnarine, David D. Thomas. University of Minnesota, Minneapolis, MN, USA. We have engineered site-directed probe pairs in the regulatory light chain (RLC) for exchange into smooth muscle heavy meromyosin (HMM) and subfragment-1 (S1), in order to examine the phosphorylation-induced changes in RLC structural states using time-resolved FRET (TR-FRET). Phosphorylation of the RLC is required for activation of contraction in smooth muscle and modulates force in striated muscle. Force development in smooth muscle is triggered by phosphorylation of the RLC’s N-terminal phosphorylation domain (PD) at Ser19, alleviating inhibitory interactions be- tween the two heads. Since crystal structures of the RLC lack the PD, the mechanism by which RLC phosphorylation allosterically triggers disruption of HMM auto-inhibition remains unresolved. Our site-directed FRET probe pairs resolved intramolecular atomic distance measurements between the Sunday, February 16, 2014 161a