Thick Filament Compliance in Passively Stretched ... - Cell Press

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Feb 13, 2017 - 5% Dextran, 2.45 μm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in ...
Monday, February 13, 2017

Platform: Skeletal Muscle Mechanics, Structure, and Regulation 893-Plat Structural Kinetics of the RLC Domain of Myosin during Activation of Skeletal Muscle Fibers by Photolysis of Caged-Calcium Luca Fusi, Elisabetta Brunello, Ziqian Yan, Malcolm Irving. Randall Division, King’s College London, London, United Kingdom. Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and cooperativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s1) increase in [Ca2þ] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25 C, 5% Dextran, 2.45 mm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12 C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK. 894-Plat Structural Changes in the Thick Filaments during Activation of Demembranated Skeletal Muscle Fibers Marco Caremani1, Luca Fusi2, Massimo Reconditi1, Gabriella Piazzesi1, Theyencheri Narayanan3, Malcolm Irving2, Vincenzo Lombardi1, Elisabetta Brunello2. 1 University of Florence, Florence, Italy, 2King’s College London, London, United Kingdom, 3European Synchrotron Radiation Facility, Grenoble, France. We determined the [Ca2þ]-dependence of the changes in thick filament structure associated with activation of small bundles of demembranated fibers from rabbit psoas muscle by X-ray diffraction at beamline ID02 of the European Synchrotron Radiation Facility. In relaxing conditions the OFF structure of the thick filament characteristic of resting intact muscle, with a strong first myosin layer line (ML1) corresponding to the quasi-helical arrangement of the myosin motors on the thick filament surface and a short backbone periodicity, measured by the spacing of the myosin-based meridional M6 reflection (SM6; Linari et al. 2015, Nature 528:276-279), was observed at temperatures above 25 C in the presence of 5% Dextran T500. To study the effects of calcium activation in these conditions whilst preserving sarcomere homogeneity and minimising the period of high temperature activation, fiber bundles were equilibrated at the required [Ca2þ] at 1 C, and X-ray diffraction data were collected 1.5s after a temperature jump to 25 C. Activation at low [Ca2þ], producing only about 20% of the maximal isometric force, was accompanied by large decreases in the intensities of the ML1 and of the myosin-based meridional M2 and M5 X-ray reflections associated with the OFF structure of the thick filament, showing that the quasi-helical arrangement of the myosin motors on the thick filament surface is lost at a relatively low level of isometric force. In contrast the changes in the interference fine structure of the myosinbased meridional M3 reflection, associated with the conformation of the myosin motors during maximal isometric contraction, and the increase in SM6 associated with the longer backbone periodicity require higher [Ca2þ]. Supported by FIRB-Futuro in Ricerca, PRIN-MIUR and Telethon (Italy), MRC and BHF (UK), and ESRF. 895-Plat Thick Filament Compliance in Passively Stretched Skeletal Muscle Weikang Ma1, Danielle Buck2, Joshua Nedrud2, Thomas C. Irving1, Henk Granzier3. 1 Biological Sciences, Illinois Institute of Technology, Chicago, IL, USA, 2 Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA,

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3 Cellular and Molecular Medicine, University of Arizona, Chicago, AZ, USA. Titin is a giant sarcomeric protein (~2.9MDa to 4.0MDa) that spans half of the sarcomere from the Z disc to the M band. Titin closely interacts with the thick filament inside the A band, and plays important roles in sarcomere development and force generation. In vertebrate muscle, multiple titin isoforms with different molecular weights exist resulting from alternative splicing of a single titin gene. Different sizes of titin lead to different overall stiffnesses of the muscle having multiple effects on muscle function. Titin with different sizes would expect to have different effects on myofilament properties. Here we used the Rbm20 mouse model to study how titins of different sizes, and hence, compliance can affect sarcomere structure with increasing sarcomere length in skeletal muscle using small angle X-ray diffraction. In this Rbm20 mouse model, homozygous mice (KO) expresses the largest titin, and heterozygous mice (HET) expresses an intermediate molecular weight titin compared to titin expressed in wild type mice. The results showed that titin-based passive tension strains thick filaments, and the titin isoform in Rbm20 KO muscle strains the thick filament the least. Our results also showed that the thick filament extensibility, and hence, compliance, in passively stretched muscle is non-linear. Meridional X-ray reflections attributed to myosin binding protein C (MyBP-C) decrease in intensity with increasing sarcomere length, consistent with the notion that MyBP-C interacts with actin and that this interaction is lost when thin filaments are pulled out of the C-zone. Our results also showed that KO muscle with most compliant titin isoform had the largest inter-filament spacing and lowest equatorial intensity ratio at slack length. Supported by R01AR060697 and P41GM103622 from the National Institutes of Health.

896-Plat Evidence for an I-Band Spring that is Tuned to the Length of the Skeletal Muscle Sarcomere Joseph D. Powers1,2, Massimo Reconditi1, Luca Fusi3, Elisabetta Brunello3, Vincenzo Lombardi1, Gabriella Piazzesi1. 1 Laboratory of Physiology, Department of Biology, University of Florence, Sesto Fiorentino, Italy, 2Department of Bioengineering, University of Washington, Seattle, WA, USA, 3King’s College London, London, United Kingdom. At the plateau of isometric contraction (force T0), the half-sarcomere compliance (Chs) is accounted for in almost equal parts by the compliance of the array of myosin cross-bridges (Ccb) and the compliance of myofilaments (Cf) in series with cross-bridges. Early during isometric force development (forces < 0.3 T0), however, Chs is less than that expected from the simplified two-element model assumed above, suggesting the presence of an elastic element in parallel with myosin motors with a compliance (Cp) ~20 times larger than Ccb at T0 (Fusi et al. J. Physiol. 592:1109-18, 2014). To identify the structural basis of Cp,Chs-force relations are determined here by imposing 4 kHz oscillations on single fibers from the tibialis anterior muscle ofR. esculenta (4 C) during force development at different sarcomere lengths (SL) from 2.15 mm (full overlap) to 3.1 mm. If Cp originates from elements in the A-band of the sarcomere, Chs should increase with SL because of the reduction of filament overlap. Instead, for SL > 2.15 mm, the Chs-force relations are found shifted downward for forces 4mm).[1] If peak stresses were to vary at very long SL (> 4mm) where titin is the sole contributor (in myofibrils) to force, then that would suggest that the freespring length of titin can be length-modulated somehow. The result being that a shorter free-spring titin would generate higher stress at matched SL compared to a myofibril where titin length was not shortened. We stretched