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FN3 domains can refold fast and even under substantial force
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Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
4789 Th, 14:30-14:45 (P42) Temperature-jump-induced (endothermic) muscle force generation is sensitive to filament sliding, demonstrating the Fenn effect K.W. Ranatunga, M.E. Coupland, G.J. Pinniger. Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, UK The active muscle force is endothermic so that force versus temperature is sigmoidal with indication of saturation at physiological temperatures (see review by Ranatunga and Coupland, 2003, Adv Exp Med & Biol 538: 441). A temperature-jump (T-jump, in <1 ms) induces a bi-phasic tension rise (phase 2b and phase 3), where phase 2b represents "endothermic force generation" (Davis and Harrington, 1987, Proc Nat Acad Sci USA 84: 975; Goldman et al., 1987; Bershitsky and Tsaturyan, 1992, J Physiol 447: 425). On the basis of its sensitivity to inorganic phosphate (Pi) and [MgADP], the endothermic force generation is a molecular step before Pi-release (Ranatunga, 1999, Proc R Soc London B 266: 1381; Coupland et al., 2005, J Physiol 567: 471). We have now begun a study to examine the sensitivity of endothermic force generation to filament sliding. Experiments were done on single skinned fibres from rabbit psoas at -10°C. A fibre was maximally Ca-activated and ramp shortening/lengthening steps of different velocities applied and a T-jump of -3°C induced by a 0.2ms laser pulse (X = 1.32~t; Ranatunga, 1996, Biophys J 71: 1905). Results show that the relative amplitude and the rate of T-jumpinduced force generation increase as the shortening velocity is increased; conversely, the amplitude decreases as the filament sliding velocity is decreased from shortening towards isometric, and is near zero (i.e. no net tension rise) during lengthening. Thus, endothermic force generation is enhanced during shortening but inhibited during lengthening; this would account for the well known Fenn effect in energy liberation in muscle. Supported by Wellcome Trust. 4796 Th, 14:45-15:00 (P42) Origin of complex force kinetics in striated muscles: cross-bridge kinetics or sarcomere dynamics? R. Stehle 1, I.A. Telley2, J. Denoth 2, G. Pfitzer 1. 1Institute of Physiology, University of Cologne, Cologne, Germany, 2Laboratory for Biomechanics, ETH Zurich Hoenggerberg, Zurich, Switzerland A common assumption in cross-bridge models derived from force transient kinetics of striated muscle is that the filament sliding in each half-sarcomeres is the same. To understand the kinetic mechanical properties of striated muscle on a systemic basis, the intrinsic dynamic behaviour of each functional element, i.e., of each half-sarcomere has to be known. We apply rapid (within ~<10 ms) solution changes to fixed-end hold, subcellular myofibrillar bundles (diameter -2 ~tm) and measure simultaneously: (i) using atomic force cantilevers the myofibril force kinetics (accuracy -5 nN), and (ii) by video fluorescence microscopy under high spatial (-10nm) and temporal (-10ms) resolution the length changes of individual half-sarcomeres. We find that some phases of force transients arise nearly completely from highly organized sarcomere dynamics, e.g., (i) the rapid phase of myofibrillar force decay following a Ca 2+removal is due to the asynchronous, sequential relaxation of individual halfsarcomeres, and (ii) also, the rapid phase 2 in force decay after sudden increase of the product Pi (inorganic phosphate) which is thought to probe directly the kinetics of the force-generating step in the cross-bridge cycle arises from sequential lengthening of sarcomeres. These examples illustrate that multi-phasic force transients of muscle fibers do not necessarily report directly transitions in the cross-bridge cycle. Instead of extending cross-bridge models based on observations of force transients only, simultaneous monitoring of sarcomere dynamics is required to elucidate the contribution of intersarcomeric coupling mechanisms to the force response. 4798 Th, 16:00-16:15 (P45) Mechanical diversity of titin and its relation to passive and active contractile properties of skeletal muscles W.A. Linke. Physiology and Biophysics Unit, University of Muenster, Germany The active and passive contractile performance of skeletal-muscle fibers largely depends on the myosin-heavy-chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern titin's importance for total passive muscle stiffness and the relationship between titin-based stiffness and active contractile parameters. To address these issues, we studied a large set of adult rabbit muscles (n = 37) for titin-size diversity, passive mechanics, and possible correlations with the fiber/MHC composition. Titin-isoform analyses showed sizes between -3300 and 3700 kDa; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, even at the single-fiber level. Fiber and single-myofibril mechanics revealed an inverse relationship between titin size and titin-based passive tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly
Oral Presentations collagen. For instance, soleus has compliant titin but its total passive stiffness is higher than that of psoas expressing stiff titins. Thus, the relative contributions of titin and collagen to total passive stiffness vary greatly among muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIBIIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type-l-fiber and MHC-I proportions. Titinbased stiffness decreased with the slow-fiber/MHC percentage. However, no relationships were found with the proportions of the different type-II-fiber/MHCII subtypes. We conclude that the active and passive mechanical properties of muscle fibers show a low correlation. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. 4800 Th, 16:15-16:30 (P45) Titin-like Ig/FN3 domains can refold fast and even under substantial force W.A. Linke 1, B. Bullard 2, M.L. Leake 3, A.E Oberhauser 4. 1Physiology and Biophysics Unit, University of Muenster, Germany, 2Department of Biology, University of York, UK, 3Clarendon Laboratory, University of Oxford, UK, , 4Department of Neuroscience; University of Texas, Galveston, USA Passive tension generation and elasticity are important properties of muscle conferred mainly by titin-like proteins. Titin's force-extension relationship has been studied at the single-molecule level using optical tweezers and the AFM. Whereas the unfolding of titin Ig-domains or fibronectin-type-3 (FN3) domains is well-studied, much less is known about domain refolding. Here we used AFM force spectroscopy to examine the folding of Ig/FN3-domains in two proteins of the titin family, kettin and projectin, which make up the elastic connecting filaments in Drosophila or Lethocerus sarcomeres. Single-protein analyses revealed that Fn3-domains are mechanically weaker (unfolding force, Fu:~ 50-150 pN) than Ig-domains (Fu:~ 150-250 pN). The refolding speed of Ig/FN3-domains was very fast (85% at 15s -1 (25°C)) and refolding was fully reversible in many stretching-relaxation cycles. Refolding seen before full relaxation suggested that the process can take place at forces well above zero. The force level at which refolding occurs was quantified using a novel force-clamp technique. Interestingly, the Ig/FN3 domains were found to refold under large forces up to -30 pN. Domain refolding was not stepwise but took place in three distinct phases: (1) rapid elastic recoil; (2) small changes in contour-length but large length fluctuations; and 3) final shortening step to the initial contour-length. Hence, these domains do not refold in a "classical" two-state process. A complex energy landscape in domain unfolding-refolding may arise from the stretching of the protein far away from the native state, which provides additional entropic effects. We conclude that these proteins of the titin family could act as folding-based springs. 4802 Th, 16:30-16:45 (P45) Titin and its associated proteins: integrating structure, mechanics, and signal transduction pathways H.L. Granzier 1, S. Labeit 2 . 1Department VCAPR Washington State University, Pullman, WA, USA, 2An~sthesiologie und Operative Intensivmedizin, Universit&tsklinikum Mannheim, Germany. In addition to myosin-based thick filaments and actin-based thin filaments, the sarcomere contains a third myofilament comprised of the giant protein titin. Titin's C-terminus anchors in the Z-disk with single molecules extending all the way to the M-line region of the sarcomere. The majority of titin's I-band region functions as a molecular spring. This spring maintains the precise structural arrangement of thick and thin filaments, and gives rise to passive muscle stiffness, an important determinant of filling of the heart. In this presentation we will focus on recent findings vis-a-vis titin's molecular spring segments that are found in different isoforms of titin. We will discuss alternative splicing and posttranslational mechanisms for adjusting the mechanical properties of titiin's spring elements, and evaluate the role(s) of these mechanisms in muscle development and disease. We will also focus on recent insights regarding the role of titin as a biomechanical sensor and signaling molecule. Many titinbinding proteins have been discovered that are part of structural complexes and biomechanical sensing pathways including those that play important roles in proteasome-dependent degradation of muscle proteins. Thus, titin is a molecular giant with multiple structural and mechanical roles in muscle. In addition, new roles are emerging in stress sensing and signal transduction, important in, for example, controlling protein turn-over.
Journal of Biomechanics 2006, Vol. 39 (Suppl 1)
4789 Th, 14:30-14:45 (P42) Temperature-jump-induced (endothermic) muscle force generation is sensitive to filament sliding, demonstrating the Fenn effect K.W. Ranatunga, M.E. Coupland, G.J. Pinniger. Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, UK The active muscle force is endothermic so that force versus temperature is sigmoidal with indication of saturation at physiological temperatures (see review by Ranatunga and Coupland, 2003, Adv Exp Med & Biol 538: 441). A temperature-jump (T-jump, in <1 ms) induces a bi-phasic tension rise (phase 2b and phase 3), where phase 2b represents "endothermic force generation" (Davis and Harrington, 1987, Proc Nat Acad Sci USA 84: 975; Goldman et al., 1987; Bershitsky and Tsaturyan, 1992, J Physiol 447: 425). On the basis of its sensitivity to inorganic phosphate (Pi) and [MgADP], the endothermic force generation is a molecular step before Pi-release (Ranatunga, 1999, Proc R Soc London B 266: 1381; Coupland et al., 2005, J Physiol 567: 471). We have now begun a study to examine the sensitivity of endothermic force generation to filament sliding. Experiments were done on single skinned fibres from rabbit psoas at -10°C. A fibre was maximally Ca-activated and ramp shortening/lengthening steps of different velocities applied and a T-jump of -3°C induced by a 0.2ms laser pulse (X = 1.32~t; Ranatunga, 1996, Biophys J 71: 1905). Results show that the relative amplitude and the rate of T-jumpinduced force generation increase as the shortening velocity is increased; conversely, the amplitude decreases as the filament sliding velocity is decreased from shortening towards isometric, and is near zero (i.e. no net tension rise) during lengthening. Thus, endothermic force generation is enhanced during shortening but inhibited during lengthening; this would account for the well known Fenn effect in energy liberation in muscle. Supported by Wellcome Trust. 4796 Th, 14:45-15:00 (P42) Origin of complex force kinetics in striated muscles: cross-bridge kinetics or sarcomere dynamics? R. Stehle 1, I.A. Telley2, J. Denoth 2, G. Pfitzer 1. 1Institute of Physiology, University of Cologne, Cologne, Germany, 2Laboratory for Biomechanics, ETH Zurich Hoenggerberg, Zurich, Switzerland A common assumption in cross-bridge models derived from force transient kinetics of striated muscle is that the filament sliding in each half-sarcomeres is the same. To understand the kinetic mechanical properties of striated muscle on a systemic basis, the intrinsic dynamic behaviour of each functional element, i.e., of each half-sarcomere has to be known. We apply rapid (within ~<10 ms) solution changes to fixed-end hold, subcellular myofibrillar bundles (diameter -2 ~tm) and measure simultaneously: (i) using atomic force cantilevers the myofibril force kinetics (accuracy -5 nN), and (ii) by video fluorescence microscopy under high spatial (-10nm) and temporal (-10ms) resolution the length changes of individual half-sarcomeres. We find that some phases of force transients arise nearly completely from highly organized sarcomere dynamics, e.g., (i) the rapid phase of myofibrillar force decay following a Ca 2+removal is due to the asynchronous, sequential relaxation of individual halfsarcomeres, and (ii) also, the rapid phase 2 in force decay after sudden increase of the product Pi (inorganic phosphate) which is thought to probe directly the kinetics of the force-generating step in the cross-bridge cycle arises from sequential lengthening of sarcomeres. These examples illustrate that multi-phasic force transients of muscle fibers do not necessarily report directly transitions in the cross-bridge cycle. Instead of extending cross-bridge models based on observations of force transients only, simultaneous monitoring of sarcomere dynamics is required to elucidate the contribution of intersarcomeric coupling mechanisms to the force response. 4798 Th, 16:00-16:15 (P45) Mechanical diversity of titin and its relation to passive and active contractile properties of skeletal muscles W.A. Linke. Physiology and Biophysics Unit, University of Muenster, Germany The active and passive contractile performance of skeletal-muscle fibers largely depends on the myosin-heavy-chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern titin's importance for total passive muscle stiffness and the relationship between titin-based stiffness and active contractile parameters. To address these issues, we studied a large set of adult rabbit muscles (n = 37) for titin-size diversity, passive mechanics, and possible correlations with the fiber/MHC composition. Titin-isoform analyses showed sizes between -3300 and 3700 kDa; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, even at the single-fiber level. Fiber and single-myofibril mechanics revealed an inverse relationship between titin size and titin-based passive tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly
Oral Presentations collagen. For instance, soleus has compliant titin but its total passive stiffness is higher than that of psoas expressing stiff titins. Thus, the relative contributions of titin and collagen to total passive stiffness vary greatly among muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIBIIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type-l-fiber and MHC-I proportions. Titinbased stiffness decreased with the slow-fiber/MHC percentage. However, no relationships were found with the proportions of the different type-II-fiber/MHCII subtypes. We conclude that the active and passive mechanical properties of muscle fibers show a low correlation. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. 4800 Th, 16:15-16:30 (P45) Titin-like Ig/FN3 domains can refold fast and even under substantial force W.A. Linke 1, B. Bullard 2, M.L. Leake 3, A.E Oberhauser 4. 1Physiology and Biophysics Unit, University of Muenster, Germany, 2Department of Biology, University of York, UK, 3Clarendon Laboratory, University of Oxford, UK, , 4Department of Neuroscience; University of Texas, Galveston, USA Passive tension generation and elasticity are important properties of muscle conferred mainly by titin-like proteins. Titin's force-extension relationship has been studied at the single-molecule level using optical tweezers and the AFM. Whereas the unfolding of titin Ig-domains or fibronectin-type-3 (FN3) domains is well-studied, much less is known about domain refolding. Here we used AFM force spectroscopy to examine the folding of Ig/FN3-domains in two proteins of the titin family, kettin and projectin, which make up the elastic connecting filaments in Drosophila or Lethocerus sarcomeres. Single-protein analyses revealed that Fn3-domains are mechanically weaker (unfolding force, Fu:~ 50-150 pN) than Ig-domains (Fu:~ 150-250 pN). The refolding speed of Ig/FN3-domains was very fast (85% at 15s -1 (25°C)) and refolding was fully reversible in many stretching-relaxation cycles. Refolding seen before full relaxation suggested that the process can take place at forces well above zero. The force level at which refolding occurs was quantified using a novel force-clamp technique. Interestingly, the Ig/FN3 domains were found to refold under large forces up to -30 pN. Domain refolding was not stepwise but took place in three distinct phases: (1) rapid elastic recoil; (2) small changes in contour-length but large length fluctuations; and 3) final shortening step to the initial contour-length. Hence, these domains do not refold in a "classical" two-state process. A complex energy landscape in domain unfolding-refolding may arise from the stretching of the protein far away from the native state, which provides additional entropic effects. We conclude that these proteins of the titin family could act as folding-based springs. 4802 Th, 16:30-16:45 (P45) Titin and its associated proteins: integrating structure, mechanics, and signal transduction pathways H.L. Granzier 1, S. Labeit 2 . 1Department VCAPR Washington State University, Pullman, WA, USA, 2An~sthesiologie und Operative Intensivmedizin, Universit&tsklinikum Mannheim, Germany. In addition to myosin-based thick filaments and actin-based thin filaments, the sarcomere contains a third myofilament comprised of the giant protein titin. Titin's C-terminus anchors in the Z-disk with single molecules extending all the way to the M-line region of the sarcomere. The majority of titin's I-band region functions as a molecular spring. This spring maintains the precise structural arrangement of thick and thin filaments, and gives rise to passive muscle stiffness, an important determinant of filling of the heart. In this presentation we will focus on recent findings vis-a-vis titin's molecular spring segments that are found in different isoforms of titin. We will discuss alternative splicing and posttranslational mechanisms for adjusting the mechanical properties of titiin's spring elements, and evaluate the role(s) of these mechanisms in muscle development and disease. We will also focus on recent insights regarding the role of titin as a biomechanical sensor and signaling molecule. Many titinbinding proteins have been discovered that are part of structural complexes and biomechanical sensing pathways including those that play important roles in proteasome-dependent degradation of muscle proteins. Thus, titin is a molecular giant with multiple structural and mechanical roles in muscle. In addition, new roles are emerging in stress sensing and signal transduction, important in, for example, controlling protein turn-over.