Mechano-Chemo-transduction in Cardiomyocytes during Beat-To Beat Contraction under Mechanical Load

Mechano-Chemo-transduction in Cardiomyocytes during Beat-To Beat Contraction under Mechanical Load

Monday, February 29, 2016 The two methods are complementary and give consistent results, which we interpret in light of recent findings on the conform...

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Monday, February 29, 2016 The two methods are complementary and give consistent results, which we interpret in light of recent findings on the conformation of flexible polyelectrolytes.

Symposium: Mechanosensing and Mechanosignaling in Muscle 923-Symp Mechanosensitive Structural States of Titin Zsolt Ma´rtonfalvi1, Pasquale Bianco1,2, Katalin Naftz1, Dorina K}oszegi1, Gyo¨rgy Ferenczy1, Miklo´s S. Kellermayer1. 1 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary, 2University of Florence, Florence, Italy. Titin, a giant, multi-domain filamentous protein has been suggested to act as a sensor of sarcomeric stress and strain. The exact mechanisms of this putative mechanosensing function are, however, yet unknown. To gain an insight into the mechanosensitive structural states we have manipulated titin with high-resolution optical tweezers and imaged structural states of extended molecules with atomic force microscopy. Discrete, stepwise transitions can be resolved in titin during stretch at forces as low as 5 pN. Multiple mechanisms and molecular regions, such as the unfolding of globular domains and the extension of unique sequences, contribute to a pattern of transitions which is sensitive to the history of contractile events. Globular domains are apparently selected for unfolding according to a spatially dispersed gradient of mechanical stabilities which emerges as a safety mechanim for protecting the sarcomere against structural disintegration under excessive mechanical conditions. A C-terminally located region corresponding to the titin kinase unfolds systematically under overstretching forces suggesting that this domain may indeed be mechanically activated in intrasarcomeric conditions. Mechanically unfolded weak domains may dynamically reorganize towards the molten-globule state, thereby yielding an extra contractility that aids saromere mechanics. Altogether, titin displays a complex pattern of history-dependent, force-driven structural transitions which, by dynamically exposing ligandbinding sites, may set the stage for the sensing of the sarcomeric mechanical status. 924-Symp Titin(S): Towards an Atomic Understanding of Mechanosensory Events in the Elastic Scaffolds of the Muscle Sarcomere Olga Mayans1, Jennifer Fleming1, Rhys Williams2, Barbara Franke1, Hang Lu3, Guy Berrian4. 1 University of Konstanz, Konstanz, Germany, 2University of Liverpool, Liverpool, United Kingdom, 3Georgia Institute of Technology, Atlanta, GA, USA, 4Emory University, Atlanta, GA, USA. The giant intra-sarcomeric filaments of the titin-like family are key orchestrators of stretch-sensing pathways that regulate muscle responses to mechanical load. Despite acute variations in the length and domain organization of these filamentous proteins across the animal biodiversity, they all comprise numerous Ig/FnIII domains linked in series and one or two kinase domains invariably located near their C-terminus. Combining the 3D-structural elucidation of multi-domain components at atomic level, in silico simulations, molecular engineering and in vivo transgenic muscle technologies, we are revealing the molecular events taking place during mechanosensing in titin-like proteins. Our findings show that the sensory role of titin is enabled by a subtle interdomain order in the chain, imposed by short linkers that sterically govern domain packing and dynamics. This modular design is sensitive to mechanical deformations but affords a ‘‘chain memory’’ mechanism for molecular recovery. The local disruption of such domain arrangements by genetic mutation leads to human myopathy. Our data show that also the kinase domains of titin-like proteins undergo elastic deformations in their regulatory, flanking segments during muscle activity in vivo. Contrary to expectations, these kinases are catalyticallydispensable for muscle function and development. Instead, they act as regulated scaffolds for the recruitment of turnover/signaling proteins onto kinase-based signalosomes. This talk will provide a molecular perspective on the stretchinduced mechanics and signaling of titin-like proteins. Zacharchenkov T, et al. (2015). Biochem Soc Trans. In press. Bogomolovas J, et al. (2014). Open Biology. 4(5):140041. Von Castelmur E, et al. (2012). P.N.A.S. 109(34):13608-13. 925-Symp Mechano-Chemo-transduction in Cardiomyocytes during Beat-To Beat Contraction under Mechanical Load Ye Chen-Izu. Pharmacology, Bioengieneering, Cardiology, University of California, Davis, Davis, CA, USA. The heart can sense and respond to mechanical load in order to maintain adequate blood flow to meet the body’s demands for oxygen and fuel. However, intrinsic mechanisms that transduce mechanical stress to biochemical reactions

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(called mechano-chemo-transduction, M-C-T) in cardiomyocytes have been understudied and remain incompletely understood. Method: We have developed an innovative Cell-in-Gel system to embed cardiomyocytes in 3D viscoelastic hydrogel to impose mechanical stresses on the single cell during excitation and contraction. Results: We found that higher load causes elevated Ca2þ transient and enhanced contractility in systole; this constitutes an auto-regulatory mechanism that increases contractile force to compensate for higher load. Nevertheless, excessive loading also causes spontaneous Ca2þ activities in diastole, which provide a substrate for arrhythmias. Furthermore, we systematically investigate the key molecules involved in M-C-T, and found that mechanical stress activates nitric oxide synthase (NOS) and Nox2 which, through NO and ROS signaling, modulate CaMKII activity. These powerful signaling pathways orchestrate the modulations of Ca2þ handling molecules to tune the cellular Ca2þ dynamics in response to mechanical stress. Perspective: We posit that this fundamental M-C-T mechanism underlies the auto-regulation of contractility under physiological loading, but also causes Ca2þ dysregulation under excessive loading (i.e. hypertension, volume overload, or when mutations disrupt M-C-T as in muscular dystrophy), which leads to mechanical stress induced arrhythmias, deleterious remodeling, and heart failure. 926-Symp Detyrosinated Microtubules Bear Load and Transmit Mechanical Force in Cardiomyocytes Patrick Robison1, Matthew Caporizzo2, Alexey Bogush1, Kenneth Margulies3, Benjamin Prosser1. 1 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 2University of Pennsylvania School of Engineering and Applied Science, Philadelphia, PA, USA, 3Department of Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, USA. The cytoskeleton plays an integral role in mechanotransduction, the conversion of mechanical forces to intracellular signals. In striated muscle, mechanotransduction through the microtubule (MT) cytoskeleton modulates redox signaling and calcium homeostasis through a pathway termed X-ROS signaling. But how do microtubules sense mechanical force, integrate and transmit that signal? Recent advances in imaging now provide the spatial and temporal resolution needed to characterize MT behavior during cardiomyocyte contraction, overcoming a major hurdle to understanding MT mechanics during the contractile cycle. These advances have allowed us to detect MTs deforming into short, sinusoidal buckles, indicating some ability to bear compressive force in contracting cardiac myocytes. Furthermore, we found that this load-bearing is highly regulated by a specific MT post-translational modification called detyrosination. Inhibiting detyrosination with pharmacological or genetic tools fundamentally alters how MTs sense and respond to mechanical force - interdigitated MTs appear to simply slide past each other in the contracting myocyte as opposed to deforming under load. Consistent with this impaired force-sensing, mechanotransduction through the network is significantly blunted when detyrosination is reduced. These findings raise a key mechanistic question- if MTs bear compressive load, how is that load applied to the MT? There are a wide variety of binding partners that could facilitate transmission of force between the contractile apparatus and the MT network. However, a complex that anchors MTs to the sarcomere has not been explicitly detailed. Here we examine potential cross-linking proteins, including the muscle intermediate filament Desmin and the microtubule motor kinesin, as candidate members of a MT anchoring complex capable of sensing the tyrosination state of a microtubule.

Platform: Voltage-gated K Channels, Mechanisms of Voltage Sensing and Gating I 927-Plat N-Arachidonoyl Taurine Rescues Diverse Long QT Syndrome-Associated Mutations in the Cardiac I)Ks Channel Sara I. Liin1, Johan E. Larsson1, Rene Barro-Soria2, Mark A. Skarsfeldt3, Bo H. Bentzen3, H Peter Larsson2. 1 Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, Sweden, 2Physiology and Biophysics, University of Miami, Miami, FL, USA, 3 Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark. The cardiac I)Ks channel is important for cardiomyocyte repolarization. More than 300 loss-of-function mutations in the genes encoding I)Ks have been identified in patients with Long QT syndrome. These mutations cause cardiac arrhythmias, such as torsades de pointes and ventricular fibrillation. How specific mutations cause arrhythmia is, however, not known in most cases and there is no approved I)Ks channel activator for treatment of arrhythmia. In this work, we study the biophysical properties and mechanism of loss of function of Long QT syndrome-associated I)Ks channel mutations expressed in