Feb 12, 2017 - The M-line complex appears as a globular head-like structure in titin ... directions and with constant velocity (1 um/s) and pressing force (1 nN).
Sunday, February 12, 2017
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598-Pos Board B363 Structure and Nanomanipulation of the Titin M-Line Complex Zsolt Martonfalvi, Dominik Sziklai, Marton Kovacs, Zsombor Papp, Miklos S. Kellermayer. Semmelweis University, Budapest, Hungary. Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In the M-line the C-terminal region of titin overlaps with that of oppositely oriented titin from the other half of the sarcomere. Furthermore, titin-binding proteins such as myomesin and M-protein localize in the M-line so as to form a complex. The M-line complex appears as a globular head-like structure in titin preparations. The exact structure and the molecular arrangement within this M-line complex is unknown. We analyzed the structure and stability of the M-line complex by using atomic force microscopy (AFM). We mechanically dissected the M-line complex of single surface-adsorbed titin oligomers by using AFM-based nanolithographic procedures. Titin was first deposited on mica so that the oligomers conformationally equilibrated on the surface. Then the M-line complex was dissected by pressing the cantilever tip into the center of the globular head and moving the tip sideways in predetermined directions and with constant velocity (1 um/s) and pressing force (1 nN). Finally, we scanned the surface so as to reveal the evoked changes. Loops of filaments with lengths up to 400 nm were pulled out of the M-line complex. The thickness of the filaments was one half that of native titin, suggesting that partial unfolding may have taken place as a result of nanodissection. Our results suggest that the titin M-line complex may have a higher order three dimensional structure involving the packaging of participating filamentous molecules. Nanodissection may be used as a tool to investigate the internal structure of stable biomolecular complexes.
3 Department of Pharmacology and Toxicology, Helwan University, Cairo, Egypt, 4Department of Surgery, The Ohio State University Wexner Medical Center, Columbus, OH, USA. Diastolic dysfunction occurs not only in diastolic heart failure, but also ubiquitously in systolic heart failure. A main determinant of diastolic passive tension is the elastic sarcomeric protein titin. The role of titin mechanics in diastolic dysfunction is not clearly understood. Here we developed a novel approach to detect mechanical properties of the elastic domain of single titin molecules from failing and non-failing human hearts. We further investigate the role of titin mechanics in heart failure by correlating clinical and biometric data from the corresponding human hearts, in conjunction with diastolic function of isolated human cardiomyocytes. Using atomic force microscopy, antibody specific tethering of the human native titin PEVKdistal Ig domain and the distal Ig domain alone was identified. Titin domains were stretched and released at different frequencies based on ventricular volume changes in the cardiac cycle. The nonlinear force tracings recorded from a series of linear stretch and release steps reflected titin viscoelasticity. Mean titin domain passive tension was measured in the range of 21.2 to 213.8 pN. The mechanical properties of the testing domains were significantly correlated with both the diastolic parameters measured in field-stimulated single cardiomyoyctes, and the biometric data of the corresponding human hearts. Our approach identified the mechanical properties of human native cardiac titin domains, and their relationship to the diastolic function of human hearts, up to single cell level. The current study provided insights into our understanding of cardiac relaxation kinetics in health and disease, contributing to the discovery of novel diagnostic and therapeutic strategies for heart failure.
599-Pos Board B364 Pre-Activation of Cardiomyocytes Determines Speed of Contraction: Role of Titin Michiel Helmes1,2, Aref Najafi1, Martijn van der Locht1, Maike Schuldt1, Ilse AE Bollen1, Max Goebel1, Coen Ottenheijm1, Jolanda van der Velden1,3, Diederik WD Kuster1. 1 Physiology, VU Medical Centre, Amsterdam, Netherlands, 2Ionoptix Llc, Westwood, MA, USA, 3ICIN Netherlands Heart Institute, Utrech, Netherlands. The giant myofilament protein titin has an extendable region that functions as a molecular spring. Cardiomyocytes have exquisite control over the length of titin, through splicing, enabling it to regulate passive stiffness. We hypothesized that titin as it sets the preload on the cardiomyocyte when stretched, together with diastolic Ca2þ pre-activates the cardiomyocyte during diastole and that this pre-activation is a major determinant for force production in the subsequent systolic phase. Via this route titin is thought to play an important role in active force development. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. In the present study we aimed to investigate the effect of long, highly compliant titin on the contractile properties of single cardiomyocytes. We measured single cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and heterozygous (HET) RBM20 deficient rats. In addition we studied detergentpermeabilized human patient samples that had known variations in titin based passive stiffness. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies (>6 Hz). HET myocytes operated at higher SL to achieve the same level of work (2.1mm vs. 1.94mm at 6 Hz). In detergent-permeabilized cardiomyocytes isolated from human and rat heart we simulated cardiac twitches by transiently (0.5 s) exposing the cell to a physiological calcium concentration of pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or prestretching the myocyte increased the kinetics of force development and thus the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a time limited contraction such as a cardiac twitch. Pre-activation was pre-load dependent as the sarcomere length to which the myocytes had to be stretched for equivalent levels of pre-activation varied with the compliance of titin.
601-Pos Board B366 Modeling and Experiment to Determine the Role of Passive Stiffness on Mechanical (Strain Rate) Control of Relaxation Charles S. Chung. Department of Physiology, Wayne State University, Detroit, MI, USA. Diastolic dysfunction is increasingly linked to exercise intolerance and heart failure-like symptoms. The serendipitous discovery of a rat with a spontaneous mutation in the RNA Binding Motif-20 (RBM20) has led to new studies showing the effects of reduced titin-based passive tension. Decreased passive stiffness in the heart is associated with increased exercise tolerance, but also slowed crossbridge attachment and detachment rates. The influence on relaxation, and specifically mechanical control of relaxation by fast end systolic stretch, is not yet known. We utilized MyoSim, a computational modeling environment, and experiments using intact trabeculae to investigate the relationship between titin-based passive stiffness and mechanical control of relaxation. We have previously obtained physiological myofilament parameters by fitting a 2-state crossbridge model to experimental data using MyoSim. These model parameters were held fixed except for passive stiffness, which was reduced by 50 and 75% to match passive stiffness in heterozygous and homozygous RBM20 mutant tissues. Isometric relaxation was slowed by 4 and 5%, respectively, but relaxation rate became 19 and 23% more sensitive to mechanical control of relaxation (slope of relationship between relaxation rate and end systolic strain rate). These data predict that titin based stiffness may modify relaxation rate. Trabeculae experiments using wildtype, heterozygous, and homozygous RBM20 mutant rats, show no significant change in the mechanical control of relaxation. However, reduced titin stiffness was associated with a later time to peak contraction (shortening). Reducing crossbridge attachment and detachment rates in the computational model parameters replicates the experimental results. These data suggest that passive stiffness may play a role on mechanical control of relaxation and confirm that crossbridge properties significantly contribute to early diastolic relaxation.
600-Pos Board B365 A Novel Approach to Identify the Role of Single Molecule Titin Mechanics in Human Heart Failure Mei-pian Chen1,2, Nancy S. Saad1,3, Benjamin D. Canan1,2, Ahmet Kilic4, Peter J. Mohler1,2, Paul M.L. Janssen1,2. 1 Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA, 2Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA,
602-Pos Board B367 Direct Observation of Strain Transmission through the Microtubule Network of Cardiomyocytes Matthew A. Caporizzo1, Brandon Kao2, Patrick Robison1, Alexey I. Bogush1, Benjamin L. Prosser1. 1 Department of Physiology, The University of Pennsylvania, Philadelphia, PA, USA, 2Department of Material Science, The University of Pennsylvania, Philadelphia, PA, USA. The stable microtubule (MT) network plays a significant role in regulating cardiomyocyte contractility and acts as a critical component for stretch-dependent activation of intracellular Ca2þ release. Our previous studies suggest that force transmission though the MT network enhances Ca2þ sparks in response to mechanical stress, yet stress transmission through the MT network has not been
