Published online before print October 4, 2001, doi: 10.1161/hh2201.099453
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© 2001 American Heart Association, Inc.
Cellular Biology |
Interaction Between PEVK-Titin and Actin Filaments
Origin of a Viscous Force Component in Cardiac Myofibrils
From the Institute of Physiology and Pathophysiology (M.K., C.N., W.A.L.), University of Heidelberg, Heidelberg; Max-Planck Institute of Medical Research (S.F.-B., D.J.M.), Heidelberg; Max-Planck Institute of Molecular Physiology (E.R., M.G.), Dortmund; and Institut für Anästhesiologie und Operative Intensivmedizin (D.L.), Universitätsklinikum, Mannheim, Germany.
Correspondence to Dr W.A. Linke, Institute of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany. E-mail wolfgang.Linke{at}urz.uni-heidelberg.de
Abstract—— The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B–cardiac PEVK was expressed in Escherichia coli and tested—along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains—for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca2+] reversed the PEVK effect. PEVK concentrations 
10 µg/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0°C, but more strongly at 30°C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca2+-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Key Words: connectin • passive tension • myofibril mechanics • myocardial viscosity • actin binding protein
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