Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements

Kaori Watanabe,Preetha Nair,Dietmar Labeit,Miklós S.Z Kellermayer,Marion Greaser,Siegfried Labeit,Henk Granzier

doi:10.1074/jbc.m200356200

Abstract

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.

Highlights

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium
To determine the validity of the deduced single molecule characteristics in the context of the sarcomere, we modeled the extensible region of cardiac titin (N2B isoform) as three serially linked worm-like chain (WLC): tandem Ig segments, the PEVK domain, and N2B unique sequence (N2B-Us) (Fig. 6A)
In atomic force microscope (AFM)-based mechanical experiments, the data may be derived from single molecules and multimolecular complexes

Summary

The abbreviations used are

N2B-Us, N2B unique sequence; WLC, worm-like chain; AFM, atomic force microscopy/microscope; pN, piconewton(s); PP, polyproline. Predict, within experimental error, the measured extensible behavior of the titin spring elements in the cardiac sarcomere as well as the passive force-sarcomere length relation of cardiac myocytes. We conclude that the serially linked WLC model with mechanically distinct springs using parameters established in this work closely describes the in vivo behavior of cardiac titin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION