Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science . 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres ( ‫ف‬ 2.0 m) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ‫ف‬ 2.0 to 2.7 m, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ‫ف‬ 2.7 m, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116 indicated that in the physiological SL range ( a ) the Ig-like domains of the tandem Ig segments remain folded and ( b ) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.T itin is a giant filamentous protein that, in addition to the thin and thick filaments, constitutes the third myofilament system of striated muscle. In the sarcomere, titin molecules span the entire 1-2-m distance from the Z-line to the M-line. Previous studies have revealed that the A-band region of the molecule is rendered inextensible due to its tight association with the thick filament, whereas the I-band region behaves elastically as the sarcomere undergoes changes in length. The elastic properties of the I-band region of titin are primarily responsible for the passive force that is generated when unactivated (i.e., passive) muscle is stretched. Passive force is present in actively contracting muscle as well, where it helps maintain the structural integrity of the sarcomere and thereby ensures efficient muscle contraction. (For recent reviews and original citations see Fürst and Gautel, 1995;Trinick, 1996;Wang, 1996;Labeit et al., 1997;Maruy...

Abstract-The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline, and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above Ϸ2.2 m SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable. (Circ Res. 1999;84:1339-1352.)Key Words: elasticity Ⅲ diastole Ⅲ myocardial compliance Ⅲ connectin Ⅲ passive force D uring diastole, while the ventricle fills, the myocardium is passively stretched. The rate of filling and the magnitude of the end-diastolic volume are significantly affected by the shape of the passive pressure-volume relation. Titin, a striated muscle-specific giant elastic protein, is largely responsible for the generation of the diastolic force in the cardiac myocyte. 1 In addition to influencing ventricular filling, titin also helps maintaining the structural integrity of the contracting sarcomere (for reviews, ...