Molecular Dissection of N2B Cardiac Titin’s Extensibility

Abstract

Titin is a giant filamentous polypeptide of multidomain construction spanning between the Z- and M-lines of the cardiac muscle sarcomere. Extension of the I-band segment of titin gives rise to a force that underlies part of the diastolic force of cardiac muscle. Titin's force arises from its extensible I-band region, which consists of two main segment types: serially linked immunoglobulin-like domains (tandem Ig segments) interrupted with a proline (P)-, glutamate (E)-, valine (V)-, and lysine (K)-rich segment called PEVK segment. In addition to these segments, the extensible region of cardiac titin also contains a unique 572-residue sequence that is part of the cardiac-specific N2B element. In this work, immunoelectron microscopy was used to study the molecular origin of the in vivo extensibility of the I-band region of cardiac titin. The extensibility of the tandem Ig segments, the PEVK segment, and that of the unique N2B sequence were studied, using novel antibodies against Ig domains that flank these segments. Results show that only the tandem Igs extend at sarcomere lengths (SLs) below approximately 2.0 microm, and that, at longer SLs, the PEVK and the unique sequence extend as well. At the longest SLs that may be reached under physiological conditions ( approximately 2.3 microm), the PEVK segment length is approximately 50 nm whereas the unique N2B sequence is approximately 80 nm long. Thus, the unique sequence provides additional extensibility to cardiac titins and this may eliminate the necessity for unfolding of Ig domains under physiological conditions. In summary, this work provides direct evidence that the three main molecular subdomains of N2B titin are all extensible and that their contribution to extensibility decreases in the order of tandem Igs, unique N2B sequence, and PEVK segment.

An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice

“…The N2B region of titin extends mostly toward the upper limit of the physiological sarcomere length of cardiac muscle (25,44). Our findings highlight what we believe is a novel role for FHL1 in modulating titin N2B function and sarcomere extensibility at the upper limits of the physiological sarcomere length, a state that could be physiologically reached in vitro through acute stretch and in vivo by pressure overload.…”

Targeted deletion of titin N2B region leads to diastolic dysfunction and cardiac atrophy

“…Analysis of the N2B-KO mice allowed us to test the hypothesis that the N2B unique sequence eliminates the necessity for unfolding of Ig domains under physiological conditions (3). In absence of the N2B element, we found that the extension of PEVK and tandem Ig segments is increased (Fig.…”

“…In addition, cardiac titin contains a third extensible region, the N2B element (2), which is absent in skeletal muscle. The N2B region extends greatly toward the upper limit of the physiological sarcomere length of cardiac muscle (3,4). It has been suggested that this extension reduces the steepness of the passive forcesarcomere length relation, decreasing the likelihood of the unfolding of Ig domains (3).…”

“…The N2B region extends greatly toward the upper limit of the physiological sarcomere length of cardiac muscle (3,4). It has been suggested that this extension reduces the steepness of the passive forcesarcomere length relation, decreasing the likelihood of the unfolding of Ig domains (3). Mutations in the N2B region can lead to dilated or hypertrophic cardiomyopathy, apparently through altered affinity to FHL2, a heart-specific member of the LIM domain gene family (5).…”

Physiological Functions of the Giant Elastic Protein Titin in Mammalian Striated Muscle

“…In cardiac muscle, the extension of the N2B sequence that occurs in the upper limit of the physiological SL decreases passive force and presumably prevents the unfolding of the Ig segment [36][37][38]. A consensus still has not been achieved on whether the unfolding/refolding of Ig segments occurs within the physiological SL.…”