Molecular dissection of N2B cardiac titin's extensibility

Titin is a giant filamentous polypeptide of multidomain construction spanni ng 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 t he diastolic force of cardiac muscle. Titin's force arises from its extensi ble I-band region, which consists of two main segment types: serially linke d immunoglobulin-like domains (tandem Ig segments) interrupted with a proli ne (P)-, glutamate (E)-, valine (V)-, and lysine (K)-rich segment called PE VK segment. In addition to these segments, the extensible region of cardiac titin also contains a unique 572-residue sequence that is part of the card iac-specific N2B element. In this work, immunoelectron microscopy was used to study the molecular origin of the in vivo extensibility of the I-band re gion of cardiac titin. The extensibility of the tandem Ig segments, the PEV K segment, and that of the unique N2B sequence were studied, using novel an tibodies against Ig domains that flank these segments. Results show that on ly the tandem Igs extend at sarcomere lengths (SLs) below similar to 2.0 mu m, and that, at longer SLs, the PEVK and the unique sequence extend as wel l. At the longest SLs that may be reached under physiological conditions (s imilar to 2.3 mu m), the PEVK segment length is similar to 50 nm whereas th e unique N2B sequence is similar to 80 nm long. Thus, the unique sequence p rovides additional extensibility to cardiac titins and this may eliminate t he 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 e xtensibility decreases in the order of tandem Igs, unique N2B sequence, and PEVK segment.