I-band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure

In cardiac muscle, the giant protein titin exists in different length isofo rms expressed in the molecule's I-band region. Both isoforms, termed N2-A a nd N2-B, comprise stretches of Ig-like modules separated by the PEVK domain . Central I-band titin also contains isoform-specific Ig-motifs and nonmodu lar sequences, notably a longer insertion in N2-B. We investigated the elas tic behavior of the I-band isoforms by using single-myofibril mechanics, im munofluorescence microscopy, and immunoelectron microscopy of rabbit cardia c sarcomeres stained with sequence-assigned antibodies. Moreover, we overex pressed constructs from the N2-B region in chick cardiac cells to search fo r possible structural properties of this cardiac-specific segment, We found that cardiac titin contains three distinct elastic elements: poly- Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends similar to 60 nm at high physiological stretch. Recruitment of all three e lements allows cardiac titin to extend fully reversibly at physiological sa rcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH2 terminus in cardiac myocytes greatly disrupte d thin filament, but not thick filament structure. Our results strongly sug gest that the NH2-terminal N2-B domains are necessary to stabilize thin fil ament integrity. N2-B-titin emerges as a unique region critical for both re versible extensibility and structural maintenance of cardiac myofibrils.