Extensibility of isoforms of cardiac titin: Variation in contour length ofmolecular subsegments provides a basis for cellular passive stiffness diversity

Titin is a giant polypeptide that spans between the Z- and M-lines of the c ardiac muscle sarcomere and that develops force when extended. This force a rises from titin's extensible I-band region, which consists mainly of three segment types: serially linked immunoglobulin-like domains (Ig segments), interrupted by the PEVK segment, and the N2B unique sequence. Recently it w as reported that the myocardium of large mammals co-expresses small (N2B) a nd large (N2BA) cardiac isoforms and that the passive stiffness of cardiac myocytes varies with the isoform expression ratio. To understand the molecu lar basis of the differences in passive stiffness we investigated titin's e xtensibility in bovine atrium, which expresses predominantly N2BA titin, an d compared it to that of rat, which expresses predominantly N2B titin. Immu noelectron microscopy was used with antibodies that flank the Ig segments, the PEVK segment, and the unique sequence of the N2B element. The extension of the various segments was then determined as a function of sarcomere len gth (SL). When slack sarcomeres of bovine atrium were stretched, the PEVK s egment extended much more steeply and the unique N2B sequence less steeply than in rat, while the Ig segments behaved similarly in both species. Howev er, the extensions normalized with the segment's contour length (i.e., the fractional extensions) of Ig, PEVK, and unique sequence segments all increa se less steeply with SL in cow than in rat. Considering that fractional ext ension determines the level of entropic force, these differences in fractio nal extension are expected to result in shallow and steep passive force-SL curves in myocytes that express high levels of N2BA and N2B titin, respecti vely. Thus, the findings provide a molecular basis for passive stiffness di versity.