Tracking of Qdot Conjugated Titin Antibodies in Single Myofibril Stretch Experiments Reveals Ig-domain Unfolding at Physiological Sarcomere Lengths

Abstract

The mechanical characteristics of titin in muscle sarcomeres were previously studied by us in single myofibril stretch experiments, where the extensibility of I-band titin segments was usually measured under static conditions. Here we investigated the behavior of I-band titin during and after stretch of single rabbit psoas myofibrils in real-time. The focus was on titin's proximal Ig-domain region, whose stretch dynamics were analyzed by labeling the myofibrils specifically in the N2A-titin domain using antibody-conjugated quantum dots, which stained the periphery of the myofibril but did not enter the myofilament lattice. Qdot labels were tracked to obtain the stretch-dependent change in epitope distance (across Z-disc) and sarcomere length (SL) over time. In contrast to what was expected from the current titin extensibility model, at sarcomere lengths of 2.5 and 3.8 μm, titin's proximal Ig-domain region elongated continuously, in proportion to the half I-band length. Already at ∼2.6 μm SL the proximal Ig-segment length exceeded the value expected if all Ig-domains remain folded. Our results suggest that Ig-domains unfold in parallel with PEVK-titin extension at physiological sarcomere lengths and under relatively low forces. By reducing the antibody-Qdot concentration, we succeeded in observing titin Ig-domain dynamics in myofibrils at the single-molecule level.