Titin Immunoglobulin Domain Refolding Produces Mechanical Work in situ

Titin’s role in providing passive force in striated muscle is well-established. Generally, titin’s elasticity is attributed to its unique PEVK region, a nearly elastic non-linear spring. The remaining length of I-band titin is largely composed of immunoglobulin (Ig) domains grouped into tandem-proximal and distal regions. Titin’s Ig domains were long thought to unfold only under high forces and refold only under near-zero forces. Recent evidence from single-molecule Ig domain constructs indicates Ig domains may unfold and refold at physiological forces. We performed a series of passive stretch-shortenings on intact rabbit psoas myofibrils to determine if Ig domain refolding may indeed occur during shortening to produce meaningful mechanical work in situ. We hypothesized refolding may occur quickly and under force, producing physiologically meaningful work. Using the established modified worm-like chain model for titin’s PEVK, and a novel myofibril immunolabelling system providing, for the first time, simultaneous sarcomere force and PEVK length measurements, we approximated the mechanical work contribution of Ig domain refolding based on estimated I-band titin segmental lengths. In stretch-shortening protocols tested, we found Ig domain refolding contributes work during shortening, accounting for up to 25.6±13.4% of energy recovered. We show Ig domain refolding occurs under forces of 5.6±3.7 pN, within physiological ranges of forces experienced by titin filaments. Our findings in intact myofibrils, where full-length titin is in its in-situ position, correspond closely to in vitro experiments using isolated titin fragments. We demonstrate Ig domain refolding is relevant to passive force and work production in situ, using isolated myofibrils.