Immunoglobulin domain unfolding and refolding in passively stretched myofibrils.

Abstract

In isolated myofibrils, passive force is attributed solely to the protein titin. Mechanically, titin is a non-linear biological spring, with two primary mechanical regions: the PEVK region, and the immunoglobulin (IG) domain region. The PEVK region is approximately elastic, and the IG domain region contains domains that unfold and refold depending on force. Under passive tension, skeletal muscle myofibrils exhibit stress-relaxation, particularly at longer lengths (>3.6 µm/sarcomere) where IG domains begin to unfold. During muscle shortening, IG domains refold, and the PEVK region shortens. We investigated the rate of IG domain unfolding and refolding in myofibrils. Paired tests on myofibrils isolated from the psoas muscle of rabbits (n=7). Passive force was measured using force cantilevers for two test protocols: a 2 s-4 s-2 s stretch, hold, release and a 2 s-0 s-2 s stretch, no-hold, release, both with a target length of 4.5μm/sarcomere. Stretches were performed using a piezoelectric motor. Both test protocols were compared to simulated values using the worm-like chain model. Differences between the two test protocols were apparent specifically in the rate of IG domain refolding. Passive force was present experimentally during the entirety of the two second shortening, with the myofibrils remaining taught over the entire duration of both tests. This behavior was not apparent in simulated tests, where myofibrils were predicted to become slack (no passive force) after 0.79 s and 0.9 s during the 2-s shortening period for the 4-s hold protocol and the no-hold protocol, respectively. This suggests IG domain refolding in myofibrils happens considerably quicker in situ than anticipated from isolated titin testing. Our data show that IG domain refolding at this higher rate likely contributes to substantial positive mechanical work during myofibril shortening for both protocols.