The Mechanical Properties of Titin within a Sarcomere

Titin is a structural protein in muscle that spans the half sarcomere from z-band to M-line. Although there are selected studies on titin’s mechanical properties from tests on isolated molecules or titin fragments, little is known about its behavior within the structural confines of a sarcomere. Here, we tested the hypothesis that titin properties might be reflected well in single myofibrils. Therefore, the purpose of this study was to measure the passive mechanical properties of isolated single myofibrils and evaluate whether these properties reflect the basic mechanical properties of the titin molecule. Single myofibrils from rabbit psoas were prepared for measurement of passive stretch-shortening cycles at lengths where passive titin forces become important. Three repeat stretch-shortening cycles with magnitudes between 1.0-3.0μm/sarcomere were performed at a speed of 0.1μm/s·sarcomere and repeated after a ten minute rest at zero force. These tests were performed in a relaxation solution (passive) and an activation solution (active) where cross-bridge attachment was inhibited with butanedione monoxime. Myofibrils behaved viscoelastically producing an increased efficiency with repeat stretch-shortening cycles, but a decreased efficiency with increasing stretch magnitudes. Furthermore, we observed a first distinct inflection point in the force-elongation curve at an average sarcomere length of 3.5μm that was associated with an average force of 68±5nN/mm -1 . This inflection point was thought to reflect Ig domain unfolding and was missing after a ten minute rest at zero force, suggesting a lack of spontaneous Ig domain refolding. These passive myofibrillar properties are consistent with those observed in isolated titin molecules, suggesting that the mechanics of titin are well preserved in isolated myofibrils, and thus, can be studied readily in myofibrils, rather than in the extremely difficult and labile single titin preparations.