Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C048

Oral Communications

Specific cleavage of the titin springs in situ uncovers titin's role in sarcomere structure and active muscle contraction

Y. Li1, A. Unger1, M. von Frieling-Salewsky1, A. Rivas-Pardo2, J. M. Fernandez2, W. A. Linke1

1. Institute of Physiology II, University of Muenster, Muenster, Germany. 2. Department of Biological Sciences, Columbia University, New York, New York, United States.

Background. The giant protein titin contributes to muscle force generation. However, titin's mechanical function in muscle is still incompletely understood, in part because no tool has been available to specifically cleave the titin springs in the sarcomere, e.g., during mechanical measurements. Methods and Results. In a new genetic mouse model, a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic I-band titin. The HaloTag-TEV cassette allowed for in-situ imaging of titin, specific proteolysis during myofibre mechanics and visualisation of successful cleavage on protein gels or tissue sections. Using permeabilized skeletal myofibre bundles dissected from mice homozygous for HaloTag-TEV titin (knock-in; KI), we measured passive force over the sarcomere-length (SL) range 2.2-3.4 µm and maximum Ca2+-triggered force (pCa 5.0) at 2.6 µm SL, in the absence or presence of TEV enzyme. Wildtype (WT) myofibres treated with TEV protease served as controls. Incubation of the skinned myofibers with TEV protease cleaved HaloTag-TEV titin specifically and completely in <1/2 hour (room temperature), but had no effect on other proteins or on titin in WT myofibres, as detected by loose protein gel electrophoresis. Titin cleavage barely affected myofibre ultrastructure, measured by electron microscopy, in the absence of a stretch or Ca2+-activation. However, mechanical stressing of TEV protease-treated KI fibres caused sarcomere destabilization and myosin-filament disarray. Interestingly, the Z-discs and M-bands were also affected, getting out of alignment and appearing staggered. In psoas and soleus fibre bundles, passive tension dropped with titin cleavage, on average by ~80% and ~60%, respectively, the remaining tension being attributable to extracellular-matrix proteins, such as collagen. Active force development at pCa 5.0 was reduced, on average by ~50%, in titin-cleaved psoas and soleus fibres, compared to controls. Large inter-sample variability in the proportion of active-force reduction was due to heterogeneity in sarcomere structural preservation. Conclusion. The HaloTag-TEV mouse allows, for the first time, a direct quantitation of titin's contribution to passive and active forces in muscle. Our findings show that intact titin springs are not only necessary for most of the passive tension generation of skeletal muscles, but also for the regular structure of all sarcomere bands and for maintaining high active Ca2+-dependant force.

Where applicable, experiments conform with Society ethical requirements