Quantifying the Titin Contribution to Muscle Force Generation Using a Novel Method to Specifically Cleave the Titin Springs in Situ

The giant protein titin contributes to muscle force generation. However, titin's mechanical role in muscle is still incompletely understood because of previous inability to specifically cleave the titin spring. To overcome this problem, we used a mouse model in which both a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic I-band titin. The Halo-TEV cassette allowed for in-situ imaging of titin, specific proteolysis during myofiber mechanics, and visualization of successful cleavage on protein gels or tissue sections. Using permeabilized skeletal myofiber bundles dissected from mice homozygous for Halo-TEV-titin, we measured passive force over the sarcomere-length (SL) range 2.2-3.4 µm and maximum Ca2+-triggered force (pCa5) at 2.6 µm SL, in the absence or presence of TEV-enzyme. Wildtype myofibers treated with TEV-protease served as additional controls. We found that incubation of myofibers with TEV-protease at room temperature cleaved genetically modified titin specifically and completely in <1 hour, but had no effect on wildtype titin. Titin cleavage barely affected myofiber ultrastructure in the absence of a stretch or Ca2+-activation; however, mechanical stressing caused sarcomere destabilization and myosin-filament disarray. In both psoas and soleus myofiber bundles, passive forces dropped with titin cleavage by ∼50%, the remaining forces presumably being attributable to extracellular-matrix proteins. Subsequent treatment of the samples with high-salt buffer (0.6 M KCl/1.0 M KI) to dissolve the titin anchorage caused a further ∼25% drop in passive force, suggesting this frequently-used extraction method consistently overestimates the titin-contribution to passive tension. Active force development was reduced, on average by ∼50%, in titin-cleaved psoas or soleus fibers, compared to controls. Our approach thus allows for a direct and reliable quantitation of titin's contribution to passive and active forces in muscle.