The Specific Cleavage of Titin Springs to Quantify the Contribution of Titin to Myocardial Passive Stiffness

The giant sarcomere protein titin bears passive load in cardiomyocytes (CMs) and altered titin stiffness occurs in heart failure. Contributions to CMs stiffness may also come from the actin and microtubular networks. Our aim was to quantify the contribution of titin to CMs viscoelasticity through specific cleavage of the titin springs in situ. We have developed a knock-in (KI) mouse model carrying a tobacco etch virus (TEV) protease-recognition site and a HaloTag in titin's elastic region. This HaloTag-TEV cassette allows for specific titin cleavage during mechanical measurements of CMs and visualization of successful cleavage. TEV-protease induced a complete, rapid, and specific cleavage of cardiac titin in skinned homozygous KI CMs, but not in wildtype. Confocal and atomic force microscopy were combined to quantify the transversal stiffness of CMs by nanoindentation. The Young's modulus of homozygous KI cells at a pre-set force of 3 nN was significantly reduced with titin-cleavage, by 26±4% (n= 26 cells), indicating cell softening. The amplitude of this decrease was similar to that found in nanoindentation measurements on human CMs upon microtubule depolymerisation (Chen et al, Nat Med 2018;24:1225-1233). Furthermore, skinned KI CMs were stretched within physiological sarcomere lengths and the passive force recorded before and after TEV-treatment. TEV-protease reduced the steady-state force by 53±8% and the viscous force by 56±7% (n=9 cells). The skinned KI CMs were also incubated first with an actin-severing, Ca2+-independent gelsolin fragment and then with TEV-protease, which reduced the passive force to ∼82% and ∼27% of the value before treatment, respectively (n=6 cells). Thus, titin is the main contributor to tensile force but the actin cytoskeleton contributes as well. High mechanical connectivity between the cytoskeletal elements is likely to occur in CMs, implicating a tensegrity structure.