Downsizing the molecular spring of the giant protein titin reveals that skeletal muscle titin determines passive stiffness and drives longitudinal hypertrophy

Abstract

Titin, the largest protein known, forms an elastic myofilament in the striated muscle sarcomere. To establish titin’s contribution to skeletal muscle passive stiffness, relative to that of the extracellular matrix, a mouse model was created in which titin’s molecular spring region was shortened by deleting 47 exons, the TtnΔ112-158 model. RNA sequencing and super-resolution microscopy predicts a much stiffer titin molecule. Mechanical studies with this novel mouse model support that titin is the main determinant of skeletal muscle passive stiffness. Unexpectedly, the in vivo sarcomere length working range was shifted to shorter lengths in TtnΔ112-158 mice, due to a ~ 30% increase in the number of sarcomeres in series (longitudinal hypertrophy). The expected effect of this shift on active force generation was minimized through a shortening of thin filaments that was discovered in TtnΔ112-158 mice. Thus, skeletal muscle titin is the dominant determinant of physiological passive stiffness and drives longitudinal hypertrophy.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues

“…Insertion of the HaloTag-TEV cassette in titin. The I-band section of titin, containing up to 100 immunoglobulin-like (Ig) domains, is a major contributor to the passive stiffness of striated muscle tissue 22,31,32 . This segment includes an alternatively spliced region that provides titin with muscle-specific, tailored mechanical properties 6,31,33 .…”

“…scenario, the contribution of domain folding to contraction will depend on the physiological range of forces experienced by titin in sarcomeres, generally estimated to be below 10 pN 10,15,32,61 .…”

Contracting striated muscle has a dynamic I‐band spring with an undamped stiffness 100 times larger than the passive stiffness

“…In future work, the comparative definition in situ of both the undamped stiffness and the subsequent relaxation process for different isoforms of titin will allow the structural basis of the functional differences to be identified. Most importantly, our approach provides a new tool for the detailed in situ description of the structural-functional basis of muscle dysfunctions related to mutations or site-directed mutagenesis in titin that alter the I-band stiffness (Granzier & Labeit, 2004;Chung et al 2011;Mateja et al 2013;Methawasin et al 2014;Brynnel et al 2018).…”

Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle

“…It is well established that the passive force magnitude and the SL at which passive force is measurable are correlated to titin size and stiffness (Brynnel et al, 2018; Mateja et al, 2013; Prado et al, 2005). Furthermore, unspecific titin degradation by trypsin or ionizing radiation in skinned fibers leads to passive force reductions proportional to the amount of titin destroyed (Higuchi, 1992; Horowits et al, 1986).…”

“…In agreement, changes to I-band titin affect active force production but are not easily explained under the current paradigm of muscle contraction (Linke, 2018; Nishikawa, 2020). For example, skeletal muscles with genetically modified I-band titin show altered mechanical properties, including muscle stiffness and force, length-dependent activation, and mechanosignaling (Brynnel et al, 2018; Buck et al, 2014; Mateja et al, 2013). A relationship between titin-based and actomyosin-based forces has been suggested to optimize the work generated by muscle contraction (Rivas-Pardo et al, 2016).…”