Work Done by Titin Protein Folding Assists Muscle Contraction

Abstract

Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6-8 pN. We use single molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but so far unrecognized contributor to the force generated by a contracting muscle.

Effects of activation on the elastic properties of intact soleus muscles with a deletion in titin

“…These include hypotheses that refolding of unfolded Ig domains might increase the speed of muscle shortening (Rivas-Pardo et al, 2016), to the idea that the PEVK region of titin sticks to thin filaments (Rode et al, 2009), and the suggestion that the crossbridges not only translate but also rotate the thin filaments, thereby winding titin upon them . Leonard and Herzog (2010) and others (Herzog et al, 2012Monroy et al, 2012;Nishikawa et al, 2012;Rassier et al, 2015) have speculated that the increase in titin-based stiffness upon muscle activation might be due to titin binding to actin or thin filaments.…”

“…When muscles are activated, an increase in muscle stiffness can be measured even before the cross-bridges begin to produce force, suggesting that a non-cross-bridge element contributes to the increased muscle stiffness (Bagni et al, 2002(Bagni et al, , 2004Rassier et al, 2015). Although titin has long been thought to contribute to muscle passive tension (Maruyama, 1976;Magid and Law, 1985;Wang et al, 1991;Linke et al, 1998), a role for titin in active muscle has increasingly been proposed (Tatsumi et al, 2001;Bagni et al, 2002;Herzog and Leonard, 2002;Leonard and Herzog, 2010;Lindstedt et al, 2002;Nishikawa et al, 2012;Rassier et al, 2015;Herzog et al, 2016;Rivas-Pardo et al, 2016). Recent work on single myofibrils stretched beyond overlap of the thick and thin filaments clearly demonstrates that titin-based stiffness increases upon Ca 2+ activation (Leonard and Herzog, 2010;Powers et al, 2014Powers et al, , 2016.…”

Biasing effects of receptor-ligand complexes on protein-unfolding statistics

“…Due to the nonlinear elasticity of linker molecules (e.g., polyethylene glycol, spacers, or unfolded protein backbone), the force loading rateḞ =Ḟ (F ) becomes a function of the force and the integral in Eq. (1) can no longer be evaluated analytically. In a standard pulling experiment, a harmonic pulling device (e.g., atomic force microscopy cantilever or optically trapped bead) is connected to the aforementioned linker molecules.…”

“…Refolding of individual titin domains is believed to assist in muscle contraction [1], stretching forces expose cryptic binding sites involved in focal adhesions [2], and mechanically stable receptor-ligand pairs govern the assembly of large extracellular machineries and adhesion of bacterial cells to their cellulosic carbon sources [3,4]. Single-molecule pulling experiments with atomic force microscopes [5], optical tweezers [6], or magnetic tweezers [7] have become widely used techniques to study such phenomena at the singlemolecule level.…”

Editorial on EMC 2017 special issue

“…Our classic view considered titin being (only) a structural filament protein that acts as a blueprint for sarcomere assembly, centers myosin in the sarcomere, and may serve as a signaling node and important determinant of myofilament passive tension. However, we may have to rethink the view on titin as a passive contributor to myofilament function, as emerging evidence suggests an additional role of titin in contributing to active force development (Rivas-Pardo et al 2016).…”