Calcium increases titin N2A binding to F-actin and regulated thin filaments

Abstract

Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A – F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A – actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A – actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin – actin binding occurs in skeletal muscle sarcomeres in vivo.

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

“…Calcium is an important regulator of muscle activation and the I91 domain in titin has been previously shown to be more stable in the presence of Ca 2+ . We hypothesized that Ca 2+ ‐enhanced stability might explain the Ca 2+ ‐enhanced binding observed between a construct of the N2A region and actin filaments . We repeated the equilibrium stability experiments in the presence of 50 μM CaCl 2 (pCa 4.3) to test this hypothesis.…”

“…In one proposed model, the N2A region of titin binds to actin upon an influx of calcium ions (~pCa 4.5) that occurs with muscle activation . This model is supported by a recent study demonstrating the binding of a recombinantly expressed N2A protein to actin and that this binding is enhanced in the presence of calcium …”

“…Upon influx of calcium in the active state, however, it has been established that p94 is activated and dissociated from titin . The N2A region has demonstrated enhanced binding to actin in the presence of calcium but the exact binding site remains unknown. The dissociation of p94 may open up a new binding site for actin during in eccentric contractions.…”

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

“…; Dutta et al . ). In future work, the definition of the structural basis of the tunability of titin stiffness in situ will be possible by combining sarcomere‐level mechanics and X‐ray diffraction in intact fibres from the frog muscle and, eventually, under the condition that the sarcomere‐level resolution is preserved, in demembranated fibres from mouse muscle with specific mutations in the relevant titin domains.…”

“…A way to test whether the undamped elasticity is due to the PEVK region could be to apply our protocols to investigate if the specific PKC-α-mediated changes in PEVK stiffness (Hidalgo et al 2009) imply similar changes in the undamped titin stiffness. Notably, other mechanisms beyond Ig domains could contribute to the structural dynamics that integrate our undamped stiffness measure and make titin a tunable spring, among them, either the presence of breakable intramolecular links that change titin contour length (Kellermayer et al 2001), or the dynamic binding of the PEVK region of titin to actin that would keep the length of the titin spring constant at different SLs by shifting the titin bonds along the actin filament Dutta et al 2018). In future work, the definition of the structural basis of the tunability of titin stiffness in situ will be possible by combining sarcomere-level mechanics and X-ray diffraction in intact fibres from the frog muscle and, eventually, under the condition that the sarcomere-level resolution is preserved, in demembranated fibres from mouse muscle with specific mutations in the relevant titin domains.…”