Roles of N2A Titin in Signaling and Muscle Mechanics

Since the discovery of titin in the late 1970's, the N2A region has been of interest as a candidate locus for participation in cellular signaling and as a mechanical hub that mediates the transition from passive to active mechanical properties of skeletal muscles. The purpose of this review is to highlight our current understanding of the role of N2A titin in skeletal muscle mechanics and signaling. We review the known binding partners that interact with N2A titin and the evidence for calcium-dependent interactions between N2A titin and actin, as well as how these interactions might be stabilized in muscle sarcomeres. Evidence from co-sedimentation studies, atomic force microscopy and in vitro motility assays suggests that titin binds to actin at pCa = 6, before cross-bridges begin to produce force. This calcium-dependent N2A-actin binding eliminates low force straightening of the proximal tandem Ig domains, which would otherwise limit titin-based forces in actively stretched muscles. The idea that cross-bridges could account for the enhanced forces and energy stored during active stretch of skeletal muscles is incompatible with the much smaller strains experienced by cross bridges and the much shorter duration of their attachments to actin. A wide variety of experiments including eccentric contraction, residual force enhancement, and stretch-shortening cycles demonstrates a role for titin in force enhancement during and after stretching of active muscles. Based on the available data from different laboratories, we have developed a model in which two proximal tandem immunoglobulin domains, Ig80 and Ig83, act at the nucleation trigger for N2A-actin binding, with the binding of I83 providing the calcium dependence that has been associated with this interaction. While these domains appear necessary for N2A-actin binding, their binding affinities are likely not sufficiently strong to withstand the high forces experienced during stretch of active muscles, requiring other stabilizing interactions. The available evidence suggests that the proximal PEVK domains and CARP/MARP1 likely stabilize N2A - actin binding in active muscles, although neither of these interactions is itself calcium-dependent. The available biophysical and muscle mechanics data support the view that titin is a tunable viscoelastic material in muscles with calcium-dependent viscoelastic properties that modulate the response of muscles to passive and active stretch.