Oxidative Stress Regulates Titin Elasticity by Affecting Ig-Domain Stability

Abstract

Background: The elasticity of titin is regulated through several mechanisms, including isoform switching and phosphorylation of unique spring elements (N2-Bus, PEVK). However, the titin springs consist mainly of immunoglobulin-like (Ig) domains, which are centrally involved in the molecular mechanism of titin elasticity. Passive force-regulating mechanisms targeting the Ig-domains of titin have not been described.Methods and Results: We have elucidated a novel oxidative stress-related mechanism regulating muscle elasticity by altering the stability of titin-Ig domains (Alegre-Cebollada et al., Cell. 2014;156:1235-46). Using single-molecule AFM force spectroscopy, force measurements of isolated skinned human myocytes, and redox proteomics, we show that I-band Ig-domains of titin are weakened by oxidative modification of cryptic cysteines. We demonstrate that mechanical unfolding of these Ig domains exposes hidden cysteines, which now become accessible to disulfide bonding or S-glutathionylation in the presence of millimolar concentrations of oxidized glutathione (GSSG). In the AFM experiments, the cysteines of unfolded titin-Ig domains preferentially formed mixed disulfides with glutathione, which prevented the refolding of these domains. Oxidation by GSSG substantially reduced the passive tension of stretched human myocytes, and the effect was fully reversible with the incubation of reduced glutathione. Exposing perfused mouse hearts to oxidative stress (0.1 mM H2O2) revealed that Ig-domains from I-band titin are preferential targets of oxidation, as monitored using ICAT labeling/mass spectrometry.Conclusions: Titin elasticity in striated muscle is modulated by oxidative stress through reversible weakening of Ig-domain stability via S-glutathionylation of buried cysteines. These titin Ig domains could also represent individual mechanosensors, whose mechanical properties determine mechano-chemical signaling processes in stressed myocytes.