Posttranslational Modification of Titin Domains as a Main Regulator of Myocardial Stiffness

Abstract

The giant protein titin is responsible for the elasticity of striated muscle cells and engages in both mechanical and signaling functions of the heart. TTN, which encodes titin, is a major human disease gene. Mutations in the myosin-bound part of titin account for a large share of familial cases of dilated cardiomyopathy. The elasticity of cardiac titin, which resides in the molecule's I-band region, is regulated by various means, including titin-isoform switching, phosphorylation of unique spring elements by different kinases, and oxidative stress-related alterations to titin. In failing human hearts, titin is hypo-phosphorylated, which appears to be due in part to a phosphorylation deficit at the cardiac-specific N2-B unique sequence and likely contributes to a pathologically increased (titin-based) diastolic stiffness. A recent focus has been the regulation of the mechanical properties of the immunoglobulin-like (Ig-)domains in the titin spring segment. New evidence suggests that a low but non-negligible number of Ig-domains unfolds-refolds within the physiological sarcomere-length range. These Ig-domains are targeted via an oxidative stress-related mechanism recently identified by single-molecule AFM force spectroscopy. If unfolded, the Ig-domains expose cryptic cysteines, which in the presence of oxidized glutathione can become S-glutathionylated. The presence of mixed disulfide(s) with glutathione weakens the mechanical stability of the unfolded Ig-domains and prevents their refolding, thus reducing titin stiffness. Incubation of stretched (skinned) human cardiomyocytes with oxidized glutathione significantly lowered their passive tension, and the effect was fully reversible on incubation with a reductant. In experimental mouse hearts exposed to oxidative stress (0.1 mM H2O2), I-band Ig-domains of titin were identified as preferential targets of oxidation using ICAT labeling/mass spectrometry. These findings establish that disrupted Ig-folding-unfolding dynamics can cause sustained but reversible changes to titin elasticity, which may also be relevant in heart disease.