Titin is a giant elastic protein expressed in the contractile units of striated muscle cells, including the sarcomeres of cardiomyocytes. The last decade has seen enormous progress in our understanding of how titin molecular elasticity is modulated in a dynamic manner to help cardiac sarcomeres adjust to the varying hemodynamic demands on the heart. Crucial events mediating the rapid modulation of cardiac titin stiffness are post‐translational modifications (PTMs) of titin. In this review, we first recollect what is known from earlier and recent work on the molecular mechanisms of titin extensibility and force generation. The main goal then is to provide a comprehensive overview of current insight into the relationship between titin PTMs and cardiomyocyte stiffness, notably the effect of oxidation and phosphorylation of titin spring segments on titin stiffness. A synopsis is given of which type of oxidative titin modification can cause which effect on titin stiffness. A large part of the review then covers the mechanically relevant phosphorylation sites in titin, their location along the elastic segment, and the protein kinases and phosphatases known to target these sites. We also include a detailed coverage of the complex changes in phosphorylation at specific titin residues, which have been reported in both animal models of heart disease and in human heart failure, and their correlation with titin‐based stiffness alterations. Knowledge of the relationship between titin PTMs and titin elasticity can be exploited in the search for therapeutic approaches aimed at softening the pathologically stiffened myocardium in heart failure patients.
The giant sarcomere protein titin is a major determinant of cardiomyocyte stiffness and contributor to cardiac strain sensing. Titin-based forces are highly regulated in health and disease, which aids in the regulation of myocardial function, including cardiac filling and output. Due to the enormous size, complexity, and malleability of the titin molecule, titin properties are also vulnerable to dysregulation, as observed in various cardiac disorders. This review provides an overview of how cardiac titin properties can be changed at a molecular level, including the role isoform diversity and posttranslational modifications (acetylation, oxidation and phosphorylation) play in regulating myocardial stiffness and contractility. We then consider how this regulation becomes unbalanced in heart disease, with an emphasis on changes in titin stiffness and protein quality control. In this context, new insights into the key pathomechanisms of human cardiomyopathy due to a truncation in the titin gene (TTN) are discussed. Along the way, we touch on the potential for titin to be therapeutically targeted to treat acquired or inherited cardiac conditions, such as HFpEF or TTN-truncation cardiomyopathy.
The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.
Aim: Dantrolene interacts with ryanodine receptors in skeletal and cardiac muscle affecting sarcoplasmic reticulum calcium release. Since dantrolene is lipophilic it could also affect intra-sarcoplasmic reticulum calcium handling proteins such as calsequestrin. This study investigated whether dantrolene (1-30 µmol/L) alters the polymerization state and the calcium binding capacity of recombinant cardiac calsequestrin and cardiac sarcoplasmic reticulum calcium handling. Methods: Human recombinant cardiac calsequestrin was used to make simultaneous measurements of turbidity (to indicate calsequestrin polymerization) and calcium binding to calsequestrin in the presence and absence of dantrolene.Caffeine-induced Ca 2+ transients were used to investigate the effects of dantrolene on sarcoplasmic reticulum calcium loading and release in saponin-permeabilized cardiomyocytes laid down in monolayers in 96-well array plates. Results: Dantrolene (1-30 µmol/L) increased the polymerization state of calsequestrin and its calcium binding capacity. In the presence of dantrolene, calsequestrin-dependent turbidity increased 2.5-11.5-fold at 1.0 mmol/L calcium added to unbuffered Ca 2+ solutions and 3-10-fold when calcium was raised from 0.06 to 30 µmol/L. The dantrolene-dependent increase in turbidity at 30 µmol/L dantrolene was associated with a 3-fold increase in the number of calcium ions bound per calsequestrin molecule at 30 and 100 µmol/L calcium. The caffeine-induced releasable calcium loaded by the sarcoplasmic reticulum at 0.63 µmol/L free calcium in permeabilized cardiomyocytes was also increased in the presence of 30 µmol/L dantrolene. Conclusion: Dantrolene alters the polymerization state and the calcium binding properties of cardiac calsequestrin and increases sarcoplasmic reticulum calcium loading in permeabilized cardiomyocytes.
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