Phosphorylating Titin’s Cardiac N2B Element by ERK2 or CaMKIIδ Lowers the Single Molecule and Cardiac Muscle Force

Abstract

Titin is a large filamentous protein that is responsible for the passive force of the cardiac sarcomere. Titin's force is generated by its I-band region, which includes the cardiac-specific N2B element. The N2B element consists of three immunoglobulin domains, two small unique sequence insertions, and a large 575-residue unique sequence, the N2B-Us. Posttranslational modifications of the N2B element are thought to regulate passive force, but the underlying mechanisms are unknown. Increased passive-force levels characterize diastolic stiffening in heart-failure patients, and it is critical to understand the underlying molecular mechanisms and identify therapeutic targets. Here, we used single-molecule force spectroscopy to study the mechanical effects of the kinases calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) and extracellular signal-regulated kinase 2 (ERK2) on the single-molecule mechanics of the N2B element. Both CaMKIIδ and ERK2 were found to phosphorylate the N2B element, and single-molecule force spectroscopy revealed an increase in the persistence length (Lp) of the molecule, indicating that the bending rigidity of the molecule was increased. Experiments performed under oxidizing conditions and with a recombinant N2B element that had a simplified domain composition provided evidence that the Lp increase requires the N2B-Us of the N2B element. Mechanical experiments were also performed on skinned myocardium before and after phosphorylation. The results revealed a large (∼30%) passive force reduction caused by CaMKIIδ and a much smaller (∼6%) reduction caused by ERK2. These findings support the notion that the important kinases ERK2 and CaMKIIδ can alter the passive force of myocytes in the heart (although CaMKIIδ appears to be more potent) during physiological and pathophysiological states.

Experimentally Increasing the Compliance of Titin Through RNA Binding Motif-20 (RBM20) Inhibition Improves Diastolic Function In a Mouse Model of Heart Failure With Preserved Ejection Fraction

“…This notion is consistent with the indistinguishable mobility of the N2BAsc isoforms expressed in the LV of the conventional and conditional Rbm20 Δ RRM mice (data not shown). The functional consequence of upregulating spring element exons is an increase in the contour length of the spring region: the tandem Ig segment by 34 × ~5 nm (increase due to Ig domain upregulation, with an estimated domain spacing of ~ 5 nm) and the PEVK region by 300× 0.35 nm (the number of upregulated amino acid residues times their maximal residue spacing of ~0.35 nm) 35 . This increased contour length explains the increased slack sarcomere length that was found (Fig.…”

Posttranslational modifications of titin from cardiac muscle: how, where, and what for?

“…Dephosphorylation of S4010, S4062, S4099, and S4185 can occur in vivo through PP5 . The phosphorylation of all these sites has been associated with a decrease in titin‐based cardiomyocyte passive tension , the dephosphorylation with an increase (Fig. A).…”

“…In theory, the phosphorylation of both segments together might cause no change in passive tension, as the mechanical effects could cancel out each other. However, mechanical experiments have demonstrated that the passive force of permeabilized cardiomyocytes decreases with ex vivo CaMKIIδ‐treatment . Moreover, passive tension was elevated in cardiomyocytes of CaMKIIδ/γ double‐knockout mouse hearts and reduced in those of CaMKIIδ‐overexpressing transgenic mouse hearts, relative to WT mouse cardiomyocytes .…”

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

“…The elastic segment of titin has been shown to be a major target of post-translational modifications. While phosphorylation of residues within the unstructured regions of titin have been shown to cause modest changes in the elasticity of titin (97–99), far more drastic changes can be achieved by targeting the Ig domains of the I-band. In a mouse model of myocardial infarction, oxidative stress due to ischemia-reperfusion injury lead to massive S-glutathionylation of sarcomeric proteins, including some very high molecular weight proteins alleged to be titin (100).…”