Cardiomyopathy-causing deletion K210 in cardiac troponin T alters phosphorylation propensity of sarcomeric proteins

Abstract

Ca2+ desensitization of myofilaments is indicated as a primary mechanism for the pathogenesis of familial dilated cardiomyopathy (DCM) associated with the deletion of lysine 210 (ΔK210) in cardiac troponin T (cTnT). ΔK210 knock-in mice closely recapitulate the clinical phenotypes documented in patients with this mutation. Considerable evidence supports the proposition that phosphorylation of cardiac sarcomeric proteins is a key modulator of function and may exacerbate the effect of the deletion. In this study we investigate the impact of K210 deletion on phosphorylation propensity of sarcomeric proteins. Analysis of cardiac myofibrils isolated from ΔK210 hearts identified a decrease in phosphorylation of cTnI (46%), cTnT (30%) and MyBP-C (32%) compared with wild type controls. Interestingly, immunoblot analyses with phospho-specific antibodies show augmented phosphorylation of cTnT-Thr203 (28%) and decreased phosphorylation of cTnI-Ser23/24 (41%) in mutant myocardium. In vitro kinase assays indicate that ΔK210 increases phosphorylation propensity of cTnT-Thr203 three fold, without changing cTnI-Ser23/24 phosphorylation. Molecular modeling of cTnT-ΔK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203–224) that lead to a more basic environment around Thr203, which may explain the enhanced PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-ΔK210 binds stronger to cTnI compared with cTnT-wt. Collectively, our observations suggest that cardiomyopathy-causing ΔK210 has far-reaching effects influencing cTnI-cTnT binding and posttranslational modifications of key sarcomeric proteins.

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

“…A 210 has enhanced affinity for cTnI provides further evidence for the long-range effects of this mutation (72).…”

A-kinase anchoring proteins: scaffolding proteins in the heart

“…Indeed, the deletion of lysine-210 of cTnT is associated with familial-dilated cardiomyopathy (52). Interestingly, this mutation, which decreased phosphorylation of cMyBP-C and cTnI (101), impairs PKA anchoring to cTnT (109). On the other hand, a polymorphism at amino acid 2906 of myospryn, which replaces a lysine with an asparagine, is associated with left ventricular hypertrophy in patients with hypertension (79).…”

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component, Troponin T

“…21). A subsequent biochemical study demonstrated that ⌬K210 caused Thr-203, located at the N terminus of the H1(T2) helix of TnT, to be more susceptible to phosphorylation by PKC-␣ when the TnT was complexed with TnI and TnC (57). These authors speculated that there might be a change in the electrostatic microenvironment near Thr-203 because of the deletion of Lys-210 (57).…”

“…A subsequent biochemical study demonstrated that ⌬K210 caused Thr-203, located at the N terminus of the H1(T2) helix of TnT, to be more susceptible to phosphorylation by PKC-␣ when the TnT was complexed with TnI and TnC (57). These authors speculated that there might be a change in the electrostatic microenvironment near Thr-203 because of the deletion of Lys-210 (57). Taken together with our results, this suggests that the modification of the conserved residues located at the N-terminal side of the H1(T2) helix appears to have a more drastic, and possibly deteriorative, effect on the Ca 2ϩ -stimulated functions of cardiac muscle fibers, although it is yet to be delineated as to whether the N terminus of the helix is involved in interaction with another component of muscle proteins or is critical for the stability of the helix.…”