PKC phosphorylation of titin's PEVK element: A novel and conserved pathway for modulating myocardial stiffness

Carlos Hidalgo, Bryan Hudson, Julius Bogomolovas, Yi Zhu, Brian Anderson, Marion Greaser, Siegfried Labeit, Henk Granzier

Research output: Contribution to journalArticle

177 Scopus citations

Abstract

RATIONALE:: Protein kinase C (PKC) regulates contractility of cardiac muscle cells by phosphorylating thin-and thick-filament-based proteins. Myocardial sarcomeres also contain a third myofilament, titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. OBJECTIVE:: The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. METHODS AND RESULTS:: Phosphorylation assays with PKCα revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin's spring elements showed that PKCα targets the proline-glutamate-valine-lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site-directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKCα (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKCα significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKCα decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and using a serially-linked wormlike chain model we show that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKCα phosphorylation. CONCLUSIONS:: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Original languageEnglish (US)
Pages (from-to)631-638
Number of pages8
JournalCirculation research
Volume105
Issue number7
DOIs
StatePublished - Sep 2009

Keywords

  • Connectin
  • Diastole
  • Passive stiffness
  • Posttranslational modification

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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