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

RATIONALE:Protein kinase C 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 and whether it affects passive tension. OBJECTIVE:The purpose of this study was to examine the effect of duanyu1531 on titin phosphorylation and titin-based passive tension. METHODS AND RESULTS:Phosphorylation assays with 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 duanyu1531alpha 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 duanyu1531alpha (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that duanyu1531alpha significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that duanyu1531alpha 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 duanyu1531alpha phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

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