Published online before print August 13, 2009, doi: 10.1161/CIRCRESAHA.109.198465
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© 2009 American Heart Association, Inc.
Molecular Medicine |
PKC Phosphorylation of Titin’s PEVK Element
A Novel and Conserved Pathway for Modulating Myocardial Stiffness
From the Sarver Molecular Cardiovascular Research Program (C.H., B.H., Y.Z., B.A., H.G.), University of Arizona, Tucson; the Institute for Integrative Pathophysiology (J.B., S.L.), Universitätsmedizin Mannheim, Germany; and the Department of Animal Science (M.G.), University of Wisconsin, Madison.
Correspondence to Henk Granzier, Department of Physiology, University of Arizona, PO Box 245217, Tucson, AZ 85724. E-mail granzier{at}email.arizona.edu
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 PKC
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 PKC 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 PKC (S11878 and S12022); when these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKC significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC 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 PKC phosphorylation.
Conclusions: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.
Key Words: connectin • diastole • passive stiffness • posttranslational modification
Related Article:
Circ. Res. 2009 105: 611-613.
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  S. H. Ahmed and M. L. Lindsey Titin Phosphorylation: Myocardial Passive Stiffness Regulated by the Intracellular Giant Circ. Res., September 25, 2009; 105(7): 611 - 613. [Full Text] [PDF]
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