期刊
CIRCULATION RESEARCH
卷 105, 期 7, 页码 631-U23出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/CIRCRESAHA.109.198465
关键词
connectin; diastole; passive stiffness; posttranslational modification
资金
- NIH [HL62881, HL77196, CA23074]
- DFG [La668/11-1]
- NIEHS [ES06694]
- University of Arizona
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 alpha 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 alpha 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 alpha (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 alpha significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC alpha 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 alpha phosphorylation. Conclusions: PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness. (Circ Res. 2009; 105: 631-638.)
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