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

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

SUBMITTER: Hidalgo C

PROVIDER: S-EPMC2764991 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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

Hidalgo Carlos C Hudson Bryan B Bogomolovas Julius J Zhu Yi Y Anderson Brian B Greaser Marion M Labeit Siegfried S Granzier Henk H

Circulation research 20090813 7

<h4>Rationale</h4>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.<h4>Objective</h4>The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension.<h4>Methods and results</h4>Phosphorylation assays wi ...[more]

PMID: 19679839

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Project description:The Proline, Glutamate, Valine and Lysine-rich (PEVK) region of titin constitutes an entropic spring that provides passive tension to striated muscle. To study the functional and structural repercussions of a small reduction in the size of the PEVK region, we investigated skeletal muscles of a mouse with the constitutively expressed C-terminal PEVK exons 219-225 deleted, the TtnΔ219-225 model (MGI: TtnTM 2.1Mgot ). Based on this deletion, passive tension in skeletal muscle was predicted to be increased by ∼17% (sarcomere length 3.0 μm). In contrast, measured passive tension (sarcomere length 3.0 μm) in both soleus and EDL muscles was increased 53 ± 11% and 62 ± 4%, respectively. This unexpected increase was due to changes in titin, not to alterations in the extracellular matrix, and is likely caused by co-expression of two titin isoforms in TtnΔ219-225 muscles: a larger isoform that represents the TtnΔ219-225 N2A titin and a smaller isoform, referred to as N2A2. N2A2 represents a splicing adaption with reduced expression of spring element exons, as determined by titin exon microarray analysis. Maximal tetanic tension was increased in TtnΔ219-225 soleus muscle (WT 240 ± 9; TtnΔ219-225 276 ± 17 mN/mm2), but was reduced in EDL muscle (WT 315 ± 9; TtnΔ219-225 280 ± 14 mN/mm2). The changes in active tension coincided with a switch toward slow fiber types and, unexpectedly, faster kinetics of tension generation and relaxation. Functional overload (FO; ablation) and hindlimb suspension (HS; unloading) experiments were also conducted. TtnΔ219-225 mice showed increases in both longitudinal hypertrophy (increased number of sarcomeres in series) and cross-sectional hypertrophy (increased number of sarcomeres in parallel) in response to FO and attenuated cross-sectional atrophy in response to HS. In summary, slow- and fast-twitch muscles in a mouse model devoid of titin's PEVK exons 219-225 have high passive tension, due in part to alterations elsewhere in splicing of titin's spring region, increased kinetics of tension generation and relaxation, and altered trophic responses to both functional overload and unloading. This implicates titin's C-terminal PEVK region in regulating passive and active muscle mechanics and muscle plasticity.

Dataset Information

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

Publications

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

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