Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate

Robrecht Thoonen, Shewit Giovanni, Suresh Govindan, Dong Lee, Guang Rong Wang, Timothy D. Calamaras, Eiki Takimoto, David A Kass, Sakthivel Sadayappan, Robert M. Blanton

Research output: Contribution to journalArticle

Abstract

Background - Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. Methods and Results - We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ)-binding domain to identify myocardial-specific PKGI antiremodeling substrates. Our screen identified cardiac myosin-binding protein-C (cMyBP-C), a cardiac myocyte-specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Conclusions - Taken together, these data support that PKGIα and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial-specific PKGIα LZ-dependent antiremodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.

Original languageEnglish (US)
Pages (from-to)1115-1122
Number of pages8
JournalCirculation: Heart Failure
Volume8
Issue number6
DOIs
StatePublished - Nov 1 2015

Fingerprint

Cardiac Myosins
Cyclic GMP-Dependent Protein Kinases
Leucine Zippers
Phosphorylation
Cardiac Myocytes
myosin-binding protein C
G-substrate
Cyclic GMP-Dependent Protein Kinase Type I
Heart Failure
COS Cells
Hypertrophic Cardiomyopathy
Ventricular Pressure
Mutant Proteins
Vasodilation
Hypotension

Keywords

  • cyclic GMP-dependent protein kinase type I
  • heart failure
  • leucine zippers
  • myosin-binding protein C
  • ventricular remodeling

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate. / Thoonen, Robrecht; Giovanni, Shewit; Govindan, Suresh; Lee, Dong; Wang, Guang Rong; Calamaras, Timothy D.; Takimoto, Eiki; Kass, David A; Sadayappan, Sakthivel; Blanton, Robert M.

In: Circulation: Heart Failure, Vol. 8, No. 6, 01.11.2015, p. 1115-1122.

Research output: Contribution to journalArticle

Thoonen, R, Giovanni, S, Govindan, S, Lee, D, Wang, GR, Calamaras, TD, Takimoto, E, Kass, DA, Sadayappan, S & Blanton, RM 2015, 'Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate', Circulation: Heart Failure, vol. 8, no. 6, pp. 1115-1122. https://doi.org/10.1161/CIRCHEARTFAILURE.115.002308
Thoonen, Robrecht ; Giovanni, Shewit ; Govindan, Suresh ; Lee, Dong ; Wang, Guang Rong ; Calamaras, Timothy D. ; Takimoto, Eiki ; Kass, David A ; Sadayappan, Sakthivel ; Blanton, Robert M. / Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate. In: Circulation: Heart Failure. 2015 ; Vol. 8, No. 6. pp. 1115-1122.
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AU - Govindan, Suresh

AU - Lee, Dong

AU - Wang, Guang Rong

AU - Calamaras, Timothy D.

AU - Takimoto, Eiki

AU - Kass, David A

AU - Sadayappan, Sakthivel

AU - Blanton, Robert M.

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