Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury

David Y. Barefield, James W. McNamara, Thomas L. Lynch, Diederik W.D. Kuster, Suresh Govindan, Lauren Haar, Yang Wang, Erik N. Taylor, John N. Lorenz, Michelle L. Nieman, Guangshuo Zhu, Pradeep K. Luther, Andras Varró, Dobromir Dobrev, Xun Ai, Paul M.L. Janssen, David A Kass, Walter Keith Jones, Richard J. Gilbert, Sakthivel Sadayappan

Research output: Contribution to journalArticle

Abstract

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Original languageEnglish (US)
Pages (from-to)236-246
Number of pages11
JournalJournal of Molecular and Cellular Cardiology
Volume129
DOIs
StatePublished - Apr 1 2019

Fingerprint

Cardiac Myosins
Calpain
Reperfusion Injury
Proteolysis
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Transgenic Mice
myosin-binding protein C
Fluorescence Microscopy
Reperfusion
Echocardiography

Keywords

  • Calpain
  • Cardioprotection
  • cMyBP-C
  • Ischemia-reperfusion injury
  • MYBPC3

ASJC Scopus subject areas

  • Molecular Biology
  • Cardiology and Cardiovascular Medicine

Cite this

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury. / Barefield, David Y.; McNamara, James W.; Lynch, Thomas L.; Kuster, Diederik W.D.; Govindan, Suresh; Haar, Lauren; Wang, Yang; Taylor, Erik N.; Lorenz, John N.; Nieman, Michelle L.; Zhu, Guangshuo; Luther, Pradeep K.; Varró, Andras; Dobrev, Dobromir; Ai, Xun; Janssen, Paul M.L.; Kass, David A; Jones, Walter Keith; Gilbert, Richard J.; Sadayappan, Sakthivel.

In: Journal of Molecular and Cellular Cardiology, Vol. 129, 01.04.2019, p. 236-246.

Research output: Contribution to journalArticle

Barefield, DY, McNamara, JW, Lynch, TL, Kuster, DWD, Govindan, S, Haar, L, Wang, Y, Taylor, EN, Lorenz, JN, Nieman, ML, Zhu, G, Luther, PK, Varró, A, Dobrev, D, Ai, X, Janssen, PML, Kass, DA, Jones, WK, Gilbert, RJ & Sadayappan, S 2019, 'Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury', Journal of Molecular and Cellular Cardiology, vol. 129, pp. 236-246. https://doi.org/10.1016/j.yjmcc.2019.03.006
Barefield, David Y. ; McNamara, James W. ; Lynch, Thomas L. ; Kuster, Diederik W.D. ; Govindan, Suresh ; Haar, Lauren ; Wang, Yang ; Taylor, Erik N. ; Lorenz, John N. ; Nieman, Michelle L. ; Zhu, Guangshuo ; Luther, Pradeep K. ; Varró, Andras ; Dobrev, Dobromir ; Ai, Xun ; Janssen, Paul M.L. ; Kass, David A ; Jones, Walter Keith ; Gilbert, Richard J. ; Sadayappan, Sakthivel. / Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury. In: Journal of Molecular and Cellular Cardiology. 2019 ; Vol. 129. pp. 236-246.
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AU - Lynch, Thomas L.

AU - Kuster, Diederik W.D.

AU - Govindan, Suresh

AU - Haar, Lauren

AU - Wang, Yang

AU - Taylor, Erik N.

AU - Lorenz, John N.

AU - Nieman, Michelle L.

AU - Zhu, Guangshuo

AU - Luther, Pradeep K.

AU - Varró, Andras

AU - Dobrev, Dobromir

AU - Ai, Xun

AU - Janssen, Paul M.L.

AU - Kass, David A

AU - Jones, Walter Keith

AU - Gilbert, Richard J.

AU - Sadayappan, Sakthivel

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N2 - Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

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