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

doi:10.1016/j.yjmcc.2019.03.006

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

^*Corresponding author for this work

Neuroscience

Research output: Contribution to journal › Article › peer-review

21 Scopus citations

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 language	English (US)
Pages (from-to)	236-246
Number of pages	11
Journal	Journal of Molecular and Cellular Cardiology
Volume	129
DOIs	https://doi.org/10.1016/j.yjmcc.2019.03.006
State	Published - Apr 2019

Funding

The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356, R01HL105826, and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs, including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.). The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356 , R01HL105826 , and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs , including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.).

Keywords

Calpain
Cardioprotection
Ischemia-reperfusion injury
MYBPC3
cMyBP-C

ASJC Scopus subject areas

Cardiology and Cardiovascular Medicine
Molecular Biology

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10.1016/j.yjmcc.2019.03.006

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Barefield, D. Y., McNamara, J. W., Lynch, T. L., Kuster, D. W. D., Govindan, S., Haar, L., Wang, Y., Taylor, E. N., Lorenz, J. N., Nieman, M. L., Zhu, G., Luther, P. K., Varró, A., Dobrev, D., Ai, X., Janssen, P. M. L., Kass, D. A., Jones, W. K., Gilbert, R. J., & 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, 129, 236-246. https://doi.org/10.1016/j.yjmcc.2019.03.006

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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.",

keywords = "Calpain, Cardioprotection, Ischemia-reperfusion injury, MYBPC3, cMyBP-C",

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

note = "Funding Information: The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356, R01HL105826, and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs, including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.). Funding Information: The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356 , R01HL105826 , and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs , including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.). Publisher Copyright: {\textcopyright} 2019 The Authors",

year = "2019",

month = apr,

doi = "10.1016/j.yjmcc.2019.03.006",

language = "English (US)",

volume = "129",

pages = "236--246",

journal = "Journal of Molecular and Cellular Cardiology",

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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

TY - JOUR

T1 - Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury

AU - Barefield, David Y.

AU - McNamara, James W.

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

N1 - Funding Information: The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356, R01HL105826, and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs, including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.). Funding Information: The authors were supported by NIH National Heart, Lung, and Blood grants, including R01HL130356 , R01HL105826 , and K02HL114749 (to S.S.); R01DC011528 (to R.J.G.); and F32HL131304 (to D.Y.B), R01-HL131517 and R01-HL136389 (to D.D.), the American Heart Association Midwest Affiliate Research Programs , including Cardiovascular Genome-Phenome Study 15CVGPSD27020012 and Catalyst 17CCRG33671128 (to S.S.), Predoctoral Fellowship 11PRE7240022 (to D.Y.B.), Postdoctoral Fellowship 13POST14720024 (to S.G.), Postdoctoral Fellowship 13POST17220009 (to D.W.D.K.), Predoctoral Fellowship 15PRE22430028 (to T.L.L.IV.), Postdoctoral Fellowship 17POST33630095 (to J.W.M), and a British Heart Foundation Programme Grant RG/11/21/29335 (to P.K.L.). Publisher Copyright: © 2019 The Authors

PY - 2019/4

Y1 - 2019/4

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.

AB - 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.

KW - Calpain

KW - Cardioprotection

KW - Ischemia-reperfusion injury

KW - MYBPC3

KW - cMyBP-C

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UR - http://www.scopus.com/inward/citedby.url?scp=85063037205&partnerID=8YFLogxK

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DO - 10.1016/j.yjmcc.2019.03.006

M3 - Article

C2 - 30862451

AN - SCOPUS:85063037205

SN - 0022-2828

VL - 129

SP - 236

EP - 246

JO - Journal of Molecular and Cellular Cardiology

JF - Journal of Molecular and Cellular Cardiology

ER -

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

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