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

doi:10.1016/j.yjmcc.2019.03.006

. 2019 Apr:129:236-246.

doi: 10.1016/j.yjmcc.2019.03.006. Epub 2019 Mar 9.

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

David Y Barefield¹, James W McNamara², Thomas L Lynch³, Diederik W D Kuster⁴, Suresh Govindan⁵, Lauren Haar⁶, Yang Wang⁶, Erik N Taylor⁷, John N Lorenz⁸, Michelle L Nieman⁸, Guangshuo Zhu⁹, Pradeep K Luther¹⁰, Andras Varró¹¹, Dobromir Dobrev¹², Xun Ai¹³, Paul M L Janssen¹⁴, David A Kass⁹, Walter Keith Jones⁶, Richard J Gilbert¹⁵, Sakthivel Sadayappan¹⁶

Affiliations

¹ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Center for Genetic Medicine, Northwestern University, Chicago, IL, USA. Electronic address: david.barefield@northwestern.edu.
² Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, USA.
³ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, IL, USA.
⁴ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Department of Physiology, Amsterdam Cardiovascular Sciences, VU University Medical Center, Amsterdam, the Netherlands.
⁵ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA.
⁶ Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, IL, USA.
⁷ Department of Physiology and Biophysics, Boston University, Boston, MA, USA.
⁸ Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
⁹ Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁰ Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, UK.
¹¹ Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary.
¹² Institute of Pharmacology, West German Heart and Vascular Center, University Duisburg-Essen, Essen, Germany.
¹³ Department of Physiology and Biophysics, Rush University, Chicago, IL, USA.
¹⁴ Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, OH, USA.
¹⁵ Research Service, Providence VA Medical Center and Brown University, Providence, RI, USA.
¹⁶ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, USA. Electronic address: sadayasl@ucmail.uc.edu.

PMID: 30862451
PMCID: PMC7222036
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 et al. J Mol Cell Cardiol. 2019 Apr.

. 2019 Apr:129:236-246.

doi: 10.1016/j.yjmcc.2019.03.006. Epub 2019 Mar 9.

Authors

Affiliations

¹ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Center for Genetic Medicine, Northwestern University, Chicago, IL, USA. Electronic address: david.barefield@northwestern.edu.
² Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, USA.
³ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, IL, USA.
⁴ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Department of Physiology, Amsterdam Cardiovascular Sciences, VU University Medical Center, Amsterdam, the Netherlands.
⁵ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA.
⁶ Department of Molecular Pharmacology and Therapeutics, Loyola University Chicago, Maywood, IL, USA.
⁷ Department of Physiology and Biophysics, Boston University, Boston, MA, USA.
⁸ Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
⁹ Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
¹⁰ Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, UK.
¹¹ Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Szeged, Hungary.
¹² Institute of Pharmacology, West German Heart and Vascular Center, University Duisburg-Essen, Essen, Germany.
¹³ Department of Physiology and Biophysics, Rush University, Chicago, IL, USA.
¹⁴ Department of Physiology and Cell Biology, The Ohio State University College of Medicine, Columbus, OH, USA.
¹⁵ Research Service, Providence VA Medical Center and Brown University, Providence, RI, USA.
¹⁶ Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA; Heart, Lung and Vascular Institute, Division of Cardiovascular Health and Disease, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH, USA. Electronic address: sadayasl@ucmail.uc.edu.

PMID: 30862451
PMCID: PMC7222036
DOI: 10.1016/j.yjmcc.2019.03.006

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.

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Figures

**Fig. 1.**
Cardiac MyBP-C is dephosphorylated and degraded in ischemic human myocardium. (A) Representative sample of human infarcted heart tissue with annotated remote [R], border zone [B], and ischemic regions [I]. (B) A representative Coomassie blue-stained SDS-PAGE of myofilament protein preparations from the remote, border, and ischemic regions of an ischemic human heart reveals clear protein degradation in the ischemic region. (C) Calpain protease activity measured in the remote, border, and ischemic regions (n = 6). (D) Western blotting with remote, border, and ischemic protein samples shows full-length and degraded 40kDa cMyBP-C (top panel) and cMyBP-C phosphorylated at residues S273, S282, and S302, with actin shown as a loading control (bottom). (E) The relative percentage of full-length and degraded 40kDa cMyBP-C in the remote, border, and ischemic regions (n = 9; # comparison of full-length cMyBP-C among tissue regions, ‡ comparison of 40kDa cMyBP-C among tissue regions using one-way ANOVA with Holm-Sidak *post-hoc* test). (F) Quantification of phosphorylated S273, S282, and S302 normalized against total full-length cMyBP-C (n = 8, top; 4, middle; 6, bottom). All data are mean ± SEM (* P<0.05; one-way ANOVA with Holm-Sidak *post-hoc* test).

**Fig. 2.**
Cardiac MyBP-C is dephosphorylated and degraded in a mouse ischemia/reperfusion model. (A) TTC-staining of I/R-injured mouse hearts showed the remote regions stained blue [R], the border region stained pink [B], and the infarct [I] region in white. The sham heart [S] was stained with TTC and blue dye and shows uniform blue staining. (B) The calpain proteolysis target α-fodrin shows degradation in protein samples from the border and ischemic regions. (C) Calpain activity was determined from sham, remote, border, and infarcted regions (n = 6, S; 6, R; 7, B; 7, I). (D) The levels of cMyBP-C from these regions were determined using Western blot with the anti-cMyBP-C^2–14 antibody, and the extent of cMyBP-C degradation was evaluated. The presence of the 40kDa cMyBP-C N’-terminal fragment was identified in the remote and infarct area. Sarcomeric actin was used as a loading control (lower panel). Western blot analysis of cMyBP-C was performed to determine the level of cMyBP-C phosphorylation using phospho-specific antibodies. (E) Quantification of total cMyBP-C and (*F–H*) phosphorylated cMyBP-C at residues S273, S282, and S302 (n = 6, total protein; 4, phospho-protein). All data are mean ± SEM (* P<0.05; one-way ANOVA with Holm-Sidak *post-hoc* test).

**Fig. 3.**
Calpain proteases degrade cMyBP-C and generate the 40kDa fragment. (A) *In silico* analysis of calpain proteolysis sites on cMyBP-C, with bar height representing the likelihood of cleavage, indicates R271 as a potential location of cleavage (dotted line), corresponding to the known sequence of the 40kDa fragment. The calpain-target site is highlighted in black. (B) Incubation of myofilaments with increasing concentration of calpain with 10 mM calcium for 1 hour at 37 °C shows a dose-dependent increase in cMyBP-C proteolysis and the generation of the 40kDa fragment with a SYPRO Ruby-stained SDS-PAGE loading control (bottom). (C) Proteolysis of cMyBP-C in myofilament fractions incubated with 1 μg calpain for 1 hour at 37 °C is prevented in the absence of calcium or in the presence of 10 nM of the calpain inhibitor MDL 28170. (D) Myofilament protein fractions demonstrate proteolysis at time points following incubation of 20 μg of total myofilament protein with 1 U μ-calpain with 10 mM calcium. (E) Western blotting for cMyBP-C shows reduction in full-length cMyBP-C and an increase in 40kDa cMyBP-C with longer calpain incubation. (F and G) The known calpain targets cTnT and cTnI show a reduction in full-length protein and an increase in degraded protein with increasing incubation time with calpain.

**Fig. 4.**
Transgenic expression of cMyBP-C with ablation of the CTS. (A) Schematic representation of the transgenic cMyBP-C construct which includes an α-myosin heavy chain promoter, N’-terminal Myc tag sequence, *Mybpc3* cDNA, and an hGh polyadenylation site. The ΔCTS construct removes the region coding for CTS, amino acids 272-TSLAGAGRR-280. (B and C) HW:BW ratios showed cardiac hypertrophy in the t/t hearts, with no changes observed in WT(t/t) or ΔCTS(t/t) compared to NTG (n = 8, 8, 7, 8). (D and E) Expression of the hypertrophic markers *Nppa* and *Myh7* normalized to *Gapdh* by qPCR showed a significant elevation in the t/t samples only (n = 3). (F) Separation of myosin heavy chain isoforms by SDS-PAGE to identify the hypertrophic marker β-myosin heavy chain showed an increase in the t/t group only. (G) The expression level of total *Mybpc3* transcript normalized to *Actb* by qPCR shows transgenic overexpression in the WT(t/t) and ΔCTS(t/t) hearts. (H) Myofilament protein fractions from NTG, t/t, WT(t/t), and ΔCTS(t/t) hearts resolved by SDS-PAGE and stained with SYPRO-Ruby showed no changes in cMyBP-C stoichiometry. (I) Western blotting of cMyBP-C from whole-heart lysate reveals normal cMyBP-C stoichiometry in the WT and ΔCTS hearts compared to NTG with no detectable cMyBP-C in the t/t hearts. (J) Two-color fluorescent Western blots of total and phospho-serine cMyBP-C. (*K–M*) Quantification of phosphorylated cMyBP-C at residues 273, 282, and 302 (n = 4). All data are mean ± SEM (* P<0.05 vs. NTG, ‡ P<0.05 vs. t/t, # P<0.05 vs. WT(t/t); one-way ANOVA with Holm-Sidak *post-hoc* test).

**Fig. 5.**
Normal cardiac transmural fiber helical progression in CTS(t/t) hearts compared to NTG, WT(t/t), and t/t ascertained by generalized Q-space MRI (GQI) with tractography. (A) Quantification of CTS helical transmural fiber progression with individual readings (points), mean readings at each location (grey line) and the 95% confidence interval (black lines) shown as the function of relative transmural depth (RTD) in the myocardium from endocardium to epicardium (Endo:Epi) (n = 4 hearts). (B) Transmural helix angle fiber progression shown as the linear regression of mean values for the NTG, t/t, WT(t/t), and CTS(t/t) hearts shown as the function of the relative transmural depth in the myocardium. The (t/t) demonstrates significantly reduced helix angle progression compared to all other groups, which indicates a pathological architectural phenotype. (* P<0.05 compared to NTG, ‡ P < 0.05 compared to t/t; one-way ANOVA on linear regression model with least squared means)

**Fig. 6.**
Transgenic expression of ΔCTS in the t/t background shows normal cardiac structure and function. (A–C) Representative parasternal long axis M-mode echocardiography images showed dilation and reduced contractility only in the t/t hearts (n = 6, 6, 4, 7). (D) Representative pressure-volume loops. (E) Pressure-volume catheterization derived ejection fraction, (F) end diastolic volumes, (G) contractility shown by preload recruitable stroke work (PRSW), and (H) relaxation (Tau) were not significantly different in WT(t/t) and ΔCTS(t/t) compared to NTG, whereas deficits were observed in t/t hearts across all parameters (n = 6, 6, 6, 7). All data are mean ± SEM (* P<0.05 vs. NTG, ‡ P<0.05 vs. t/t; one-way ANOVA with Holm-Sidak *post-hoc* test).

**Fig. 7.**
Prevention of calpain proteolysis of cMyBP-C reduces infarct size following ischemia-reperfusion injury. (A) Western blot of cMyBP-C from NTG, WT(t/t), and ΔCTS(t/t) myofilaments incubated with and without calpain in 1.25 mM calcium shows the generation of the N’-terminal 40 kDa cMyBP-C proteolysis fragment. The fragment appears at a higher weight in the WT(t/t) sample due to the presence of a Myc tag. (B) Quantification of the percent of 40kDa cMyBP-C fragment to total cMyBP-C (n = 3) (* P<0.05; two-way ANOVA). (C) Echocardiography results from NTG, WT(t/t), and ΔCTS(t/t) mice pre- and post-I/R show preserved systolic function in ΔCTS(t/t) mice but not in NTG or WT(t/t) controls. Additionally, NTG and WT(t/t) hearts showed ventricular wall thinning following I/R, whereas this was not apparent in ΔCTS(t/t) hearts (n=5) (horizontal black bars represent p<0.05 between indicated groups; two-way ANOVA). (D) Example tissue sections of NTG, WT(t/t), and ΔCTS(t/t) hearts stained with TTC and Phthalo Blue pigment solution following I/R injury. After cutting into cross sections, tissue mass, area at risk, and infarct areas were measured. Blue myocardium represents the remote area, red stained myocardium represents the area-at-risk, and the white regions are infarcted tissue. (E) Quantification of these areas show the area at risk was similar between the three groups. (F) The % infarct area was significantly reduced in ΔCTS(t/t) hearts. (n = 5, NTG; 6, WT(t/t); 6, ΔCTS(t/t)). Horizontal black bar represents p<0.05 between indicated groups; one-way ANOVA). All data are mean ± SEM.

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[1] Finegold JA, Asaria P, Francis DP, Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisation and United Nations, Int J Cardiol 168(2) (2013) 934–45. - PMC - PubMed

[2] Finegold JA, Asaria P, Francis DP, Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisation and United Nations, Int J Cardiol 168(2) (2013) 934–45. - PMC - PubMed

[3] Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, E.S.C.A.A.H.A.W.H.F.T.F.f.U.D.o.M.I. Joint, C. Authors/Task Force Members, Thygesen K, Alpert JS, White HD, Biomarker S, Jaffe AS, Katus HA, Apple FS, Lindahl B, Morrow DA, E.C.G. Subcommittee, Chaitman BR, Clemmensen PM, Johanson P, Hod H, Imaging S, Underwood R, Bax JJ, Bonow JJ, Pinto F, Gibbons RJ, Classification S, Fox KA, Atar D, Newby LK, Galvani M, Hamm CW, Intervention S, Uretsky BF, Steg PG, Wijns W, Bassand JP, Menasche P, Ravkilde J, Trials, S. Registries, Ohman EM, Antman EM, Wallentin LC, Armstrong PW, Simoons ML, Trials, S. Registries, Januzzi JL, Nieminen MS, Gheorghiade M, Filippatos G, Trials, S. Registries, Luepker RV, Fortmann SP, Rosamond WD, Levy D, Wood D, Trials, S. Registries, Smith SC, Hu D, Lopez-Sendon JL, Robertson RM, Weaver D, Tendera M, Bove AA, Parkhomenko AN, Vasilieva EJ, Mendis S, E.S.C.C.f.P. Guidelines, Bax JJ, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, Funck-Brentano C, Hasdai D, Hoes A, Kirchhof P, Knuuti J, Kolh P, McDonagh T, Moulin C, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Tendera M, Torbicki A, Vahanian A, Windecker S, Document R, Morais J, Aguiar C, Almahmeed W, Arnar DO, Barili F, Bloch KD, Bolger AF, Botker HE, Bozkurt B, Bugiardini R, Cannon C, Lemos J. de, Eberli FR, Escobar E, Hlatky M, James S, Kern KB, Moliterno DJ, Mueller C, Neskovic AN, Pieske BM, Schulman SP, Storey RF, Taubert KA, Vranckx P, Wagner DR , Third universal definition of myocardial infarction, J Am Coll Cardiol 60(16) (2012) 1581–98. - PubMed

[4] Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD, E.S.C.A.A.H.A.W.H.F.T.F.f.U.D.o.M.I. Joint, C. Authors/Task Force Members, Thygesen K, Alpert JS, White HD, Biomarker S, Jaffe AS, Katus HA, Apple FS, Lindahl B, Morrow DA, E.C.G. Subcommittee, Chaitman BR, Clemmensen PM, Johanson P, Hod H, Imaging S, Underwood R, Bax JJ, Bonow JJ, Pinto F, Gibbons RJ, Classification S, Fox KA, Atar D, Newby LK, Galvani M, Hamm CW, Intervention S, Uretsky BF, Steg PG, Wijns W, Bassand JP, Menasche P, Ravkilde J, Trials, S. Registries, Ohman EM, Antman EM, Wallentin LC, Armstrong PW, Simoons ML, Trials, S. Registries, Januzzi JL, Nieminen MS, Gheorghiade M, Filippatos G, Trials, S. Registries, Luepker RV, Fortmann SP, Rosamond WD, Levy D, Wood D, Trials, S. Registries, Smith SC, Hu D, Lopez-Sendon JL, Robertson RM, Weaver D, Tendera M, Bove AA, Parkhomenko AN, Vasilieva EJ, Mendis S, E.S.C.C.f.P. Guidelines, Bax JJ, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, Funck-Brentano C, Hasdai D, Hoes A, Kirchhof P, Knuuti J, Kolh P, McDonagh T, Moulin C, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Tendera M, Torbicki A, Vahanian A, Windecker S, Document R, Morais J, Aguiar C, Almahmeed W, Arnar DO, Barili F, Bloch KD, Bolger AF, Botker HE, Bozkurt B, Bugiardini R, Cannon C, Lemos J. de, Eberli FR, Escobar E, Hlatky M, James S, Kern KB, Moliterno DJ, Mueller C, Neskovic AN, Pieske BM, Schulman SP, Storey RF, Taubert KA, Vranckx P, Wagner DR , Third universal definition of myocardial infarction, J Am Coll Cardiol 60(16) (2012) 1581–98. - PubMed

[5] Benjamin EJ, Virani SS, Callaway CW, Chamberlain AM, Chang AR, Cheng S, Chiuve SE, Cushman M, Delling FN, Deo R, de Ferranti SD, Ferguson JF, Fornage M, Gillespie C, Isasi CR, Jimenez MC, Jordan LC, Judd SE, Lackland D, Lichtman JH, Lisabeth L, Liu S, Longenecker CT, Lutsey PL, Mackey JS, Matchar DB, Matsushita K, Mussolino ME, Nasir K, O’Flaherty M, Palaniappan LP, Pandey A, Pandey DK, Reeves MJ, Ritchey MD, Rodriguez CJ, Roth GA, Rosamond WD, Sampson UKA, Satou GM, Shah SH, Spartano NL, Tirschwell DL, Tsao CW, Voeks JH, Willey JZ, Wilkins JT, Wu JH, Alger HM, Wong SS, Muntner P, American E Heart Association Council on, C. Prevention Statistics, S. Stroke Statistics, Heart Disease and Stroke Statistics-2018 Update: A Report From the American Heart Association, Circulation 137(12) (2018) e67–e492. - PubMed

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

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

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