N-terminal fragment of cardiac myosin binding protein C modulates cooperative mechanisms of the thin filament activation in the atria and ventricles

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Abstract

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C lies on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both the kinetics of the ATP hydrolysis cycle and the lifetime of the cross bridge, as well as the calcium regulation of actin-myosin interaction, thereby modulating the contractile function of the myocardium. The role of cMyBP-C in atrial contraction is poorly studied. We examined the effect of the N-terminal C0-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of nucleotide, ATP, and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less strongly than ventricular myosin, and the C0-C2 fragment makes these differences in myosin isoforms more pronounced.

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About the authors

A. M. Kochurova

Institute of Immunology and Physiology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com
Russian Federation, 620049 Yekaterinburg

E. A. Beldiia

Institute of Immunology and Physiology of the Russian Academy of Sciences; Ural Federal University

Email: dvshchepkin@gmail.com
Russian Federation, 620049 Yekaterinburg; 620002 Ekaterinburg

V. V. Nefedova

Research Center of Biotechnology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com

Bach Institute of Biochemistry

Russian Federation, 119071 Moscow

N. S. Ryabkova

Lomonosov Moscow State University; HyTest Ltd.

Email: dvshchepkin@gmail.com

Department of Biochemistry, Faculty of Biology

Russian Federation, 119234 Moscow; 20520 Turku, Finland

D. S. Yampolskaya

Research Center of Biotechnology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com

Bach Institute of Biochemistry

Russian Federation, 119071 Moscow

A. M. Matyushenko

Research Center of Biotechnology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com

Bach Institute of Biochemistry

Russian Federation, 119071 Moscow

S. Y. Bershitsky

Institute of Immunology and Physiology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com
Russian Federation, 620049 Yekaterinburg

G. V. Kopylova

Institute of Immunology and Physiology of the Russian Academy of Sciences

Email: dvshchepkin@gmail.com
Russian Federation, 620049 Yekaterinburg

D. V. Shchepkin

Institute of Immunology and Physiology of the Russian Academy of Sciences

Author for correspondence.
Email: dvshchepkin@gmail.com
Russian Federation, 620049 Yekaterinburg

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2. Fig. 1. Electropherograms of the preparation of the C0-C2 fragment of cMyBP-C and myosin heavy chains. a – Electropherogram of the preparation of the C0-C2 fragment of cMyBP-C: 1 – molecular weight marker; 2 – preparation of the C0-C2 fragment. b – Electropherogram for determining the composition of myosin heavy chain isoforms. PAGE is stained with SYPRO Ruby (Thermo Fisher Scientific, USA). The image was obtained using a GS-800 densitometer (Bio-Rad, USA). MHC – myosin heavy chains; 1 – myosin from the left ventricle; 2 – molecular weight marker (Mark12™ Unstained Standard, Invitrogen™, USA); 3 – myosin from the left atrium

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3. Fig. 2. Effect of 500 nM C0-C2 fragment of cMyBP-C on the Ca2+ dependence of the sliding velocity of thin filaments on atrial (a) and ventricular (b) myosin in an in vitro motile system. Experimental data (mean ± standard deviation) were approximated by the Hill equation (1), the parameters of the equation are given in Table 2.

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4. Fig. 3. Effect of the C0-C2 fragment of cMyBP-C on the dependence of the sliding velocity of thin filaments on the concentration of atrial (a) and ventricular (b) myosin in the in vitro motile system. The experimental values ​​of the velocities (mean ± standard deviation) were approximated by the Hill equation (1). The myosin concentrations at which the filament velocity was halved (Cmyosin) for ventricular myosin at pCa 4 were 97.0 ± 1.5 nM and 62.4 ± 1.2* nM without the C0-C2 fragment and with its addition, respectively; at pCa 6, these values ​​were 95.4 ± 0.6 nM and 76.7 ± 1.4 * nM. For atrial myosin, the Cmyosin values ​​at pCa 4 were 111.2 ± 1.2 nM and 67.6 ± 1.3* nM; at pCa 5.5 – 121.6 ± 1.3 nM and 37.5 ± 0.5* nM. The symbol * indicates a significant difference in the Cmyosin value in the presence of the C0-C2 fragment from that in its absence (p < 0.05)

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5. Fig. 4. Effect of the C0-C2 fragment of cMyBP-C on the dependence of the sliding velocity of F-actin (a and b) and regulated thin filaments at pCa 4 (c and d) on atrial (a, c) and ventricular (b, d) myosin on the ATP concentration in the in vitro motile system. Experimental data (mean ± standard deviation) were approximated by the Hill equation (1). The CATP values ​​for the dependence of the F-actin sliding velocity on the ATP concentration in the IPS on ventricular myosin were 55.2 ± 0.1 μM and 6.5 ± 0.1* μM, and on atrial myosin – 42.8 ± 0.1 μM and 26.6 ± 0.1* μM without and in the presence of the C0-C2 fragment, respectively. The CATP values ​​for the dependence of the sliding velocity of thin filaments on the ATP concentration for ventricular myosin were 92.5 ± 2.5 μM and 86.4 ± 4.5 μM, and for atrial myosin – 53.9 ± 0.5 μM and 56.2 ± 2.5 μM. The symbol * indicates the difference in the CATP value in the presence of the C0-C2 fragment from that in its absence (p < 0.05)

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6. Fig. 5. Effect of the C0-C2 fragment of cMyBP-C on the dependence of the sliding velocity of F-actin (a and c) and regulated thin filaments at pCa 4 (b and d) on atrial (a, b) and ventricular (c, d) myosin on the ADP concentration in the in vitro motile system. Experimental data (mean ± standard deviation) for F-actin (a, b) and thin filaments (d) with the C0-C2 fragment are approximated by a linear function; experimental data for F-actin and thin filaments without the C0-C2 fragment with both myosin isoforms (a, c) and for thin filaments with ventricular myosin are approximated by a logistic function (2). The CADP values ​​without the C0-C2 fragment for the dependence of the F-actin sliding velocity on atrial myosin on the ADP concentration were 3.03 ± 0.35 mM, and for the dependence of the thin filament velocity – 3.46 ± 0.23 mM. The CADP values ​​without the C0-C2 fragment for the dependence of the F-actin sliding velocity on ventricular myosin on the ADP concentration were 1.84 ± 0.2 mM; for thin filaments, the CADP value without the C0-C2 fragment was 2.12 ± 0.15 mM, and in its presence – 1.00 ± 0.25 mM

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7. Fig. 6. Effect of ADP on the calcium dependence of the sliding velocity of thin filaments on ventricular (a) and atrial (b) myosin in an in vitro motile system. Experimental values ​​of the velocities (mean ± standard deviation) are approximated by the Hill equation (1). The parameters of the Hill equation are presented in Table 3.

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