(C-91) The Role of N-Terminal Cardiac Myosin-Binding Protein C in Controlling Myosin Head States

Introduction:Myosin is a key protein in muscle contraction. Myosin heads have been discovered to exist in two different potential states.^1–7 The two states are commonly referred to as disordered relaxed state or relaxed state (RX) and super-relaxed state (SRX); when in SRX they are unable to bind to actin. The populations have different ATP turnover rates in which the RX is tenfold faster than the SRX; SRX is also more energy efficient than RX.^1,4–7 The ratio of these populations can regulate muscle force through energy consumption and available myosin heads.^2,8,9

Cardiac myosin-binding protein C (cMyBP-C) plays important roles in myofilament regulation.^10–13 The N-terminal region (C0-C2) is a point of interest since it has been shown to play regulatory roles by interacting with myosin-S2 and actin.^11–13

This report seeks to improve our understanding of cMyBP-C and its role in muscle contraction by questioning if the N-terminal region (C0-C2) of cMyBP-C affects myosin heads to control the populations of RX and SRX in cardiac tissue. To accomplish this task, we used cardiac myofibrils from wild type and N-terminal deletion rats with and without Ca2+ and mavacamten to investigate the relationship between the populations of RX and SRX myosin heads and the N-terminus region of cMyBP-C. The significance of this research is to provide a direct correlation between N-terminal cMyBP-C and myosin populations, which may have therapeutic potentials for treatment of heart failure.
Materials and Methods: Two types of myofilbrils will be used, one is wild type the other is C0-C2 cMyBP-C Deletion. The stopped-flow experiments were performed with a KinTek stopped-flow spectrometer with the chamber temperature set to 20oc for all trials. The myofibrils are prepared with fiber preparation buffer overnight containing 6 mM imidazole, 8 mM Mg-acetate, 70 mM K-propionate, 5 mM EGTA, 7 mM ATP, 1 mM NaN3, and pH 7.0 and then homogenized through mechanical means in myofibril stopped flow buffer containing 106mM K-acetate, 50mM Imidazole, 12mM Mg-acetate, 2mM EGTA, and pH 7.0. The myofibrils are then incubated with 10 µM MANT-ATP for ~1 minute. Experiments were conducted by rapidly mixing equal volume of 250 µM ATP and the incubated 100 nM myofibrils. Data was recorded on three scales each with 1000 data points: 25, 200, and 500 seconds.

The collected data were fit using Sigma Plot to produce a double exponential, the two phases show the populations of RX and SRX and the two rates show the tenfold difference in turnover.¹

Results, Conclusions, and Discussions: The relaxed wild type (WT) myofibrils showed two populations: 62.2% for RX and 37.8% for the SRX. Apparently, high population of SRX is a feature of the relaxed myofibrils. The addition of CaCl2 to the myofibrils, which causes the muscle to activate through the Ca2+-troponin binding, significantly changed the ratio of RX to SRX. The relative proportion of the RX increased to 80.1% and the SRX decreased to 19.9%. Adding mavacamten to WT fibers reduced the RX population to 41.1% and increased the SRX to 58.9%. These results show that the population of myosin heads change from SRX to RX when muscle is activated in order to generate force at a quicker rate.

Comparing to the relaxed WT-myofibrils, deletion of C0-C2 of MyBPC shifted the ratio of RX and SRX to 78.33% for RX and 21.67% for the SRX. The addition of calcium chloride to the deletion myofibrils increased the relative proportion of RX to 99.73% and the SRX decreased to 0.27%. The addition of mavacamten to deletion fibers had a lessened effect, changing RX and SRX to 61.1% and 38.9% respectively. These results show a significant loss of the SRX when compared to the wild type population which has been seen when the whole protein has been removed as well as a reduced effect of the inhibitory drug mavacamten.⁸

Our results showed a significant difference in the populations of RX and SRX in N-terminal deletion cMyBP-C myofibrils when compared to wild type. This suggests that the N-terminal domain of cMyBP-C plays important roles in controlling SRX, which may impact cardiac muscle regulation and activation, thus affecting muscle force generation.

Stopped-flow is a unique way to monitor the rapid changes to myosin heads that occur during the turnover of ATP. Our results strongly suggest that the N-terminal region of cMyBP-C may participate in myofilament regulation and activation through its interaction with myosin heads and regulating myosin head RX/SRX populations, especially when muscle is activated.






Acknowledgements (Optional):

References (Optional): (1) Walklate, J.; Kao, K.; Regnier, M.; Geeves, M. A. Exploring the Super-Relaxed State of Myosin in Myofibrils from Fast-Twitch, Slow-Twitch, and Cardiac Muscle. Journal of Biological Chemistry 2022, 298 (3), 101640. https://doi.org/10.1016/j.jbc.2022.101640.

(2) Hooijman, P.; Stewart, M. A.; Cooke, R. A New State of Cardiac Myosin with Very Slow ATP Turnover: A Potential Cardioprotective Mechanism in the Heart. Biophysical Journal 2011, 100 (8), 1969–1976. https://doi.org/10.1016/j.bpj.2011.02.061.

(3) Nag, S.; Trivedi, D. V. To Lie or Not to Lie: Super-Relaxing with Myosins. eLife 2021, 10, e63703. https://doi.org/10.7554/eLife.63703.

(4) Cooke, R. The Role of the Myosin ATPase Activity in Adaptive Thermogenesis by Skeletal Muscle. Biophys Rev 2011, 3 (1), 33–45. https://doi.org/10.1007/s12551-011-0044-9.

(5) Stewart, M. A.; Franks-Skiba, K.; Chen, S.; Cooke, R. Myosin ATP Turnover Rate Is a Mechanism Involved in Thermogenesis in Resting Skeletal Muscle Fibers. Proceedings of the National Academy of Sciences 2010, 107 (1), 430–435. https://doi.org/10.1073/pnas.0909468107.

(6) Wilson, C.; Naber, N.; Pate, E.; Cooke, R. The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle. Biophysical Journal 2014, 107 (7), 1637–1646. https://doi.org/10.1016/j.bpj.2014.07.075.

(7) Naber, N.; Cooke, R.; Pate, E. Slow Myosin ATP Turnover in the Super-Relaxed State in Tarantula Muscle. Journal of Molecular Biology 2011, 411 (5), 943–950. https://doi.org/10.1016/j.jmb.2011.06.051.

(8) McNamara, J. W.; Singh, R. R.; Sadayappan, S. Cardiac Myosin Binding Protein-C Phosphorylation Regulates the Super-Relaxed State of Myosin. Proceedings of the National Academy of Sciences 2019, 116 (24), 11731–11736. https://doi.org/10.1073/pnas.1821660116.

(9) McNamara, J. W.; Li, A.; Lal, S.; Bos, J. M.; Harris, S. P.; Velden, J. van der; Ackerman, M. J.; Cooke, R.; Remedios, C. G. dos. MYBPC3 Mutations Are Associated with a Reduced Super-Relaxed State in Patients with Hypertrophic Cardiomyopathy. PLOS ONE 2017, 12 (6), e0180064. https://doi.org/10.1371/journal.pone.0180064.

(10) Harris, S. P.; Bartley, C. R.; Hacker, T. A.; McDonald, K. S.; Douglas, P. S.; Greaser, M. L.; Powers, P. A.; Moss, R. L. Hypertrophic Cardiomyopathy in Cardiac Myosin Binding Protein-C Knockout Mice. Circulation Research 2002, 90 (5), 594–601. https://doi.org/10.1161/01.RES.0000012222.70819.64.

(11) Schlossarek, S.; Mearini, G.; Carrier, L. Cardiac Myosin-Binding Protein C in Hypertrophic Cardiomyopathy: Mechanisms and Therapeutic Opportunities. Journal of Molecular and Cellular Cardiology 2011, 50 (4), 613–620. https://doi.org/10.1016/j.yjmcc.2011.01.014.

(12) Sadayappan, S.; de Tombe, P. P. Cardiac Myosin Binding Protein-C: Redefining Its Structure and Function. Biophys Rev 2012, 4 (2), 93–106. https://doi.org/10.1007/s12551-012-0067-x.

(13) Sadayappan, S.; de Tombe, P. P. Cardiac Myosin Binding Protein-C as a Central Target of Cardiac Sarcomere Signaling: A Special Mini Review Series. Pflugers Arch - Eur J Physiol 2014, 466 (2), 195–200. https://doi.org/10.1007/s00424-013-1396-8.