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• W3109736363 abstract "A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions. A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions. Vertebrate striated muscle contraction results from cyclic interactions between myosin heads on the thick filaments and actin monomers on the thin filament. Emerging evidence in the last decade suggests that, in addition to Ca2+-mediated regulatory mechanisms within the thin filaments, the strength of muscle contraction may also be tuned by mechanisms intrinsic to myosin on the thick filaments (1Alamo L. Koubassova N. Pinto A. Gillilan R. Tsaturyan A. Padrón R. Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.Biophysical Rev. 2017; 9: 461-480Crossref PubMed Scopus (19) Google Scholar, 2Alamo L. Pinto A. Sulbarán G. Mavárez J. Padrón R. Lessons from a tarantula: new insights into myosin interacting-heads motif evolution and its implications on disease.Biophysical Rev. 2017; 10: 1465-1477Crossref PubMed Scopus (20) Google Scholar, 3Trivedi D.V. Adhikari A.S. Sarkar S.S. Ruppel K.M. Spudich J.A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light.Biophysical Rev. 2017; 10: 27-48Crossref PubMed Scopus (68) Google Scholar, 4Spudich J.A. Hypertrophic and dilated cardiomyopathy: four decades of basic research on muscle lead to potential therapeutic approaches to these devastating genetic diseases.Biophys. J. 2014; 106: 1236-1249Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5Spudich J.A. Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Pflugers Archiv Eur. J Physiol. 2019; 471: 701-717Crossref PubMed Scopus (47) Google Scholar, 6Spudich J.A. The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy.Biochem. Soc. T. 2015; 43: 64-72Crossref PubMed Scopus (74) Google Scholar). Specifically, myosin heads in relaxed thick filaments are thought to exist in an equilibrium between two functional states (7Nag S.N. Trivedi D.V. To lie or not to lie: super-relaxing with myosins, eLife.2020, AcceptedGoogle Scholar, 8McNamara J.W. Li A. Remedios C.G.D. Cooke R. The role of super-relaxed myosin in skeletal and cardiac muscle.Biophysical Rev. 2014; 7: 5-14Crossref PubMed Scopus (74) Google Scholar, 9Cooke R. The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle.Biophysical Rev. 2011; 3: 33-45Crossref PubMed Scopus (59) Google Scholar): (1) the disordered relaxed (DRX) state, in which myosin is free and ready to interact with actin and has an average ATP turnover time of <10 s and (2) the super-relaxed (SRX) state, in which myosin is unavailable for interaction with actin and has a prolonged ATP turnover time of >100 s. Many studies have started showing that the availability of the myosin heads to form actin cross bridges can be controlled by altering this DRX–SRX equilibrium, which in turn can regulate the strength of striated muscle contraction, all of which has been discussed in a recent report by Nag and Trivedi (7Nag S.N. Trivedi D.V. To lie or not to lie: super-relaxing with myosins, eLife.2020, AcceptedGoogle Scholar). The original discovery of the SRX state was based on single ATP turnover experiments in skinned rabbit fast and slow skeletal muscles (10Stewart M.A. Franks-Skiba K. Chen S. Cooke R. Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers.P Natl. Acad. Sci. U. S. A. 2009; 107: 430-435Crossref PubMed Scopus (133) Google Scholar), which was later confirmed by studies in skeletal and cardiac muscle systems from other species (8McNamara J.W. Li A. Remedios C.G.D. Cooke R. The role of super-relaxed myosin in skeletal and cardiac muscle.Biophysical Rev. 2014; 7: 5-14Crossref PubMed Scopus (74) Google Scholar, 11Hooijman P. Stewart M.A. Cooke R. A new state of cardiac myosin with very slow ATP turnover: a potential cardioprotective mechanism in the heart.Biophys. J. 2011; 100: 1969-1976Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 12Naber N. Cooke R. Pate E. Slow myosin ATP turnover in the super-relaxed state in tarantula muscle.J. Mol. Biol. 2011; 411: 943-950Crossref PubMed Scopus (48) Google Scholar, 13Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.P Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (117) Google Scholar, 14Colson B.A. Petersen K.J. Collins B.C. Lowe D.A. Thomas D.D. The myosin super-relaxed state is disrupted by estradiol deficiency.Biochem. Bioph Res. Co. 2014; 456: 151-155Crossref PubMed Scopus (18) Google Scholar, 15Phung L.A. Karvinen S.M. Colson B.A. Thomas D.D. Lowe D.A. Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice.Plos One. 2018; 13: e0199062Crossref PubMed Scopus (11) Google Scholar, 16McNamara J.W. Singh R.R. Sadayappan S. Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin.P Natl. Acad. Sci. Usa. 2019; 116: 11731-11736PubMed Google Scholar, 17McNamara J.W. Li A. Smith N.J. Lal S. Graham R.M. Kooiker K.B. Dijk S. J. van Remedios C.G.D. Harris S.P. Cooke R. Ablation of cardiac myosin binding protein-C disrupts the super-relaxed state of myosin in murine cardiomyocytes.J. Mol. Cell Cardiol. 2016; 94: 65-71Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Nelson S.R. Li A. Beck-Previs S. Kennedy G.G. Warshaw D.M. Imaging ATP Consumption in resting skeletal muscle: one molecule at a time.Biophys. J. 2020; 119: 1050-1055Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The structural basis for this biochemical SRX state is unclear because of a lack of high-resolution atomic-level structure of the myosin. At best, the functional SRX state of myosin has been loosely correlated to the structural interacting-heads motif (IHM) state of myosin in which the two myosin heads assymetrically interact with one another while folding back onto the proximal tail (1Alamo L. Koubassova N. Pinto A. Gillilan R. Tsaturyan A. Padrón R. Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.Biophysical Rev. 2017; 9: 461-480Crossref PubMed Scopus (19) Google Scholar, 2Alamo L. Pinto A. Sulbarán G. Mavárez J. Padrón R. Lessons from a tarantula: new insights into myosin interacting-heads motif evolution and its implications on disease.Biophysical Rev. 2017; 10: 1465-1477Crossref PubMed Scopus (20) Google Scholar, 3Trivedi D.V. Adhikari A.S. Sarkar S.S. Ruppel K.M. Spudich J.A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light.Biophysical Rev. 2017; 10: 27-48Crossref PubMed Scopus (68) Google Scholar). In the muscle thick filaments, it has been hypothesized that, in addition to myosin interactions with titin and myosin-binding protein C (MyBPC), the folded-back myosin IHM state is stabilized by several intramolecular interactions among various subdomains of myosin such as the regulatory light chain (RLC), heavy meromyosin (HMM), light meromyosin (LMM), subfragment 1 (S1), and subfragment 2 (S2) that give rise to S1–S1, S1–S2, RLC–RLC, S1–LMM, and S2–LMM interactions (1Alamo L. Koubassova N. Pinto A. Gillilan R. Tsaturyan A. Padrón R. Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function.Biophysical Rev. 2017; 9: 461-480Crossref PubMed Scopus (19) Google Scholar, 2Alamo L. Pinto A. Sulbarán G. Mavárez J. Padrón R. Lessons from a tarantula: new insights into myosin interacting-heads motif evolution and its implications on disease.Biophysical Rev. 2017; 10: 1465-1477Crossref PubMed Scopus (20) Google Scholar, 3Trivedi D.V. Adhikari A.S. Sarkar S.S. Ruppel K.M. Spudich J.A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light.Biophysical Rev. 2017; 10: 27-48Crossref PubMed Scopus (68) Google Scholar). If these interactions underlying the IHM state also give rise to the functional SRX state, then it is essential to build an experimental model that can capture most of these features. In previous studies, shorter myosin models such as full-length HMM, 25-hep HMM (HMM containing 25 heptad repeats of the S2), 2-hep HMM (HMM containing 2 heptad repeats of the S2), and S1 were all shown to form the SRX state (13Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.P Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (117) Google Scholar, 19Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.P Natl. Acad. Sci. Usa. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). However, in comparison to 2-hep HMM or S1, not only the SRX population was higher in full-length HMM and 25-hep HMM, but also the DRX–SRX equilibrium was more sensitive to electrostatic perturbations (13Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.P Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (117) Google Scholar, 19Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.P Natl. Acad. Sci. Usa. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). These observations suggested that the myosin SRX state is more stable in the presence of two S1 heads and the extended portion of the proximal tail. Importantly, myosin under physiological conditions does not exist in these short forms but present in an extensive filamentous form formed by the coiled-coil interactions of distal tails, highlighting an essential role for the extended tail in the myosin function. The present study focuses on using reconstituted thick filaments that mimic the structural environment close to the native thick filaments for functional characterization of myosin in an in vitro setting. There are two different ways to study myosin thick filaments. The first, called the native thick filaments, involves thick filaments extracted from the native muscle by removing the thin filaments using gelsolin treatment (20Hidalgo C. Padrón R. Horowitz R. Zhao F.-Q. Craig R. Purification of native myosin filaments from muscle.Biophys. J. 2001; 81: 2817-2826Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 21Zhao F.-Q. Craig R. Woodhead J.L. Head-head interaction characterizes the relaxed state of Limulus muscle myosin filaments.J. Mol. Biol. 2008; 385: 423-431Crossref PubMed Scopus (49) Google Scholar), and the second, referred to as the reconstituted or synthetic thick filaments (STFs), involves spontaneous self-assembly of myosin into bipolar thick filaments by lowering the ionic strength of the buffer containing full-length myosin (22Huxley H.E. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.J. Mol. Biol. 1963; 7: 281-308Crossref PubMed Scopus (1014) Google Scholar, 23Davis J.S. Assembly Processes in vertebrate skeletal thick filament formation.Annu. Rev. Biophys. Bio. 1988; 17: 217-239Crossref Scopus (44) Google Scholar). Of these two, the first model provides a native-like environment to study the myosin thick filament in the presence of MyBPC, titin, and other thick filament–associated proteins. The latter, STFs, represents a simpler model that captures essential interactions pertinent to myosin alone while retaining the 14.3-nm myosin subunit periodicity and a 43-nm axial periodicity as in native filaments (24Pollard T.D. Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation.J. Cell Biol. 1975; 67: 93-104Crossref PubMed Scopus (56) Google Scholar, 25Koretz J.F. Structural studies of synthetic filaments prepared from column-purified myosin.Biophys. J. 1979; 27: 423-432Abstract Full Text PDF PubMed Scopus (19) Google Scholar). Also, electron microscopy structures confirm a bipolar myosin arrangement and the bare zone in both native thick filaments and STFs (22Huxley H.E. Electron microscope studies on the structure of natural and synthetic protein filaments from striated muscle.J. Mol. Biol. 1963; 7: 281-308Crossref PubMed Scopus (1014) Google Scholar, 26D’Haese J. Hinssen H. Structure of synthetic and native myosin filaments from Amoeba proteus.Cell Tissue Res. 1974; 151: 323-335Crossref PubMed Scopus (6) Google Scholar, 27Condeelis J.S. The self-assembly of synthetic filaments of myosin isolated from Chaos carolinensis and Amoeba proteus.J. Cell Sci. 1977; 25: 387-402Crossref PubMed Google Scholar). In addition, the 3-fold rotational symmetry found in native thick filaments is also preserved in STFs (28Emes C.H. Rowe A.J. Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle.Biochim. Biophys. Acta Bba - Protein Struct. 1978; 537: 125-144Crossref PubMed Scopus (27) Google Scholar). The STF model also provides a bottom-up approach to study the underlying biology of thick filaments and their function, starting from full-length myosin, offering advantages in different contexts. For example, this model permits the controlled addition of binding partners such as MyBPC to build a more complex system (29Koretz J.F. Effects of C-protein on synthetic myosin filament structure.Biophys. J. 1979; 27: 433-446Abstract Full Text PDF PubMed Scopus (60) Google Scholar). Also, this system allows us to recombinantly study mutations in the rod domain of the myosin that causes several skeletal and cardiac myopathies (30Wolny M. Colegrave M. Colman L. White E. Knight P.J. Peckham M. Cardiomyopathy mutations in the tail of β-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes.J. Biol. Chem. 2013; 288: 31952-31962Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 31Moore J.R. Leinwand L. Warshaw M D. The myosins: Exploration of the Development of our current understanding of these mutations in the motor.Circ. Rec. 2014; 111: 375-385Crossref Scopus (124) Google Scholar). Finally, different studies have proposed cooperative myosin activation in thick filaments by studying several physiological perturbations such as an increased ADP:ATP ratio (10Stewart M.A. Franks-Skiba K. Chen S. Cooke R. Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers.P Natl. Acad. Sci. U. S. A. 2009; 107: 430-435Crossref PubMed Scopus (133) Google Scholar, 15Phung L.A. Karvinen S.M. Colson B.A. Thomas D.D. Lowe D.A. Age affects myosin relaxation states in skeletal muscle fibers of female but not male mice.Plos One. 2018; 13: e0199062Crossref PubMed Scopus (11) Google Scholar, 32Xu S. Offer G. Gu J. White H.D. Yu L.C. Temperature and Ligand dependence of conformation and Helical order in myosin filaments.Biochemistry. 2003; 42: 390-401Crossref PubMed Scopus (57) Google Scholar), RLC phosphorylation (3Trivedi D.V. Adhikari A.S. Sarkar S.S. Ruppel K.M. Spudich J.A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light.Biophysical Rev. 2017; 10: 27-48Crossref PubMed Scopus (68) Google Scholar), strong actin binding of myosin and Ca2+ binding to thick filaments (33Alamo L. Ware J.S. Pinto A. Gillilan R.E. Seidman J.G. Seidman C.E. Padrón R. Effects of myosin variants on interacting-heads motif explain distinct hypertrophic and dilated cardiomyopathy phenotypes.Elife. 2017; 6: e24634Crossref PubMed Scopus (90) Google Scholar, 34Sa N. Tomasic I. Gollapudi S. Nag S. Myosin regulatory light chain: a major player in Defining the ‘OFF’ state of cardiac myosin.Circ Res. 2019; 125: A340Crossref Google Scholar, 35Podlubnaya Z. Kaķkol I. Moczarska A. Stȩpkowski D. Udaltsov S. Calcium-induced structural changes in synthetic myosin filaments of vertebrate striated muscles.J. Struct. Biol. 1999; 127: 1-15Crossref PubMed Scopus (19) Google Scholar), which are important physiological aspects that can be easily studied in a system containing assembled myosin filaments that preserves most of the myosin molecular interactions typically present in thick filaments. However, the use of such reconstituted thick filaments for functional studies, especially in relevance to the low energy–consuming SRX state of myosin, has been sparse. In this study, using shorter cardiac myosin models such as HMM and S1 as well as STFs made of cardiac full-length myosin, we tested the hypothesis that several intramolecular interactions within the myosin molecule that are known to stabilize the IHM state of myosin may also underlie the SRX state. We provide evidence that many electrostatic and cooperative interactions that underlie the myosin SRX state are preserved in STFs than in HMM and S1. Using STFs, we then show that many pathophysiological insults of hypertrophic cardiomyopathy (HCM) such as R403Q myosin mutation, hyper-phosphorylation of myosin light chains, and increased ADP:ATP ratio destabilize the myosin SRX population. Interestingly, such destabilizing effect of ADP on the SRX state is only seen in STFs made of WT myosin, but not in S1, HMM, or STFs made of mutant R403Q myosin. In addition, we show that a cardiac myosin inhibitor, mavacamten (36Green E.M. Wakimoto H. Anderson R.L. Evanchik M.J. Gorham J.M. Harrison B.C. Henze M. Kawas R. Oslob J.D. Rodriguez H.M. Song Y. Wan W. Leinwand L.A. Spudich J.A. McDowell R.S. et al.A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.Science. 2016; 351: 617-621Crossref PubMed Scopus (291) Google Scholar, 37Kawas R.F. Anderson R.L. Ingle S.R.B. Song Y. Sran A.S. Rodriguez H.M. A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle.J. Biol. Chem. 2017; 292: 16571-16577Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which is currently in phase III clinical trials for treating HCM, reduces the cardiac muscle contractility by shifting the myosin DRX–SRX equilibrium more towards the SRX state, consistent with some previous reports (13Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.P Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (117) Google Scholar, 19Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.P Natl. Acad. Sci. Usa. 2018; 115: E7486-E7494Crossref PubMed Google Scholar, 38Toepfer C.N. Garfinkel A.C. Venturini G. Wakimoto H. Repetti G. Alamo L. Sharma A. Agarwal R. Ewoldt J.F. Cloonan P. Letendre J. Lun M. Olivotto I. Colan S. Ashley E. et al.Myosin Sequestration regulates sarcomere function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of hypertrophic cardiomyopathy.Circulation. 2020; 141: 828-842Crossref PubMed Scopus (79) Google Scholar). However, using concentration-dependent responses in this study, we show that this ability of mavacamten to promote myosin into the SRX state is greater in STFs than in HMM and S1. We also found that the potency of mavacamten in promoting myosin into the SRX state is similar in STFs made of healthy atrial WT, healthy ventricular WT, or disease-causing ventricular mutant (R403Q) myosins. We further demonstrate that the mavacamten-bound SRX heads in STFs can be reversibly recruited into the DRX state via the ADP-mediated cooperative activation of thick filaments. This mechanism is not reproduced in either HMM or S1. A schematic showing the full-length myosin structure with the different subdomains is shown in the top panel of Figure 1A. The HMM domain of the myosin used in this study was prepared by chymotryptic cleavage of full-length myosin and consists of the two S1 domains connected to the ∼46-heptad S2 domain. Each myosin S1 head consists of a motor domain, essential light chain (ELC), and RLC subunits. The S1 used in this study is the short S1 that includes the motor domain and the ELC but not the RLC. Full-length myosin reconstitutes into bipolar myosin filaments in a buffer of lower ionic strength (<150 mM) referred hereafter as STFs (bottom panel of Fig. 1A). To evaluate whether bovine cardiac (Bc) shorter myosin fragments, S1 and HMM, differ from STFs in their biochemical properties, we first measured the basal ATPase activity in these myosin systems. The resulting measurements did not show significant differences among the three myosin systems (Fig. 1B). The basal ATPase activities for BcS1, BcHMM, and BcSTFs were 0.016 ± 0.02 (n = 8), 0.017 ± 0.001 (n = 6), and 0.018 ± 0.001 (n = 8) s-1, respectively. Similarly, the basal ATPases measured in other myosin models such as porcine cardiac STFs made of WT and R403Q myosin were 0.026 ± 0.001 (n = 12) and 0.023 ± 0.001 (n = 12), respectively. Next, we measured basal ATPase activity in various myosin systems in response to increasing concentrations of mavacamten. A comparison of these responses did not show any obvious differences between systems in the potency of mavacamten on myosin ATPase inhibition (Fig. 1C). Statistical analysis using one-way ANOVA confirmed that the concentrations of mavacamten required to attain IC50 in the basal ATPase activity were not significantly different between the three myosin groups. The IC50 values in BcS1, BcHMM, and BcSTFs were 0.76 ± 0.06 (n = 8), 0.57 ± 0.06 (n = 6), and 0.82 ± 0.06 μM (n = 8), respectively, which are consistent with those reported for S1 and HMM in previous reports (19Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.P Natl. Acad. Sci. Usa. 2018; 115: E7486-E7494Crossref PubMed Google Scholar, 36Green E.M. Wakimoto H. Anderson R.L. Evanchik M.J. Gorham J.M. Harrison B.C. Henze M. Kawas R. Oslob J.D. Rodriguez H.M. Song Y. Wan W. Leinwand L.A. Spudich J.A. McDowell R.S. et al.A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.Science. 2016; 351: 617-621Crossref PubMed Scopus (291) Google Scholar, 37Kawas R.F. Anderson R.L. Ingle S.R.B. Song Y. Sran A.S. Rodriguez H.M. A small-molecule modulator of cardiac myosin acts on multiple stages of the myosin chemomechanical cycle.J. Biol. Chem. 2017; 292: 16571-16577Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). These results suggest that BcSTFs behaves the same way as BcHMM or BcS1 in the enzymatically coupled ATPase experiments . It is well established that the IHM state of myosin, which could also underlie the SRX state, involves a complex set of intermolecular and intramolecular interactions that are electrostatic in nature (3Trivedi D.V. Adhikari A.S. Sarkar S.S. Ruppel K.M. Spudich J.A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light.Biophysical Rev. 2017; 10: 27-48Crossref PubMed Scopus (68) Google Scholar, 39Nag S. Trivedi D.V. Sarkar S.S. Adhikari A.S. Sunitha M.S. Sutton S. Ruppel K.M. Spudich J.A. The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Nat. Struct. Mol. Biol. 2017; 24: 525-533Crossref PubMed Scopus (91) Google Scholar, 40Robert-Paganin J. Auguin D. Houdusse A. Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition.Nat. Commun. 2018; 9: 4019Crossref PubMed Scopus (44) Google Scholar). Therefore, in the next series of experiments, we perturbed these electrostatic interactions in BcS1, BcHMM, and BcSTFs by progressively increasing the ionic strength (KCl) of the buffer and examined the effect on parameters derived using single ATP turnover kinetic experiments. The fluorescence decay profile obtained during the chase phase in the single ATP turnover kinetic experiments characteristically depicted two phases, a fast phase followed by a slow phase (Fig. S1 in Supporting information). Therefore, a biexponential function was fitted to estimate four different parameters—Afast, kfast, Aslow, and kslow—where A represents the % amplitude and k represents the observed ATP turnover rate of each phase (10Stewart M.A. Franks-Skiba K. Chen S. Cooke R. Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers.P Natl. Acad. Sci. U. S. A. 2009; 107: 430-435Crossref PubMed Scopus (133) Google Scholar, 13Anderson R.L. Trivedi D.V. Sarkar S.S. Henze M. Ma W. Gong H. Rogers C.S. Gorham J.M. Wong F.L. Morck M.M. Seidman J.G. Ruppel K.M. Irving T.C. Cooke R. Green E.M. et al.Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.P Natl. Acad. Sci. U. S. A. 2018; 115: E8143-E8152Crossref PubMed Scopus (117) Google Scholar, 19Rohde J.A. Roopnarine O. Thomas D.D. Muretta J.M. Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin.P Natl. Acad. Sci. Usa. 2018; 115: E7486-E7494Crossref PubMed Google Scholar). Afast and kfast characterize myosin in the DRX state, whereas Aslow and kslow characterize it in the SRX state. A qualitative comparison, as shown in Figure 2A, suggests that the response of BcSTFs and BcHMM to increasing KCl concentration was distinct from that of BcS1 (see Fig. S2, A-C, in Supporting information for representative raw traces). For example, Aslow, which represents the % amplitude of the myosin SRX population, progressively decreased" @default.
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• W3109736363 title "Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems" @default.
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