Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen

Abstract

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure. Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure. Cardiac myosin-binding protein C (cMyBP-C, Fig. 1) has been identified as a therapeutic target for systolic or diastolic dysfunction in heart failure and cardiomyopathy. cMyBP-C may contribute to heart failure due to hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). HCM is a common heart disease, affecting around 1:500 individuals, for which there are few therapeutic treatments (1Spudich J.A. Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.Pflugers Arch. 2019; 471: 701-717Crossref PubMed Scopus (30) Google Scholar). In HCM the heart typically becomes enlarged, hypercontractile, and unable to relax effectively. The systolic contraction is preserved, but diastolic relaxation is diminished. HCM is associated with heart failure, arrhythmias, and cardiac death at any age. The primary cause of HCM is most often a missense mutation in one of several sarcomeric proteins or the truncation of cMyBP-C. In addition to cMyBP-C, the sarcomeric proteins associated with HCM include those associated with the thick (myosin) and thin (actin) filaments, including myosin, troponin, tropomyosin, leiomodin, and titin. DCM results from missense mutations in some of the same sarcomeric genes as for HCM (but not cMyBP-C truncations) as well as Z-disk proteins (2Arimura T. Ishikawa T. Nunoda S. Kawai S. Kimura A. Dilated cardiomyopathy-associated BAG3 mutations impair Z-disc assembly and enhance sensitivity to apoptosis in cardiomyocytes.Hum. Mutat. 2011; 32: 1481-1491Crossref PubMed Scopus (98) Google Scholar). DCM can also be caused by mutations in nonsarcomeric genes, such as those encoding proteins of the nucleus, ion channels, and cytoskeleton (3Hershberger R.E. Hedges D.J. Morales A. Dilated cardiomyopathy: The complexity of a diverse genetic architecture.Nat. Rev. Cardiol. 2013; 10: 531-547Crossref PubMed Scopus (465) Google Scholar). Any one of these mutations leads to dilation of the ventricular chambers and defects in systolic contraction. Numerous studies have demonstrated that increasing or decreasing PKA-mediated phosphorylation of cMyBP-C allows for tuning cardiac contraction and relaxation through phosphorylation-sensitive interactions with actin and myosin. These interactions and their effects are complex, and it is not yet fully understood how they are integrated. Binding to actin is proposed to activate the thin filament in low calcium conditions by repositioning tropomyosin to the unblocked position, thereby stimulating contraction (4Mun J.Y. Previs M.J. Yu H.Y. Gulick J. Tobacman L.S. Beck Previs S. Robbins J. Warshaw D.M. Craig R. Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 2170-2175Crossref PubMed Scopus (82) Google Scholar, 5Risi C. Belknap B. Forgacs-Lonart E. Harris S.P. Schroder G.F. White H.D. Galkin V.E. N-terminal domains of cardiac myosin binding protein C cooperatively activate the thin filament.Structure. 2018; 26: 1604-1611.e1604Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Binding to actin has also been observed to shift F-actin to a more ordered state, similar to the effect of myosin binding to actin (6Colson B.A. Rybakova I.N. Prochniewicz E. Moss R.L. thomas D.D. Cardiac myosin binding protein-C restricts intrafilament torsional of actin in a Natl. Acad. Sci. U. S. A. Scopus Google Scholar, B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). ordered may also myosin binding to actin. cMyBP-C to actin is proposed to a on the of the filaments, thereby relaxation S. S. Harris S.P. of cardiac binding protein-C on actin are with a J. Full Text Full Text PDF PubMed Scopus Google Scholar, C. J. of by myosin-binding protein C can be by its binding to the thin Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. of and by myosin binding protein 2019; PubMed Scopus Google Scholar). cMyBP-C is also for relaxation S. E. C. A. S. T. Cardiac myosin-binding protein C is for relaxation in PubMed Scopus Google Scholar). cMyBP-C binding to myosin has been to the state, A. S. Harris S.P. R. of cardiac myosin binding protein-C the of myosin in Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. S. J.A. The myosin and the basis of hypercontractility caused by hypertrophic cardiomyopathy PubMed Scopus Google Scholar). by decreasing and interactions with the thin and thick filaments B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, Harris S.P. The myosin-binding protein C to F-actin in a phosphorylation-sensitive Full Text Full Text PDF PubMed Scopus Google Scholar, B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar, the of the of cardiac myosin binding protein C with in an PubMed Scopus Google Scholar, in that cause hypertrophic cardiomyopathy the with the of myosin-binding PubMed Scopus Google can have at low calcium M.J. J.Y. Previs Gulick J. Robbins J. Craig R. Warshaw D.M. Myosin-binding protein C an in cardiac PubMed Scopus Google relaxation by the by cMyBP-C binding to actin J. A. of and by myosin binding protein 2019; PubMed Scopus Google Scholar, A. S. Gulick J. Previs M.J. Robbins J. Warshaw D.M. binding of cardiac myosin binding protein-C to actin and of of the Cardiol. Full Text Full Text PDF PubMed Scopus Google to enhance relaxation G.F. R. of cardiac Myosin-binding protein-C is a of diastolic Heart PubMed Scopus Google myosin binding due to in actin (6Colson B.A. Rybakova I.N. Prochniewicz E. Moss R.L. thomas D.D. Cardiac myosin binding protein-C restricts intrafilament torsional of actin in a Natl. Acad. Sci. U. S. A. Scopus Google Scholar, B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google and myosin binding and contraction by myosin from the S. S. J.A. The myosin and the basis of hypercontractility caused by hypertrophic cardiomyopathy PubMed Scopus Google Scholar, S. Cardiac myosin binding protein-C phosphorylation the of Natl. Acad. Sci. U. S. A. 2019; Google Scholar). by B.A. D.D. of cardiac myosin-binding protein C effects of phosphorylation on protein Natl. Acad. Sci. U. S. A. PubMed Scopus Google and M.J. J.Y. Previs Gulick J. Robbins J. Warshaw D.M. Craig R. and calcium myosin-binding protein and Natl. Acad. Sci. U. S. A. PubMed Scopus Google that phosphorylation a of N-terminal cMyBP-C that binding to actin and myosin to modulate contraction and in phosphorylation of cMyBP-C have been observed in heart failure and HCM including those by mutations in cMyBP-C and sarcomeric proteins cMyBP-C binding protein C phosphorylation in hypertrophic and heart Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. of cardiac myosin binding protein-C phosphorylation in heart Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, cMyBP-C with drugs that mimic phosphorylation and that modulate its binding to actin or myosin is a to cardiac muscle in heart failure and cardiomyopathy. small-molecule of myosin have been to activate or cardiac muscle contraction in heart failure and HCM R.L. R. R. myosin potential therapeutic for systolic heart 2011; PubMed Scopus Google Scholar, R.L. M.J. R. J.A. small-molecule of contractility hypertrophic cardiomyopathy in PubMed Scopus Google Scholar). some are such as proteins may as cMyBP-C as an target for cMyBP-C small-molecule has been to in this is that high-throughput on cMyBP-C have not been in actin or myosin-binding such as are and in the of that can be of fluorescence lifetime R.L. J. R. T. D.D. FRET assay 2013; PubMed Scopus Google now allows for high-throughput for lifetime and for that are and We a fluorescence lifetime assay for the of F-actin with N-terminal cMyBP-C domains C0-C2 B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). The time-resolved fluorescence assay a reduction in lifetime of to actin at when with reduction was and with actin binding of C0-C2 as by The of the assay was demonstrated by in binding due to phosphorylation and mutations to HCM and In the we have used this a fluorescence lifetime to screen the of We this library it is a diverse of pharmacologically active compounds that target and with compounds fluorescence at A. S. E. J. D.D. FRET for high-throughput fluorescence lifetime 2018; Google Scholar, A. A. R. J. J. of PubMed Scopus Google we labeled actin with the fluorescent Fluor which was by a screen identified three compounds that to binding to actin in the micromolar range. We these compounds for in the assay and their in and titration calorimetry (ITC) the micromolar of the three compounds to cMyBP-C. phosphorescence anisotropy (TPA) that the compounds not bind to actin and that they we a novel assay as an to C0-C2 binding to actin. We used this assay to the effects of the identified on actin binding. is the first assay to identify compounds that specifically bind to cMyBP-C and modulate its interactions with actin. The results of this for the assay in of be in the of for cardiac muscle We have a assay actin. The of to actin was reduced C0-C2 binding. a to binding in a to of compounds that modulate actin-cMyBP-C binding B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). a of compounds at the used to S. S. Harris S.P. of cardiac binding protein-C on actin are with a J. Full Text Full Text PDF PubMed Scopus Google Scholar). this we the of C0-C2 binding to the actin was labeled with that are at the of a A. S. E. J. D.D. FRET for high-throughput fluorescence lifetime 2018; Google Scholar). of Fluor and Fluor actin in the of cMyBP-C N-terminal were by to fluorescence lifetime and C and Fig. on actin lifetime decreased with increasing C0-C2 binding. effect was reduced with phosphorylated C0-C2, binding a in lifetime C0-C2 binding to we used this for the of binding with AF568-actin that at C0-C2 the lifetime is to phosphorylation by was to compounds that in binding similar to those phosphorylation, we these of AF568-actin and C0-C2 to the B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google we that these to binding of which is similar to that in the muscle We the library The screen was in with of AF568-actin and C0-C2 screen the effect of the compounds on the lifetime of AF568-actin and the effect of the compounds on the lifetime of AF568-actin in the of C0-C2 The factor for this screen was as the of this assay that the lifetime of AF568-actin by more of the of were from the of the and compounds from the first and The in lifetime of AF568-actin due to effects of on was by the effects of on AF568-actin from the effects on C0-C2 binding reduced AF568-actin compounds with C0-C2 binding to actin the lifetime of from were and The compounds three compounds in common and that were not and in of the same library also these three compounds in the of these that were not identified in any of the and the was the with an in lifetime that was the compounds not due to effects on AF568-actin was the and aurintricarboxylic was the These three compounds were in Using the same conditions as in the primary we the of of the three compounds on the AF568-actin lifetime observed in the three compounds effect on AF568-actin C0-C2 reduced the lifetime of and three in a to the lifetime observed for AF568-actin is consistent with three compounds inhibiting C0-C2 binding to actin. The of for of the compounds as by is In a assay C0-C2, we the effects of the compounds on and phosphorylated AF568-actin was to C0-C2 phosphorylated by a decreased lifetime of was observed the was with and three compounds lifetime to that of AF568-actin In the of C0-C2 was used in the assay that effects of compounds on phosphorylated C0-C2 be as phosphorylation of C0-C2 the of binding to of effects on the of and of binding to are as and was with protein is from at C0-C2 and is from at in a are as and was with protein is from at C0-C2 and is from at binding of these three compounds to cMyBP-C was by titration of C0-C2 with of the three identified compounds are shown in binding with at an of C0-C2 that there is the compounds and the binding in with a of of and of of but not or interactions with actin were in and results binding of the compounds to C0-C2 that the compounds bind to C0-C2, and this its binding with actin. We to effects on binding a assay F-actin was by and the of C0-C2 to the F-actin in the was three compounds caused C0-C2 to the of any bind to the used for the assay. was the the are with to the compounds cause C0-C2 to bind to the we the compounds C0-C2 and cause it to We three drugs for C0-C2 to by in the the of protein or the drugs and NF023, we not but for we a reduction in due to that some C0-C2 which to the that with assay for this an to we an FRET assay to C0-C2 binding to actin Actin was labeled with the fluorescent and with increasing of C0-C2 labeled with the at in the Binding of to in and binding for and phosphorylated C0-C2 that this was These binding with around and are in with the binding and those by B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). same conditions as the screen and for the in Fig. and and binding of and phosphorylated C0-C2 The three compounds with actin as well as with of F-actin with and with or We this by effects on by TPA of actin labeled at with of the three compounds we identified in this screen as by we a that bind actin and its anisotropy was one of the compounds from the due to effects on in the TPA assay was used to the three compounds for their to C0-C2 binding to actin. We that actin are by C0-C2 binding (6Colson B.A. Rybakova I.N. Prochniewicz E. Moss R.L. thomas D.D. Cardiac myosin binding protein-C restricts intrafilament torsional of actin in a Natl. Acad. Sci. U. S. A. Scopus Google Scholar, B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). the effect of C0-C2 binding on anisotropy anisotropy was from to in the of of the three compounds the C0-C2 effects on F-actin anisotropy to Fig. C and consistent with the compounds binding to C0-C2 and it from binding to actin. The which C0-C2 domains binding to actin B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google identified the first three compounds of inhibiting C0-C2 interactions with actin. of this a and TPA that these three compounds bind to C0-C2, inhibiting its to bind to actin. we have identified the first three compounds that Three compounds from were identified in of the That three of the were identified the of of the in screen not effects in the screen and are In of the same not or the same three but were in the We conclude that is and for compounds this The reproducible of that of of compounds a of compounds for was to the identified compounds interactions by binding to one or three compounds were to bind C0-C2 and not actin by a of and TPA binding to C0-C2 in the of actin but not binding to actin at the for binding were similar to the by the assay. The a consistent of with for binding and The were the We have not this but that the of the results 1) that of the compounds bind to is by the binding of one of this the the and The an of binding interactions with C0-C2, the may be on the to actin binding. there may be a of the drugs for C0-C2 to actin for C0-C2 in a FRET to C0-C2 to its with F-actin labeled with by Using this we were to the reduced binding when C0-C2 was phosphorylated with assay confirmed the reduction of or phosphorylated interactions by three compounds. of to binding was by the compounds as they a of C0-C2 or C0-C2 binding to the used in this assay and In addition to we interactions of the three compounds with actin of the three compounds that they were not binding to actin. C0-C2 binding to F-actin its anisotropy as and three compounds confirmed that the effects were due to their binding to C0-C2 and its binding to actin. with results for compounds that modulate interactions Prochniewicz E. D.D. time-resolved compounds that modulate and 2018; Full Text Full Text PDF PubMed Scopus Google most of the identified compounds a on the TPA of actin binding to cardiac but not to actin may be to have effects those binding to the actin in be to these compounds that bind to cardiac also bind to The from that these compounds may bind to C0-C2 in a that they bind to domains such as and it be to the effects on C0-C2 binding to myosin. to be the effects of these compounds on cardiac muscle but the used in this that the assay is for of compounds to identify of its We now have, for the first time, a validated focused on cMyBP-C, a known key factor in heart failure. The results from this screen that similar thin filaments tropomyosin and be In we observed that tropomyosin C0-C2 binding and as from studies C. Belknap B. Forgacs-Lonart E. Harris S.P. Schroder G.F. White H.D. Galkin V.E. N-terminal domains of cardiac myosin binding protein C cooperatively activate the thin filament.Structure. 2018; 26: 1604-1611.e1604Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). of C0-C2 interactions with tropomyosin the or of may be identified in these similar high-throughput on lifetime of to myosin C0-C2 binding be and we are Actin was from muscle by in as in B.A. N-terminal in cardiac myosin-binding protein C Cardiol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). actin was labeled at with Fluor and in with Fluor was on of in was to a of and actin was by the addition of a of and a of by at for was to a of a in was for at and at the shown in in three actin was and was and effects were the same at and the results were actin was labeled with with was at a of for at and at was by the addition of a of dye or was by the actin through F-actin and as in B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). to on of actin in was with to to the actin phosphorescence actin was labeled at with (6Colson B.A. Rybakova I.N. Prochniewicz E. Moss R.L. thomas D.D. Cardiac myosin binding protein-C restricts intrafilament torsional of actin in a Natl. Acad. Sci. U. S. A. Scopus Google Scholar, E. D.D. of actin active with effects of and myosin PubMed Scopus Google Scholar). The of by was and as by and labeled were by the addition of F-actin was in against encoding E. for the C0-C2 of cMyBP-C with N-terminal and were from we C0-C2 that it a at a in the the in C0-C2 were to in proteins at and and was to were in the cMyBP-C C0-C2 a were confirmed by in E. and of C0-C2 protein were as B.A. N-terminal in cardiac myosin-binding protein C Cardiol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). C0-C2 by was to C0-C2 as B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google and to and and at was labeled with in was first with the for at and was a in to a of was for at and by the addition dye was by against The of was as by C0-C2 was with C0-C2 at for is the to phosphorylation as by of proteins with B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar). lifetime were a fluorescence lifetime B.A. N-terminal in cardiac myosin-binding protein C Cardiol. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. D.D. and lifetime-based FRET in to identify small-molecule of Google by F-actin or with was with a and was with a was with a and was with and The was that the of the and the were The observed was as B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar, R.L. J. R. D.D. of high-throughput time-resolved FRET in 2014; PubMed Scopus Google Scholar). The compounds were in and were In of compounds was an compounds were with the first and with of and used for The of the compounds was These assay were a and at were to In actin or with C0-C2 in was by a the assay the compounds. were at for the with the were were of C0-C2, and compounds. to that from the and a of AF568-actin that lifetime with the binding of C0-C2 in the of were in the and as they observed for well were with the to the lifetime 1) by to a B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar, R.L. J. R. D.D. of high-throughput time-resolved FRET in 2014; PubMed Scopus Google Scholar). The of the of the fluorescent dye to actin at to the is the fluorescence and is the fluorescence lifetime when to or of assay was from on as by the of assay a assay B.A. high-throughput fluorescence lifetime-based assay for binding of myosin-binding protein C to PubMed Google Scholar, D.D. D.M. R.L. to small-molecule of calcium Google Scholar, for in and of PubMed Scopus Google and are the of the and and and are the of the and the was AF568-actin with AF568-actin with C0-C2 and was a it was in the compounds in of independent of the compounds were those that the lifetime of AF568-actin when C0-C2 was of C0-C2 that AF568-actin lifetime when C0-C2 was not by to that of the to were first from the in the from of compounds from the first screen and compounds from the Any in lifetime by a on AF568-actin was from the effect of the on AF568-actin when C0-C2 was The for compounds was The compounds were in to a which was in were at were from the a The same of as for the was in the of the was the S. The of its in PubMed Scopus Google and are in the and in the of the is the C is the is the for which of effect is and is the of The titration of compounds binding to cMyBP-C was on an in was C0-C2 in the a in was by with The was with for of and binding were by of the binding is to binding was the In labeled on with was with increasing of or labeled to with of were for and at of was for The binding was from from of actin and of effects by was at with and at was and in to or in with at that first effect in the for and and for The same were in the of These were with to and TPA was at to anisotropy B.A. cardiac myosin-binding protein C restricts actin in a and phosphorylation-sensitive 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). three drugs were for C0-C2 to by in the the of protein or the drugs in these is of due to from or of C0-C2 was with of NF023, or for and were a to The same was with or of C0-C2 and of NF023, or are as and was with protein are the T. in and as of has been and by the of in this to some as in B. A. C. a on this The We for with the fluorescence lifetime T. A. and B. A. C. T. A. C. A. R. A. and B. A. C. T. A. and B. A. C. T. A. C. and B. A. C. T. A. C. A. R. A. and B. A. C. T. A. and B. A. C. T. A. C. and B. A. C. T. A. and B. A. C. T. A. C. A. R. A. and B. A. C. and C. and B. A. C. T. and B. A. C. B. A. C. was by of and B. A. and a of Heart in the of and B. A. The is the of the and not the of the of

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