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Effects of hypertrophic and dilated cardiomyopathy mutations onpower output by human β-cardiac myosinJames A. Spudich1,*, Tural Aksel1, Sadie R. Bartholomew1, Suman Nag1, Masataka Kawana1,Elizabeth Choe Yu1, Saswata S. Sarkar1, Jongmin Sung1, Ruth F. Sommese1, Shirley Sutton1, Carol Cho1,Arjun S. Adhikari1, Rebecca Taylor1, Chao Liu1, Darshan Trivedi1 and Kathleen M. Ruppel1,2,*

ABSTRACTHypertrophic cardiomyopathy is the most frequently occurringinherited cardiovascular disease, with a prevalence of more thanone in 500 individuals worldwide. Genetically acquired dilatedcardiomyopathy is a related disease that is less prevalent. Both arecaused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere,including human β-cardiac myosin, the motor protein that powersventricular contraction. Despite numerous studies, most performedwith non-human or non-cardiac myosin, there is no clear consensusabout the mechanism of action of these mutations on the function ofhuman β-cardiac myosin. We are using a recombinantly expressedhuman β-cardiac myosin motor domain along with conventional andnew methodologies to characterize the forces and velocities of themutant myosins compared with wild type. Our studies are extendingbeyond myosin interactions with pure actin filaments to include theinteraction of myosin with regulated actin filaments containingtropomyosin and troponin, the roles of regulatory light chainphosphorylation on the functions of the system, and the possibleroles of myosin binding protein-C and titin, important regulatorycomponents of both cardiac and skeletal muscles.

KEY WORDS: Cardiac myosin, Cardiomyopathy mutations,Force–velocity curves

Historical perspectiveDozens of laboratories, working over the course of more than acentury and using a wide variety of approaches, have contributedimportantly to our understanding of muscle contraction (Rall,2014). The muscle sarcomere may well be the most studied proteincomplex in cell biology, partially due to its extraordinarily highdegree of structural organization.I (J.A.S.) was asked to preface my talk at the 2015 Journal of

Experimental Biology Symposium on Muscle: Molecules to Motionwith a brief historical perspective of my laboratory’s specificcontributions to research on actin and myosin, and how thosecontributions led to our current focus on understanding the molecularbasis of hypertrophic and dilated cardiomyopathy. My laboratory,over the last several decades, has been devoted to developing both invivo and in vitro systems to understand the molecular basis of energytransduction by the myosin family of molecular motors (Spudich,2011, 2012). Our interests have included the roles of myosins in non-muscle cells and the molecular basis of muscle contraction. Essential

for our research was the development of in vitro motility assays forpurified actin and myosin in the 1980s (Kron and Spudich, 1986;Spudich et al., 1985). We used these assays to show that the globularhead ofmyosin, known as subfragment-1 (S1), is themotor domain ofthe myosin molecule (Toyoshima et al., 1987), and, together withmolecular genetic approaches, we provided early strong functionalevidence that the light-chain binding region of the S1 acts as aswinging lever arm during chemo-mechanical coupling (e.g. Shihet al., 2000; Uyeda et al., 1996). Our study on changing the lever armlength and even removing the light-chain binding region altogetherwas an earlywork that showed that the heavy chain, truncated after theconverter domain (Fig. 1, myosin head structure on the right), is the‘motor domain’ of themolecule, and the lever arm amplifies allostericmovements in the motor domain to obtain a larger stroke size – thedistance myosin moves an actin filament per ATP hydrolyzed (Uyedaet al., 1996). Using the physics of laser trapping, we simplified themotility assay to the single molecule level, which allowed themeasurement of the stroke size (∼10 nm) as well as the intrinsic forceproduced by the motor (a few piconewtons) (Finer et al., 1994). Withthese tools in hand, and after studying a variety ofmyosinmotor typesover several decades, my laboratory is now fully focused on one of themost clinically important members of the myosin family of molecularmotors, human β-cardiac myosin.

Human cardiomyopathy mutations are a leading cause ofcardiac deathThe human β-cardiac myosin motor is a major site in the sarcomerefor hundreds of missense mutations that result in a spectrum of heartdiseases called cardiomyopathies. Genetically based hypertrophiccardiomyopathy (HCM), an autosomal dominant inherited disease(Geisterfer-Lowrance et al., 1990; Seidman and Seidman, 2000,2001), is clinically characterized by thickening of the ventricularwalls with a resultant decrease in ventricular chamber size thatoccurs without a predisposing cause. Systolic performance of theheart is preserved or even increased, but relaxation capacity isdiminished. HCM affects more than one in 500 individuals (Harveyand Leinwand, 2011; Maron, 2011; Maron et al., 1995; Semsarianet al., 2015). Dilated cardiomyopathy (DCM), in contrast, ischaracterized by thinning of the heart walls, enlargement of theventricular chambers, and weakness of the cardiac muscle, resultingin an inability to raise the cardiac output to meet the demands ofeven modest exercise (Hershberger et al., 2013).

There are reported to be more than 300 pathogenic mutations inβ-cardiac myosin (Buvoli et al., 2008; Colegrave and Peckham,2014; Seidman and Seidman, 2001; Walsh et al., 2010). Most ofthese are HCM mutations in the globular head domain, myosin S1(Colegrave and Peckham, 2014). The other major site in thesarcomere for HCM-causing mutations is a regulatory proteinknown as myosin binding protein-C (MyBP-C). MyBP-C is thought

1Department of Biochemistry, Stanford University School of Medicine, Stanford, CA94305, USA. 2Department of Pediatrics (Cardiology), Stanford University School ofMedicine, Stanford, CA 94305, USA.

*Authors for correspondence ( jspudich@stanford.edu; kruppel@stanford.edu)

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to put a break on the contraction of the muscle, which can beregulated by its phosphorylation (Chow et al., 2014; Kensler et al.,2011; Seidman and Seidman, 2000; van Dijk et al., 2014). A smallerbut significant set of mutations are found in the tropomyosin–troponin complex. Tropomyosin (Tm) and the three troponinsubunits, troponin T (TnT), troponin I (TnI), and troponin C (TnC),form the Ca2+-regulatory complex of the sarcomere. The actin,myosin, tropomyosin, and the three troponins constitute a six-component system that is considered to be the fundamentalregulated contractile complex of the sarcomere (Fig. 2). MyBP-C,which is in the vicinity of the myosin heads in the sarcomere, shouldbe added as a seventh component of the fundamental regulatedcomplex. Furthermore, the giant protein titin is also in the vicinity ofthe myosin heads in the sarcomere and has been shown to carryDCMmutations (Gerull et al., 2002; Herman et al., 2012; LeWinterand Granzier, 2013, 2014).Much more is known about the role of the Tm.Tn regulatory

complex in cardiac contraction than that of the regulatory proteinsMyBP-C and titin. There is good evidence that domains of MyBP-Cnear the N terminus bind to the regulated actin filament while its Cterminus binds to the myosin thick filament backbone (Fig. 2), thusplaying a regulatory role in muscle contraction (Chow et al., 2014;Kensler et al., 2011; van Dijk et al., 2014). Studies also suggest that

MyBP-C binds to myosin S2, the coiled-coil domain just C-terminalto the globular S1 head, as well as to the regulatory light chain(RLC) of the myosin S1 (Harris et al., 2004; Kampourakis et al.,2014; Pfuhl and Gautel, 2012; Ratti et al., 2011). Titin has beenthought of as an element involved in passive tension in thesarcomere (Granzier and Irving, 1995), but as discussed below,MyBP-C and titin may both contribute importantly to the regulationof ensemble force production by the myosin.

The force–velocity curve is fundamental to musclecontractionAs described above, clinically HCM mutations are associatedwith hypercontractility, whereas DCM mutations are associatedwith hypocontractility. This has led to the general hypothesis thatHCM mutations are hypercontractile and DCM mutations arehypocontractile at the fundamental molecular level. Many importantstudies have contributed to our current understanding of the effectsof some HCM and DCM mutations on various functions of thepurified actomyosin complex, usually its ATPase activity or itsvelocity in the in vitro motility assay (for review, see Moore et al.,2012; Sivaramakrishnan et al., 2009). Only a few studies haveattempted to explore the effects of the mutations on the power outputgenerated by a reconstituted actin–myosin complex (Aksel et al.,2015; Debold et al., 2007; Greenberg et al., 2010; Sommese et al.,2013b). Ultimately, the molecular basis of changes in power outputis what one needs to understand.

Power (P) output comes directly from the force–velocity (F–V)curve of muscle contraction, as P=F×v (Fig. 3). The force axis onthis curve is related to the load on the muscle; the force the muscleproduces must overcome the load. Ventricular function isinfluenced by the two types of loads that are applied to cardiacmuscle. One is preload, the load applied by the volume of bloodfilling the left ventricle that results in stretching of the myofibrilsprior to the initiation of ejection. The other is afterload, the load dueto pressure in the systemic circulation during contraction when theaortic valve is open. The afterload is the primary load that the heartneeds to overcome to eject blood out of the left ventricle.

A somewhat oversimplified but useful view of velocity (v) andensemble force (Fensemble) breaks these down into five parameters, f,d, ts, tc, andNt (Fig. 3). The velocity at any load is a reflection of d/ts,the distance moved per strongly-bound state time of the motor toactin. Fensemble is the intrinsic force f of each motor domainmultiplied by the total number of myosin heads in a force-producing

vo=d/ts

0 2 4 6 8 10 12 14Lever arm length (nm)

measured from putative fulcrum point

Fulcrumpoint

Actin

0

1

2

3

4

Slid

ing

velo

city

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)

Fig. 1. Sliding velocity as a function of leverarm length. The Dictyosteium discoideummyosin II gene was genetically engineered toproduce four forms of S1 with different lever armlengths (shown on the right), expressed inD. discoideum, and purified as describedpreviously (Uyeda et al., 1996). The normalD. discoideum myosin S1 has two light chains.Constructs containing zero light chains and onelight chain were expressed, as well as onecontaining three light chains. The latter wascreated by inserting an additional essential lightchain (magenta) binding domain into the myosin IIheavy chain. The fulcrum was inferred from theintercept of the straight line with the x-axis. vo,unloaded sliding velocity; d, stroke size; ts,strongly bound state time. Data are from Uyedaet al. (1996).

List of symbols and abbreviationsd distanceDCM dilated cardiomyopathyELC essential light chainf intrinsic forceFensemble ensemble forceHCM hypertrophic cardiomyopathyMyBP-C myosin binding protein-CNt total number of functionally available headsRLC regulatory light chainS1 subfragment-1sS1 short subfragment-1tc total cycle timeTm tropomyosinTnC troponin CTnI troponin ITnT troponin Tts strongly bound state timev velocityWT wild type

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state at any moment. The latter is the total number of headsfunctionally available (Nt) times the duty ratio. The duty ratio forthose heads available to interact with the actin is ts/tc, where ts is thestrongly bound state time of myosin on the actin at a particular loadand tc is the total cycle time of the actin-activated myosinchemomechanical cycle. The weakly bound states of the motorare the ATP- and ADP-Pi-bound states, while the strongly boundstates are the ADP-bound states (Fig. 4). Thus the ensemble force inthe sarcomere is Fensemble=f (ts/tc)Nt.We have been examining these individual parameters for several

myosin HCM and DCMmutations studied in a recombinant form ofhuman β-cardiac myosin (short S1 or sS1) that consists of residues1–808 (the motor domain elongated by the essential light chain[ELC] binding domain) co-expressed with the human cardiacventricular ELC (MYL3) (Fig. 1, second myosin head image).

Changes in intrinsic force, cycle time and strongly boundstate time are generally small for HCM and DCM mutationsAlthough several-fold changes in intrinsic force, cycle time orstrongly bound state time have been reported in studies of HCM andDCM using non-human myosins compared with wild type (WT),we have found only small (10–50%) changes in any of theseparameters for the HCM mutant forms we have studied using thehuman β-cardiac sS1 (R403Q, R453C, R719W, R723G, andR663H). In general, these relatively small changes are notsurprising, as most patients with HCM do not begin to exhibitsymptoms until at least the third decade of life (and often muchlater).

The DCM mutants (I201T, A223T, R237W, S532P, and F764L)showed somewhat larger changes compared withWT than the HCMmutants. M531R, a left ventricular non-compaction mutant, showedthe largest change, the tc of which was decreased 1.7-fold comparedwith WT (Aksel et al., 2015).

In general, we are finding that the DCM mutations all behavesimilarly; for example, they all increase tc. In contrast, the HCMmutations are variable, some appearing more hypocontractile withregard to these parameters and others slightly hypercontractile.These variabilities led us to develop a metric that relates to Fensemble

more directly, using a loaded in vitro motility assay.

An ensemble force metric derived from a loaded in vitromotility assayOne way to measure actin gliding velocities at different loads isusing a loaded in vitro motility assay in which increasingconcentrations of an actin binding protein is utilized as the load-bearing molecule (Bing et al., 2000; Greenberg and Moore, 2010;

Power

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Per

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vel

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Per

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Fensemble=f(ts/tc)Nt

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Fig. 3. Schematic plot of a force–velocity (F–V) curve for heart muscle.The power curve is generated bymultiplying the force times the velocity at eachpoint along the F–V curve. The grey zone is the region of the power curvethought to dominate systolic contraction.

Tn complex ActinTm

S2MyBP-C

S1

Fig. 2. Schematic drawing to scale of essential components of thesarcomere. Tropomyosin (Tm) and the three subunits of troponin (Tncomplex) decorate the actin filament. The domains of myosin are the S1 head(only one of two are shown for clarity), the coiled-coil S2, and the lightmeromyosin that forms the thick filament (bottom grey platform). TheN-terminal domains of myosin binding protein-C (MyBP-C) bind to the thinactin filament and the C-terminal domains bind to the thick myosin filament.Titin (not shown) traverses in between the thin and thick filaments, closelyassociated with the thick filament backbone. Interactions among titin, MyBP-C,S1, and S2 are possible and perhaps regulatory, but have not been fullyexplored.

5. ATP hydrolysis

3. ADP release

4. ATP binding

2. Power strokePi release

1. Actin binding

T

D

D.Pi

D

Fig. 4. Schematic figure of the key steps of the actin-activated myosinchemomechanical cycle. The total cycle involves five key steps. The pre-stroke states of S1 are shown on the right and the post-stroke states on the left.Step 1: The pre-stroke S1 with bound ADP (D) and phosphate (Pi) undergoes aweak to strong transition in binding to actin. This is the rate-limiting step in thecycle and determines the total cycle time (tc), which is 1/kcat, where kcat is theturnover number, or the number of substrate molecules each enzyme siteconverts to product per unit time. Step 2: While tightly bound to actin, the leverarm swings about its fulcrum point (black circle) to the right to its post-strokeposition (black arrow), moving the actin filament to the left (bold blue arrow) withrespect to themyosin thick filament. Step 3: ADP release frees the active site forbinding of ATP (T). This step is related to the velocity of the actin filament slidingas the filament cannot move any faster than the myosin heads can let go. Step4: ATP binding weakens the interaction of the S1 to actin. Although the ATP-bound heads still maintain a weak affinity for the actin, this state is typicallydrawn as dissociated from the actin filament. Step 5: ATP hydrolysis results incocking of the head into the pre-stroke state (Shih et al., 2000).

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Haeberle and Hemric, 1995; Warshaw et al., 1990). This assay hasbeen used to compare power output and maximal force generationbetween myosin variants (Greenberg et al., 2010; Haeberle, 1994;Malmqvist et al., 2004) and between different solution conditions(Greenberg andMoore, 2010; Janson et al., 1992). The load-bearingmolecule often used is α-actinin.The α-actinin assay does not work well, however, with the small

motor domain sS1, presumably because of the small size of thisdomain compared with that of two-headed myosin constructs(Aksel et al., 2015). To obtain information about the relativeensemble forces of mutant and WT human β-cardiac sS1, wedeveloped an utrophin-based loaded in vitro motility assay (Akselet al., 2015) (Fig. 5A). The actin-binding protein utrophin is used toput a load on the actin filaments that the myosin must overcome. Toachieve proper control of myosin and load molecule ratios, atruncated utrophin construct of similar size to sS1 is engineeredto anchor on a glass surface via the same attachment used for sS1.Both the velocity of the filaments and the percentage of mobilefilaments decrease as utrophin concentration is increased. These twoparameters can be combined into what we call the percent timemobile (Fig. 5B). This parameter is a proxy for the ensemble forcegenerated by the motor molecule (Aksel et al., 2015). Importantly,to facilitate these experiments, we also developed a rapid, objectivefilament-tracking software called FAST (Aksel et al., 2015).(For details of the FAST classes and routines, see the FASTpackage available at http://spudlab.stanford.edu/fast-for-automatic-motility-measurements/. FAST is currently available for both Linux

[Ubuntu 14.04] and Mac operating systems. Academic license forFAST is available for free on http://spudlab.stanford.edu/fast-for-automatic-motility-measurements/.)

We have used the utrophin loaded motility assay and FAST tocharacterize human α- and β-cardiac sS1 (Aksel et al., 2015). Asseen in Fig. 5B, the amount of utrophin needed to overcome themotility of human α-cardiac sS1 is considerably less than thatneeded to overcome the human β-cardiac sS1, consistent with α-cardiac sS1 being a weaker motor (producing less Fensemble) thanβ-cardiac sS1 (Malmqvist et al., 2004). We have also applied thisapproach to mutants that show opposite mechanical and enzymaticphenotypes with respect to each other, the hypercontractile leftventricular non-compaction mutant M531R and the hypocontractileDCM mutant S532P (Aksel et al., 2015). By this test, M531Ris a stronger motor than WT, while S532P is a weaker motor. Wealso showed that omecamtiv mecarbil, a previously discoveredcardiac specific myosin activator (Malik et al., 2011), increasesβ-cardiac sS1 Fensemble generation as judged by this loaded in vitromotility assay (Aksel et al., 2015). We are now applying thisapproach to more than 15 cardiomyopathy mutant forms of humanβ-cardiac sS1.

HCM and DCM mutations in the tropomyosin–troponincomplex are Ca2+-sensitizing and Ca2+-desensitizing,respectivelyThe tropomyosin–troponin complex is the Ca2+ sensor in themuscle and is composed of tropomyosin and three troponinsubunits: (1) TnC, which binds Ca2+; (2) TnI, which binds actinand tropomyosin and is the inhibitory subunit; and (3) TnT,which binds tropomyosin and communicates the Ca2+ signal fromTnC to tropomyosin (Brown and Cohen, 2005; Kobayashi andSolaro, 2005). In the absence of Ca2+, tropomyosin is in aposition along the actin filament that blocks the binding ofmyosin. Upon Ca2+ binding to TnC, the inhibitory region of TnIdissociates from tropomyosin, allowing the tropomyosin to moveazimuthally on the actin filament, revealing the myosin bindingsites on actin. Mutations in these proteins have been studied usingvarious methods, including: animal models, skinned fibers intowhich mutant proteins have been exchanged, and reconstituted invitro molecular studies using endogenous and/or recombinantproteins such as ATPase, fluorescence, and motility assays (e.g.Miller et al., 2001; Mirza et al., 2005).

Extensive studies by many investigators have yieldedimportant advances in understanding the effects of HCM- andDCM-causing mutations on a reconstituted six-componentsystem consisting of actin, myosin, tropomyosin, and the threetroponin subunits (for reviews, see Hitchcock-DeGregori, 2008;Kobayashi and Solaro, 2005; Moss et al., 2004; Tardiff, 2011;Willott et al., 2010). Most biochemical thin filament studies havebeen performed using non-human cardiac myosin (e.g. Szczesnaet al., 2000; Venkatraman et al., 2005, 2003) or skeletal myosin(e.g. Kobayashi et al., 2013; Lin et al., 1996; Mirza et al., 2005;Tobacman et al., 1999). More recent studies have used humanβ-cardiac myosin, where some specific differences inbiochemical parameters were found compared with earlier non-human myosin studies (Bloemink et al., 2014; Deacon et al.,2012; Gupte et al., 2015; Sommese et al., 2013b). Regardless ofthe source of the mammalian myosin, however, the generalparadigm holds true that HCM mutations in tropomyosin ortroponin are sensitizers to Ca2+ (e.g. Fig. 6 shows HCMmutations for three TnI mutations), while DCM mutations aredesensitizers (Willott et al., 2010).

A

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Humanβ-cardiac sS1

Utrophin

SNAP-PDZ18

Utrophin concentration (nmol l–1)

Tim

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0 0.2 0.4 0.6 0.8

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Alpha

Fig. 5. Cartoon of the loaded in vitro motility assay and data obtained forhuman α- and β-cardiac sS1. (A) Myosin sS1 (red) and utrophin (green) areanchored to a microscope cover slip on the surface via the same attachmentsystem involving binding of a C-terminal eight-residue peptide that specificallybinds to SNAP-PDZ18 (light blue), which coats the surface. The utrophin puts aload on the actin filament and slows its gliding velocity. (B) Loaded in vitromotility percent time mobile data for α- (red) and β- (blue) human cardiac sS1.Solid lines show the best stop-model fit (Aksel et al., 2015) to the percent timemobile data. Dashed lines mark values determined from the fit that reflect therelative force generation by the two motors; a lower x-intercept means weakerforce. Data are from Aksel et al., (2015).

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The myosin mesa and a possible role for MyBP-C and titin inregulating thenumberof functionally availablemyosin headsin the sarcomereBesides β-cardiac myosin, the other major site in the sarcomere forHCM mutations is the regulatory protein MyBP-C (Buvoli et al.,2008; Seidman and Seidman, 2011; Walsh et al., 2010). MyBP-C isthought to put a break on the contraction of the muscle, which can beregulated by its phosphorylation (Chow et al., 2014; Kensler et al.,2011; Seidman and Seidman, 2000; van Dijk et al., 2014). There isgood evidence that domains of MyBP-C near the N terminus bind tothe regulated actin filament, whereas its C terminus binds to themyosin thick filament backbone (Fig. 2), playing a regulatory role inmuscle contraction (Chow et al., 2014; Kensler et al., 2011; van Dijket al., 2014). MyBP-C is in the A-band region of the sarcomere inclose proximity to the myosin heads (Fig. 2). Another regulator, thegiant molecule titin (Granzier and Irving, 1995; LeWinter andGranzier, 2013, 2014), extends through the A-band region, running

from the Z-disc, to which actin filaments are bound, to the M-line,the center of the sarcomere. Thus, each half sarcomere has titinmolecules running its entire length, where the titin plays animportant role in passive tension of the sarcomere (Granzier andIrving, 1995) as well as other roles (LeWinter and Granzier, 2014).

A recent report made the suggestion that MyBP-C and/or otherproteins in the vicinity of the myosin heads may play a role insequestering some of the heads and taking them out of thefunctionally available pool, reducingNt and thus Fensemble (Spudich,2015). The concept of MyBP-C removing myosin heads from thefunctioning pool of heads in the sarcomere has been suggestedpreviously, with evidence for binding of MyBP-C to S2 and theRLC (for reviews, see Kampourakis et al., 2014; Moss et al., 2015).But there may be a site on the motor domain of S1, which haspreviously been overlooked, that participates in such sequestrationof myosin heads. This site has been called the myosin mesa, whichis highly conserved among the cardiac myosins, from mouse α-cardiac to human β-cardiac myosin (Spudich, 2015). The mesa hasmany more arginine residues on its surface than lysine residues, theopposite of usual protein surfaces (Warwicker et al., 2014).Arginines are known to play a role in transient interactionsbetween different proteins (Jones et al., 2000). Thus, the cluster ofeight arginine residues on the mesa, R169, R204, R249, R403,R442, R453, R652, and R663, could be involved in transientinteractions between the mesa and another protein or protein domain(e.g. MyBP-C, titin, myosin S2) (Fig. 7, blue-labeled amino acids).Strikingly, all eight of these arginines are sites of HCM mutations(Alfares et al., 2015). A number of other non-argininecardiomyopathy mutations are also on the mesa in the vicinity ofthese arginines (Fig. 7). Besides a potential MyBP-C interactionwith the myosin mesa, titin could be another player in such aninteraction. Indeed, Granzier and colleagues reported that certaintitin domains in the A-band of the sarcomere have conserved surfacepatterns and show weak interaction with myosin S1 (Muhle-Gollet al., 2001).

An intriguing hypothesis is that the myosin mesa HCMmutationsreduce the affinity of S1’s putative binding partner(s) (e.g.MyBP-C,titin, myosin S2), releasing those heads to now be involved in thecontractile process. This would result in hypercontractility of the

A

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Myosin mesa Converter

M531R

M539L

V606M1263T

N444S

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M539L

V606MI263T

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R652G R663H

R169G

R453CR204H

R249QR442C

R652G R663H

R169G

R453CR204H

R249QR442C

R403Q

R403Q

Fig. 7. Locations of 17 hypertrophic cardiomyopathymutations in the myosin catalytic domain. (A) Cartoonshowing two groups of HCMmutations. Four mutations arein the converter domain (dark green), and 13 are on or verynear the myosin mesa (bright green). Sixteen of the HCMmutations were chosen because they have beendocumented to be the cause of HCM in families carryingthese mutations (Alfares et al., 2015). The 17th is M531R,which, while only documented in one family, is a leftventricular non-compaction mutant myosin that appears tobe hypercontractile in our studies. (B) The top view of themesa showing 13 mutations on or near the mesa surface.The residues labeled in blue denote the positively chargedamino acids. (C) The same view as in B except the surfacecharge distribution is shown. I263T, N444S, and M531Rare slightly below the surface in acidic pockets, while theremainder of the mutations are on or very near the surfaceand most are arginine residues, seven of which form aparticularly large domain of positive charge on the right halfof the mesa as viewed.

WT

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Fig. 6. Effects of TnI mutations on Ca2+ regulation of actin-activatedmyosin S1 ATPase. Percent of the maximal wild-type (WT) ATPase activity(100%) is plotted as a function of the negative logarithm of the Ca2+

concentration. WT (black) is compared with R21C (blue), S166F (green) andΔK177 (red). Maximum WT activity was set to 100% and all other activitieswere normalized to this. Mean activities±standard error are plotted. Myosin S1was prepared by proteolytic cleavage of bovine cardiac myosin.

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muscle, which is characteristic of HCM clinically. Because themyosin motor domain is highly allosteric and there may be otherbinding sites for the putative interacting partner, this molecularmechanism for HCM hypercontractility may extend to other HCMmutations as well, and this mechanism could be a unifyinghypothesis as towhyHCMmutations are hypercontractile clinically.

Conclusion and future perspectivesOur laboratory is focused on changes in biochemical andbiomechanical parameters in HCM and DCM mutant forms ofhuman β-cardiac myosin. Our studies use both the two-componenthuman system of actin and myosin (Aksel et al., 2015; Sommeseet al., 2013b) and the six-component reconstituted human system:actin, myosin, Tm, TnC, TnI, and TnT (Gupte et al., 2015;Sommese et al., 2013a). From the considerations described here andelsewhere (Spudich, 2015), it is possible that many of the HCMmutations in the motor domain of myosin alter the effects of aputative MyBP-C, titin or myosin S2 interaction with the mesasurface. Thus, reconstitution of the eight-component system is likelyto be necessary to fully understand the effects of the HCMmutations. This may equally apply to the DCM mutations.Similarly, the effects of small molecule activators and inhibitors

on the human cardiac ventricular system (e.g. Malik et al., 2011)will likely have to be understood at the eight-component level inmolecular studies to correlate effects seen in the reconstitutedsystem with those observed in skinned fibers, cardiomyocytes, thewhole organ, and animals.

AcknowledgementsWe thank Allan Borrayo for technical assistance inmaintaining cell cultures and viruspreparations.

Competing interestsJ.A.S. is a founder of and owns shares in Cytokinetics, Inc., and MyoKardia, Inc.,biotech companies that are developing therapeutics that target the sarcomere.

Author contributionsJ.A.S. and K.M.R. developed the general concepts and approaches of the researchprogram. T.A. developed the loaded in vitromotility assay. J.S. built the laser trap forsingle molecule analysis. S.N., M.K., A.A., S.S.S., T.A., J.S., R.F.S., S.R.B., E.C.Y.,S.S., R.T., C.C., C.L., D.T., and K.M.R. performed experiments and, together withJ.A.S., analyzed the data. S.R.B. carried out the experiments shown in Fig. 6. J.A.S.prepared the manuscript, which S.N., M.K., A.A., S.S.S., T.A., J.S., R.F.S., S.R.B.,E.C.Y., S.S., R.T., C.C., C.L., D.T., and K.M.R. edited prior to submission.

FundingThis work was supported by the National Institutes of Health [GM33289 andHL117138 to J.A.S.]; the Human Frontiers Science Program [to J.A.S.]; a NationalInstitutes of Health Cellular, Biochemical, andMolecular Sciences Training Grant [toS.R.B. and C.L.]; a Stanford Interdisciplinary Graduate Fellowship [to R.F.S.]; aStanford Dean’s Postdoctoral Fellowship [to S.N.]; the National Science FoundationGraduate Research Fellowship Program [to S.R.B.]; and the Lucile Packard ChildHealth Research Institute Postdoctoral Grant [to A.S.A.]. Deposited in PMC forrelease after 12 months.

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