1521-0103/353/1/159–168$25.00 http://dx.doi.org/10.1124/jpet.114.222224THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:159–168, April 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

The Small-Molecule Fast Skeletal Troponin Activator,CK-2127107, Improves Exercise Tolerance in a Rat Modelof Heart Failure s

Darren T. Hwee, Adam R. Kennedy, James J. Hartman, Julie Ryans, Nickie Durham,Fady I. Malik, and Jeffrey R. JasperCytokinetics Inc., South San Francisco, California

Received December 17, 2014; accepted February 11, 2015

ABSTRACTHeart failure–mediated skeletal myopathy, which is character-ized by muscle atrophy and muscle metabolism dysfunction,often manifests as dyspnea and limb muscle fatigue. We havepreviously demonstrated that increasing Ca21 sensitivity of thesarcomere by a small-molecule fast skeletal troponin activatorimproves skeletal muscle force and exercise performance inhealthy rats and models of neuromuscular disease. Theobjective of this study was to investigate the effect of a novelfast skeletal troponin activator, CK-2127107 (2-aminoalkyl-5-N-heteroarylpyrimidine), on skeletal muscle function and ex-ercise performance in rats exhibiting heart failure–mediatedskeletal myopathy. Rats underwent a left anterior descendingcoronary artery ligation, resulting in myocardial infarction anda progressive decline in cardiac function [left anterior de-scending coronary artery heart failure (LAD-HF)]. Comparedwith sham-operated control rats, LAD-HF rat hindlimb and

diaphragm muscles exhibited significant muscle atrophy.Fatigability was increased during repeated in situ isokineticplantar flexor muscle contractions. CK-2127107 produceda leftward shift in the force-Ca21 relationship of skinned, singlediaphragm, and extensor digitorum longus fibers. Exerciseperformance, which was assessed by rotarod running, waslower in vehicle-treated LAD-HF rats than in sham controls(116 6 22 versus 193 6 31 seconds, respectively; mean 6S.E.M.; P 5 0.04). In the LAD-HF rats, a single oral dose ofCK-2127107 (10 mg/kg p.o.) increased running time comparedwith vehicle treatment (2836 47 versus 1166 22 seconds; P50.0004). In summary, CK-2127107 substantially increasesexercise performance in this heart failure model, suggestingthat modulation of skeletal muscle function by a fast skeletaltroponin activator may be a useful therapeutic in heartfailure–associated exercise intolerance.

IntroductionDespite advances in device and pharmacological therapy,

heart failure (HF) continues to be a leading cause of morbidityand mortality. Although cardiac dysfunction is the centralphenotypic abnormality, the compensatory hemodynamic,autonomic, and neurohormonal responses to this dysfunctionultimately lead to amultisystem syndrome that contributes tooverall morbidity (Piepoli et al., 2010a,b). Skeletal muscle isamong the organ systems affected by HF, as changes inmuscle mass, myocyte calcium handling, capillary density,muscle fiber type, mitochondrial structure, and oxidativecapacity have been observed in patients and animal models ofHF (Drexler et al., 1992; Perreault et al., 1993; Schieffer et al.,1995; Anker et al., 1997; Delp et al., 1997; Duscha et al., 1999;Ward et al., 2003). These alterations contribute to decreased

muscle strength and endurance (Pina et al., 2003; Toth et al.,2010) and, collectively, this skeletal myopathy is believed tobe a substantial contributor to exercise intolerance anddyspnea, resulting in disability and diminished quality of lifein patients with HF (Pina et al., 2003).The sarcomere is the basic contractile unit of skeletal

muscle. Within the sarcomere, the calcium ion (Ca21) is a keyregulatory signaling molecule, as the binding of Ca21 totroponin C in the troponin complex permits tropomyosinmovement, consequent actin-myosin interaction, and musclecontraction (Brooks, 2004). The magnitude of Ca21 releaseincreases proportionally with the level of neuromusculartransmission and, in turn, at submaximal levels of neuro-muscular activation, the force of contraction increases propor-tionally with calcium release (Chin, 2010).Modulation of the sarcomere is an emerging therapeutic

target of interest to treat heart and skeletal muscle–relateddiseases (Ochala, 2010; Malik et al., 2011; Kho et al., 2012a).We have demonstrated that the small molecule tirasemtiv(formerly CK-2017357) specifically binds to fast skeletaltroponin and slows the off rate of Ca21 from troponin, thus

All research studies were financially supported by Cytokinetics.D.T.H. and A.R.K. contributed equally to this work.dx.doi.org/10.1124/jpet.114.222224.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: ANCOVA, analysis of covariance; CK-2127107, 2-aminoalkyl-5-N-heteroarylpyrimidine; DMSO, dimethyl sulfoxide; EDL,extensor digitorum longus; HF, heart failure; ITC, isothermal titration calorimetry; LAD, left anterior descending coronary artery; SERCA,sarcoendoplasmic reticulum Ca21 ATPase.

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sensitizing the complex to Ca21 ions (Russell et al., 2012).This mechanism of action leads to a significantly higher forceoutput at submaximal levels of motor nerve stimulation inanimals and humans (Russell et al., 2012; Hansen et al.,2014). Tirasemtiv and its structural analogs have demon-strated efficacy in improving muscle strength in preclinicaldisease models of neuromuscular origin, including a mousemodel of nemaline myopathy (Lee et al., 2013; Ottenheijmet al., 2013), rat model of myasthenia gravis (Russell et al.,2012), and mouse model of amytrophic lateral sclerosis (Hweeet al., 2014). Furthermore, in clinical trials, tirasemtivimproved respiratory function and other measures of skeletalmuscle strength and endurance in patients with amytrophiclateral sclerosis (Shefner et al., 2012), myasthenia gravis, andexercise-limiting calf claudication (Bauer et al., 2014).Increasing sarcomeric calcium sensitivity by a fast skeletal

troponin activator might mitigate the skeletal muscle dys-function observed in HF. In this study, we examined the effectof CK-2127107, a novel fast skeletal troponin activator that isstructurally distinct from tirasemtiv in rats with inducedsystolic heart failure that exhibited characteristics of skeletalmyopathy and substantially reduced exercise performance.

Materials and MethodsSmall Molecule CK-2127107. CK-2127107 (2-aminoalkyl-5-N-

heteroarylpyrimidine) (Yang et al., 2011), was identified andoptimized as a small-molecule fast skeletal troponin activatorcandidate. The CK-2127107 used in these experiments was synthe-sized at Cytokinetics (South San Francisco, CA).

Myofibril Isolation and Protein Purification. Slow skeletal,fast skeletal, and cardiac myofibrils were prepared from slow bovinemasseter, fast rabbit psoas, and bovine heart muscle, respectively, aspreviously described (Solaro et al., 1971; Young and Davey, 1981;Herrmann et al., 1993). Skeletal isoforms of myosin, actin, troponin,and tropomyosin were purified from rabbit psoas muscle, and cardiacisoforms were purified from the bovine heart, as previously described(Margossian and Lowey, 1982; Pardee and Spudich, 1982; Potter,1982; Smillie, 1982). Troponin-tropomyosin–regulated actin myosinwas reconstituted according to published methods (Ebashi et al.,1967; Spudich and Watt, 1971).

ATPase Assays. Myosin ATPase activity was measured aspreviously described (De La Cruz and Ostap, 2009; Russell et al.,2012). Fast and slow skeletal myofibril steady-state activity wasmeasured in a buffer containing 12 mmol/l 1,4-piperazinediethane-sulfonic acid (Pipes), 2 mmol/l MgCl2, 60 mmol/l KCl, and 1 mmol/ldithiothreitol at a pH of 6.8. Activity was measured at 25°C ina pyruvate kinase and lactate dehydrogenase–coupled enzymesystem. This assay regenerates myosin-produced ADP into ATP byoxidizing NADH, producing an absorbance change at 340 nm.A SpectraMax plate reader (Molecular Devices, Sunnyvale, CA)monitored the change in absorbance as a function of time in thepresence of varying concentrations of CK-2127107.

Measurement of Calcium Release from Troponin. Calciumrelease from the troponin complex was measured using an adaptedprotocol from Rosenfeld and Taylor (1985). Quin-purified rabbitskeletal troponin was exchanged into 1� PM12 buffer (12 mmol/lK-Pipes, 2 mmol/l MgCl2, pH 6.8) by passage over a P-6DG column(Bio-Rad, Hercules, CA) that was pre-equilibrated in that buffer.Troponin concentrations were determined by UV absorbance (278 nm)in 6 mol/l guanidine-HCl using an extinction coefficient of 0.45 fora 1 mg/ml solution. Quin-2 (Invitrogen, Carlsbad, CA) was dissolvedin dimethylformamide and diluted in 1� PM12 just prior to use.Transient fluorescence measurements were made using an SF-61DXstopped-flow fluorimeter (Hi-Tech Scientific, Bradford-on-Avon, UK),

with excitation provided by a monochromator (337 nm, 10-mm slitwidth) and emission measured through a glass filter (495-nm longpass). Fluorescence intensities were translated into calcium concen-trations using calcium standard curves prepared in the presence ofequivalent concentrations of dimethyl sulfoxide (DMSO) and CK-2127107,as were used in the transient reactions. Released calcium concen-trations were expressed as the final concentration after mixing.

Isothermal Titration Calorimetry. Isothermal titration calo-rimetry (ITC) was performed in a Micro-Cal VP-ITCmicrocalorimeter(GE Healthcare, Inc., Piscataway, NJ) at 25°C. Titrations wereconducted by filling the sample chamber with 25 mmol/l rabbit fastskeletal troponin complex in 12 mmol/l Pipes, 100 mmol/l KCl,250 mmol/l CaCl2, 3% DMSO, and 5 mmol/l b-mercaptoethanol. Theligand solution that was injected into the sample chamber contained300 mmol/l CK-2127107 in a buffer consisting of 12 mmol/l Pipes,100 mmol/l KCl, 250 mmol/l CaCl2, 3% DMSO, and 5 mmol/lb-mercaptoethanol. Ligand injections were made every 300 secondsat a volume of 10 ml. To correct for the heats of dilutions of CK-2127107,the stable heat signal from the injections near the end of the experimentwas subtracted from the remaining values. All data collection andanalysis was conducted using the modified Origin software (OriginLab,Northampton,MA) includedwith the instrument using a single bindingsite model. Troponin concentrations were determined by UV absor-bance (278 nm) in 6 mol/l guanidine-HCl using an extinction coefficientof 0.45 for a 1-mg/ml solution.

Sham and Heart Failure Rats. Rats used in this study weremaintained in accordance with the Guide for the Care and Use ofLaboratory Animals of the Institute (Seventh Edition, NationalResearch Council) and under the supervision of the CytokineticsInstitutional Animal Care and Use Committee. Ligation of the leftanterior descending coronary artery was performed in femaleSprague-Dawley rats at Charles River Laboratories (Raleigh, NC).Sham surgeries were also performed with the same surgicalpreparation, except that ligation with the suture material was notperformed. Sham (n 5 25) and left anterior descending coronaryartery (LAD) heart failure (LAD-HF) (n5 21) rats were shipped to theinvestigators’ animal facility. After 3 days of acclimation, cardiacfunction was assessed by echocardiography in all rats. Cardiacfunction was measured longitudinally at 2, 4, 7, and 10 weeks afterarrival.

Histology. Sham and LAD-HF hearts were excised, weighed,fixed in 10% formalin for 24 hours, and embedded in paraffin. Heartsections were stained with Masson’s Trichome for visualization ofcollagen deposition. The diaphragm, soleus, and extensor digitorumlongus (EDL) were excised, cleaned, pinned to a corkboard, and frozenin melting isopentane. Serial frozen cross sections were cut at 10 mmand stained for myosin ATPase after preincubation at a pH of 4.35.Digital images were obtained under 200� total magnification(Olympus BX41; Olympus America, Melville, NY) and analyzed byAxiovision software (Zeiss, Jena, Germany). Stained fibers wereclassified as type I, IIa, or II b/x and measured for the individualmyofiber cross-sectional area (mm2).

Western Blot Analysis. Soleus muscle was homogenized in a celllysis buffer (Life Technologies, Grand Island, NY). Fifty microgramsof protein was prepared in a Laemmli sample loading buffer,separated by SDS-PAGE, and transferred to a nitrocellulose mem-brane. The membranes were incubated overnight at 4°C in a blockingbuffer with sarcoendoplasmic reticulum Ca21 ATPase 2a (SERCA2a;1:1000; ThermoScientific,Waltham,MA) and glyceraldehyde 3-phosphatedehydrogenase (1:12,500; Cell Signaling Technology, Danvers, MA).Following three rinses in 1� Tris-buffered saline/Tween 20, mem-branes were incubated with corresponding fluorescent secondaryantibodies (1:10,000; LI-COR Biosciences, Lincoln, NE). After threerinses, membranes were visualized with a LI-COR infrared imag-ing system. Image quantification was performed using Odyssey2.1 software (LI-COR).

Skinned Muscle Fibers Force–pCa Relationship. Muscletissue for in vitro skinned fiber studies was prepared using an

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adapted protocol based on Lynch and Faulkner (1998). Briefly, ratdiaphragm, soleus, and EDL muscle from sham and LAD-HF ratswere rapidly dissected, rinsed in physiologic saline, and thenincubated in a skinning solution (125 mmol/l K-propionate, 20 mmol/limidazole, 5 mmol/l EGTA, 2 mmol/l MgCl2, 2 mmol/l ATP, pH 7.0)supplemented with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO)for 30 minutes at 4°C. The buffer was then changed to a storagesolution (125 mmol/l K-propionate, 20 mmol/l imidazole, 5 mmol/lEGTA, 2 mmol/l MgCl2, 2 mmol/l ATP, 50% glycerol, pH 7.0) andstored at 220°C for later use.

For skinned fiber analysis, single muscle fibers were dissectedfrom larger segments of tissue in a rigor buffer at 4°C (20 mmol/l4-morpholinepropanesulfonic acid, 5 mmol/l MgCl2, 120 mmol/lpotassium acetate, 1 mmol/l EGTA, pH 7.0). The fibers were thensuspended between a 400A force transducer (Aurora Scientific,Aurora, ON, Canada) and a fixed post and secured with 2–4 ml of a5% solution of methylcellulose in acetone. Fibers were then incubatedat 10°C in a relaxing buffer (20 mmol/l 4-morpholinepropanesulfonicacid, 5.5 mmol/l MgCl2, 132 mmol/l potassium acetate, 4.4 mmol/l ATP,22 mmol/l creatine phosphate, 1 mg/ml creatine kinase, 1 mMdithiothreitol, 44 ppm antifoam 204, pH 7.0) and the baseline tensionwas adjusted. Tension was generated in each fiber by changing thefiber buffer over to a relax buffer supplemented with 1 mmol/l EGTAand a 15 mmol/l solution of calcium chloride and calculated using aweb resource (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm).Relax buffer pCA concentrations ranged from 8 (low Ca21 activationand fiber tension) to 4 (saturated Ca21activation andmaximum tension).CK-2127107 was added to these buffers from a DMSO solution (finalDMSO concentration 5 1%).

In Situ Muscle Contractile Characteristics. Isometric andisokinetic hindlimb force was measured in sham and LAD-HF rats inthe presence and absence of CK-2127107 (10 mg/kg i.v. in 50%polyethylene glycol:40% Cavitron [Ligand Pharmaceuticals Inc., LaJolla, CA]:10% dimethylacetamide). Rats were placed under anesthe-sia with isoflurane (1–5%). One incision was made on the midthighregion of the right leg to expose the sciatic nerve. To preventcocontraction of the ankle dorsiflexors, an additional incision wasmade lateral to the patella to isolate and sever the peroneal nerve.Rats were then placed on a temperature-maintained in situ muscleanalysis rig (Model 806C; Aurora Scientific). The knee was immobi-lized in a clamp between two sharpened screws, and the foot wastaped to a footplate attached to a force transducer (Aurora Scientific).Stainless steel needle electrodes (0.10 mm) were hooked around theexposed sciatic nerve. Sham and LAD-HF rats were treated withvehicle or CK-2127107 (10 mg/kg i.v.). Muscle contractile propertieswere assessed by applying an electrical current (under supramaximalvoltage conditions) to the nerve and recording the force generated bythe muscle via a servomotor. An isometric force–frequency relation-ship (10–150 Hz, 1-millisecond pulse width, 350-millisecond trainduration) was assessed with the ankle joint at 90° of flexion. Anisokinetic force-velocity relationship in response to a 30-Hz stimula-tion was assessed over a range of 0–20.1 radians/s. The fatigueproperties of the ankle plantar flexor muscles were assessed byrepeated isokinetic contractions (30-Hz stimulation, 3.1 rad/s, onceper second) over a 10-minute period.

Diaphragm Force–Frequency Relationship. Diaphragm con-tractile force was measured by electrical field stimulation in an organbath system (Radnoti, Monrovia, CA). The diaphragm and lastfloating rib from sham and LAD-HF rats were excised, rinsed inphysiologic saline, and placed in a temperature controlled water-jacketed chamber (26–27°C) containing Krebs-Henseleit buffer(118 mmol/l NaCl, 10 mmol/l glucose, 4.6 mmol/l KCl, 1.2 mmol/l KH2PO4,1.2 mmol/l MgSO4·7H2O, 24.8 mmol/l NaHCO3, 2.5 mmol/l CaCl2, 50 mg/ltubocurarine, 50 U/l insulin, pH 7.4) that was continuously aeratedwith 95% O2/5% CO2. After 10 minutes of equilibration, vertical stripsspanning the floating rib to the central tendon were cut fromdiaphragms. Braided silk sutures were tied at the central tendon andfloating rib and attached to a force transducer between two platinum

electrodes. Diaphragm strips were set to a length that producedmaximum twitch tension (Lo). The force-frequency profile of themuscle was obtained by stimulating the muscle at frequenciesbetween 10 and 150 Hz (Grass Stimulator [Grass Technologies,Warwick, RI], 800-millisecond train duration, 0.6-millisecond pulsewidth). CK-2127107 was suspended in DMSO and directly addedinto the bath.

Rotarod Exercise Assay. Exercise performance was assessedin sham and LAD-HF rats utilizing a rotarod protocol developedat Cytokinetics. At least 3 days after echocardiography, rotarodperformance was assessed at weeks 4, 7, and 10 to observe the declineof exercise performance. The rotarod assay consisted of a training daythat was immediately followed by an assessment day. The trainingday consisted of two sessions: a constant speed of 10 rpm for 5minutesin themorning and an accelerating speed of 14–16 rpm over 5minutesin the afternoon. On the assessment day, rats were run again at anincreasing speed from 14 to 16 rpm over 5 minutes. Rats were thenimmediately run at a constantly accelerating rate from 12 to 25 rpmover the course of 10 minutes. The time to fall was recorded, with thetest terminated at 600 seconds. Once exercise-intolerant LAD-HF ratswith a fractional shortening of less than 25% were identified, shamand LAD-HF rats were advanced to assessment of rotarod runningunder therapeutic intervention.

A blinded cross-over study design was employed to investigate theeffect of CK-2127107 (10 mg/kg p.o.) and its vehicle [19.3% poly-ethylene glycol:80% (15%) Captisol, pH 3, 0.2% Tween 80, 0.5%hydroxypropyl methylcellulose] dosed 30 minutes prior to evaluatingrotarod performance. At each assessment time point, all rats weredosed with either CK-2127107 or vehicle on the first day, and on thenext day, each rat received the converse treatment. Each dayconsisted of a 5-minute primer session, whereby rats were run at anincreasing speed from 14 to 16 rpm. Rats were then run at a constantlyaccelerating rate from 12 to 25 rpm over the course of 10minutes. Timeto fall was recorded, with the test being terminated at 600 seconds.Only LAD-HF rats that ran less than 300 seconds on their vehicle dayat week 10 and thus exhibited signs of exercise intolerance wereincluded in the study.

Statistical Analyses. Statistical analyses between mean aver-ages employing one-way analysis of variance and unpaired t testswere performed as appropriate using GraphPad Prism softwareversion 6.03 (La Jolla, CA). Repeated measures analysis of covariance(ANCOVA) was performed with SAS software version 9.2 (Cary, NC).Unless noted otherwise, results are expressed as mean 6 S.E.M.Significance was set at P values , 0.05.

ResultsCK-2127107 Selectively Binds to and Sensitizes the

Fast Skeletal Troponin Complex to Calcium. CK-2127107(Yang et al., 2011) was identified and optimized as a small-molecule fast skeletal troponin activator candidate utilizinghigh-throughput screens of type II, fast skeletal myofibrils.Using purified cardiac, slow skeletal, and fast skeletal myofi-brils, the specificity, potency, and mechanism of action ofCK-2127107 was characterized through a series of biochemicalassays.Calcium activates the myosin ATPase activity of skinned

(detergent permeabilized) myofibrils. The midpoint of theATPase-calcium curve relationship is defined as its halfmaximal activation (pCa50) and is a measure of the sensitivityof the sarcomere to calcium. The addition of either 2.5, 10, or40 mmol/l CK-2127107 to fast skeletal rabbit psoas myofibrilsresulted in a concentration-dependent leftward shift of themyosin ATPase-calcium relationship, with the ATPaseactivity pCa50 shifting from a control value of 5.67 to 5.99

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(2.5 mmol/l), 6.23 (10 mmol/l), and 6.55 (40 mmol/l) (Fig. 1A).The muscle-type selectivity of CK-2127107 was examinedusing skinned myofibrils prepared from different muscles,i.e., rabbit psoas (fast skeletal), bovine masseter (slowskeletal), and bovine heart (cardiac), as previously described(Russell et al., 2012). CK-2127107 selectively activated fastskeletal myofibrils (EC50 5 3.4 mmol/l; maximal activation53.6-fold). CK-2127107 had no effect on slow skeletal orcardiac myofibrils, demonstrating its selectivity for fastskeletal muscle (Fig. 1B).Having established the specificity of CK-2127107 for fast

skeletal myofibrils, we leveraged that property to identify thespecific target of CK-2127107 in the fast skeletal sarcomere.Individual components of the sarcomere, including the thinfilament proteins actin, troponin, tropomyosin, and the thickfilament protein myosin can be purified from different sources(fast, slow, or cardiac) and reconstituted in different combi-nations to generate similar ATPase activity and calciumdependence as intact skinned myofibrils. Using cardiacmyosin as a probe for thin filament activation, CK-2127107increased its cardiac myosin ATPase activity in only thoseheterologous reconstituted versions of the thin filament thatcontained fast skeletal troponin, demonstrating that CK-2127107

interacts specifically with the fast skeletal troponin complex(Fig. 1C).The observed selectivity and activity in heterologous

reconstituted assays suggested that CK-2127107 interactsdirectly with fast skeletal troponin. This interaction wasverified using ITC to directly characterize its binding in-teraction. Titration of CK-2127107 into a solution of purifiedrabbit fast skeletal troponin complex (TnT/TnI/TnC) resultedin an exothermic reaction that fit a single site binding model(Supplemental Fig. 1). Pooled data from several experimentsindicated that CK-2127107 bounded to fast skeletal troponinwith high affinity (Kd 5 3.0 6 0.06 mmol/l; n 5 5), and witha stoichiometry consistent with a single binding site pertroponin complex (n 5 1.24 6 0.016). One potential mecha-nism for sensitizing the sarcomere to calcium is throughstabilization of the calcium-troponin complex. Stabilizationslows the rate of calcium dissociation from the troponincomplex, thus prolonging the time troponin spends in theactive (open) conformation. The rate of calcium dissociationfrom troponin was monitored by rapidly mixing the calcium-saturated, fast skeletal troponin complex with Quin-2,a fluorescent calcium chelator whose fluorescence intensityincreases when it binds calcium. When CK-2127107 was

Fig. 1. Characterization of the fast skeletal troponin activator CK-2127107. (A) The ATPase activity of skinned rabbit fast skeletal myofibrils wasmeasured using a pyruvate kinase/lactate dehydrogenase–coupled assay. In the presence of CK-2127107, the control pCa50 of 5.67 (DMSO; circles) shiftsto 5.99 (2.5 mmol/l; squares), 6.23 (10 mmol/l; triangles), and 6.55 (40 mmol/l; inverted triangles). Plotted are the means 6 S.E.M. for two experiments.(B) Concentration-response ATPase activity analysis was performed at a tissue-specific pCa25, with fast skeletal (rabbit), slow skeletal (bovine), andcardiac (bovine) detergent-skinned myofibrils (n = 4 per group). Data were fitted using a four-parameter concentration-response equation, resulting ina calculated fast skeletal maximal fold activation of 3.6 and an EC50 of 3.4 mmol/l. Slow skeletal and cardiac myofibril activation was insufficient toproduce reliable EC50 estimates. (C) Activation of the thin filament (as indicated by an increase in cardiac myosin ATPase observed at a fixed calciumconcentration) is found in the presence of mixed thin filaments containing the fast skeletal isoform of troponin but not the cardiac isoform of troponin.The tropomyosin isoform did not substantially affect themyosin ATPase rate, indicating that the fast skeletal troponin complex is required for compoundactivity. (D) CK-2127107 (green line) slows release of Ca2+ from skeletal troponin. Calcium release wasmonitored using the fluorescence enhancement ofQuin-2 upon binding to calcium.

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tested in this assay, the calcium release rate slowed (from 24.1to 9.8 s21), consistent with a stabilization of the Ca21-bound,“active” conformation of troponin (Fig. 1D). Thus, CK-2127107increases calcium sensitivity, and by slowing the calciumrelease rate from troponin, exerts a leftward shift on the force-calcium relationship of muscle.Skeletal Muscle Limb Atrophy Occurs in Rats with

Systolic Dysfunction Following Myocardial Infarction.As previously reported (Pfeifer et al., 2001; Koh et al., 2003),skeletal muscle atrophy and exercise intolerance occurs in therat LAD-HF model produced by ligation of LAD. Ligation ofthe LAD to produce myocardial infarction was performed on8-week-old female Sprague-Dawley rats, and cardiac functionwas serially assessed by echocardiography every 2–3 weeksfollowing surgery. Compared with rats undergoing a shamsurgery, significant decreases in fractional shortening (49.662.2% versus 28.3 6 3.0%; P , 0.0001) were observed in LAD-HF rats 2 weeks after ligation, and cardiac function continuedto gradually decline during the next 8 weeks (Fig. 2A). Sham

and LAD-HF hearts were collected postmortem for mass andhistologic assessment. Heart mass, normalized to body mass,was significantly higher in LAD-HF rats than in the shamcontrols (4.3 6 0.1 versus 3.5 6 0.01 mg heart/g body mass;P , 0.0001; Fig. 2B), which is consistent with the pathologiccardiac hypertrophy that typically occurs in myocardialinfarction–induced heart failure (Rubin et al., 1983; Purdhamet al., 2008). Masson’s trichrome stains of heart sections fromsham controls and LAD-HF rats revealed extensive collagendisposition distal to the ligation site in the left ventricle ofLAD-HF rats (Supplemental Fig. 2).We evaluated the skeletal muscle mass and myofiber size

characteristics of sham andLAD-HF rats. The hindlimbmusclemass, normalized to body mass, was not significantly differentbetween sham and LAD-HF groups (soleus: 0.496 0.01 versus0.48 6 0.01 mg muscle/g body mass; plantaris: 1.2 6 0.04versus 1.1 6 0.04 mg muscle/g body mass). By mass, therewas a trend for smaller muscles in LAD-HF rats comparedwith sham rats (sham versus LAD-HF soleus: 160 6 4 versus

Fig. 2. Coronary LAD artery ligation produces cardiac dysfunction and skeletal muscle atrophy. (A) Echocardiography revealed significant decreases inleft ventricular fractional shortening over time in LAD-HF rats (n = 13–21 group; ****P , 0.0001 versus sham). (B) Heart mass (normalized to bodymass) is significantly higher in LAD-HF rats, suggestive of pathologic hypertrophy (21–25 rats/group; ****P , 0.0001 versus sham). (C) LAD-HFplantarflexor muscles (soleus and plantaris) had a significantly smaller myofiber area and exhibited a leftward shift in fiber size distribution (n = 5–7 pergroup; *P , 0.05).

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1486 4 mg; P5 0.059; plantaris: 3726 11 versus 3476 8mg;P5 0.09). We also evaluated the myofiber cross-sectional areain histologic sections from hindlimb EDL, soleus, andplantaris muscles. The soleus and plantaris myofiber areawas significantly smaller in LAD-HF rats than in shamcontrol rats (sham versus LAD-HF soleus: 3039 6 122.9 mm2

versus 2338 6 245 mm2; plantaris: 3025 6 230 versus 2349 6176 mm2; n5 5–8 per group; P, 0.05; Fig. 2C), demonstratingthat the condition of heart failure led to changes in skeletalmuscle myofiber morphology.Soleus Muscle Sarcoendoplasmic Reticulum Ca21

ATPase Protein Is Reduced in LAD-HF Rats. Soleusmuscle SERCA is responsible for the reuptake of calcium ions

from the cytoplasm into the sarcoplasmic reticulum. Previousstudies have suggested that decreases in SERCA protein dueto HFmay contribute to calcium dysregulation and overall HFskeletal myopathy (Simonini et al., 1999; Bueno et al., 2010;Middlekauff et al., 2012). In this study, SERCA2a proteinlevels in LAD-HF soleus muscle was significantly lower thanin sham soleus muscle (Supplemental Fig. 4).CK-2127107 Increases Ca21 Sensitivity in Fast Skel-

etal Muscle Fibers and Increases Muscle Force inResponse to Submaximal Stimulation Frequencies inLAD-HF Rats. Fast muscle fibers from EDL and slowmuscle fibers from soleus muscle from sham and LAD-HFrats were harvested and chemically skinned to assess the

Fig. 3. CK-2127107 increases Ca2+ sensitivity, submaximal force, and fatigue resistance in situ. (A) CK-2127107 (3 mmol/l) caused a leftward shift in theforce-Ca2+ relationship in skinned sham and LAD-HF fast fiber EDL muscle. No changes were observed in slow fiber soleus muscle with CK-2127107treatment. (B) LAD-HF plantarflexor muscles produced a significantly lower peak isometric force (*P , 0.05 versus sham). CK-2127107 significantlyincreased submaximal isometric tension up to 60-Hz stimulation in LAD-HF plantarflexor muscles (†P , 0.01 versus vehicle). (C) CK-2127107significantly increased the force-isokinetic velocity relationship in both sham and LAD-HF plantarflexor muscles at 30-Hz stimulation (†P , 0.001versus respective vehicle groups). (D) LAD-HF plantarflexor muscles produced significantly less isokinetic force during a fatigue-inducing repeatedstimulations protocol (*P , 0.01 versus sham). (E) CK-2127107 significantly increased work in both sham and LAD-HF plantarflexor muscles inresponse to 300 repeated stimulations (bar graph: mean 6 S.E.M.; *P , 0.05; **P , 0.01; ****P , 0.0001).

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single fiber force–pCa relationship. CK-2127107 (3 mmol/l)produced a significant leftward shift of the force-pCa relation-ship in both sham and LAD-HF EDL muscle fibers (pCa5sham: DMSO versus CK-2127107 is 5.496 0.06 versus 5.8560.05; LAD-HF: DMSO versus CK-2127107 is 5.56 0.04 versus5.86 6 0.03; n 5 7–9 per group; Fig. 3A), consistent with thecalcium sensitizing effect of CK-2127107. As expected, givenits selectivity for fast skeletal muscle troponin, CK-2127107(10mmol/l) did not have an effect on the force-pCa relationshipof slow fibers from soleus muscle (Fig. 3A).Significant decreases in isometric and isokinetic leg muscle

force and increased muscle fatigability have been previouslyobserved in heart failure patients (Clark et al., 1997;Harrington et al., 1997; Gielen et al., 2012). The in situisometric and isokinetic force characteristics of the rat ankleplantarflexor muscles in sham and LAD-HF rats weremeasured following sciatic nerve stimulation. Isometricmuscle force was significantly lower in LAD-HF rat plantar-flexor muscles at stimulation frequencies from 60 Hz up totetanus (maximum activation) at 150 Hz (Fig. 3B) Adminis-tration of CK-2127107 (10 mg/kg i.v.) significantly increased

plantar flexor isometric force in response to subtetanic nervefrequency stimulations to the same extent in both sham andLAD-HF rats (Fig. 3B). Compared with vehicle treatment,CK-2127107 increased the isometric force to the greatestmagnitude in response to the 30-Hz stimulation frequency.Thus, to provide the most sensitive assessment of CK-2127107,30 Hz was selected for subsequent in situ muscle functionevaluation. The isokinetic plantarflexor force output at 30-Hzstimulation was similar between vehicle-treated sham andLAD-HF rats. CK-2127107 significantly increased the isokineticforce to the same extent in both sham and LAD-HF rats (Fig. 3C).Thus, increasing sarcomere Ca21 sensitivity by CK-2127107treatment increases both the isometric and isokinetic force atan equal, submaximal, stimulation frequency in the presenceor absence of heart failure.As a measure of muscle fatigability in situ, the sham and

LAD-HF ankle plantar flexor muscle groups were subjected to300 repetitive isokinetic contractions (30-Hz stimulation, 3.1rad/s) over a period of 10 minutes. Administration ofCK2127107 (10 mg/kg i.v.) significantly increased initial forceat 30 Hz in both LAD-HF and sham animals (Fig. 3D). After

Fig. 4. CK-2127107 increases Ca2+ sensitivity and submaximal force in LAD-HF diaphragms with characteristics of skeletal myopathy. (A) LAD-HFmyofiber area was significantly smaller than sham myofibers. Fiber size distribution reveals a leftward shift in LAD-HF diaphragm fiber size (n = 6–7per group; *P, 0.05). (B) Skinned LAD-HF diaphragm fibers exhibit reduced Ca2+ sensitivity compared with sham diaphragm fibers, with a significantrightward shift in the LAD-HF force-calcium relationship (*P , 0.05 based on pEC50 values), demonstrating reduced calcium sensitivity in LAD-HFfibers. CK-2127107 (3 mM) significantly increased the force-pCa relationship in both sham and LAD-HF diaphragm fibers. (C) LAD-HF diaphragmmuscles demonstrate a lower isometric peak force (*P , 0.05 versus sham). CK-2127107 significantly increased force in LAD-HF diaphragm muscle atsubmaximal frequencies of stimulation (†P , 0.05 versus untreated LAD-HF diaphragm, *P , 0.05).

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300 contractions, LAD-HF muscle produced less total workthan sham muscles (6.74 6 0.25 versus 7.62 6 0.24 J/g; P ,0.05; Fig. 3E), suggesting increased fatigability in LAD-HFskeletal muscles. Sham and LAD-HF rats treated withCK-2127107 (10 mg/kg i.v.) significantly increased total workand attenuated fatigue throughout the entire protocol (sham:7.62 6 0.24 versus 9.82 6 0.53 J/g; P , 0.01; LAD-HF: 6.74 60.25 versus 10.58 6 0.49 J/g; P , 0.0001; Fig. 3, D and E).CK-2127107 Significantly Increases Ca21 Sensitivity

and Submaximal Diaphragm Force. Respiratory muscleweakness has been highlighted as a specific contributor todyspnea and exercise intolerance in heart failure patients(Meyer et al., 2001). The diaphragm is a primary muscleinvolved in respiration, and dysfunction of this muscle isspecifically a noted complication of heart failure (Meyer et al.,2001; van Hees et al., 2008). In this study, the LAD-HFdiaphragm myofiber area was significantly smaller than thatof sham diaphragms (1534 6 114.7 versus 1141 6 77.9 mm2;P , 0.05; Fig. 4A; Supplemental Fig. 3).We chemically skinned and isolated single diaphragm

muscle fibers to assess the difference in the force-calciumrelationship between sham and LAD-HF diaphragms. Com-pared with sham diaphragm fibers, there was a significantrightward shift in the LAD-HF force-calcium relationship(sham DMSO versus LAD-HF DMSO: 5.57 6 0.02 versus5.5 6 0.02; P , 0.05 by F test; Fig. 4B), indicating reducedcalcium sensitivity in LAD-HF fibers. CK-2127107 (3 mmol/l)caused a significant leftward shift in the force-calcium re-lationship, restoring the calcium sensitivity lost to heart failure(Fig. 4B).Given the atrophy and reduced calcium sensitivity in LAD-

HF diaphragms, the isometric force output was measuredin sham and LAD-HF diaphragms ex vivo by electrical fieldstimulation. The LAD-HF diaphragm muscle produceda lower force compared with sham diaphragms at stimula-tion frequencies ranging from 40 to 80 Hz (P, 0.05 by t testwithin each stimulation frequency; Fig. 4C). CK-2127107(30 mmol/l) significantly increased force in both sham andLAD-HF diaphragms, with the effect being more prominentat stimulation frequencies less than or equal to 30 Hz (P ,0.05 by analysis of variance within each stimulation fre-quency; Fig. 4C). The leftward shift in the force-frequencyrelationship is consistent with the leftward shift observedin the force-calcium relationship of skinned muscle fibers(Fig. 4C).CK-2127107 Significantly Improves Rotarod Perfor-

mance in Exercise-Intolerant LAD-HF Rats. Previousinvestigators demonstrated reduced exercise performance inan LAD-infarct model of heart failure (Pfeifer et al., 2001;Koh et al., 2003; Allen et al., 2008). We used an acceleratingrotarod to assess exercise performance in sham and LAD-HFrats 16 weeks after coronary artery ligation. LAD-HF rats hada significantly shorter running time on an acceleratingrotarod than sham rats (116 6 22 versus 193 6 31 seconds,P, 0.05, by repeated measures ANCOVA; Fig. 5), indicating adecrease in exercise performance. A single dose of CK-2127107(10 mg/kg p.o.) increased performance time by approximately150% in LAD-HF rats (116 6 22 versus 2836 47 seconds, P 50.0004, by repeated measures ANCOVA). There was nodifference between sham and LAD-HF groups treated withCK-2127107 (sham versus LAD-HF was 274.8 6 31 versus283 6 47 seconds).

DiscussionChronic HF patients are often limited by breathlessness

and muscular fatigue, and HF is typically associated withskeletal muscle dysfunction and exercise intolerance (Wilsonet al., 1995). The extent of impaired ventricular systolicfunction in HF patients does not correlate well with theseverity of dyspnea and exercise intolerance (Gibbs et al.,1990; Harrington and Coats, 1997). Therefore, improvementssolely in cardiac function as a consequence of therapy may notnecessarily reverse the existing skeletal muscle abnormalitiesand functional deficits in HF patients. Thus, the developmentof skeletal muscle–specific therapies may be useful in improvingheart failure–mediated exercise intolerance.Left anterior descending coronary artery ligation was used

to produce myocardial infarction and consequent HF in rats,as this model has been shown to have significant reductions inexercise capacity (Yamaguchi et al., 1999; Pfeifer et al., 2001;Koh et al., 2003). Alterations in skeletal muscle Ca21

sensitivity and handling have also been noted with heartfailure (Ward et al., 2003; Kho et al., 2012b), and pharmaco-logical calcium sensitizers have previously been shown toincrease calcium sensitivity in diaphragms from heart failurerats (van Hees et al., 2011). In this study, LAD-HF ratsexhibited significant and progressive heart dysfunction andsubsequently demonstrated characteristics of skeletal myop-athy, including myofiber atrophy and changes in myofibrilcalcium sensitivity and handling in diaphragm and hindlimbmuscles at rest. Compared with sham rats, these skeletalmuscle changes contributed to significant decreases in iso-metric diaphragm force output and increased hindlimbmuscle fatigability in situ and exercise intolerance in vivoby rotarod assessment.CK-2127107 is a novel small molecule fast skeletal troponin

activator currently in human clinical trials for conditions ofmuscle weakness and fatigue. CK-2127107 increased sarco-meric Ca21 sensitivity in skinned LAD-HF skeletal fibersin vitro, increased force output at submaximal stimulation

Fig. 5. CK-2127107 significantly increases rotarod performance in LAD-HF rats with exercise intolerance. LAD ligation resulted in a reducedrotarod running time compared with sham-operated control rats (1936 31versus 116 6 32 seconds; P = 0.042; mean 6 S.E.). CK-2127107–treatedsham rats ran longer than vehicle-treated sham rats (2756 31 versus 1936 31 seconds; P = 0.022). LAD-HF rats treated with CK-2127107 increasedtheir rotarod running time approximately 2.5-fold compared with vehicletreatment (283 6 47 seconds, P = 0.0004, by ANCOVA; *P , 0.05, ***P ,0.001), completely normalizing exercise capacity in LAD-HF rats.

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frequencies in the diaphragm ex vivo and hindlimb muscle insitu, and significantly improved rotarod performance in LAD-HF rats in vivo, thus displaying efficacy on skeletal musclefunction at multiple levels of investigation. These resultsdemonstrated that increasing sarcomere Ca21 sensitivity isa potentially unique therapeutic approach to ameliorate heartfailure–mediated deficits in skeletal muscle strength andfunction.One mechanism for fatigue in skeletal muscle is a reduction

in sarcoplasmic reticular Ca21 release and uptake that thenresults in decreased force production (Allen et al., 2008).Fatiguing exercise can impair SERCA function, which canaffect both uptake and storage of calcium. In the absence ofaltered troponin calcium sensitivity, decreased Ca21 releasefrom the sarcoplasmic reticulum would lead to diminishedtroponin activation and subsequently diminished force pro-duction (Blanchard and Solaro, 1984; Nosek et al., 1987;Westerblad and Allen, 1996; Chin and Allen, 1998; Allenet al., 2008, 2011). The fatigue experienced by exercisingheart failure patients is associated with an increased in-organic phosphate to phosphocreatine ratio and a lowerintracellular pH relative to control subjects (Massie et al.,1987; Mancini et al., 1989), which conceivably affectssarcoplasmic Ca21 release, sensitivity, and force output. Inthe current study, we observed an increase in muscle fatiguein situ and significantly reduced exercise performance time inLAD-HF rats. CK-2127107 reversed these deficits, suggestingthat increasing troponin Ca21 sensitivity can compensate forthe presumed reduction in SERCA function in HF.Inspiratory muscle weakness is noted in 30–50% of heart

failure patients (Meyer et al., 2001; Ribeiro et al., 2012), andindices of diaphragm dysfunction and structural abnormali-ties have been noted in both patients and animal models ofheart failure (Meyer et al., 2001; van Hees et al., 2008; Wonget al., 2011). LAD-HF rats in this study exhibited significantdiaphragm atrophy and decreased sarcomeric Ca21 sensitiv-ity. CK-2127107 increased Ca21 sensitivity and causeda leftward shift in the isometric force frequency relationshipin LAD-HF diaphragm muscle. Patients with heart failureand inspiratory muscle weakness have an improved cardio-respiratory response to exercise following inspiratory muscletraining (Winkelmann et al., 2009; Lin et al., 2012). Thus,much like the specific training of the respiratory muscles, it ispossible that increased diaphragmmuscle Ca21 sensitivity byCK-2127107 may improve respiratory function during exer-cise and might thereby improve overall exercise tolerance.The present study demonstrates that a single dose of CK-

2127107 can substantially improve exercise performance ina model of mild to moderate heart failure. Increasing thecalcium sensitivity of fast skeletal muscle with the attendantincreases in responsiveness to neuromuscular input andfatigue resistance appear to underlie this improvement inexercise performance affected by CK-2127107. These findingssuggest that skeletal muscle dysfunction rather than cardiacmuscle dysfunction may play a greater role in exerciseintolerance early in the course of heart failure. Overall, thesefindings have important implications for understanding howmuscle fatigue and weakness contribute to reduced exerciseperformance in heart failure. They further suggest a potentialtherapeutic role for small molecule fast skeletal troponinactivators as a means to improve skeletal muscle function andexercise performance in heart failure.

Authorship Contributions

Participated in research design: Hwee, Kennedy, Hartman, Ryans,Durham, Malik, Jasper.

Conducted experiments: Hwee, Kennedy, Hartman, Ryans,Durham.

Performed data analysis: Hwee, Kennedy, Hartman, Ryans,Durham, Malik, Jasper.

Wrote or contributed to the writing of the manuscript: Hwee,Kennedy, Hartman, Durham, Malik, Jasper.

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