1521-0103/353/3/480–489$25.00 http://dx.doi.org/10.1124/jpet.114.221861THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:480–489, June 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Ranolazine Reduces Remodeling of the Right Ventricle andProvoked Arrhythmias in Rats with Pulmonary Hypertension s

John T. Liles, Kirsten Hoyer, Jason Oliver, Liguo Chi, Arvinder K. Dhalla,and Luiz BelardinelliDepartment of Biology, Gilead Sciences, Inc., Fremont, California

Received December 8, 2014; accepted March 12, 2015

ABSTRACTPulmonary arterial hypertension (PAH) is a progressive diseasethat often results in right ventricular (RV) failure and death. Duringdisease progression, structural and electrical remodeling of theright ventricle impairs pump function, creates proarrhythmicsubstrates, and triggers for arrhythmias. Notably, RV failure andlethal arrhythmias are major contributors to cardiac death inpatients with PAH that are not directly addressed by currentlyavailable therapies. Ranolazine (RAN) is an antianginal, anti-ischemic drug that has cardioprotective effects in experimentaland clinical settings of left-sided heart dysfunction. RAN also hasantiarrhythmic effects due to inhibition of the late sodium currentin cardiomyocytes. We therefore hypothesized that RAN couldreduce themaladaptive structural and electrical remodeling of theright ventricle and could prevent triggered ventricular arrhythmias

in themonocrotaline rat model of PAH. Indeed, in both in vivo andex vivo experimental settings, chronic RAN treatment reducedelectrical heterogeneity (right ventricular-left ventricular actionpotential duration dispersion), shortened heart-rate corrected QTintervals in the right ventricle, and normalized RV dysfunction.Chronic RAN treatment also dose-dependently reduced ventricularhypertrophy, reduced circulating levels of B-type natriuretic peptide,and decreased the expression of fibrotic markers. In addition, theacute administration of RAN prevented isoproterenol-inducedventricular tachycardia/ventricular fibrillation and subsequentcardiovascular death in rats with established PAH. These resultssupport the notion that RAN can improve the electrical andfunctional properties of the right ventricle, highlighting its potentialbenefits in the setting of RV impairment.

IntroductionPulmonary arterial hypertension (PAH) is a progressive and

fatal disease characterized by vascular remodeling and vaso-constriction of the pulmonary arterial circulation that oftenresults in right ventricular (RV) failure and death. The pro-gressive vasculopathy leads to intraluminal narrowing andobstruction of the resistance vasculature, leading to sustainedincreases in pulmonary vascular resistance and pulmonaryarterial pressure. In an attempt to compensate for the increasedafterload and wall stress, the right ventricle undergoes struc-tural and electrical remodeling (Malenfant et al., 2013; Simon,2013), creating proarrhythmic substrates and triggers for arrhyth-mias (Benoist et al., 2011, 2012; Rocchetti et al., 2014). Althoughthe extent of the vascular pathology in PAH can determinemorbidity and mortality, RV dysfunction is now recognized asthe key determinant of disease outcome (Voelkel et al., 2012;Archer et al., 2013; Rain et al., 2013, 2014). The central role ofRV pathology in disease progression is underscored by evidence

showing that RV failure is the primary cause of death inpatients with PAH, and by reports that lethal arrhythmias area major contributor to sudden cardiac death (SCD) in thesepatients (Rajdev et al., 2012; Voelkel et al., 2012; Olsson et al.,2013; Rain et al., 2013, 2014; Rich et al., 2013).Current PAH therapies, consisting mainly of prostanoids,

phosphopdiesterase type-5 inhibitors, and endothelin receptorantagonists, reduce pulmonary vascular resistance throughpreferential vasodilation of the pulmonary circulation (Abrahamet al., 2010; van de Veerdonk et al., 2011). This strategy oftargeting the vasoconstrictive component of PAH improvessymptoms and slows disease progression, but mortality ratesremain high and functional impairment of RV functionremains a significant problem (van de Veerdonk et al., 2011;Archer et al., 2013; Guihaire et al., 2013;Malenfant et al., 2013;Rain et al., 2013). There is no current PAH therapy thatdirectly targets the structural and electrical consequences ofRV remodeling (Voelkel et al., 2012; Archer et al., 2013; Olssonet al., 2013).Ranolazine (RAN) is aFood andDrugAdministration–approved

antianginal drug with anti-ischemic activity that has also beenshown to have antiarrhythmic properties due to inhibition ofthe late sodium current (INa) in cardiomyocytes (Hale et al.,2008; Antzelevitch et al., 2014). As a result of extensive clinical

This research was supported by Gilead Sciences, Inc. All of the authors areemployees of Gilead Sciences.

J.T.L. and K.H. contributed equally to this work and are co-first authors.dx.doi.org/10.1124/jpet.114.221861.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: APD, action potential duration; BNP, B-type natriuretic peptide; CTGF, connective tissue growth factor; ECG, electrocardiogram;ERP, effective refractory period; INa, sodium current; ISO, isoproterenol; LV, left ventricular; MAP, monophasic action potential; MAPD, monophasicaction potential duration; MCT, monocrotaline; PAH, pulmonary arterial hypertension; QTc, heart-rate corrected QT; RAN, ranolazine; RV, rightventricular; SCD, sudden cardiac death; TGF-b, transforming growth factor-b; VF, ventricular fibrillation; VT, ventricular tachycardia.

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and experimental studies, the cardioprotective effects of RANin settings of left-sided heart dysfunction are well established(Rastogi et al., 2008; Aistrup et al., 2013; Maier et al., 2013).Although the left and right ventricles have notable and welldefined differences (i.e., structural, functional, electrical, andembryological), common mechanisms such as ischemia, Ca21

overload, oxidative stress, and fibrosis are implicated in thepathology of both left- and right-sided heart failure (Bogaardet al., 2009; Borgdorff et al., 2013; Simon, 2013; Toischer et al.,2013). Pathologic processes that are known to increase late INa

(Voelkel et al., 2012; Freund-Michel et al., 2013; Shryock et al.,2013; Toischer et al., 2013) are also implicated in the pro-gression of RV failure in PAH (Voelkel et al., 2012; Freund-Michel et al., 2013; Shryock et al., 2013; Toischer et al., 2013). Itwas recently reported that RAN reduced RV mass, improvedRV performance, and increased exercise capacity in 10 patientswithWorld Health Organization group 1 PAH and symptoms ofangina (Shah et al., 2012). It has also been reported that RANcan reduce RVhypertrophy in a rat model in which RV pressureoverload is induced independently from changes in pulmonaryarterial pressures by pulmonary artery banding (Fang et al.,2012). In addition, a recent study in a 3-week monocrotaline(MCT) rat model showed that RAN, when initiated 2 days afterMCT injection, prevented RV structural and electrical remodel-ing by suppressing late INa (Rocchetti et al., 2014). MCT isknown to consistently induce pulmonary vascular remodeling inthe rat within the first 4–7 days after injection, with peakincreases in pulmonary pressure occurring at 4 to 5 weeks(Bruner et al., 1983; Rosenberg and Rabinovitch, 1988; Hoornand Roth, 1992; Lappin and Roth, 1997; Guihaire et al., 2013).In our study, RAN treatment was started at day 7 after

MCT injection to allow for the vasculopathy to develop beforeintervention, and all end points were assessed 4 weeks afterMCT administration. We used a combination of in vivo and exvivo approaches to determine whether treatment with RANcould halt the development of the PAH phenotype in a modelthat reliably reproduces RV impairment and major pathologicfeatures of the human disease, and to extend recent findingsby determining whether RAN treatment prevents the in-duction of arrhythmias/SCD in rats with established PAH.

Materials and MethodsAnimals. Male Sprague-Dawley rats (250–300 g; Charles River,

Hollister, CA) were used in this study. Experiments were performedunder protocols approved by the Institutional Animal Care and UseCommittee of Gilead Sciences (Association for Assessment and Accred-itation of Laboratory Care International accreditation no. 001135).Animal use conformed to the National Institutes of Health guidelines(publication no. 85-23, revised 1996). Animals received a single sub-cutaneous injection ofMCT (60mg/kg bodyweight) to induce PAHwithin28 days, and control rats received an equal volume of solvent (vehicle,1ml/kg bodyweight). All animals had free access to standard rodent chow(Purina 5001; Nestle Purina Pet Care Co., Fairburn, GA) and water forthe first week after MCT injection; thereafter, subsets of rats wereswitched to a diet containing either 0.25%RANor 0.5%RANbyweight todetermine the effect of chronic RAN administration during PAH de-velopment (Research Diets, New Brunswick, NJ). Plasma levels of RANachieved in this study using chronic oral administration (0.25%, 1 to2mM; 0.5%, 4–7mM) arewithin the therapeutic range (6–8mM). At theseconcentrations, RAN selectively inhibits late INa versus other targets(Hale et al., 2008; Zhao et al., 2011).

For the in vivo studies, rats were divided into four groups thatreceived the following: 1) vehicle injection and normal diet (sham), 2)

MCT injection and normal diet (PAH), 3) MCT injection and diet con-taining 0.25%RAN, and 4)MCT injection and diet containing 0.5%RAN.

Rats used in the ex vivo studies (isolated heart) were separated intofour treatment groups: 1) vehicle injection and normal diet (sham), 2)vehicle injection and diet containing 0.5% RAN (sham plus 0.5%RAN), 3) MCT injection and normal diet (PAH), and 4) MCT injectionand diet containing 0.5% RAN (PAH plus 0.5% RAN). For all studies,Purina 5001 (Nestle Purina Pet Care Co.) was used as the standard

Fig. 1. Effect of chronic administration of RAN to rats with PAH. At4 weeks after MCT injection, the rats developed significant RV hypertrophy(A), increased RV developed pressure (B), and increased plasma BNP levels(C) compared with sham animals. Chronic administration of RAN starting1 week after the injection of MCT dose-dependently decreased RV hyper-trophy, RV developed pressure, and plasma BNP levels. Data are presentedasmeans6S.E.M. (one-way analysis of variance followed byNewman–Keulspost hoc analysis with all pairwise comparisons; n = 6–10). *P , 0.05 versussham; †P , 0.05 versus MCT; ‡P , 0.05 versus 0.25% RAN.

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rodent chow, and the only difference between diets was the concentra-tion of RAN (0% for sham and 0.25 or 0.5% by weight for respective PAHexperiments).

In Vivo Measurements. At 4 weeks after MCT administration,rats were anesthetized using isoflurane (Sigma-Aldrich, St. Louis,MO), and they were then intubated and ventilated. Body temperaturewas measured using a rectal thermometer and was maintained at37°C using a heating pad. After a stabilization period, pulmonaryhemodynamics were assessed using a Millar catheter (SPR-839; MillarInstruments, Houston, TX) inserted into the right ventricle and ad-vanced into the pulmonary artery. Thereafter, the same SPR-839catheter was slowly pulled back into the right ventricle to also acquireRV hemodynamics. A separate Millar catheter (SPR-869) was insertedinto the right common carotid artery for measurement of systemic ar-terial pressure. After allowing for stabilization from surgical prepara-tion, data were continuously recorded on a personal computer usinga pressure-volume unit (model MPVS-300; ADInstruments, ColoradoSprings, CO). In addition, an electrocardiogram (ECG) was recordedusing subdermal needle electrodes (25 gauge; ADInstruments) in thelead II configuration.

B-type natriuretic peptide (BNP) (AbCam Inc., Cambridge, MA)protein analysis in plasma was performed according to the manu-facturer’s instructions. In brief, a 1:4-fold dilution and a 1:10-folddilution of plasma for BNP and tissue inhibitor of metalloproteinase-1, respectively, were incubated for 2 hours at room temperature usingthe manufacturer’s assay reagents. Both BNP and tissue inhibitor ofmetalloproteinase-1 protein concentrations were calculated fromtheir assay standard curve optical density values.

Tissue Collection and Morphologic Measurements. At theconclusion of hemodynamic procedures, rats were euthanized sothat blood and organs could be collected for further evaluation. Theheart was dissected into atria, right ventricle, and left ventricle(including the septum), plotted dry with gauze, and weighed. RVhypertrophy was determined by the ratio of RV weight divided bytibia length (RV/TL). This measurement has the advantage of beingunaffected by body weight and left ventricular (LV) mass changes(Yin et al., 1982).

Measurements of Fibrosis and InflammationMarkers. Paraffin-embedded cross sections (5 mm) of the right ventricle were pre-pared from paraformaldehyde-fixed tissues and were stained withMasson’s trichrome for tissue collagen and then imaged. The mRNAgene expression of collagen 1a1, connective tissue growth factor(CTGF), and transforming growth factor-b (TGF-b) in RV tissue wasdetermined using Luminex xMap technology according to the manu-facturer’s instructions (Luminex Inc., Austin, TX). Briefly, approx-imately 50mg tissue was lysed in homogenizing buffer (Affymetrix Inc.,Santa Clara, CA) and then incubated with an mRNA detection probepanel (Affymetrix Inc.) at 54°C overnight. On the following day, tissuelysates and mRNA detection probe cocktail were hybridized withamplifier reagents in accordance with the manufacturer’s instructions.Data were collected using laser-detected Luminex SD equipment andwere analyzed by Masterplex CT software (Luminex Inc.).

Langendorff Perfusion. At 28 days after MCT injection, ratswere anesthetized (60 mg/kg ketamine/xylazine i.p.), and hearts wereisolated, mounted on a Langendorff perfusion system, and perfused atconstant pressure (70mmHg) with Krebs-Henseleit buffer containing

the following: 118mMNaCl, 25mMNaHCO3, 11mMglucose, 4.2 mMKCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.5 mM EDTA, and 1.5 mMCaCl2. The buffer was continuously gassed with 95% O2 and 5% CO2

(pH 7.4) at 37°C. The effluent of the Thebesian veins was drained viapolyethylene tubing (PE-50) placed at the LVapex. Isovolumic contractileperformance was measured using a water-filled balloon insertedinto the left ventricle (for more information, see the SupplementalData and Supplemental Table 1). Hearts were paced at 5 Hz using abipolar electrode placed on the epicardial wall of the right ventricleand connected to a stimulator (S88 stimulator; Grass Technologies,Warwick, RI).

Measurements of Electrophysiological Parameters in theIsolated Heart. Pseudo ECGs were obtained with Ag-AgCl monopolarECG electrodes (Harvard Apparatus, Holliston, MA) and theheart-rate corrected QT (QTc) intervals were calculated according tothe Bazett formula.Monophasic action potentials (MAPs) were recordedat the epicardial surfaces of the left and right ventricles using pressure-contact mini MAP electrodes (Harvard Apparatus). Monophasic actionpotential duration (MAPD) was measured at 30, 50, and 90% repo-larization. The interventricular dispersion of repolarization was definedas RV – LV MAPD90. The ventricular effective refractory period (ERP)was determined as the shortest S1 to S2 interval generating anMAP bystimulating hearts using an S1 to S2 protocol: S1 pulses were con-tinuously applied at an interval of 200milliseconds, and an S2 stimuluswas introduced at various coupling intervals that were reduced from100 to 50 milliseconds at 10-millisecond decrements and from 48 to 30milliseconds at 2-millisecond decrements.

To trigger ventricular arrhythmia, hearts paced at 6 Hz underwentburst pacing (50 Hz for 1 second), at which time the electrical pacingwas stopped to allow the heart to resume its intrinsic rate. Stimulusintensity during burst pacing was increased until tachycardia orfibrillation was induced (Benoist et al., 2011).

Induction of Arrhythmias in PAH Rats and Isolated Heartsduring Acute RAN Treatment. In in vivo experiments, cardiacarrhythmia was induced with isoproterenol (ISO, 0.1 mg/kg i.v.; ISOchallenge) in a group of rats 49–60 days after MCT or vehicle injection.To determine the acute antiarrhythmic effect of RAN, a subgroup ratswith PAH received RAN (8 mg/kg bolus, followed by 10 mg/kg per hourinfusion i.v.) 10 minutes before the ISO challenge.

In isolated hearts from rats with established PAH, arrhythmia wasprovoked with the above-mentioned burst pacing protocol (50 Hz,1 second, increasing stimulus intensity). Ten rat hearts were treatedwith 10mMRAN10minutes before and during the burst pacing protocol.

Data were acquired on a PowerLab 8/30 device (ADInstruments)attached to a computer andwere analyzedwith LabChart Pro7 software(ADInstruments).

Chemicals. RAN was provided by Gilead Sciences Inc. (FosterCity, CA). All other reagents, including MCT, were obtained fromSigma-Aldrich.

Statistical Analyses. All data are expressed as means 6 S.E.M.Statistical analyses were performed using one-way analysis of vari-ance, followed by either the Newman–Keuls post hoc test or Tukey posthoc test for multiple comparisons. The Fisher’s exact test was used foranalyzing the differences in the incidences of arrhythmias (Prism 6.00;GraphPad Software, La Jolla, CA). A P value of ,0.05 was consideredstatistically significant.

TABLE 1Effect of chronic treatment with RAN on systemic blood pressure and heart rate in animals with MCT-induced PAHMeasurements were performed at 28 days after injection of MCT (60 mg/kg body weight). One week after MCT injection,two groups of rats were switched to a diet containing either 0.25 or 0.5% RAN by weight.

Sham PAH PAH Plus0.25% RAN

PAH Plus0.5% RAN

Mean arterial pressure (mm Hg) 95 6 6.9 89 6 7.6* 100 6 5.2 99 6 1.7Heart rate (beats/min) 291 6 17 292 6 12 313 6 13 300 6 21

*P , 0.05 versus sham.

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ResultsChronic Administration of RANReducesMCT-Induced

RV Hypertrophy and Normalizes RV Performance. Theeffects of 0.25 and 0.5% RAN in diet on RV hypertrophy, RVdeveloped pressure (in millimeters of mercury), and plasmalevels of BNP (in picograms per milliliter) in MCT rats aresummarized in Fig. 1. At 4 weeks, as expected, MCT admin-istration caused RV hypertrophy (9.8 6 0.8 versus 6.2 60.1 mg/mm, RV/TL), increased RV developed pressure (68 6 7versus 24 6 0.3 mm Hg), and increased plasma BNP levels3-fold (527 6 113 versus 173 6 89 pg/ml) compared with shamcontrols (Fig. 1). Chronic treatment with RAN (0.25 and 0.5%),starting at 1 week after the administration of MCT, dose-dependently reduced RV hypertrophy (7.8 6 0.7 and 5.7 6 0.3mg/mm), RV developed pressure (51 6 6 and 34 6 2 mm Hg),and BNP plasma levels (266 6 114 and 57 6 17 pg/ml). RANhad no effect on systemic blood pressure or heart rate (Table 1).Chronic Administration of RANReducesMCT-Induced

PAH. Pulmonary hemodynamic measurements were acquireddirectly from the proximal pulmonary artery using a solid-state catheter (SPR-839) at 4 weeks after MCT injection.MCT administration induced significant increases in systolicpulmonary arterial pressure (68 6 6 versus 23 6 1 mm Hg),mean pulmonary arterial pressure (40 6 3 versus 17 61 mmHg), and diastolic pulmonary arterial pressure (2261 versus10 6 1 mm Hg) compared with sham controls (Fig. 2). Chronictreatment with RAN (0.25 and 0.5%), starting at 1 week afterthe injection of MCT, dose-dependently reduced systolicpulmonary arterial pressure (53 6 5 and 34 6 1 mm Hg),mean pulmonary arterial pressure (326 2 and 246 1 mmHg),and diastolic pulmonary arterial pressure (18 6 1 and 15 6 1mm Hg; Fig. 2).Chronic Administration of RAN Reduces RV Collagen

Deposition and Fibrotic Gene Expression. To evaluatethe effect of RAN treatment on the development of cardiac fibrosisand remodeling, RV samples were collected 4 weeks after theinjection ofMCT for histology and gene expression patterns in theright ventricle (Fig. 3). RV sections stained with Masson’strichrome showed more pronounced collagen deposition fromanimals with PAH compared with sham controls and withanimals treated with 0.5% RAN (Fig. 3, A–C). Right ventriclesfrom rats with MCT-induced PAH had increased expression ofthemRNA transcripts for collagen 1a1, CTGF, andTGF-b (Fig. 3,D–F, respectively). RAN dose-dependently reduced the expres-sion of these fibrotic genes in the right ventricle (Fig. 3, D–F).Chronic Administration of RAN Reduces Electrical

Remodeling in Hearts Isolated from Rats with PAH.Hearts were isolated 4 weeks after the injection of MCT fromrats treated with vehicle (standard rodent chow) or 0.5%RAN,or from sham animals with and without RAN treatment. Theeffect of chronic treatment with RAN on the RV MAPD, theventricular heterogeneity in repolarization (RV-LV MAPD90),and electrophysiological parameters was determined, andthese data are summarized in Figs. 4 and 5. Sham animals onstandard chow had RV MAPDs of 17 6 2.2 milliseconds, 24 62.8 milliseconds, and 50 6 3.8 milliseconds measured at 30,50, and 90% repolarization, respectively. RV MAPD30 andMAPD50 increased 2-fold in hearts from animals with PAHcompared with sham controls (37 6 1.7 versus 17 6 2.2 milli-seconds and 476 3.5 versus 246 2.8milliseconds, respectively)and MAPD90 increased 1.6-fold (83 6 6.0 versus 50 6 3.8

milliseconds; Fig. 4B). The prolongation of theMAPDs observedat 4 weeks after the injection of MCT was significantly less inhearts from rats with PAH treated with 0.5%RAN (236 1.7, 316 2.1, and 62 6 3.5 milliseconds for MAPD30, MAPD50, andMAPD90, respectively).In addition, the interventricular dispersion in MAPD90

was markedly increased in hearts from rats with PAH com-pared with the sham animals (17 6 5.5 versus 26.1 6 2.0milliseconds), whereas RAN treatment normalized dispersion

Fig. 2. Effect of chronic administration of RAN to rats with MCT-induced PAH. Systolic (A), mean (B), and diastolic (C) pulmonary arterialpressures were measured at 4 weeks after MCT injection. PAH causedsignificant increases in systolic, mean, and diastolic pulmonary arterialpressures compared with control (sham) animals. One week after MCTinjection, two groups of rats were switched to a diet with either 0.25 or0.5% RAN by weight, which dose-dependently decreased pulmonaryarterial pressures. Data are presented as means 6 S.E.M. (one-wayanalysis of variance followed by Newman–Keuls post hoc analysis withall pairwise comparisons; n = 6–10). *P , 0.05 versus sham; †P , 0.05versus MCT; ‡P , 0.05 versus 0.25% RAN. PAP, pulmonary arterialpressure.

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values (22 6 2.4 milliseconds; Fig. 4C). Consistent with theMAP data in isolated hearts, MCT-induced prolongation ofthe QTc interval (236 6 9.7 versus 155 6 7.1 milliseconds)was significantly decreased in animals with PAH chronicallytreated with 0.5% RAN (198 6 7.8 milliseconds; Fig. 5A).ERP, which was increased in hearts from rats with PAHcompared with sham controls (78 6 3.5 versus 51 6 6.8milliseconds), was also normalized in hearts from rats withPAH treated with 0.5% RAN (546 6.6 milliseconds; Fig. 5B).Treatment with 0.5% RAN in sham animals had no (signifi-cant) effect on any of these electrophysiological parametersmeasured.Effect of Chronic Administration of RAN on Arrhyth-

mogenesis in Hearts Isolated from Rats with PAH. Theeffect of RAN on the proarrhythmic potential of PAH wasdetermined using isolated hearts from rats treated with vehicleor 0.5% RAN at 4 weeks after the injection of MCT. Burstpacing for 1 second at 50 Hz induced ventricular tachycardia(VT)/ventricular fibrillation (VF) in 50% of hearts isolated fromrats with PAH (three of six hearts). By contrast, VT/VFwas notinducible in hearts isolated from RAN-treated rats (P 5 0.2)and was only induced in one of six hearts isolated from shamanimals (Fig. 6A). Representative tracings of MAP, ECG,and LV pressure during arrhythmia induction are shown inFig. 6, B and C. Tracings from hearts isolated from sham andRAN-treated animals with PAH show that the hearts re-turned to normal sinus rhythm and function almost im-mediately after burst pacing, and a triggered VF episode isevident in tracings from a heart of a rat with PAH withoutRAN (Fig. 6, B and C).

Acute Effect of RAN on Arrhythmogenesis in PAHRats and Isolated Hearts. Both in vivo and ex vivoexperiments were conducted to determine the effect of acuteadministration of RAN on arrhythmia induction in the settingof established RV remodeling (Table 2), and these data aresummarized in Fig. 7. Of note, hemodynamics were continuallymeasured to ensure that no changes occurred with the doses ofRAN used in these studies. In anesthetized rats with PAH,arrhythmias were induced by a bolus injection of ISO (0.1mg/kgi.v.; Fig. 7A). Administration of ISO to animals with establishedPAH caused VT/VF and subsequent death in 70% of animals(7 of 10), whereas ISO induced arrhythmias in only 17% of shamanimals (one of six). Furthermore, RAN (8mg/kg bolus, 10mg/kgper hour infusion i.v.) given 10 minutes before the ISOchallenge completely prevented ISO-induced arrhythmias anddeath (zero of five; P50.026 versus without RAN treatment;Fig. 7A). Fig. 7B shows representative tracings of pulmonaryarterial pressure, blood pressure, and an ECG in a rat withPAH 6 minutes after an ISO injection that triggered VF.Similar results were obtained in hearts isolated from rats at

day 28 after the administration of MCT (PAH) when VT/VFwas triggered using burst pacing for 1 second at 50 Hz. A totalof three of six hearts from the PAH group exhibited VT/VF.The electrical stimulation protocol provoked arrhythmias intwo hearts, whereas one heart exhibited spontaneous VT/VFbefore the electrical stimulation protocol. Acute administra-tion of RAN (10 mM) 10 minutes before the burst stimuluscompletely prevented the induction of VT/VF (0 of 10) inhearts from rats with PAH (P 5 0.09 versus without RANtreatment; Fig. 7C). In the sham group, the same stimulation

Fig. 3. Representative images of the right ventricle stained with Masson’s Trichrome from sham (A), MCT plus vehicle (B), andMCT plus 0.5% RAN (C)at 4 weeks after the injection of MCT; fibrosis is colored blue. RAN dose-dependently reduced the PAH-induced increases in mRNA expression ofcollagen1a1 (D), CTGF (E), and TGF-b (F) in the right ventricle. Data are presented as means 6 S.E.M. (one-way analysis of variance followed bya Bonferroni multiple comparisons test; n = 5–9). *P, 0.05 versus sham; †P, 0.05 versus MCT; ‡P, 0.05 versus 0.25% RAN. GAPDH, glyceraldehyde3-phosphate dehydrogenase; RFU, relative fluorescence unit. Original magnification, 100�.

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protocol induced arrhythmias in 2 of 11 hearts (18%).Representative tracings of MAP, LV pressure, and an ECGfrom one RAN-treated PAH heart are shown in Fig. 7D.Taking all different experiments together, VT/VF could not

be induced in PAH rats treated chronically or acutely withRAN, whereas 50–70% of the PAH rats developed provokedVT/VF in the absence of RAN.

DiscussionThis study shows that chronic treatment with RAN reduces

maladaptive structural and electrical remodeling of the rightventricle and prevents triggered ventricular arrhythmias inrats with MCT-induced PAH. Chronic administration of RAN

initiated 1 week after the injection of MCT led to decreasedRV hypertrophy and improved pulmonary hemodynamics ina dose-dependent manner. Circulating levels of BNP, a clini-cally validated biomarker of RV failure, were also significantlydecreased in rats treated with RAN, indicating improved RVfunction (McLaughlin et al., 2013). Hearts isolated from ratswith PAH showed a 50% induction in electrical stimulation–induced VT/VF, whereas arrhythmia was not inducible inhearts isolated from PAH rats treated with RAN (acute andchronic). Furthermore, the prolonged action potential duration(APD), ERP, and QTc intervals measured in the right ventricleof animals with PAH were significantly reduced after chronicRAN treatment. In vivo studies using rats with alreadyestablished PAH and RV remodeling demonstrated that theacute administration of RAN significantly reduced ISO-inducedVT/VF and associated cardiovascular death. The results fromthese in vivo and ex vivo (isolated heart) experiments supportthat RAN reduces the arrhythmogenic substrate formation inthe right ventricle presumably by decreasing electrical hetero-geneity (reduced RV-LV APD dispersion) and by decreasingcardiac fibrosis (structural remodeling), which results in anoverall improvement in the electrical properties of the rightventricle.

Fig. 4. Effect of chronic administration of RAN on the MAP and re-polarization abnormalities in the right ventricle of isolated hearts fromrats with PAH. (A) Superimposed MAP of hearts from sham (blue), PAH(black), and PAH plus 0.5% RAN (red) animals showing differences inMAP duration. (B) RV MAP duration at 30, 50, and 90% repolarizationwas significantly prolonged in hearts from rats with PAH and was reducedwith 0.5% RAN (0.5% RAN by weight in chow) treatment (n = 5 to 6). (C)Electrical heterogeneity defined as interventricular dispersion (RV-LVAPD90) was significantly increased in hearts from rats with PAH andnormalized with chronic 0.5% RAN treatment compared with isolatedhearts from normal (sham) rats (n = 5 to 6). Data are presented as means6 S.E.M. (one-way analysis of variance followed by Tukey post hocanalysis with all pairwise comparisons). *P, 0.05 versus sham; †P, 0.05versus PAH only.

Fig. 5. Effect of chronic administration of RAN on electrophysiologicalparameters. In isolated hearts from rats with PAH, surface ECGs revealedprolonged QTc interval calculated according to the Bazett formula (n = 6 inall groups; A) and increased ERP (n = 4–6; B) compared with normalhearts. Chronic 0.5% RAN treatment, which began at 1 week after theinjection of MCT, showed decreased QTc interval and normalized ERPvalues at 4 weeks after MCT injection. Data are presented as means 6S.E.M. (one-way analysis of variance followed by Tukey post hoc analysiswith all pairwise comparisons). *P , 0.05 versus sham; **P , 0.02 versussham; †P , 0.05 versus PAH only.

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The Effect of RAN on RV Structural and FunctionalRemodeling in Rats with PAH. The present understand-ing of maladaptive ventricular remodeling was established inexperimental and human studies of left-sided heart failure;however, many of the same mechanisms have now beendescribed in right-sided heart failure (Borgdorff et al., 2013;Simon, 2013). Importantly however, there are prominentembryological, structural, and metabolic differences thatpreclude the extrapolation of data between the two ventricles(Bogaard et al., 2009; Simon, 2013).This study was designed to investigate the effect of chronic

treatment with RAN in an animal model of PAH that led to

RV remodeling, electrical abnormalities, and failure. A sig-nificant increase in pulmonary arterial pressure after MCTinjection was accompanied by RV hypertrophy, increased RVfibrosis, and impaired RV performance compared with shamanimals (Figs. 1–3). In the right ventricle of ratswith PAH,RANdose-dependently slowed the development of ventricular hyper-trophy and normalized the expression of collagen, CTGF, andTGF-b (Figs. 1 and 3). In addition, in hearts isolated from ratswith PAH, cardiac performance of the right ventricle was nor-malized by RAN treatment (Supplemental Table 1). The resultsof this study evaluating the effects of chronic treatment withRAN are in good agreement with the results from a smallclinical study evaluatingRAN in patientswith PAH (Shah et al.,2012) and with the recent report that RAN prevents MCT-induced pulmonary hypertension, RV hypertrophy, and RVfibrosis (Rocchetti et al., 2014). Our results are also con-sistent with the study conducted by Fang et al. (2012), whichdemonstrate that RAN treatment improves RV performanceas measured by exercise capacity and cardiac index in ratssubjected to RV pressure overload by pulmonary arterybanding.BNP, a well characterized biomarker that is predictive of

RV performance, mortality, and clinical outcomes in patientswith PAH (Sztrymf et al., 2010; McLaughlin et al., 2013;Vonk-Noordegraaf et al., 2013), was significantly increased inrats with PAH and was decreased by 0.5% RAN (Fig. 1C).BNP is produced by and released from ventricular cardio-myocytes (Bruneau et al., 1997), and plasma levels of BNP areclosely linked to the severity of ventricular remodeling anddysfunction (Sztrymf et al., 2010; McLaughlin et al., 2013).The effect of RAN in reducing BNP in our study is consistentwith the benefits of RAN in animal models of left-sided heartdisease as well as with the amelioration of RV failure in thisanimal model (Aistrup et al., 2013; Guihaire et al., 2013;McLaughlin et al., 2013).The Effect of RAN on RV Electrical Remodeling in

Rats with PAH. Cardiac arrhythmias are major contributorstomorbidity andmortality in patients withPAH, can contributeto deteriorations of cardiac function, and are not adequatelyaddressed by current therapies (Rajdev et al., 2012; Rich et al.,2013). Importantly, a recent prospective clinical study reportedthat the restoration of sinus rhythm using antiarrhythmictherapy was associated with a reduction in circulating BNPlevels and improved survival (Olsson et al., 2013).In the present study, the hearts from rats that received

MCT had electrophysiological changes in the right ventriclethat were proarrhythmic (e.g., prolongation of QTc intervaland heterogeneity of APD) and that had been reported inhumans with PAH (Rajdev et al., 2012; Rich et al., 2013;Tanaka et al., 2013). As shown in Figs. 4 and 5, chronictreatment with RAN reduced the PAH-associated electro-physiological abnormalities. In addition, ventricular hetero-geneity in repolarization (RV-LV APD90), a major contributorto the electrophysiological substrate (i.e., re-entry) leading tothe occurrence of lethal arrhythmias, was also decreased byRAN.It was recently demonstrated that the preventative admin-

istration of RAN blocks late INa enhancement in RV myocytesand partially prevents the initiation of MCT-induced RVremodeling (Rocchetti et al., 2014). In that study, RANadministration was associated with improvements in Ca21

handling, decreases in t-tubule disarray, and reduced incidence

Fig. 6. Effect of chronic administration of RAN on electrical stimula-tion–induced arrhythmias in isolated hearts from rats with PAH (4 weeksafter the injection of MCT). (A) Summarized data of incidences of VT/VFtriggered by burst pacing in hearts isolated from normal, PAH, or PAHplus 0.5% RAN (P = 0.2, Fisher’s exact test). (B and C) Representativetracings of RV MAP, ECG, LVP, and of electrical stimulation depictedbefore, during, and after burst pacing (1 second, 50 Hz) experiments inisolated hearts from rats with PAH (B) and rats with PAH treated with0.5% RAN in diet by weight (C). LVP, left ventricular pressure.

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of delayed afterdepolarizations in RV myocytes (Rocchettiet al., 2014). Our data extend these findings by demonstratingthat treatment with RAN decreases the proarrhythmicsubstrate of the remodeled right ventricle and results indecreased incidences of provoked arrhythmias and protec-tion from ISO-induced VT/VF. Our results from studiesusing chronic and acute RAN treatment in a model of RVdysfunction are also consistent with the well described

antiarrhythmic effects of RAN (Aistrup et al., 2013; Shryocket al., 2013) and support the therapeutic strategy of normalizingthe QTc interval and APD to reduce the arrhythmogenesisof diseased hearts.Although reports of documented VT/VF are relatively rare

in patients with PAH (approximately 8%) compared with thereported incidence of bradycardia (45%), electromechanicaldissociation (28%), andasystole (15%),VT/VFremainsa clinically

TABLE 2Summary of pulmonary hemodynamics, RV hypertrophy, and heart rate data from animals subjected toISO challengeAll rats with PAH had a similar PAH phenotype before the administration of ISO (0.1 mg/kg i.v.). Of note, ISO inducedsimilar increases in heart rate across groups.

Sham PAH

Baseline systolic pulmonary pressure (mm Hg) 25 6 4 41 6 9*RV hypertrophy (mg/mm) 6.7 6 0.7 10.5 6 2*Baseline heart rate (beats/min) 315 6 55 280 6 30*Increase in heart rate after ISO challenge (beats/min) 108 6 28 96 6 30

*P , 0.05 versus sham.

Fig. 7. Acute administration of RAN in vivo prevents triggered VT/VF in hearts from rats with PAH. Summary data of VT/VF incidences andsubsequent SCD in rats with PAH in the presence or absence of RAN (8 mg/kg bolus, followed by 10 mg/kg per hour infusion, i.v.) are shown (*P , 0.03versus PAH only, Fisher’s exact test). (A) The administration of ISO (0.1 mg/kg, i.v.) to rats with established PAH caused VT/VF. (B) Depicted arerepresentative tracings of pulmonary arterial pressure (PAP), systemic blood pressure (BP), and ECG at 7minutes after an ISO bolus injection (0.1mg/kg,i.v.) inducing VF in a rat with PAH (in vivo). Summary data of incidences of VT/VF in the absence or presence of 10 mM RAN are shown for ex vivoexperiments. VT/VF was electrically triggered by burst pacing (1 second, 50 Hz). (C) In the PAH group, one heart exhibited spontaneous VT/VF, shown asgray in the black bar. (D) Representative tracings of RV MAP, ECG, LVP, and electrical stimulation before, during, and after burst pacing in isolatedhearts from rats with PAH perfused with 10 mM RAN are depicted (ex vivo). LVP, left ventricular pressure.

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relevant risk for many patients with PAH (Hoeper et al., 2002;Vonk-Noordegraaf et al., 2013). In this study, a higher suscep-tibility to provoked VT/VF was observed in vivo (induced in 70%)and in isolated hearts from rats with PAH (induced in 50%)compared with normal hearts (induced in approximately 18%;Fig. 6). VT/VF inducibility was not observed with chronic RANtreatment and was prevented by acute administration of RAN toanimals with established RV remodeling (Fig. 7, A and B). Ofnote, a single incident of SCD due to an episode of spontaneousVT/VF was captured during telemetric recording in a consciousrat severalweeks after the injection ofMCT (Supplemental Fig. 1).These data are in good agreement with those of Benoist et al.(2011), who demonstrated that MCT administration resultedin a higher incidence of provoked arrhythmias and sustainedVT/VF compared with normal rat hearts.Limitations. Late INa was not directly measured in this

study; however, there is extensive clinical and experimentalevidence to support late INa inhibition as the primary mech-anism for the cardioprotective effects of RAN (Maier et al., 2013;Shryock et al., 2013). Notably, Rocchetti et al. (2014) recentlydemonstrated that late INa is enhanced in the right ventricle ofMCT rats and is blocked by RAN. In addition to inhibiting lateINa, RAN has been reported to inhibit adrenergic receptors andto partially inhibit fatty acid oxidation at high concentrations(.100 mM; Minotti, 2013). Although these mechanisms cannotbe completely excluded, it is unlikely that they are majorcontributors to the efficacy of RAN in MCT-induced PAH,because the plasma levels of RAN achieved in our study havebeen shown to selectively inhibit late INa versus all othertargets (Hale et al., 2008; Zhao et al., 2011). Of note, steady-state plasma levels of RAN (#10 mM) had no effect on ISO-induced increases in heart rate in our acute studies, indicatingthe lack of antiadrenergic effects.In this study, RAN, an antianginal, anti-ischemic drug known

to have beneficial effects in models of LV dysfunction, wasefficacious in reducing both structural and electrical remod-eling of the right ventricle in an animal model of PAH. Inin vivo experiments, chronic administration of RAN dose-dependently decreased pulmonary artery pressure, RVhypertrophy, RV dysfunction, and the expression of fibrosismarkers (collagen 1a1, CTGF, and TGF-b) in the rightventricle. These findings were supported with ex vivo ex-periments showing that RAN decreased electrical remodelingof the right ventricle, as indicated by reduced repolarizationabnormalities and by decreases in the prolonged ERP and QTcintervals. In in vivo experiments, we showed that acute RANtreatment prevented triggered arrhythmias in hearts of ratswith PAH (Fig. 7). In conclusion, the administration of RANduring PAH development decreased adverse structural remod-eling and reduced the electrophysiological abnormalities of thediseased right ventricle, providing evidence that the inhibitionof late INa may have beneficial effects in the setting of RVimpairment due to PAH.

Acknowledgments

The authors thank Dr. Sarah Fernandes for expert assistance withthe telemetry study.

Authorship Contributions

Participated in research design: Liles, Hoyer, Chi, Dhalla,Belardinelli.

Conducted experiments: Liles, Hoyer, Oliver.

Performed data analysis: Liles, Hoyer, Oliver.Wrote or contributed to the writing of the manuscript: Liles, Hoyer,

Chi, Dhalla, Belardinelli.

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Address correspondence to: Kirsten Hoyer, Department of Biology, GileadSciences, Inc., 7601 Dumbarton Circle, Fremont, CA 94555. E-mail: kirsten.hoyer@gilead.com

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