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The Myosin Activator Omecamtiv Mecarbil Increases Myocardial Oxygen Consumption and Impairs Cardiac Efficiency Mediated by Resting Myosin ATPase Activity Bakkehaug et al: Oxygen Wastage of Omecamtiv Mecarbil Jens Petter Bakkehaug, MD1; Anders Benjamin Kildal, MD1,3; Erik Torgersen Engstad, MD1; Neoma Boardman, PhD1; Torvind Næsheim, MD2; Leif Rønning, DVM1; Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Ellen Aasum, PhD1; Terje S. Larsen, PhD1; Truls Myrmel, MD, PhD2,3; Ole-Jakob How, PhD1 Cardiovascular Research Group, 1Institute of Medical Biology and 2Institute of Clinical Medicine, M ediciine ne, Faculty of Health Sciences, Arctic University Un niv versity of Norway, Norwa way wa y, Tromsø, Norway and 3 Departmentt of Cardiothoracic Caard dio i tho oraacic and nd Vascular Vascullarr Surgery, Surrgeery, H Heart earrt andd L ea Lung ung C Clinic, liinicc, U University niveersi siity y Hosp Ho spit sp ital it al ooff No Nort rth rt h No Norw rway rw y, Tr Trom om msø s , No Norw rway rw ay Hospital North Norway, Tromsø, Norway Correspondence to Ole-Jakob How Cardiovascular Research Group, Institute of Medical Biology Faculty of Health Sciences, Arctic University of Norway, Tromsø, Norway Phone: +47 98821821 Fax: +47 77645440 E-mail: [email protected] DOI : 10.1161/CIRCHEARTFAILURE.114.002152 Journal Subject Codes: Basic science research:[130] Animal models of human disease, Heart failure:[11] Other heart failure, Myocardial biology:[107] Biochemistry and metabolism, Myocardial biology:[105] Contractile function, Treatment:[118] Cardiovascular pharmacology 1 Abstract Background—Omecamtiv mecarbil (OM) is a novel inotropic agent that prolongs systolic ejection time (SET) and increases ejection fraction (EF) through myosin ATPase activation. We hypothesized that a potentially favorable energetic effect of unloading the left ventricle, and thus reduction of wall stress, could be counteracted by the prolonged contraction time and ATP-consumption. Methods and Results—Post-ischemic left ventricular dysfunction was created by repetitive left coronary occlusions in 7 pigs (7 healthy pigs also included). In both groups, SET and EF Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 increased after OM (0.75 mg/kg loading for 10 min, followed by 0.5 mg/kg/min continuous infusion). Cardiac efficiency was assessed by relating myocardial oxygen consump ption o consumption (MVO2) to the cardiac work indices, stroke work and pressure-volume area. To circumvent ciirc rcum umve um vent ve nt potential efficiency assessed pote po tent te ntia nt iall ne ia nneurohumoral uroh ur o umoral reflexes, cardiac efficie enc ncy y was additionally ass ses esse s d in ex vivo mouse hear rts and and d isolated iso sola late la teed my myoc ocar oc ardi ar dial di al m itoc it o ho h nd driia. OM OM im impa p ir ired ed ccardiac ardi ar diac di acc efficiency; eff fffic icie ienc ie ncy; nc y; there the heree hearts myocardial mitochondria. impaired n hhealthy eallthy ea lt y aand nd ppost-ischemic ost-isch hemi emic pigs, pigs, wa w % and and 23% % in iincrease creease se in un unlo oad ded MVO MVO2 in wass a 31% unloaded respectively. Also, the oxygen n ccost ost of the ccontractile os o trrac on actilee function fun u ction was increased by 63% and 46% respectively. The in healthyy and ppost-ischemic ost-ischemic ppigs, igs, ig g , respe p cti tively. y T he iincreased ncreased d unloaded MVO2 was confirmed in OM treated mouse hearts and explained by an increased basal metabolic rate. Adding the myosin ATPase inhibitor, 2,3-Butanedione monoxide, abolished all surplus MVO2 in the OM treated hearts. Conclusions—Omecamtiv mecarbil, in a clinically relevant model led to a significant myocardial oxygen wastage related to both the contractile and non-contractile function. This was mediated by that OM induces a continuous activation in resting myosin ATPase. Key Words: contractility; acute heart failure; inotropic agent; myocardial metabolism; myocardial oxygen consumption 2 Omecamtiv mecarbil (OM) is a novel synthetic cardiac inotrope with a unique mechanism of action. It is classified as a myosin activator, discovered through high-throughput screening with a cardiac myosin ATPase bioassay 1. The compound has been identified as potentially strengthening of the cardiac muscle, and is presently being tested in chronic heart failure patients in a phase 2b trial (www.clinicaltrials.gov NLM identifier: NCT01786512). Initial studies argue that OM works independently of the calcium transient through an increase in the number of myosin heads interacting with the actin filament 2. Analogously, this has been described as “more hands pulling on the rope” 3. OM extends systolic ejection and augments Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 left ventricular (LV) shortening, thereby increasing LV ejection fraction. This can potentially prolong n ed SET improve cardiac efficiency through reduction off LV wall stress. Conversely, a prolonged hee ccardiac ardi ar diac di ac may impair efficiency since systole is the primary energy-consuming phase of the cycle reflecting activation. OM cycl cy clee re cl refl flec fl e ti ting ng prolonged myosin ATPase activ ivat iv atio at i n. Administration of O M in a canine eart ea rt fai ailu ai lure lu ree, indi in ndiica cate ted te d no iincrease ncreease nc s in my m yocar ardi ar dial di al ooxygen xyge xy gen ge n co cons nsum ns umptio um ion io n (M (MVO VO2) an model off hheart failure, indicated myocardial consumption andd n tthis hiis study, st , hhowever, owev ow ver, tth us sug gge gested ed imp pro oved ca card r iac ef eefficiency fiiciiency y ffollowing ollo owingg O M tr reatm ment 4. IIn thus suggested improved cardiac OM treatment MVO2 was not related to cardia cardiac iaac wo w work, rk, norr was was su ssubstrate bsstr trat a e metabolism assessed. Thus, the aim clarify cardiac energetic of the ppresent resent studyy has been to cl larify ify tthe he card diac energe g ti tic and d metabolic pr pprofile ofile of OM. Besides healthy pigs, we have used a clinically relevant pig model of post-ischemic left ventricular dysfunction 5, an ex vivo working mouse heart model without neurohumoral influence 6 and isolated mitochondria from the mouse myocardium. Our hypothesis has been that OM has a neutral effect on myocardial energy consumption as the favourable effects of reduced wall stress potentially can be counteracted by the prolongation of systole through the myosin ATPase activity. 3 Methods Experimental Animals The experimental protocols were approved by the local steering committee of the National Animal Research Authority at the Faculty of Health, UIT The Arctic University of Norway. Twenty-two castrated male domestic pigs weighing 28 ± 6 kg were adapted to the animal department for 5 to 7 days and fasted overnight before experiments, with free access to water. Additionally, 49 female NMRI mice from Charles Rivers Laboratories (Wilmington, MA, USA) were used in this study. All mice received chow and drinking water ad lib, and were Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 housed at 23 ºC. Anaesthesia and surgical preparation In vvivo ivo iv o pig pig were premedicated with intramuscular mg/kg Ketamine (Pfizer The pigs w ere pr er rem emed edic ed icaated w ic ith it h in ntr t amus uscu us ular iinjections njject cttio ions ns ooff 200 m g/kg g/ kg K etam min inee (P (Pfi fize fi zeer AS AS,, Norway) atropine Pharma, No N rway)) and a d 1 mgg of an of atro opiine n (Nycomed (Ny Nyco Ny omed Ph Pharm ma, Norway). Noorw way y). Anaesthesia An naesthesiia was was induced ind n ucced byy Norway). After inhalation of isoflurane (Abbot, t, N orway) y). Af A teer en eendotracheal dootr trac a heal intubation an intravenous injection mg/kg sodium Sweden) mg/kg fentanyl inje j ction of 10 mg g/kg g pe ppentobarbital ntobarbi bittall sod di m ((Abbott, dium Abbo Ab b tt tt,, S weden) d ) and 0.01 mg/ g kg g fentany yl (Hameln Pharmaceuticals, Germany) were given, and the animals where normoventilated. An introducer sheath catheter was placed in the left internal jugular vein, and anaesthesia was maintained throughout the experiment by a continuous infusion of 4.0 mg/kg/h pentobarbital sodium, 0.02 mg/kg/h fentanyl, and 0.3 mg/kg/h midazolam (B. Braun, Germany). The circulating volume was maintained by a 20 mL/kg/h continuous infusion of 0.9% NaCl supplemented with 1.25 g/L glucose. The animals received 2500 IU heparin and 5 mg/kg amiodarone (Sanofi-Synthelabo, Sweden) to avoid blood clotting of catheters and cardiac arrhythmias. 4 The surgical preparation of the open chest pig model has been described in detail previously 5. In brief, a 7.5 Fr Swan-Ganz catheter was placed in the pulmonary artery via the jugular vein. A 7 Fr manometer-pressure catheter (Millar MPVS Ultra, Houston,Tx,USA) in the left ventricle. Arterial pressure was measured via a vascular catheter in the abdominal aorta. A 7 Fr balloon catheter was introduced to the inferior vena cava for preload reduction. Following medial sternotomy, the pericardium was removed and the coronary arteries and pulmonary trunk were dissected free to place transit-time flow probes (Medi-stim, Norway) for measurements of coronary blood flow and cardiac output. Three sonomicrometry crystals Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 (Sonometrics corporation, trx 4, Canada) were placed in the myocardium to measure long GE, axis and short axis myocardial shortening. Epicardial echocardiography (Vivid I, GE E, USA) blood was used to calibrate the crystal dimensions to ventricular volumes. Myocardial vvenous enou en ouss bl ou bloo ood oo d wass sa wa samp mpled mp d fr from a catheter placed in the great greaat cardiac ca vein via the cor ron onar a y sinus (after sampled coronary ligating the thee hemiazygous hem miaazygo zygo gouss vvein). ein) ei n). n) Ex vivo mouse Isolated pperfused erfused mouse hearts were used used d for for assessmentt off unloaded unlloaded MVO2 and myo myocardial y cardial substrate oxidation as described previously 7. In brief, the mice were anesthetized with 10 mg sodium pentobarbital i.p. The hearts were quickly excised and the aorta was cannulated and initially perfused retrogradely (Langendorff) with recycled Krebs-Henseleit bicarbonate buffer containing 5 mM glucose and 0.4 mM palmitate bound to 3% bovine serum albumin. Subsequently, hearts were perfused either in unloaded retrograde mode using for assessment of unloaded MVO2 6 or in the working heart mode for assessment of myocardial glucose and fatty acid oxidation rates by the use of radiolabeled isotopes 7. In the retrograde perfused unloaded hearts, the ventricular cavity was vented by inserting a 25 G steel cannula through the apex of the heart, allowing drainage of any 5 perfusate trapped in the LV lumen. In the working heart perfusions, the left atrium was cannulated with a 16 G steel cannula connected to a preload reservoir ensuring forward perfusion through the aortic valve. Aortic and filling pressures were set to column heights of 55 and 8 mmHg, respectively. Electrodes were placed on the right atrium for electrical pacing at 7 Hz, and cardiac temperature was maintained at 37 °C throughout in both perfusion modes. Experimental protocols Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 One in vivo pig, two ex vivo mouse heart and one isolated mitochondrial protocol were carried out (Figure 1). All protocols were conducted with a repeated measures design gnn with Omecamtiv measurements at baseline and following the administration of OM or vehicle. Om mec ecam amti am tiv ti v mecarbil formulated meca me carb ca rbil rb il ((Selleck Seell llec e k Chemicals, USA) was formul ulat ul ated at e for all experimentss aass a solution of 1 mmol/L sterile water, with NaOH mg/mL with th 550 0 mm mmol ol/L ol /L citrate cit ittra rate te iin n st ster e il i e wa w ter, aadjusted djjuste ted te d to ppH H 55.0 .0 wi w th hN aOH H 8. Wi With th nd d human hum u an n sstudies tu udies 33,8,8 we de ddecided ecide ded de d onn a dose dosse target targeting tin ng a 220% 0% in increase ncr creaasee in rre ferencee to t an nimaal 22,4,4 and reference animal SET. Thus being at a clinical re relevant level, providing significant ele leva v nt level l, pr rov ovid i in ng a sign g ificant hemodynamic output 3,8. model negative SET was defined in the ppig ig g mod dell as the th he time time bbetween etween t ppeak eak k po ppositive sitive and ppeak eak nega g tive derivatives of LV pressure (dP/dtmax and dP/dtmin, respectively) 4 and in the mouse model as the time between minimum aortic pressure and the dicrotic notch. In vivo pig In a dose-response protocol (n=3), intravenous infusion of OM showed a linear increase in SET, cardiac output and ejection fraction until maximum dose of 1 mg/kg (Figure 2). Based on this data the following dose was selected for the main protocol; a bolus dose of 0.75mg/kg over 10 minutes followed by a continuous infusion of 0.5 mg/kg/h. This corresponds to a plasma concentration of 500-1000 ng/ml 3,8. 6 The effect of OM on cardiac energetics was then assessed in healthy pigs (n=9) and in pigs with post-ischemic LV dysfunction (n=10). Following surgery and a 30-min stabilization period, baseline measurements were performed. In the post-ischemic group, acute heart failure was induced using our ischemia-reperfusion model 5. In short, this protocol uses repetitive coronary occlusion and reperfusion episodes with a total of approximately 20 minutes of accumulated ischemia. The occlusion affects ~80% of the LV and induces a reproducible acute impairment of LV function which remains stable for several hours. Then pigs were stabilized for 30-60 minutes before performing post-ischemic measurements. Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Afterwards, OM infusion was initiated and the final recordings were carried out after 20 induct cttion of minutes. The healthy group had an identical experimental protocol without the induction ischemia-reperfusion injury. Ex vivo mo mouse ous use The dose-response relationship working (n=3), T e dose Th e-rresspo onsee re elatio onsh onsh ship off OM M wass studied studiied d inn ex vivo viivo o wo ork king mouse mousee hhearts eartss (n n=33), using a range of OM concentrations ng/mL concentrat atio at io ons n from 100 10 ng ng/m / L to 1200 ng/mL. ngg/mL. An increased SET of 15-20% was obtained at 400 ng/ml subsequent ng/ g mll off OM, g/ OM, which whi hich h was selected sellected t d for the subsequ q ent pr pprotocols. otocols. In the Langendorff perfusions (n=30), baseline unloaded MVO2 measurements were made after 20 minutes of stabilization, before OM (n=17) or vehicle (n=13) was added to the recirculating buffer. After a second stabilization period, new unloaded MVO2 measurements were performed. Then extracellular K+ concentration was raised to 16mM to electrically arrest the heart, which allowed measurement of basal MVO2. Oxygen cost of excitationcontraction coupling was calculated as the difference between MVO2 measured before and after cardioplegia. In a sub study (n= 7, each group) 20 mM of the myosin ATPase inhibitor 2,3-Butanedione monoxide (BDM) (Sigma Aldrich, USA) was added after basal MVO2 measurements. After stabilization, MVO2 was measured in the resting heart with inhibited 7 myosin ATPase activity. The working heart perfusions (n=14) were used for assessment of myocardial glucose and fatty acid oxidation. Both before and after OM (n=8) or vehicle (n=6) administration, 5 consecutive samples of the perfusion buffer were taken with seven minutes interval. Hemodynamic values were recorded simultaneously. Isolated mitochondrial respiration Mitochondria were extracted from mouse hearts (n=4) using the method of Palmer et al. 9 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 with slight modification. Briefly, the heart was cut in small pieces, homogenized and treated with trypsin (5 mg/ml, 1 ml/g) in isolation buffer, followed by differential centrifugation. centrifuga gaati t on. Mitochondria were suspended in preservation buffer f (Oroboros, Innsbruck, Austri ria) ri a) aatt a Austria) concentration ice Mitochondrial conc co ncen nc entr en trat tr a io on of o 4 μl/mg tissue and stored on ic ce fo for 1-3 hours. Mitocho ond ndrial respiration was measured d uusing singg a C si lark la rk-t rk -typ -t ypee el yp elec ectr ec t od tr ode (O Oxy x grap ph--2k kO robo ro boro bo r s In IInstruments) strrume st rume ment nts) iin n bo both th tthe hee Clark-type electrode (Oxygraph-2k Oroboros aab sence and a d presence an prresen ncee of 200 200 0 nM OM. OM. Malate Mallatte (2 mM) mM) and and pyruvat py yruvvatt (5 mM) mM M) w erre use ed as as absence were used substrates. ADP (200 μM) wass added add d ed to ac chi h ev ve ma m xima xi m l mitochondria oxidative achieve maximal ph p osph p oryl y ation (O ((OXPHOS) XPHOS)) cap pacit i y. it y M easurementt was pperformed erfo f rmed at 37 ºC in 2 ml Mir05 phosphorylation capacity. Measurement mitochondrial respiration medium, adjusted to pH 7.1. Mitochondrial LEAK respiration was measured in presence of substrates, but absence of ADP. State 3 respiration was defined at maximal OXPHOS after adding ADP. State 4 respiration was recorded when all added ADP was converted to ATP, and state oligo after adding the ATPase blocker oligomycin (4 μg/ml). P/O ratio was calculated by measuring the mitochondrial oxygen consumption used to deplete 200 μM ADP (ATP produced per oxygen atom reduced by the respiratory chain). 8 Left ventricular function and energetics In vivo pig LV pressure, sonomicrometric blood flow and vascular pressure signals were recorded, digitized and analysed using ADI LabChart Pro software (Dunedin, New Zealand). At baseline, and after interventions, the LV end diastolic volume (EDV) was calculated from epicardial short axis ultrasound data (Vivid i, GE, USA) using the Teicholz formula, EDV= >7/(2.4+EDD)@ · (EDD3). The ESV was calculated by subtracting stroke volume (SV; from Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Time-transit flow probe on the pulmonary artery) from the LV end diastolic volume (EDV). The short- and long-axis sonomicrometric crystals were converted to a composite output using the Area length (Bullet) formula 10, Volume=5/6 · Area · Length. The compos composite osit os ite it sonomicrometric output was calibrated against ESV and EDV at each intervention intervention. n. To assess ass sses e s cardiac es caard rdia i c efficiency by the work- MVO O2 relationship, reelationship, 6-8 recordings recorrdin dings of varying steady-statee work parameters, coronary work k levels, l ve le vels ls, hemodynamic ls hem he mody dyn dy nam micc pa ara ram meteerss, co orona ro ary y blood blo ood d flow flo ow (CBF) (CBF BF)) and BF an nd blood bloood bloo s mpling sa ng w eree perf rfforrmed. d. P r lo re oad was wass reduced red ed ducced stepwise steepw wisse by by inflating infllatiing ing thee bballoon allloo oon n ca ath theeteer iin n sampling were performed. Preload catheter differeent nt levels lev evel elss of mechanical el mec e ha hani nica ni call work ca work and and their corresponding oxygen the caval vein to obtain different consumption detail cons co nsum ns umpt um ptio pt ion io n as ddescribed escr es crib cr ibed ib ed iin n de deta tail ta il previously pre revi viou vi ousl ou sly sl y 11. LV mechanical mec echa hani ha nica ni call wo ca work rk w was as ccalculated alcu al cula cu late la ted te d as stroke work (SW) 12 and pressure-volume area (PVA) 13. In brief, PVA consists of the area bounded by the pressure-volume loop (stroke work; SW) and the triangular area limited by the line of the end-systolic and end-diastolic pressure-volume relations, as obtained by a transient vena cava occlusion. The Y-intercept in the PVA-MVO2 relation indicates the myocardial oxygen cost not related to pump function (unloaded MVO2). 1/slope indicates the energy cost for contractile work (contractile efficiency). LV CBF was calculated from the formula LVCBF = CBF/W · LVW 14; where LVCBF and CBF are left ventricular and total CBF, respectively. W and LVW are total myocardial and LV myocardial weight, respectively. LV myocardial oxygen consumption was calculated from the formula MVO2 = 9 (LVCBF · avdO2 · Hb · 1.39)/HR · 20.2; where MVO2 is LV myocardial oxygen consumption, avdO2 is the difference between aortic and myocardial venous oxygen saturations, Hb is hemoglobin in grams per liter, 1.39 is a constant (mL O2 /g Hb), and HR is heart rate. To convert MVO2 to mechanical energy equivalents the factor 20.2 J/mL O2 was used. Ex vivo mouse Coronary flow (CF) was derived from timed measurements of coronary effluent 6. Fiber optic Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 probes (FOXY-AL 300; Ocean Optics, Duiven, Netherlands) were used to measure partial oxygen pressure (pO2) in the arterial and venous coronary buffer. These were placed proximally in the aortic line and in the pulmonary trunk, respectively. MVO2 wass then then calc ca lcul lc ulat ul ated at ed by by the t e following equation: MVO2 = >pO th calculated >pO2 (coronary inflow) - pO2 (coronary Bunssen solubility Bu sol olub ubil ub ilit il ity it y coefficient effluent)@ · Bunsen co oeffi ef ici c ent of of O2 · C CF F 6. Metabolism In vvivo ivo iv o pig pig Methods for assessing myocardial glucose and free fatty acids (FFA) oxidation by isotopic tracers are described in details previously 11. In brief, isotope-labelled oleic acid and glucose was dissolved in 50mL of plasma obtained from the pig to give a final radioactivity of 0.126 MBq/mL and 1.014 MBq/mL of [U-14C)] glucose and [9,10-3H] oleic acid, respectively. Infusion of isotopes was started 30 min before administration of OM with a bolus of 30 ml/h for 15 min, continued by steady-state infusion at 8 ml/h throughout the experiment. Arterial and coronary sinus blood samples were drawn simultaneously before and 20 minutes after OM administration. Five 1-ml samples for 14CO2 determination were transferred to airtight 14CO2 trapping vials. Four 1.25-ml aliquots were cold-centrifuged, and the plasma was 10 immediately frozen. Plasma was stored at -70 °C and analysed later for determination of 3H2O and substrate levels. The content of 3H2O in plasma was determined by Folch extraction 15. The 14CO2 content of the blood was assessed by a diffusion method, as described by Wisneski and associates 16. Aliquots of blood (with trapped 14CO2) or plasma water (with 3H2O) were then mixed with scintillation fluid, and the radioactivity was determined on a beta-scintillation counter (Packard 1900 TR Liquid Scintillation Analyzer; Packard Instruments BV-Chemical Operations, Groningen, the Netherlands). Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Ex vivo mouse Glucose and FFA oxidation in the ex vivo working hearts were measured simultan simultaneously aneo an eous eo usly us ly aass g 14CO2 released by described previously 17. Glucose oxidation was determined by measuring the metabolism glucose, measuring metabo b li bo lism sm of of [U [U--14C)] C))] gl gluc ucos uc ose, os e, and andd FFA FFA F oxidation ox xid datiion bby y me easur urin ur ing in g 3H2O re rele released leas le ased as e ffrom ed rom ro m palmitate. Cardiac output was obtained perfusate CF. [[9,10[9 ,10-3H] pa alm mitatte. Card diaac outp tp put u w as ob btaained aass tthe he sum um of ao aortic per rfussate tee flow w and and C F. At the end of the perfusion, hearts hea eaart rtss were fro oze z n an and total tota to tall dry ta y mass was determined. frozen Statistical analysis All data are presented as means ± standard deviation (SDs), unless stated otherwise. Withingroup effects of energetic data and between-group effects of hemodynamic data were analyzed using a linear mixed-models approach with a restricted maximum likelihood method and the subject identifier as the random effect. Within-group effects of hemodynamic data were assessed by one-way repeated measures analysis of variance (ANOVA). We performed Wilcoxon signed rank test to compare means of metabolic data from pigs and substrate metabolism in working mouse hearts. Measurements of MVO2 in unloaded retrograde perfused hearts and measurements of mitochondrial respiratory data were assessed 11 using the Mann-Whitney Wilcoxon test. Multiple comparisons were adjusted for by Bonferroni correction. P-values <0.05 were regarded as statistically significant, and all analyses were conducted in SPSS 22.0 (Chicago, USA). Results A total of 19 pigs were used in the in vivo study. 14 pigs were included for analysis of energetics and hemodynamics in the healthy (n=7) and the post-ischemic LV dysfunction (n=7) group. 3 pigs were excluded due to hemodynamic collapse following induction of Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 myocardial ischemia with sustainable need of vasopressor and 2 pigs were excluded due to led surgical complications. 49 mice were used in the ex vivo study. A calibration error le ed to (Langendorff) exclusion of one heart. 30 mice were included in the retrograde perfusion (Langen en ndo dorf rff) rf f) prot pr otoc ot ocol oc ol ffor orr aassessment s essment of unloaded MVO2. In ss protocol n tthe he working heart perfusi perfusion sion si o protocol for on ntt of of substrate subs su bsttrat bs atee oxidation at oxid oxid idat atio at ion io n 144 m icce were were included, incclu ude ded, d while d, whi h lee iin n th the mitochondrial mittoch mi hon ondr dria dr iall ia assessment mice r spirator re orry aassessment ssessm ment 4 m men ice we ic ere r iincluded. nccludeed. respiratory mice were LV function In vivo pig hearts Cardiac effects of the ischemia-reperfusion protocol are shown in Table 1. The effects of the accumulated 17±5 minutes of ischemia are compatible with post-ischemic LV stunning5,18 with reduced SV, SW and ejection fraction, and a concomitant increase in HR and mean pulmonary arterial pressure. The pigs received a OM dose targeting a clinically relevant increase in SET of 20% 3,8. This dose resulted in a 16 ± 4% and 20 ± 6% increase in SET in healthy and post-ischemic hearts, respectively. At the same time the HR was unaffected (Table 1). OM reduced diastolic filling time (DFT) with approximately 11%. These changes in the cardiac cycle can be illustrated by comparing the ratio of SET/DFT before and after 12 OM resulting in a 31% and 35% increase in the healthy and the post-ischemic group, respectively. Simultaneously OM induced a substantial increase in coronary blood flow (CBF) in both healthy and post-ischemic pigs (Table 1). OM caused the left ventricle to work at lower volumes in both healthy and postischemic hearts (Figure 3) coupled with an increased ejection fraction (Table 1). However, the enhanced LV emptying was accompanied by an impaired ventricular filling as seen by a reduced EDV, and an increased tau. Thus, the net result of these alterations was an unchanged SV following OM treatment in both healthy and post-ischemic hearts. Also, Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 perfusion pressure in the systemic and pulmonary circulation was unchanged by OM. Ex vivo mouse hearts working administration. In ex vvivo ivo iv ow orki or k ng mouse hearts, SET increased ed 16 16 ± 4% after OM admi mini mi n stration. Cardiac ni output was as uunchanged nchaang nc nged ed iin n these th hes esee he hear arts ar ts. ts hearts. metabolism Cardiac efficiency and metab bol olis im is In vivo pi ppig g hearts Figure 4 shows the effect of OM on the relation between mechanical work and MVO2 in typical experiments with healthy and post-ischemic hearts. Figure 5 presents all data points used in the statistical analysis of the OM effect on the work-MVO2 relationship. In therapeutically relevant doses, OM had a negative effect on cardiac efficiency as measured by increased MVO2 relative to cardiac work (Table 2). The impaired energetic state by OM was observed in both healthy and post-ischemic hearts over a large range of workloads (Figure 5). Unloaded oxygen cost (y-intercept of the PVA-MVO2 relation) increased by 31% and 23% in healthy and post-ischemic pig hearts, respectively. OM also impaired contractile 13 efficiency as seen by a 63% (healthy) and 46% (post-ischemic) increase in the slope of the PVA-MVO2 regression (Figure 5, Table 2). OM only marginally affected the relative substrate consumption with a trend towards more glucose utilization at the expense of FFA (Figure 6A). The uptake of lactate, glucose and FFA were not affected by OM (Figure 6B). Ex vivo mouse hearts Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 OM increased unloaded MVO2 in ex vivo mouse hearts similar to the extrapolated Yintercept in the PVA-MVO2 relation in pig protocols. This was attributed to a 63 ± 31% excitation-contraction increase in basal metabolic rate, while oxygen cost of excitation contraction coupling g was basal MVO unaffected. Adding the myosin ATPase inhibitor, BDM, abolished all surplus bas sal a M VO2 iin n small thee OM treated th tre r atted hearts (Figure 7). There was a sm smal a l increase in the ratee ooff glucose oxidation with OM M in n tthe he eex x vi vivo vo working wor orki king ki ng hearts hea eart r s (F Fig igu ure 6C 66C) C) wh whil ilee th il thee mi mito toch chon ch on ndr dria i l re ia esp spir irat ir atio at ion io n in (Figure while mitochondrial respiration iis olated mitochondria mito och hond driia from m mouse mouse see hearts hea earts was ea wa unaffected unnafffect cted ct ed byy OM M (Figure (F Figuree 66D). D)). T heree was was noo isolated There difference in any of the respiratory respiraato tory ry states orr mitochondrial mit itoc ocho oc ond ndri r al efficiency shown by unchanged P/O ratio. Discussion The main finding of this study is that Omecamtiv mecarbil contributes to a significantly increased myocardial oxygen cost in both healthy and post-ischemic stunned hearts. OM increases oxygen consumption due to energetically inefficient left ventricular function and increased oxygen consumption by non-contractile processes. The increase in MVO2 was mediated by hyperactivity in myosin ATPase. 14 Effect on cardiac function OM’s mechanism of action on the contractile apparatus is difficult to classify. An important characteristic of inotropy is accelerating the contraction and relaxation (measured as an increase in dP/dt max and min), indices that are only marginally affected by OM as it extends the systolic contraction rather than reinforcing it. On the other hand, OM increases ejection fraction similarly to classic inotropes by improved emptying in systole. Such reduction of wall stress is considered as energetically favourable as systolic wall stress is a main Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 determinant of MVO2 19. However, the oxygen cost of a prolonged systolic phase may outweigh the beneficial effect of a reduced wall stress. We found no significant changes in i ling il SV and cardiac output. This can potentially be explained by a reduced LV diastolic ffilling and increased Tau. Shen et al. 4 found a small increase in SV in dogs given OM, an and d th this is could coul co uldd be caused ul cau use sed by a reduced afterload as seen n bbyy a concomitant reduction reducttio ion n in vascular resistance in in these thesse do th dogs gs. gs dogs. The observed may increase T h rreduced he ed duceed ccontractile ontrraccti t le efficiency eff ffic ff i ien ncy ob observ ved dm ay y bee ccaused aussed d by an n uunfeasible nffeaasiblee in ncreeas ease diastolic time. The of SET at the expense of diastol olic ol icc filling g tim me. T h ffact he actt that EDV becomes smaller, despite ac unchanged unchange g d pr ppreload eload and HR,, suggest sugg ggestt that gg th hatt OM OM induces i du in d ces myocardial myo y card dial constrain in late diastole. However our stunning model of acute heart failure does not have the characteristics of diastolic dysfunction. Thus the interpretation that OM impairs diastolic performance demands caution. Studies that address potential effects of OM in tachycardia and reduced coronary reserve should be carried out. It is unclear how the LV filling and coronary perfusion are affected in the heart when there is a shortened diastole and concomitantly elevated HR. Thus, a thorough assessment of the impact of OM in a relevant model of diastolic dysfunction seems warranted. 15 Effect on cardiac energetics Our findings are in contrast with a previous study on dogs by Shen et al 4. These authors found that OM significantly improved cardiac efficiency by enhancing LV function without a corresponding change in MVO2 4. They reported a non-significant increase in MVO2 after 24 hours of OM infusion (MVO2 from 3 ml/min to 4 ml/min). However, a formal evaluation of cardiac efficiency require measurements of MVO2 and total cardiac work (i.e. PVA) over a wide range of workloads 13. Such an analysis can separate the energy consumption (MVO2) Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 in unloaded MVO2 (the y-intercept of the regression line), reflecting the oxygen cost of excitation-contraction coupling and/or basal metabolism, and contractile efficiency (increased slope of the regression line). In both the healthy and the post ischemic pig hearts we oobserved bserved post-ischemic a pronounced impairment in cardiac efficiency following OM infusion. This was ev evid id den entt by evident increase incr in crea cr ease ea se ooff bo both t the y-intercept and the slope of of the the PVA-MVO2 regression. regress sssio ion. n in ex vvivo ivo iv o mo mou mouse usee T ffurther urth t err eelucidate th luci lu cida ci datee the da the energetics, ene nerg rgetticcs, rg s, w eaasurred uunloaded nloa nl o de ded MV MVO O2 in To wee m measured hhe arts. He ere r we co onf nfiirmed d th that the he el lev vated d yy-intercept -intterrcep ep pt as a sseen eeen inn tthe he pigs pig gs al lso w as eevident vide vi deentt hearts. Here confirmed elevated also was by direct measurements of unlo oad aded e MVO2 in unloaded n mice. mi . When W en the hearts were arrested by Wh cardioplegia cyclic myocyte cardiopl p eg gia ((i.e. i.e. blocking g the cy yclic li ddepolarization ep polarizatio ti n off tthe h myo he y cy yte membrane)) we observed that the surplus unloaded oxygen could be attributed to an increased basal metabolic rate, and not due to the calcium handling in excitation-contraction coupling. This observation is compatible with the proposed action of OM; activation of myosin ATPase used in the sliding contraction of the myofilaments and not the traditional increased inotropy-controlled intracellular calcium transients 20. Changes in substrate metabolism do not appear to be the explanation for the increased oxygen consumption in hearts perfused with OM. Only a marginal switch towards glucose oxidation following administration of OM was seen in both the pig and mouse heart, reaching significance only in the mouse protocol. However, glucose is in fact a more “oxygen sparing” substrate compared to fatty acids due to an increased P/O 16 ratio 21. Such a switch should therefore potentially counteract the observed inefficient energetic state in OM-perfused hearts. An increased basal metabolic rate could also be caused by an altered mitochondrial respiration i.e. mitochondrial uncoupling in the oxidation phosphorylation 22. However, we did not observe any effect on the efficiency in isolated myocardial mitochondria that was incubated with OM. Combined with the missing alteration in substrate metabolism on both the organ and subcellular levels, this warrants another explanation for the increased oxygen cost in non-contracting hearts. As OM is a nonbiological compound without integrated receptor, signal molecule and ionic effectors, a Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 distinct possibility is that the myosin ATPase is activated with no relation to the electrophysiological cycling of the myocardium and therefore is still active in nonnon contracting and arrested hearts. T h ooxygen he x gen waste caused by the drug iss li xy like k ly to be a by-produc ct of o its interaction The likely by-product with myosin. myosiin. A chemical che hemi mica mi call precursor ca prec pr ecur ec urso ur sorr of OM so M was was discovered disscove vere ve red re d through thro th ough a high-throughput high hi gh-t gh - hrrou ough ghpu gh putt pu ssc reening g aimed aime med att compounds me co ompoounnds increasing inc ncre nc reassing cardiac caardiaac sarcomere sarrco come mere re ATPase AT TPaase ac ctiv vity ty y 1. Thiss screening activity compound was refined to reduce redu uce its its t toxicity, y and y, and thee ffully u ly ul y developed OM showed a 40% increased activityy of the myosin my yosin i ATPase ATP TPase att only onlly 0. 00.58μM 58μM 58 μ 1 ddemonstrating μM emonstratingg that OM is a po ppotent tent activator of myosin ATPase. The contribution of the resting rate of myosin ATPase to basal metabolism is generally regarded as small or non-existent in normal myocardium 23. Most of these studies were done with the cross-bridge inhibitor 2,3-butanedione monoxime (BDM). The specificity of BDM as a myosin inhibitor is not known and some researchers have found that it also affects the excitation-contraction coupling 23. Ebus and Stienen examined saponinskinned cardiac trabeculaes from rats without BDM 24. They were able to show that 40% of the basal activity remained after removing the contribution of all membrane bound ATPase activity by stripping away membranes with Triton X-100. This suggests that myosin ATPase has a large role in determining unloaded MVO2 of basal metabolic rate. This is in line with 17 our observation that BDM abolished all surplus MVO2 caused by OM treatment. As this assessment was conducted on arrested hearts with no oxygen cost for excitation-contraction coupling, suggests that OM induces myocardial oxygen waste mediated by hyperactivity in resting myosin ATPase. Conclusions This study shows that Omecamtiv mecarbil leads to a significant oxygen waste in the myocardium independent of neurohumoral factors. The oxygen waste can be explained by a Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 combination of a reduced contractile efficiency and increased energy consumption in the non-contracting non contracting muscle, as mediated by hyperactivity in resting myosin ATPase. An given assessment of OM’s effect on cardiac energetics in humans is of major interest giv iven iv en tthe he ongoing failure trial ongo on goin go ing in g cl cclinical in nic ical a trials of the drug in heart failu ure ppatients. atients. A clinical tria iaal (www.cli liniica calt ltrial lt alss.go al gov go v NL NLM M id iden enti en tifi ti fieer: NC fi NCT0 T 074 4857 57 79) aaddressing ddreess dd ssin ng th his iissue ssuee w as tterminated ermi er mina mi nate na tedd te (www.clinicaltrials.gov identifier: NCT00748579) this was wi w th only y tw wo ppatients atieent ents inc clu ude d d. with two included. A kn Ac k owle l dggements t Acknowledgements The authors acknowledge Trine Lund for assisting with ex vivo mouse hearts and mitochondrial respiration protocols. Sources of Funding This work was financially supported by the health authorities of North Norway and the Norwegian Council of Cardiovascular Diseases. Disclosures None. 18 References 1. 2. 3. Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Morgan BP, Muci A, Lu P-P, Qian X, Tochimoto T, Smith WW, Garard M, Kraynack E, Collibee S, Suehiro I, Tomasi A, Valdez SC, Wang W, Jiang H, Hartman J, Rodriguez HM, Kawas R, Sylvester S, Elias KA, Godinez G, Lee K, Anderson R, Sueoka S, Xu D, Wang Z, Djordjevic N, Malik FI, Morgans DJ. Discovery of omecamtiv mecarbil the first, selective, small molecule activator of cardiac Myosin. ACS Med Chem Lett. 2010;1:472–7. Malik FI, Hartman JJ, Elias K a, Morgan BP, Rodriguez H, Brejc K, Anderson RL, Sueoka SH, Lee KH, Finer JT, Sakowicz R, Baliga R, Cox DR, Garard M, Godinez G, Kawas R, Kraynack E, Lenzi D, Lu PP, Muci A, Niu C, Qian X, Pierce DW, Pokrovskii M, Suehiro I, Sylvester S, Tochimoto T, Valdez C, Wang W, Katori T, Kass DA, Shen YT, Vatner SF, Morgans DJ. Cardiac myosin activation: a potential therapeutic approach for systolic heart failure. Science. 2011;331:1439–43. Teerlink JR, Clarke CP, Saikali KG, Lee JH, Chen MM, Escandon RD, Elliott L, Bee R, Habibzadeh MR, Goldman JH, Schiller NB, Malik FI, Wolff AA. Dose-dependent augmentation of cardiac systolic function with the selective cardiac myosin activator, omecamtiv mecarbil: a first-in-man study. Lancet. 2011;378:667–675. Vatner Shen YT, Malik FI, Zhao X, Depre C, Dhar SK, Abarzúa P, Morgans DJ, Vatn ner SF. Improvement of cardiac function by a cardiac Myosin activator in conscious conscio ous dogs dog ogss with with systolic heart failure. Circ Heart Fail. 2010;3:522–7. Korvald C, Elvenes OP, Aghajani E, Myhre ES, Myrmel T. Postischemic mechanoenergetic dysfunction mech me han anoe o nergetic inefficiency is related d to to contractile dysfunctio on an aand d not altered metabolism. Physiol. metabo me b lism. Am J Physiol Heart Circ Phy ysiiol. 2001;281:2645–2653. 2001;281:2 264 6 5–26 26 653. Boardman DL, Aasum E.. In Increased Boar rdm dman n N, N, Hafstad Hafs Ha f taad AD, fs AD Larsen La n TS, TS, S Severson Seve verson on nD L, A assum E Incr rease sed se d O2 ccost osst of basal me metabolism excitation-contraction coupling from diabetic metabo oliism aand nd excit itat it a io on-con ntrraction n cou oupl plin pl ing in hhearts in eaarts fr rom m ttype ype 2 di yp iab bettic mice. Heart Circ Physiol. miice c . Am m J Physiol Physiol oll H eart C irrc P hyssio ol. 2009;296:H1373–H1379. 200 09;2 ;296 ;2 96:H 96 H13733–H H13799. Aasum Hafstad AD, Severson Larsen TS. Age-dependent Aasu Aa suum E, H afst af stad st a A D, S everso ev son so n DL DL,, La Lar rsen rsen nT S. A ge-d ge -dep -d pen ende dent de nt cchanges hang ha ngges iin n metabolism, contractile fu function, ischemic func n tion, and d is sch chem e ic sensitivity sen e sitivity y in hearts from db/db mice. Diabetes. 2003;52:434–441. 2003;52:434–4 –441 –4 41. 41 Tsyrlin Cleland JG,, Teerlink JR, R, Senior Seniior R, Nifontov Nifontov EM EM,, Mc Mc Murray Murray y JJ,, Lang g CC,, Tsy yrlin VA, Greenberg BH, Mayet J, Francis DP, Shaburishvili T, Monaghan M, Saltzberg M, Neyses L, Wasserman SM, Lee JH, Saikali KG, Clarke CP, Goldman JH, Wolff AA, Malik FI. The effects of the cardiac myosin activator, omecamtiv mecarbil, on cardiac function in systolic heart failure: a double-blind, placebo-controlled, crossover, doseranging phase 2 trial. Lancet. 2011;378:676–683. Palmer JW, Tandler B, Hoppel CL. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem. 1977;252(23):8731–8739. Helak JW and Reichek N. Quantitation of human left ventricular mass and volume by two-dimensional echocardiography: in vitro anatomic validation. Circulation. 1981;63.1398–1407. Korvald C, Elvenes OP, Myrmel T. Myocardial substrate metabolism influences left ventricular energetics in vivo. Am J Physiol Heart Circ Physiol. 2000;278:1345–1351. Sarnoff SJ, Berglund E, Welch GH, Case RB, Stainsby WN, Macruz R. Ventricular function: I. Starling’s law of the heart studied by means of simultaneous right and left ventricular function curves in the dog. Circulation .1954;9:706–718. Suga H. Ventricular energetics. Physiol Rev. 1990;70:247-277. 19 14. 15. 16. 17. 18. 19. Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 20. 21. 22. 23. 23 24. Domenech RM, Hoffmann M, Julien IE. Total and regional coronary blood flow measured by radioactive microspheres in conscious and anestesized dogs. Circulation Research. 1969;25;581–596. Folch J, Lees M, Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497-509. Wisneski JA, Gertz EW, Neese RA, Mayr M. Myocardial metabolism of free fatty acids. Studies with 14C-labeled substrates in humans. J Clin Invest. 1987;79:359–366. Aasum E, Belke DD, Severson DL, Riemersma RA, Cooper M, Andreassen M, Larsen TS. Cardiac function and metabolism in Type 2 diabetic mice after treatment with BM 17.0744, a novel PPAR-activator. Am J Physiol Hear Circ Physiol. 2002;28:949–957. Müller S, How OJ, Jakobsen Ø, Hermansen SE, Røsner A, Stenberg TA, Myrmel T. Oxygen-wasting effect of inotropy: is there a need for a new evaluation? An experimental large-animal study using dobutamine and levosimendan. Circ Heart Fail. 2010;3:277–285. Strauer BE. Myocardial oxygen consumption in chronic heart disease: Role of wall stress, hypertrophy and coronary reserve. Am J Cardiol. 1979;44:730–740. Anderson RL, Sueoka SH, Rodriguez HM, Lee KH, Kawas R, Morgan BP, Sakowicz R, Jr DJM, Malik F, Elias KA. In vitro and in vivo efficacy of the cardiac myosin activator CK CK-1827452. 1827452. Mol Bio Cell. 2005;16 (Abstract #1728) Opie LH. Metabolism of free fatty acids, glucose and catecholamines in acute accut utee myocardial infarction. Am J Cardiol. 1975;36:938–953. Gustafsson AB, Gottlieb RA. Heart mitochondria: gates of life and death. Cardiovasc Res. Res 2008;77:334–343. 200 008;77:334–343. Gibbs CL, Metabolism. G ibbs C ib L, Loiselle DS. Cardiac Basal Me etaabolism. Jpn J Ph Physiol. l 2001;51:399–426. Ebus Stienen Origin ATPase Ebu buss JP bu JP, St Stie iene ie nen ne n GJ GJ.. Or Orig igin ig in ooff cconcurrent onc ncurrrentt A nc TP Pas asee activities acti ac t viiti tiess in in skinned skin sk inne in n d cardiac card ca rdia rd iacc ia trabeculae rat. Physiol. trabeccullae from from m rat t. J Physi iol.. 1996;492(pt 1996;49 92(p pt 33):675–87. ):67 675– 67 5–8 5– 87 87. 20 Table 1. Hemodynamics Healthy hearts group Post-ischemic LV dysfunction group Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Baseline Healthy OM Baseline Post-ischemia OM Mean arterial pressure, mmHg 86 ± 7 85 ± 9 83 ± 8 84 ± 6 71 ± 6* ‡ 65 ± 5 * ‡ Mean pulmonary arterial pressure, mmHg 21 ± 2 22 ± 2 22 ±2 20 ± 1 25 ± 2* 25 ± 4 * Heart rate, beats/min 81 ± 14 91 ±14 94 ±15 82 ± 13 120 ± 31* ‡ 118 ± 33 * ‡ Cardiac output, put, l/min 2.56 ± 0.29 2.80 ± 0.37 2.83 ± 0.52 2.72 ± 0.37 2.85 ± 0.46 33.10 .100 ± 0. .1 0.53 53 * Systemic vascular asculaar yn · s resistance, dyn 2509 25 0 ± 309 09 09 2273 ± 332 2190 ± 323 * 2321 ± 423 18 1880 880 ± 569* 1570 ± 509 *† ‡ Stroke work, k, mmHg · ml 2770 ± 531 2686 26686 ± 6652 522 26266 ± 791 22828 8288 ± 4465 82 65 17 1752 752 2 ± 33 339* 39* ‡ 18 1800 800 ± 4588 * ‡ Stroke volume, me, ml 32 ± 5 31 ± 6 31 ± 8 3344 ± 6 25 ± 7* 28 ± 8 * End-diastolic volume, ml 69 ± 10 70 ± 10 61 ± 12 *† 68 ± 4 68 ± 16 56 ± 17 *† End-systolic volume, ml 37 ± 5 39 ± 6 30 ± 8 † 34 ± 3 42 ± 12 28 ± 9 † Ejection fraction, % 46 ± 2 44 ± 4 51 ± 8 50 ± 5 39 ± 7* 54 ± 12 † Coronary blood flow, ml/beat 1.25 ± 0.29 1.28 ± 0.27 1.68 ± 0.53 *† 1.30 ± 0.33 1.39 ± 0.35 1.65 ± 0.31* † Tau, msec 31 ± 2 31 ± 2 37 ± 4*† 32 ± 1 35 ± 8 46 ± 12*† Diastolic filling time, msec 512 ± 110 442 ± 88 390 ± 75*† 494 ± 112 311 ± 106* ‡ 280 ± 107 * ‡ 5 /cm 21 Systolic ejection time, msec 242 ± 8 229 ± 14 266 ± 22 † 255 ± 15 224 ± 30* 270 ± 45 † End-diastolic pressure, mmHg 13 ± 3 11 ± 2 10 ± 2 * 13 ± 3 13 ± 7 9 ± 4 *† dPdtmax, mmHg/s 1523 ± 344 1773 ± 613 2157 ± 630*† 1500 ± 249 1460 ± 429 1918 ± 734 dPdtmin, mmHg/s 2656 ± 510 2912 ± 738 3173 ± 903 2743 ± 416 2114 ± 881 1929 ± 587* Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Hemodynamic data were assessed in both the healthy and the post-ischemic LV dysfunction group (n=7 in both groups, except dPdtmax/min, and tau (both n=6)). There were three post-ischemia hemodynamic measurements in each group. Baseline, with or without post ischemiaa and subsequently after administration of Omecamtiv (OM). dPdt max/min denotes peak ak ppositive osit os itiv it ivee iv and negative pressure. time an d ne nega gati ga t vee dderivative erivative of left ventricular pressu sure su re. Systolic ejection tim re me ddenotes me enotes time dP/dt max Diastolic filling time refer time minus systolic between dP P/d /dt ma ax an and d mi min. n. D iast ia stol st olic ol i fil lli ling ng tim me refe ferr to fe to ccardiac ardiiac a ccycle y le tim yc me mi minu nuss sy nu syst sto st olic ic ejection e ection time. ej tim i e. * p<0.05 p< <0.0 05 vs. vs. s baseline, basellin ine,, † p<0.05 p<0 0.0 05 vs. vss. before befo forre re drug, dru rug, analysed anaaly ysed d by by one-way onee-way y repeated repeeatted measures ANOVA. and ‡ p<0. .05 bbetween etween hhealthy ealt ea lthy lt hy aand nd ppost-ischemic ost-ischemic LV dysfunction group p<0.05 meas me asur urem emen ents ts,, an anal alys y ed bby ys y li line near ar m ixed ix ed m odel od el aanalysis. naly na ly ysi sis. s. measurements, analysed linear mixed model 22 Table 2. Left ventricular energetics Healthy Post-ischemic LV dysfunction Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 A Healthy Healthy +OM Postischemia Post-ischemia +OM Pressure-volume area, J/beat/100g 0.78 ± 0.15 0.53 ± 0.15 0.49 ± 0.11 0.46 ± 0.16 MVO2, J/beat/100g 1.25 ± 0.29 1.38 ± 0.39 1.07 ± 0.29 1.33 ± 0.30 Y-intercept 0.22 ± 0.12 0.29 ± 0.14 * 0.31 ± 0.09 0.38 ± 0.12* Slope 1.58 ± 0.38 2.58 ± 0.60* 1.57 ± 0.36 2.30 ± 0.78* R2 0.97 ± 0.02 0.95 ± 0.02 0.95 ± 0.01 0.96 ± 0.02 Stroke work, J/beat/100g 0.49 ± 0.11 0.40 ± 0.13 0.27 ± 0.04 0.28 ± 0.08 08 MVO MV O2, J/beat/100g J beeat/1 J/ /1 100 00g 1.25 ± 0.29 1. 1 38 ± 0.39 1.38 1.07 ± 0.299 1.33 ± 0.30 Y-intercept 0. .32 ± 0.09 0.09 0.32 0.4 40 ± 0.16* 0..16 .16* 6* 0.40 0.3 36 ± 0.1 11 0.36 0.11 47 ± 0.12* 0.1 12* 0.47 Sl S ope Slope 2.11 2. 1 ± 0.37 0.37 2.9 98 ± 0.42* 0.42 42* 2* 2.98 2.6 63 ± 0.61 2.63 3.32 32 ± 1.000 R2 0.98 0. 9 ± 0.01 0.0 01 0.96 0. 96 ± 00.02 .0 02 0.96 6 ± 0.02 0.96 ± 0.0 0.03 03 B d lleft f h ddata show h the h relationship l i hi bbetween myocardial di l oxygen consumption i ((MVO2) and The ventricular (LV) mechanical work over a large range of workloads for both the healthy (n=7 pigs) and the post-ischemic LV dysfunction (n=7) group. LV mechanical work is presented in 1A) as pressure-volume area and in 1B) as stroke work. Y-intercept indicates MVO2 for myocardial processes not related to pump function (unloaded MVO2), while the 1/slope indicates the energy cost for contractile work (contractile efficiency. R2 is the coefficient of determination. * p<0.05 vs. Healthy/Post-ischemic LV dysfunction, analysed by linear mixed model analysis. 23 Figure Legends Figure 1. Outline of experimental protocols. Figure 2. Dose-response protocol in pigs (n=3). Omecamtiv (2,25 mg/min) was given continuously and hemodynamic recordings where obtained at different timepoints during a 16 min period. The accumulated OM dose at these timepoints is given on the x-axis. Data (means ± SD) are reported as % change from baseline. Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Figure 3. Illustration of typical pressure-volume loops showing the effect of Omecamtiv on left ventricular (LV) function. The loops are based on mean volumes and pressure given giv ven in Table 1. Left panel shows loop from healthy animals, and right panel from animals animal alss wi al with th ppostostos tischemic LV isch is chem ch emic em ic L V dy ddysfunction. sfunction. F gure 44.. Example Fi Ex xam mplee of of twoo experiments exp x erim men ntss showing sho owing the th he relation rel elaatio at on between betw ween left lefft ventricular ven ntrric i ular arr (LV) (LV LV) Figure mechanical work and myocardi dial di al ooxygen xy yge g n co cons sum umpt p io ion. n LV mechanical work is presented in n. myocardial consumption. the up ppe p r pa ppanel nel as ppressure-volume ressure-volu l me area (PVA) (PVA)) and (PVA d iin n tthe h lower he lower panel panel as stroke work (SW). ( W)). (S upper Left panels are data from one healthy pig and right panels are data from one pig with postischemic LV dysfunction. Data are obtained at various workloads before (ż) and after (•) infusion of Omecamtiv (OM). Y- Intercept represents unloaded MVO2, i.e., energy used for excitation-contraction coupling and basal metabolism. 1/slope of the regression line represents the contractile efficiency of the heart. Figure 5. Pooled scatters of LV mechanical work-MVO2 relationship from all experiments. Left panels are from the healthy pigs (n=7) and right panels are from pigs with post-ischemic LV dysfunction (n=7). Omecamtiv (OM) impairs cardiac efficiency as displayed by a 24 significant increase in Y-intercept and slope in all panels, except for only increased Yintercept in the lower right panel. * p<0.05 vs. No drug for Y-intercept, and † p<0.05 vs. No drug for slope (Linear mixed model analysis). See figure 4 for an extended legend. Figure 6. Dot plots presenting data inn all panels. Mean values are presented as circles crossed by a horizontal line. Panel A: Substrate oxidation rate of glucose and free fatty acids (FFA) before and after Omecamtiv (OM) in healthy pig hearts to the left and post-ischemic pig hearts to the right. Means compared by Wilcoxon signed-rank test. Panel B: Myocardial Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 uptake rate of glucose, lactate and FFA in the same animals as panel A (Wilcoxon signed rank test). Panel C: Glucose and FFA oxidation rate assessed in ex vivo working micee hearts treated with OM (n=8) and in time-matched controls (n=6). White columns are base ba seli se line li ne/v ne /veh /v hic icle l ; black columns are OM. * p < 0.05 0.05 0 vs. pre-administrat tio ion n of OM or vehicle baseline/vehicle; pre-administration on signed sig ign ned d rank rank ra nk test). test) t).. Panel t) Pane Pa nell D: In ne In vitro vit i ro resp piraati tion on of of mitochondria miito toch hondr ondr dria ia isolated isoola late ted te d fr from om m icee ic (Wilcoxon respiration mice h arts inc he cub u ateed with with h OM M tto o the le left,, aand nd AD ADP/o oxy ygen en rratio atiio (P/O O rratio) atio) to o th he right. ri M eaanss hearts incubated ADP/oxygen the Means illco c xon test t. compared by Mann-Whitney W Wilcoxon test. Figure 7. MVO2 in ex vivo mice hearts retrograde perfused in Langendorff mode. Dot plots presenting data inn all panels. Mean values are presented as circles crossed by a horizontal line. Left Panel; There is significant increase in unloaded MVO2 and oxygen consumption from basal metabolism in hearts treated with Omecamtiv (n=17) compared to time-matched controls (n=13). Oxygen cost for excitation-contraction (E-C) coupling was unaffected by OM. Right Panel; Addition of 2,3-butanedione monoxime (BDM) abolished surplus basal MVO2 in the OM treated hearts (n=7) while no effect of BDM was seen in the controls (n=7). * p<0.05 vs. Omecamtiv, † p<0.05 vs. without BDM, analysed by Mann-Whitney Wilcoxon test. 25 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 The Myosin Activator Omecamtiv Mecarbil Increases Myocardial Oxygen Consumption and Impairs Cardiac Efficiency Mediated by Resting Myosin ATPase Activity Jens Petter Bakkehaug, Anders Benjamin Kildal, Erik Torgersen Engstad, Neoma Boardman, Torvind Næsheim, Leif Rønning, Ellen Aasum, Terje S. Larsen, Truls Myrmel and Ole-Jakob How Downloaded from http://circheartfailure.ahajournals.org/ by guest on June 11, 2017 Circ Heart Fail. published online May 29, 2015; Circulation: Heart Failure is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2015 American Heart Association, Inc. All rights reserved. Print ISSN: 1941-3289. Online ISSN: 1941-3297 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circheartfailure.ahajournals.org/content/early/2015/05/29/CIRCHEARTFAILURE.114.002152 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation: Heart Failure can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. 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