[Ca ] to left ventricular relationships before and after ischemia in

Cardiovascular Research 59 (2003) 912–925www.elsevier.com/ locate/cardiores

21C ardiotonic drugs differentially alter cytosolic [Ca ] to leftventricular relationships before and after ischemia in isolated guinea

qpig heartsa,f a a,e a,bQun Chen , Amadou K.S. Camara , Samhita S. Rhodes , Matthias L. Riess ,

a a,b,c,d,e ,*Enis Novalija , David F. StoweaDepartment of Anesthesiology, Anesthesiology Research Laboratories, The Medical College of Wisconsin, Milwaukee, WI 53226,USA

bDepartment of Physiology, Anesthesiology Research Laboratories, The Medical College of Wisconsin, Milwaukee, WI 53226,USAcCardiovascular Research Center, Anesthesiology Research Laboratories, The Medical College of Wisconsin, Milwaukee, WI 53226,USA

dResearch Service, Veterans Affairs Medical Center, Milwaukee, WI 53295,USAeDepartment of Biomedical Engineering, Marquette University, Milwaukee, WI 53223,USA

fXuzhou Medical College, Xuzhou 221002,China

Received 24 March 2003; received in revised form 17 July 2003; accepted 21 July 2003

Abstract

21Objective: Cardiotonic agents may differentially alter indices of the cytosolic [Ca ] / left ventricular pressure (LVP) relationship when21given before and after ischemia. We measured and calculated systolic–diastolic [Ca ], systolic–diastolic LVP, velocity ratios (VRs)

21 21 21d[Ca ] /dt to dLVP/dt (VR ), d[Ca ] /dt to dLVP/dt (VR ), and area ratio (AR, area Ca ] /area LVP per beat) beforemax max max min min min

and after 30 min global ischemia in guinea pig hearts.Methods: Hearts were perfused with levosimendan, dobutamine, dopamine, or21 21digoxin. Ca transients were recorded by indo-1 fluorescence via a fiber optic probe placed on the LV free wall. [Ca ] /LVP loops were

21acquired by plotting LVP time as a function of [Ca ] at multiple time points during the cardiac cycle.Results: Ischemia reperfusion21 21increased [Ca ] and decreased contractility and relaxation and produced a flatter and broader [Ca ] /LVP loop. All drugs shifted the

21 21[Ca ] /LVP loop rightward and upward when given before and after ischemia. Dobutamine increased [Ca ] and contractility more than21other drugs. Digoxin increased [Ca ] the least but increased contractility similar to dopamine and levosimendan. Before ischemia

dopamine and digoxin both decreased VR and VR , whereas dobutamine increased VR , but not VR , and levosimendan had nomax min min max

effect on VR. VR and VR were markedly elevated after ischemia, but again decreased with dopamine and digoxin; dobutaminemax min

again increased VR , but not VR , and levosimendan decreased both VR and VR . Before ischemia dopamine and digoxin bothmin max max min

decreased AR, dobutamine increased AR, and levosimendan had no effect; after ischemia AR was markedly elevated but dopamine anddigoxin decreased AR, dobutamine increased AR, and levosimendan decreased AR.Conclusion: Although each drug enhanced

21 21contractility and relaxation both before and after ischemia by increasing cytosolic [Ca ] and Ca flux, dopamine and digoxin improved,21and dobutamine worsened responsiveness to Ca , i.e., velocity ratio and area ratio, whereas levosimendan had no net effect before

ischemia but improved responsiveness after ischemia. 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Keywords: Calcium-pressure loops; Cardiotonic drugs; Digoxin; Dopamine; Dobutamine; Guinea pig

qPortions of this work have appeared in abstract formBiophys J 1 . Introduction2002;82:66A, 653A and 654A;FASEB J 2002;16:A857;Anesthesiology2001;95:A622.

Positive inotropic agents such as catecholamines are*Corresponding author. Medical College of Wisconsin, MilwaukeeRegional Medical Center, M4280, 8701 Watertown Plank Road, Mil- frequently used to treat acute heart failure or to facilitatewaukee, WI 53226, USA. Tel.:11-414-456-5722; fax:11-414-456- contractility and relaxation in stunned hearts after cardiac6507.

E-mail address: [email protected](D.F. Stowe). Time for primary review 21 days.

0008-6363/03/$ – see front matter 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.doi:10.1016/S0008-6363(03)00524-8

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913Q. Chen et al. / Cardiovascular Research 59 (2003) 912–925

surgery. Most cardiotonic agents in current use, including equilibrated with 97% O , 3% CO and containing, in2 21 1 21 21 2cardiac glycosides, catecholamines and phosphodiesterase mmol / l, Na 137, K 4.5, Mg 2.4, Ca 1.25, Cl 134,

2 2(PDE) inhibitors, ultimately act to enhance contractility HCO 15.5, H PO 1.2, glucose 11.5, pyruvate 2, man-3 2 4

and relaxation by increasing the cyclic change in cytosolic nitol 16, EDTA (ethylenediaminetetraacetic acid) 0.05,21 21[Ca ] during the cardiac cycle[1–3]. However if cyto- probenecid 0.1, and insulin 5 units / l. Extracellular [Ca ]

21solic [Ca ] exceeds an optimum there is no further was half-normal so that positive inotropic drugs had aincrease in contractility and diastolic tonus and better range of contractile responses starting at a lowerdysrhythmias can occur[4]. In contrast to these commonly LVP.used cardiotonic agents, so-called calcium sensitizers, such Phasic LVP was measured with a transducer connectedas levosimendan[1,4,5], are believed to increase contrac- to a thin, saline-filled latex balloon inserted into the lefttility in part by improving responsiveness of the contractile ventricle through the mitral valve from a cut in the left

21apparatus to Ca . Specifically, levosimendan is thought to atrium. Developed LVP was defined as systolic LVP minus21bind to troponin C in a Ca -dependent manner to stabilize diastolic LVP. The first derivative of LVP, dLVP/dt, was

the Ca–troponin C bond and enhance contractility at a derived on line and maximum and minimum values21given [Ca ] [6,7]. Levosimendan also opens myocyte determined. Balloon volume was adjusted to maintain a

K channels[8], which may lead to a cardioprotective diastolic LVP of zero mmHg during the initial controlATP21effect, and may increase cAMP and myocyte [Ca ] as an period so that any increase in diastolic LVP indicated an

inotropic mechanism by selectively inhibiting PDE III[9]. increase in left ventricle (LV) wall stiffness or diastolic21Contractile responsiveness to Ca can be altered by contracture. Two pairs of bipolar electrodes were placed in

pharmacologic and pathologic conditions. Hypothermia each heart to monitor intracardiac electrograms from which[10], b-adrenergic agonists[11], PDE inhibitors [3], and spontaneous atrial heart rate (HR) was determined from theischemia reperfusion injury[12–15]each decrease contrac- right atrial beat-to-beat interval.

21 1 Coronary inflow was measured at constant temperaturetile responsiveness to Ca . Digoxin, a sarcolemmal Na ,1 (37.1 8C) with a self-calibrating in-line, ultrasonic flowK -ATPase inhibitor, dobutamine, ab -receptor activator1

1 1 21meter. CF and coronary effluent Na , K , Ca , PO ,[16,17], dopamine, ab and dopamine receptor activator 21

PCO , and pH were measured off-line with an intermittent-[18], and levosimendan[19,20] are variably effective for 2

ly self-calibrating analyzer system (Radiometertreating acute and chronic heart failure and stunnedCopenhagen ABL 505, Copenhagen, Denmark). Coronarymyocardium [3,18,21]. However, if one of these drugs

21 sinus effluent was collected through a cannula inserted intoincreases [Ca ] above a threshold, and especially if21 the right ventricle through the pulmonary artery after[Ca ] is already elevated due to myocyte injury, cardiac

ligating the venae cavae. Coronary outflow (coronaryfunction will most likely be impaired. Because the selectedsinus) O tension was also measured continuously on-linedrugs are known to enhance contractility by different 2

with an O Clark-type electrode placed in the pulmonarymechanisms, we postulated that they differentially alter the 221 artery. Because myocardial metabolism is altered bycyclic [Ca ] / left ventricular pressure (LVP) relationship

cardiotonic drugs and ischemia reperfusion[18,21,23],weand have different effects on this relationship after is-also measured myocardial O consumption (MVO ) andchemia reperfusion injury. To test this we simultaneously 2 2

21 cardiac efficiency. MVO was calculated as (coronarymeasured cytosolic [Ca ] and LVP in guinea pig isolated 2

flow/g)3(arterial PO2venous PO )324ml O /ml at 760hearts and constructed several indices of this relationship 2 2 2

mmHg; cardiac efficiency was calculated as developedto explore and compare differences.LVP3HR/MVO .2

2 .2. Measurement of cytosolic and non-cytosolic free212 . Methods Ca in isolated hearts

2 .1. Isolated heart preparation and measurements We have described details of our method to monitor andcalibrate indo-1 fluorescence signals as a measure of

21The investigation conformed to theGuide for the Care cytosolic [Ca ] in the left ventricle of isolated heartsand Use of Laboratory Animals (National Institutes of [10,14,15,22,23,25–27].Experiments were carried out in aHealth No. 85-23, revised 1996). Prior approval was light-blocking Faraday cage. Briefly, the heart was partial-obtained from the Medical College of Wisconsin Animal ly immobilized by hanging it from the aortic cannula, theStudies Committee. Our preparation and measurements pulmonary artery catheter, and the left ventricular balloonhave been described in detail[10,14,15,22–27].In brief, catheter. The heart was immersed continuously in the bathhearts were isolated from anesthetized guinea pigs (300– at 378C. The distal end of a trifurcated silica fiberoptic350 g) and perfused with crystalloid solution by the cable was placed gently against the LV epicardial surfaceLangendorff method. Hearts were perfused at 55 mmHg through a hole in the bath to excite the tissue with lightand 378C with a modified Krebs–Ringer (KR) solution filtered at 350 nm and to record emitted light filtered at

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Q. Chen et al. / Cardiovascular Research 59 (2003) 912–925914

385 and 456 nm. A rubber O ring was placed over the 2 .3. Protocolfiberoptic tip to seal the hole and netting was appliedaround the heart for optimal contact with the fiber optic tip. Forty hearts were divided randomly and equally amongBackground auto fluorescence was determined for each five groups: non-treated ischemia controls, dobutamine,heart after initial perfusion and equilibration at 378C. dopamine, digoxin and levosimendan groups. Each experi-

Each heart was then loaded with indo-1 AM for 20–30 ment lasted 210 min (Fig. 1). Initial background (beforemin with the re-circulated KR solution at a final indo-1 indo-1 loading) measurements were obtained after 30 minAM concentration of 6mM. Residual interstitial indo-1 of stabilization. After loading and residual washout ofAM was washed out by perfusing the heart with standard indo-1, on-line recordings were sampled and stored every 1perfusate for at least another 20 min. Additional experi- to 5 min during drug perfusion, normothermic ischemiaments (three hearts for each of the five groups) were and reperfusion. The 30-min period of ischemia wasundertaken to assess changes in tissue autofluorescence effected by clamping the coronary inflow cannula. Drugsdue to changes in the redox state (primarily a measure of were perfused at the approximate ED concentrations for50

NADH) and drug autofluorescence. None of the drugs LVP established from pilot studies. Dobutamine (4mM),exhibited a significant change in autofluorescence; is- dopamine (8mM), digoxin (1 mM) or levosimendan (1chemia and reperfusion as noted previously, caused anmM) was perfused for 2 min 30 min before ischemia (at 80increase and decrease in NADH as we have reported min) in each of the drug groups and again for 2 min at thepreviously [28], these values were subtracted from the same concentration beginning at 30 min reperfusion (atbasal fluorescent signal obtained with indo-1. 170 min). Each drug produced a steady-state and sub-

The fluorescence emissions at 385 and 456 nm (F and maximal increases in LVP beginning by at least the first385

F ) were recorded using a modified luminescence spec- minute of perfusion and lasting over the second minute.456

trophotometer (SLM Aminco-Bowman II, Spectronic In- All measured variables returned statistically to controlstruments, Urbana, IL, USA). The LV region of the heart values between drug infusions. LVP and dLVP/dt, cor-was excited with light filtered at 350 nm through the onary flow, and coronary sinus oxygen tension (PO ) were2

in-going fibers of the optic bundle. The arc lamp shutter recorded continuously. All analog signals were digitizedwas opened only for 2.5 s recording intervals to prevent (PowerLab /8 SP; ADInstruments, Castle, Hills, Aus-

photobleaching. Emission fluorescence was collected by tralia) and recorded at 125 Hz (Chart & Scope v3.63,fibers of the remaining two limbs of the cable and filtered ADInstruments, Castle Hills, Australia) on Power

by square interference filters at 385 nm and 456 nm. The Macintosh G4 computers (Apple, Cupertino, CA, USA)F /F ratio remains stable during the 3-h course of for later analysis using MATLAB (Mathworks, Natick,385 456

these studies indicating no change in effective measured MA, USA) and Microsoft Excel (Microsoft, Redmond,21[Ca ]. After indo-1 loading developed LVP was not WA, USA) software.

21significantly altered in non-ischemic hearts over this time We obtained simultaneous [Ca ] and LVP recordings atperiod. designated time points. Exposure at the 350 nm excitation

Fig. 1. Experimental protocol. Hearts were perfused and stabilized with Krebs–Ringer’s (KR) solution for 30 min, loaded with indo-1 AM for 30 min, andresidual indo-1 AM washed out for 20 min. In treated groups, 8mM dopamine, 4mM dobutamine, 1mM levosimendan, or 1mM digoxin was perfused for2 min before ischemia (time 80 min) and at 30 min reperfusion (time 170 min). CON hearts were perfused only with KR at the same time points. Heartswere subjected to 30 min global ischemia at 378C (time 110 to 140 min) and reperfused for 60 min (time 140 to 200 min). MnCl (50 mmol / l) was2

21perfused at the end (time 200–210 min) to quench non-cytosolic Ca .

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wavelength light was 55 s. Customized software was ischemia.Table 2 summarizes drug-induced changes indeveloped in MATLAB for off-line signal processing of coronary flow, O consumption and cardiac efficiency2

recorded data. LVP and fluorescence data were digitally before and after ischemia. Each drug increased heart rate,21 21low-pass filtered using a fourth-order bi-directional Butter- systolic [Ca ], diastolic [Ca ] and systolic–diastolic

21worth filter at 25 Hz. Data were analyzed for peak systolic, [Ca ] compared to baseline and 30 min reperfusionpeak diastolic, and systolic–diastolic LVP (mmHg) and except for digoxin, which did not increase systolic or

21 21 21 21[Ca ] (nM). First derivatives of [Ca ] (d[Ca ] /dt) and systolic–diastolic [Ca ] after ischemia. There were fewLVP (dLVP/dt) were derived on-line and values for significant differences in the relative changes from controls

21 (30 min reperfusion and baseline) induced by drugs afterd[Ca ] /dt and dLVP/dt (contractility and peak ratemax max21 21 ischemia than before ischemia.of Ca influx), as well as d[Ca ] /dt and dLVP/dtmin min

21 Each drug increased systolic LVP and systolic–diastolic(relaxation and peak rate of Ca efflux) were determined.21 LVP, and decreased diastolic LVP compared to baselineArea [Ca ] and area LVP (systolic–diastolic time inte-

and 30 min reperfusion, but the relative changes from theirgral), i.e., total LV pressure (potential work) and total21 30 min reperfusion or baseline values were comparable orcytosolic [Ca ] averaged during one beat were computed.

21 smaller for systolic LVP when drugs were given afterThe index of d[Ca ] /dt to dLVP/dt (velocitymax max21 ischemia than before ischemia. Each drug increased cor-ratio,VR ), was used to assess the fastest cytosolic Camax

onary flow before and after ischemia expect for digoxininflux to generate maximal contractility, and the index of21 which did not alter flow; dobutamine increased coronaryd[Ca ] /dt to dLVP/dt (velocity ratio, VR ), wasmin min min

21 flow more after ischemia than before ischemia. Each drug,used to assess the fastest cytosolic Ca efflux that21 except for digoxin, increased MVO . All drugs increasedallowed maximal relaxation. The index of area [Ca ] / 2

cardiac efficiency compared to baseline and 30 minarea LVP (area ratio, AR) was used to assess the net21 reperfusion, respectively; this increase was greater byamount of cytosolic free Ca moved in and out to

dobutamine largely because of the higher heart rate.generate the cardiac (potential) work over one beat.Averaged inflow (arterial) pH was 7.4560.01, pO wasConcentration–response curves were not obtained in this 2

695612 mmHg, and pCO was 2062 mmHg. Controlstudy, so direct comparisons among drugs for a given 2

venous (v) pH , pO , and pCO values were 7.3660.01,variable were not considered valid. However, the velocity v 2v 2v

250616, and 3062, respectively. All drugs decreased pHand area ratios were utilized to compare responses to drugs v

and pO , and increased pO , 30 min before and afterbecause the ratios normalized the individual values for 2v 2v21 ischemia but the effect was greatest (P,0.5) forCa and LVP for each drug. Ratios were calculated from

dobutamine before (7.2260.02, 149612, 3462) and afterraw rather than from grouped data.(7.2560.07, 112618, 3664) ischemia. There were no

1appreciable differences among the drugs for venous [Na ],2 .4. Statistical analysis 1 21[K ], or [Ca ].

21Fig. 2displays average [Ca ] /LVP loops before 30 minAll data were expressed as mean6standard error of

ischemia (80 min) and at 30 min reperfusion in absencemean (S.E.M.). One-way analysis of variance for repeated

and presence of the four positive inotropic drugs. At 30 measures (Super Anova 1.11 software for Macintoshmin reperfusion, the cardiac loop was lower, smaller and

from Abacus Concepts, Berkeley, CA, USA) was used toshifted slightly rightward than before ischemia. Before

assess within group differences over time at selected timeischemia each drug increased loop size and shifted the loop

points: 80 min (baseline) versus peak response of drugsincreasingly rightward in the order digoxin, dopamine,

given before ischemia, reperfusion 2 min (142 min), 30levosimendan, dobutamine; dobutamine increased loop

min (170 min), and peak response of drugs given after 30height (systolic LVP) more than other drugs. After is-

min reperfusion (Fig. 1). Two-way analysis of variancechemia loop height was increased by each drug to approxi-

was used to assess among group differences at baseline, 30mately the level observed before ischemia without drugs.

min reperfusion and the peak response of drug givenFig. 2A shows that after ischemia dopamine continued to

before and after ischemia. IfF-values for the analysis ofshift the cardiac loop rightward but it was smaller than

variance were significant, Tukey’s multiple-comparisonbefore ischemia.Fig. 2Bshows that dobutamine (DB) after

post-hoc tests was used to differentiate within or amongischemia, as before, shifted the cardiac loop far rightward

group differences. Differences among means were consid-compared to drug-free controls but that the loop was much

ered significant whenP,0.05.smaller (flatter) after ischemia.Fig. 2C shows thatlevosimendan (LV) after ischemia, as before, shifted thecardiac loop rightward but that it was smaller after

3 . Results ischemia.Fig. 2D shows that digoxin (DG) after ischemiashifted the cardiac loop only slightly rightward compared

Table 1summarizes drug-induced changes in heart rate, to drug-free baseline and reperfusion controls.21 21cytosolic [Ca ], and mechanical function before and after Fig. 3 displays maximal time derivatives of Ca and

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T able 121Cardiac effects of different positive inotropic agents, given before ischemia (BI) and after ischemia (AI), on cytosolic [Ca ] and mechanical indices in

isolated guinea pig hearts

Group Baseline Drug BI RP 2 min RP 30 min Drug AI(units) (% increase) (units) (units) (% increase)

21Heart rate (beats min )CON 22065 261 148624* 22367 161DA 21166 2366† 155617* 21666 1763‡DB 21366 4862† 152614* 20666 5065‡LV 20966 1362† 157613* 21968 965‡DG 21065 1162† 142618* 21763 1167‡

21Systolic [Ca ] (nM)CON 242620 764 646635* 260629 2565DA 226624 2565† 526646* 242624 963‡DB 21666 134613† 562626* 240620 128622‡LV 238622 48612† 576628* 256622 39616‡DG 232614 1866† 610635* 242618 669

Systolic LVP (mmHg)CON 4562 562 3165* 2962* 063DA 4963 3066† 3564* 3562* 1162‡§DB 4763 95616† 3563* 2962* 4467‡§LV 4862 4167† 3164* 2861* 3265‡§DG 5166 2862† 3062* 3263* 1962‡§

21Diastolic [Ca ] (nM)CON 12464 465 208614* 100612 2564DA 12068 862† 156613* 94612 263DB 11262 3466† 164616* 90610 4269*LV 11266 762† 172622* 10064 9612DG 12268 363 182620* 92612 3610

Diastolic LVP (mmHg)CON 961 2967 1662* 1562* 21168‡DA 861 22067† 2163* 1762* 21463‡DB 961 22566† 2264* 1862* 22664‡LV 962 22367† 2162* 1862* 21362‡DG 861 22367† 1961* 1762* 21365‡§

21Systolic–diastolic [Ca ] (nM)CON 130618 964 378629* 262628* 267DA 118612 4469† 306628* 154612* 1464‡§DB 10464 245635† 356628* 152616* 179630‡LV 126618 67615† 314627* 170612* 52619‡DG 11068 34610† 378629* 150610* 9613

Systolic–diastolic LVP (mmHg)CON 3661 2462 1565* 1661* 364DA 3963 61612† 1463* 1862* 3569‡DB 3763 157627† 1362* 1762* 14368‡LV 3662 84619† 1063* 1163* 71613‡DG 4066 5666† 1462* 1563* 70610‡

Values are means6S.E. for baseline, RP 2 min and RP 30 min; Values (in bold) are percent change (means6S.E.) for drug BI vs. baseline and drug AIvs. RP 30 min;n58 for each group; baseline, perfusion 80 min; RP, reperfusion; drug BI is drug before ischemia; drug AI is drug given drug afterischemia; CON, control; DA, dopamine; DB, dobutamine; LV, levosimendan; DG, digoxin.* P,0.05 vs. baseline within each group; †P,0.05, drug BI vs. baseline; ‡P,0.05, drug AI vs. RP 30 min; §P,0.05, drug AI vs. drug BI.

21LVP, d[Ca ] /dt (A) and dLVP/dt (B), at baseline Part of this effect was due to increased heart rate by thesemax max

before ischemia and during peak responses to drugs before drugs, especially dobutamine. After 2 min reperfusionischemia, at 2 and 30 min reperfusion, and during peak dLVP/dt decreased markedly in each group comparedmax

21responses of drugs after 30 min reperfusion. Before to baseline values, while d[Ca ] /dt was higher thanmax

ischemia dLVP/dt increased with each drug while baseline values; by 30 min reperfusion dLVP/dt wasmax max21 21d[Ca ] /dt increased with each drug except digoxin. equivalent to baseline values, while d[Ca ] /dt re-max max

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T able 2Cardiac effects of different positive inotropic agents, given before ischemia (BI) and after ischemia (AI), on metabolic indices in isolated guineapig hearts

Group Baseline Drug BI RP 2 min RP 30 min Drug AI(units) (% increase) (units) (units) (% increase)

Coronary flow (ml /min/g)CON 9.360.3 2362 7.660.7* 5.760.4* 2662DA 10.560.6 1767† 7.560.7* 6.960.5* 1364‡DB 10.460.9 1866† 8.960.6* 5.860.4* 4965‡§LV 10.960.6 1166† 7.960.6* 5.660.4* 1463‡DG 10.560.6 2464 7.760.5* 5.860.4* 165

21 21MVO (0.1 ml min g )2

CON 11667 565 94614 7069* 2565DA 132610 1568† 112612 9969* 1966‡DB 110610 49612† 109616 7666* 4664‡LV 11466 41610† 9368 6166* 36610‡DG 120614 13611 10267 7267* 13611

21 21Cardiac efficiency (mmHg beat 0.1ml g )CON 5164 1366 1368* 4666 19611DA 4766 2066† 2367* 4064 44613‡DB 5969 147626† 3061* 4761 99616‡LV 4762 46618† 1867* 4867 2667‡DG 5460 48610† 1767* 5168 39614‡

MVO , Myocardial O consumption; cardiac efficiency5LVDP3HR/MVO ; temperature was 378C. Values are means6SE.2 2 2

* P,0.05 vs. baseline within each group; †P,0.05, drug BI vs. baseline; ‡P,0.05, drug AI vs. RP 30 min; §P,0.05, drug AI vs. drug BI. SeeTable 1for other details.

mained higher than baseline values. Each drug given after sponsiveness by these drugs after ischemia. Dobutamine21ischemia increased d[Ca ] /dt and dLVP/dt com- did not change VR before and after ischemia comparedmax max max

pared to 30 min reperfusion, but increases in dLVP/dt to the baseline and 30 reperfusion controls; this indicatesmax

were much smaller than before ischemia.Fig. 4 displays no change in contractile responsiveness.21d[Ca ] /dt (A) and dLVP/dt (B) at the same time Changes in VR (Fig. 6) were qualitatively similar tomin min min

points asFig. 3. These changes were inverse but quali- findings ofFig. 5 except that VR was relatively greatermin21tatively similar to findings of d[Ca ] /dt and dLVP/ than VR after ischemia and that dobutamine increasedmax max

21dt , except that at 2 min reperfusion d[Ca ] /dt was VR before and after ischemia. In general, this indicatesmax min min21 21about four times greater than d[Ca ] /dt , i.e., velocity worsened relaxation responsiveness to Ca efflux bymax

21 21of Ca efflux was slower than velocity of Ca influx. dobutamine, improved relaxation responsiveness to dopa-21Figs. 5–7display the ratios of d[Ca ] /dt to dLVP/ mine and digoxin before ischemia, and to dopamine,max

21dt (VR ), d[Ca ] /dt to dLVP/dt (VR ), and digoxin and levosimendan after ischemia.Fig. 7 showsmax max min min min21area [Ca ] /area LVP (AR) at baseline, during peak decreased AR by dopamine and digoxin before ischemia

response of drugs given before ischemia, at 30 min and by dopamine, digoxin and levosimendan after is-reperfusion, and during peak response of drugs given after chemia. Dobutamine increased this ratio before and after30 min reperfusion. These ratios allow a qualitative ischemia compared to its baseline and 30 min reperfusion

21comparison of the relative effects of each drug before and controls. This indicates the net amount of Ca moved inafter ischemia.Fig. 5 shows that dopamine and digoxin and out of the myoplasm to produce (potential) contractiledecreased VR , whereas dobutamine and levosimendan work during a cardiac cycle is improved by dopamine andmax

did not affect the VR before ischemia compared to digoxin before ischemia, and by these drugs andmax

baseline values; this indicates improved contractile respon- levosimendan after ischemia, and worsened by dobutamine21siveness to Ca influx to dopamine and digoxin.VR at before and after ischemia.max

30 min reperfusion was much higher than before ischemiain each group, indicating a worsened contractile respon-

21siveness to Ca influx. However, prior treatment with 4 . Discussioneach of these drugs resulted in a better return in contractileresponsiveness compared to the control group. As before This study shows that the cardiotonic drugs dopamine,

21ischemia, dopamine and digoxin, and in addition digoxin, levosimendan and dobutamine shift the [Ca ] /levosimendan, each decreased VR after 30 min reperfu- LVP loop rightward and upward and increase loop area;max

sion but these values were higher than before ischemia. differences among loops were greatest with dobutamine atThis indicates improved but substandard contractile re- the single concentrations selected. After ischemia and 30

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21Fig. 2. Average cytosolic [Ca ] /LVP loops (6–10 cardiac cycles per heart; eight hearts per group). Loop shape and position acquired by instantaneous21 21plot of LVP and [Ca ] coordinates over one averaged beat. Loop area acquired by integration of [Ca ] /LVP over one beat. Reperfusion after ischemia

shifted the cardiac loop slightly rightward and downward. Dopamine (panel A) and especially dobutamine (panel B) given before and after ischemia shiftedthe cardiac loop rightward and upward. Levosimendan (panel C) and digoxin (panel D) given before and after ischemia shifted the cardiac loop rightwardand upward, the rightward more so by levosimendan.

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21Fig. 3. d[Ca ] /dt (A) and dLVP/dt (B) at baseline, during peak response of drugs before ischemia, at 2 and 30 min reperfusion, and during peakmax max21response of drugs after ischemia. Reperfusion increased d[Ca ] /dt but decreased dLVP/dt in each group. Each drug significantly increasedmax max

21d[Ca ] /dt before and after ischemia, but increased dLVP/dt less after ischemia than before.max max

21min reperfusion the [Ca ] /LVP loop returned nearly to its with any drug reduced the decreases in these responses.pre-ischemia baseline location but remained flatter. Drugs Dopamine and digoxin enhanced these responses beforehad similar effects on loop location and shape as before ischemia and these drugs and levosimendan did so after

21ischemia although loops were flatter. Computation of ischemia. Total LVP generated per total change of [Ca ]velocity and area ratios allowed comparison of drug during one cycle was enhanced by dopamine and digoxin

21contractility and relaxation responses to Ca before and before ischemia and by all but dobutamine after ischemiaafter ischemia. Ischemia reperfusion injury caused a (decreased AR). This index was increased threefold bytwofold decrease in the contractile response (increased ischemia reperfusion injury (increased control AR) butcontrol VR ) and a fourfold decrease in the relaxation prior drug treatment with dopamine, digoxin andmax

response (increased control VR ) but prior treatment levosimendan attenuated these increases indicating im-min

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21Fig. 4. d[Ca ] /dt (A) and dLVP/dt (B) at baseline, during peak response of drugs before ischemia, at 2 and 30 min reperfusion, and during peakmin min21response of drugs after ischemia. Reperfusion increased d[Ca ] /dt before and after ischemia, but increased dLVP/dt less after ischemia than before.min min

21proved LVP per change in [Ca ]. Dobutamine, dopamine reperfusion injury markedly worsens responsiveness, (c)and levosimendan, but not digoxin, increased coronary prior treatment with any of these positive inotropic drugsflow before and after ischemia. Each drug, especially attenuates depression of these responses after ischemia, (d)dobutamine and levosimendan, increased O consumption levosimendan more effectively improves responsiveness2

and cardiac efficiency, but there were no significant after than before ischemia, and (e) dobutamine does notdifferences in the relative increases in MVO and cardiac alter responsiveness before or after ischemia. Therefore,2

efficiency between drugs given before and after ischemia. cardiotonic drugs that have different mechanisms of action21This indicated that ischemia reperfusion does not blunt appear to have different effects on the Ca /LVP relation-

metabolic responses to drugs after ischemia reperfusion ship.21injury. We have examined the cyclic Ca /LVP relationship in

Overall, this study suggests (a) dopamine and digoxin detail in two recent studies. In one, the phasic change in21similarly improve ratios of contractility, relaxation, and Ca (system input) was used to model actinomyosin

21total LVP responsiveness to cytosolic Ca , (b) ischemia cross-bridge kinetics by best fitting the corresponding best-

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21Fig. 5. Ratio of d[Ca ] /dt to dLVP/dt (VR ) at baseline, during peak response of drugs before ischemia, at 30 min reperfusion, and during peakmax max max

response of drugs after ischemia. Reperfusion alone increased VR in each group. Dopamine and digoxin decreased VR when given before and aftermax max

ischemia, whereas dobutamine and levosimendan did not alter VR before ischemia; levosimendan decreased VR only when given after ischemia. Themax max

relative change in VR was greater when drugs were given after ischemia than before. Dobutamine induced the highest increase in VR .max max

21Fig. 6. Ratio of d[Ca ] /dt to dLVP/dt (VR ) at baseline, during peak response of drugs before ischemia, at 30 min reperfusion, and during peakmin min min

response of drugs after ischemia. Reperfusion alone increased VR in each group. Dopamine and digoxin decreased VR when given before and aftermin min

ischemia, whereas dobutamine and levosimendan did not alter VR before ischemia; levosimendan decreased VR only when given after ischemia. Themin min

relative change in VR was greater when drugs were given after ischemia than before. Dobutamine induced the highest increase in VR .min min

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21Fig. 7. Ratio of area Ca to area LVP (AR) at baseline, during peak response of drugs before ischemia, at 30 min reperfusion, and during peak response ofdrugs after ischemia. Reperfusion alone increased AR in each group. Dopamine and digoxin decreased AR when given before and after ischemia, whereasdobutamine increased AR and levosimendan did not alter AR before ischemia; levosimendan decreased AR only when given after ischemia. The relativechange in AR was greater when drugs were given after ischemia than before. Dobutamine induced the highest increase in AR; this increase was similar tothat of the control group.

fit phasic change in isovolumetric LVP (system output) at endogenous catecholamines or acetylcholine release oreffects.different temperatures[26]. In another, we constructed

21 We selected one concentration near the EC for eachseveral dynamic and static indices of the Ca /LVP 50

drug. We did not conduct concentration response curves torelationship and identified those indices that were most21 compare efficacy of these drugs, so we could not directlyuseful to describe drug-induced alterations in the Ca /

compare effects of different drugs on altering cytosolicLVP relationship[27].21[Ca ] and contraction and relaxation independently. Thus

for much of the results (Table 2, Figs. 3,4) we comparedchanges in drug responses within a drug group only. The4 .1. Selection and comparison of cardiotonic drugsthree ratios (VR , VR , and AR) furnish normalizedmax min

21Criteria for selecting and comparing these four agents values for indices of LVP and Ca and so allow com-were that (a) they stimulated different sarcolemmal re- parison among groups. We selected these ratios for com-ceptors (b, dopamine) or altered enzyme function (Na/ parison because they each yield information on the cost of

21KATPase, troponin C/PDE III) to increase myoplasmic performing work, i.e., velocity of Ca influx and efflux21Ca , (b) they produced a sub-maximal contractile re- required to produce the velocity of contraction and relaxa-

21sponse, (c) they could be easily washed out, and (d) they tion, as well as the net amount of Ca utilized to producedid not interfere with the fluorescent probe used to a net contractile effect over the cardiac cycle. Thus an

21measure Ca . We chose the isolated heart model so that increase in any ratio meant that contractile responsivenessthe influence of cardiac preload and afterload, blood-borne was decreased, and vice versa. We found that most drugsfactors, and autonomic nervous function would be mini- enhanced responsiveness and that ischemia reperfusionmized. We did not pace hearts because it is difficult to injury attenuated responsiveness. Moreover, these drugsdestroy the SA and AV nodes and a faster rate for the remained capable of enhancing responsiveness, albeit at acontrol hearts would obscure the influence of heart rate on lesser response level, after ischemia. Dobutamine alone didthe measured variables in the treated groups. Digoxin and not alter these indices of responsiveness before or afterlevosimendan each had a direct effect to increase heart rate ischemia.

21by approximately 12%. This suggests that they have direct It should be noted that the Ca /LVP loops and the21effects on SA node automaticity or that they modulate above analyses of various indices of [Ca ] plotted against

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21LVP do not furnish any temporal information. The variable 4 .3. Effects of cardiotonic drugs on cytosolic [Ca ] toreductions in cycle length induced by the different drugs LVP relationships

21likely alter the rates of entry and exit or Ca and so therates of LVP development and relaxation. The heart rate Cardiotonic drugs have been shown to improve contrac-effect is normalized to some degree by analysis of the tility and relaxation of stunned myocardium[18,30,31].

21ratios of an index of Ca to the same index of LVP. We These drugs work principally by increasing phasic cyto-21have recently addressed in detail the effect of altering heart solic [Ca ]; some may also enhance myocardial respon-

21 21rate on several static and dynamic indices of the Ca / siveness to Ca . Dobutamine was shown to enhanceLVP relationship[26] and on modeling the actin myosin contractility in in vivo infarcted mice hearts[17] andcross bridge interaction in the isolated beating heart[27]. during cardiac hypothermia[32] and to improve contractile

and metabolic function in regionally ischemic hearts[21].Our study furnishes more information on differences

214 .2. Ischemia reperfusion and cytosolic [Ca ] /LVP between dobutamine and other inotropic drugs.relationship Dobutamine did not increase VR and VR when givenmax min

before or after ischemia compared to baseline and 30 minIschemia reperfusion injury is known to alter the myop- reperfusion. This demonstrates that dobutamine increases

21 21lasmic [Ca ] /LVP relationship but this has not been Ca influx and efflux in proportion to increases inexamined in detail. Global ischemia for 20 min followed myocardium contractility and relaxation both before andby 20 min reperfusion in isolated rat hearts at 378C led to after ischemia. Dobutamine also caused the highest ARmyocardium stunning and decreased maximal extracellular among drugs given either before or after ischemia; this

21 21 21Ca -activated force and myocardial Ca responsiveness suggests that dobutamine increases cytosolic [Ca ] much[12]. Stunned myocytes isolated from pig hearts subjected higher than LVP compared to other drugs so that

21to 90 min regional ischemia and 60 min reperfusion dobutamine had the lowest [Ca ] efficiency. Our resultsdisplayed impaired contractile function and decreased suggest that although dobutamine is quite effective at

21 21Ca transients[29]. We reported that 30 min global augmenting contractility, the costs in terms of Caischemia at 378C and 60 min reperfusion increased loading, particularly during diastole after ischemia are

21cytosolic [Ca ] and decreased contractility in isolated high, and it does not enhance responsiveness like the other21beating guinea pig hearts[14,23]. Moreover, the [Ca ] / drugs.

LVP response curves generated by changing extracellular Levosimendan is reported to be a sensitizer at lower21[Ca ] before and after ischemia exhibited a reduced doses but not at higher doses[9,31]. Sato et al.[9] showed

maximal contractile response and a rightward shift of the that 0.1mM levosimendan increased contractility of right-21normalized [Ca ] /LVP relationship indicating reduced ventricular papillary muscles and single ventricular rabbit

maximal activated contractile force and reduced sensitivity cardiomyocytes and that these effects were not associated21 21to Ca [14,23]. In the present study we found that with increased cytosolic [Ca ] as assessed in aequorin

ischemia reperfusion significantly increased VR , VR and indo-1 loaded cells, respectively. Levosimendan ismax min21and AR. This demonstrated that ischemia reperfusion thought to enhance binding of Ca to troponin C without

21 21 21increased Ca influx, efflux and total cytosolic [Ca ] but increasing diastolic Ca unlike other positive inotropesdecreased contractility, relaxation and total contractile [33]. However, 3mM levosimendan similarly increased

21force over a cardiac cycle. both contractility and cytosolic [Ca ] as well as cAMPIschemia reperfusion injury causes not only cardiac concentration[9]. Other reports also show that levosimen-

dysfunction in this model but also infarction. In other dan increased cAMP concentration and contractility[5,31].studies under similar conditions we showed approximately A higher cAMP level may blunt myofilament sensitivity to

2150–60% left ventricular infraction and 40% recovery of [Ca ]. A milrinone-induced increase in LV contractility21systolic–diastolic LVP after 1–2 h reperfusion in control was associated with higher [Ca ] in guinea pig hearts[3].

hearts [14,15,23,24]. For this study positive inotropic We selected a midrange levosimendan concentration (1agents were given before ischemia. This could precondi- mM) that increased systolic–diastolic LVP by approxi-tion hearts. Our results do not indicate any difference in mately 80% and dLVP/dt by threefold; because it also

21contractile (dLVP/dt ) or relaxant (dLVP/dt ) effects significantly increased systolic Ca , it is probable thatmax min

between the control group and prior drug treated groups at levosimendan’s PDE III inhibitory effect overrides the21 21 2130 min reperfusion. However, responsiveness to Ca was Ca troponin C interaction[9]. The increase in [Ca ]

improved similarly in each treated group suggesting some was much less than for dobutamine, but was greater than21preservation of the Ca /LVP relationship after ischemia for dopamine and digoxin. It was interesting that

21reperfusion injury. These results confirm and extend levosimendan was more effective in utilizing Ca forprevious results that ischemia reperfusion decreases contractility only after ischemia reperfusion injury. This

21 21myocardial Ca sensitivity and increases Ca influx and suggests that levosimendan causes a lesser increase in21efflux associated with impaired contractility and relaxation. Ca influx and efflux relative to increases in contractility

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and relaxation after ischemia than before ischemia. Dr. Ming Tao Jiang, Dr. Leo Kevin, Jim Heisner, andLevosimendan also increased heart rate, as did each of the Anita Tredeau for help in this study.drugs. Thus it is clear that levosimendan can have othereffects in isolated hearts.

Dopamine was shown to improve mechanical andR eferences

metabolic function in post-ischemic isolated rat hearts21[18]. Digoxin was reported to increase [Ca ] less when

[1] B links JR, Endoh M. Modification of myofibrillar responsiveness tocompared to milrinone, also a PDE inhibitor, in isolated 21Ca as an inotropic mechanism. Circulation 1986;73:III85–98.guinea pig hearts[3]. In our model digoxin was as [2] B user PT, Wu SY, Parmley WW, Jasmin G, Wikman-Coffelt J.effective as dopamine in enhancing myocardial responsive- Distinct modulation of myocardial performance, energy metabolism,

2121 and [Ca ] transients by positive inotropic drugs in normal andness to Ca . Both dopamine and digoxin decreased i

severely failing hamster hearts. Cardiovasc Drugs Ther 1995;9:151–VR and VR before ischemia and after ischemia. Thismax min 157.21indicates these drugs induce relative increases in Ca[3] I shisu R, Abe Y, Onishi K et al. Changes in calcium transient and

influx and efflux that were less than the increases in left ventricular function during positive inotropic stimulation anddLVP/dt and dLVP/dt . Thus dopamine and digoxin myocardial ischemia in indo-1-loaded beating guinea pig heart. Jmax min

21 Pharmacol Toxicol Methods 1996;35:55–61.had comparable effects to enhance utilization of Ca to[4] C apogrossi MC, Houser SR, Bahinski A, Lakatta EG. Synchronousgenerate contractile force both before and after ischemia.

21 occurrence of spontaneous localized calcium release from theBoth drugs had a moderate effect to increase [Ca ],sarcoplasmic reticulum generates action potentials in rat cardiac

particularly after ischemia and reperfusion, so that myocar- ventricular myocytes at normal resting membrane potential. Circ21dial responsiveness to Ca was markedly better than for Res 1987;61:498–503.

[5] D u Toit EF, Muller CA, McCarthy J, Opie LH. Levosimendan:levosimendan.effects of a calcium sensitizer on function and arrhythmias andIn conclusion, brief cardiac perfusion by each car-cyclic nucleotide levels during ischemia/ reperfusion in the Langen-diotonic agent increased mechanical and metabolic func-dorff-perfused guinea pig heart. J Pharmacol Exp Ther21tion by increasing cytosolic [Ca ] when given either 1999;290:505–514.

21before ischemia or after ischemia. Ca responsiveness [6] H aikala H, Kaivola J, Nissinen E et al. Cardiac troponin C as atarget protein for a novel calcium sensitizing drug, levosimendan. Jwas not altered by dobutamine but was enhanced byMoll Cell Cardiol 1995;27:1859–1866.dopamine and digoxin before ischemia and afterward also

[7] S orsa T, Heikkinen S, Abbott MB et al. Binding of levosimendan, aby levosimendan. It was interesting that drug treatmentcalcium sensitizer, to cardiac troponin C. J Biol Chem

before ischemia helped to preserve the association of 2001;276:9337–9343.21contractility and Ca after ischemia, i.e., that drug effects [8] Y okoshiki H, Katsube Y, Sunagawa M, Sperelakis N. The novel1were relatively well preserved after ischemia. Taken calcium sensitizer levosimendan activates the ATP-sensitive K

channel in rat ventricular cells. J Pharmacol Exp Thertogether, our results suggest that digoxin and dopamine1997;283:375–383.increase myocardial contractility with a smaller increase in

[9] S ato S, Talukder MA, Sugawara H, Sawada H, Endoh M. Effects of21[Ca ] than other drugs, and significantly increase myofila- 21levosimendan on myocardial contractility and Ca transients in21ment Ca sensitivity. This study in isolated hearts al- aequorin-loaded right-ventricular papillary muscles and indo-1-lowed us to better understand the mechanisms and differ- loaded single ventricular cardiomyocytes of the rabbit. J Mol Cell

21 Cardiol 1998;30:1115–1128.ences among inotropic drugs on the Ca /LVP relation-[10] S towe DF, Fujita S, An JZ et al. Modulation of myocardial functionship during the cardiac cycle. In in vivo blood perfused 21and [Ca ] sensitivity by moderate hypothermia in guinea pig

and innervated hearts the effects of these drugs given over isolated hearts. Am J Physiol Heart Circ Physiol 1999;277:H2321–a longer time period is likely modified from our observa- 2332.

21tions, but the overall effect of phasic cytosolic Ca on [11] M arban E, Rink TJ, Tsien RW, Tsien RY. Free calcium in heart21muscle at rest and during contraction measured with Ca -sensitivebeat-to-beat contractility and relaxation is probably quite

microelectrodes. Nature 1980;286:845–850.similar. Thus, if these isolated heart results can be carried[12] G ao WD, Atar D, Backx PH, Marban E. Relationship between

over to the clinical arena, they would suggest that digoxin intracellular calcium and contractile force in stunned myocardium.21and dopamine are relatively more efficacious than Direct evidence for decreased myofilament Ca responsiveness and

levosimendan, and especially dobutamine, in enhancing altered diastolic function in intact ventricular muscle. Circ Res21 1995;76:1036–1048.contractility without inducing a large increase in [Ca ],

21 [13] G ao WD, Atar D, Liu Y et al. Role of troponin I proteolysis in theparticularly just after ischemia when [Ca ] is quitepathogenesis of stunned myocardium. Circ Res 1997;80:393–399.

elevated. [14] V aradarajan SG, An JZ, Novalija E, Smart SC, Stowe DF. Changes1 21in [Na ] , compartmental [Ca ], and NADH with dysfunction afteri

global ischemia in intact hearts. Am J Physiol Heart Circ Physiol2001;280:H280–293.A cknowledgements

[15] V aradarajan SG, An JZ, Novalija E, Stowe DF. Sevoflurane beforeor after ischemia improves contractile and metabolic function while

The research was supported in part by National Heart, 21reducing myoplasmic Ca loading in intact hearts. AnesthesiologyLung, and Blood Institute grants R01-HL-58691 and R01- 2002;96:125–133.5T32 GM-08377. The authors thank Dr. Gopu Varadarajan, [16] M ori M, Takeuchi M, Takaoka H, Yokoyama M. Lusitropic effects

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21of a Ca sensitization with a new cardiotonic agent, MCI-154, on [25] S towe DF, Varadarajan SG, An JZ, Smart SC. Reduced cytosolic21diseased human hearts. Cardiovasc Res 1995;30:915–922. Ca loading and improved cardiac function after cardioplegic cold

[17] W iesmann F, Ruff J, Engelhardt S et al. Dobutamine-stress magnetic storage of guinea pig isolated hearts. Circulation 2000;102:1172–resonance microimaging in mice: acute changes of cardiac geometry 1177.and function in normal and failing murine hearts. Circ Res [26] R hodes S, Ropella KM, Audi SH et al. Cross-bridge kinetics

212001;88:563–569. modeled from myoplasmic [Ca ] and LV pressure at 178C, and[18] H offmeister HM, Schaper J, Beyer ME, Kazmaier S, Seipel L. after 378C and 178C ischemia. Am J Physiol Heart Circ Physiol

Effects of positive inotropic stimulation on postischemic myocar- 2003;284:H219–H229.dium with graded dysfunction. Cardiovasc Res 1997;33:332–340. [27] R hodes S, Ropella KM, Camara AKS, et al. How inotropic drugs

21[19] R ump AF, Acar D, Klaus W. A quantitative comparison of func- alter dynamic and static indices of cyclic myoplasmic [Ca ] totional and anti-ischaemic effects of the phosphodiesterase-inhibitors, contractility relationships in intact hearts. J Cardiovasc Pharmacol,amrinone, milrinone and levosimendan in rabbit isolated hearts. Br J in press.Pharmacol 1994;112:757–762. [28] R iess ML, Camara AK, Chen Q et al. Altered NADH and improved

[20] R ump AF, Acar D, Rosen R, Klaus W. Functional and antiischaemic function by anesthetic and ischemic preconditioning in guinea pigeffects of the phosphodiesterase inhibitor levosimendan in isolated intact hearts. Am J Physiol Heart Circ Physiol 2002;283:H53–60.rabbit hearts. Pharmacol Toxicol 1994;74:244–248. [29] K im SJ, Kudej RK, Yatani A et al. A novel mechanism for

21[21] Y i KD, Downey HF, Bian X, Fu M, Mallet RT. Dobutamine myocardial stunning involving impaired Ca handling. Circ Resenhances both contractile function and energy reserves in hypo- 2001;89:831–837.perfused canine right ventricle. Am J Physiol Heart Circ Physiol [30] K orbmacher B, Sunderdiek U, Selcan G, Arnold G, Schipke JD.2000;279:H2975–2985. Different responses of non-ischemic and post-ischemic myocardium

1 1 21[22] A n JZ, Varadarajan SG, Camara A et al. Blocking Na /H towards Ca sensitization. J Mol Cell Cardiol 1997;29:2053–2066.1 21exchange reduces [Na ] and [Ca ] load after ischemia and [31] K ristof E, Szigeti G, Papp Z et al. The effects of levosimendan oni i

improves function in intact hearts. Am J Physiol Heart Circ Physiol the left ventricular function and protein phosphorylation in post-2001;281:H2398–2409. ischemic guinea pig hearts. Basic Res Cardiol 1999;94:223–230.

[23] A n JZ,Varadarajan SG, Novalija E, Stowe DF. Preconditioning with [32] R iishede L, Nielsen-Kudsk F. Myocardial effects of adrenaline,21ischemia or sevoflurane reduces cytosolic [Ca ] loading and isoprenaline and dobutamine at hypothermic conditions. Pharmacol

21improves Ca responsiveness after global ischemia in isolated Toxicol 1990;66:354–360.hearts. Am J Physiol Heart Circ Physiol 2001;281:H1508–H1523. [33] H aikala H, Linden I-B. Mechanisms of action of calcium-sensitizing

[24] N ovalija E, Fujita S, Kampine JP, Stowe DF. Sevoflurane mimics drugs. J Cardiovasc Pharmacol 1995;26(suppl. 1):S10–S19.ischemic preconditioning effects on coronary flow and nitric oxiderelease in isolated hearts. Anesthesiology 1999;91:701–712.

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