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Molecular and Cellular Biochemistry 207: 71–75, 2000.© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

The effects of high-dose epinephrine combinedwith isoprenaline on isolated rabbit heart andcardiomyocytes after cardioversion of ventricularfibrillation

Lihua Zhao,1 Wenjie Zhang,2 Weihua Zhang1 and Yang Zheng1

1Department of Cardiovascular Diseases, The First Teaching Hospital, Norman Bethune University of Medical Sciences,Changchun; 2Department of Physiology, School of Basic Medical Sciences, Norman Bethune University of MedicalSciences, Changchun, People’s Republic of China

Abstract

The effects of high-dose epinephrine (HDE) combined with isoprenaline (Iso) on myocardial hemodynamics of isolated rabbitheart were studied. The electrophysiology and L-type Ca2+ channel of single ventricular myocyte after cardioversion ofventricular fibrillation were determined. The results suggest that parameters of hemodynamics were significantly enhanced byHDE+Iso than that of HDE (p < 0.01). The OS and V

max of HDE+Iso increased 83.7 and 10.15% respectively compared to HDE

alone. The APF of HDE+Iso is much more rapid than that of HDE (138.38 ± 9.96 vs. 55.58 ± 8.63 min–1, p < 0.001). The APD50

and APD90

of HDE+Iso were significantly decreased; HR was increased (134.16 ± 1.48 vs 62.20 ± 6.25 min–1); and the amplitudecurrent of through L-type Ca2+ channel was reduced but was significantly higher than the control. We conclude that HDE+Isocan improve the hemodynamics and improve electrophysiology of cardiomyocytes after cardioversion of ventricularfibrillation which is likely interrelated with I

Ca. The combined use of epinephine and isoprenaline for cardiopulmonary

resuscitation of primary ventricular fibrillation may be beneficial when employed in clinical situations. (Mol Cell Biochem207: 71–75, 2000)

Key words: high-dose epinephrine, isoprenaline, ventricular fibrillation, hemodynamics, action potential, L-type Ca2+ channel

Abbreviations: 4-AP – 4-aminopyridine; AP – action potential; APD – action potential duration; APF – action potentialfrequency; ATP – adrenosine triphosphate; ± dp/dt

max – maximum rate of increasing (decreasing) of left ventricular pressure;

ECG – electrocardiograph; EGTA – ethylene glycol bis (2-aminoethgl ether) –N, N, N′,N′,-tetraacetic acid; HEPES –hydroxyethyl piperzine ethanesulfonic acid; HR – heart rate; LVEDP – left ventricular end-diastolic pressure; LVSP – leftventricular systolic pressure; OS – overshoot; TEA – tetraethyl-ammonium; TTX – tetrodotoxin; V

max – action potential upstroke

Introduction

The beneficial effect of high-dose epinephrine (HDE) for theresuscitation of primary ventricular fibrillation is well-documented [1, 2]. In recent years, it was reported that high-dose epinephrine combined with isoprenaline (HDE+Iso)could make the resuscitation more successful by theimprovement of spontaneous circulation [3]. However, there

is not many reports on the hemodynamics and cardiomyocyteelectrophysiological mechanism pertaining to this mode oftreatment. In this study, we analyzed the parameters of hemo-dynamics, action potential and L-type Ca2+ channels of theisolated rabbit heart and cardiomyocytes , in order to studythe effects of high-dose epinephrine combined with iso-prenaline on the electrophysiology of cardiomyocytes and theisolated heart after cardioversion of ventricular fibrillation.

Address for offprints: L. Zhao, Department of Cardiovascular Diseases, The First Teaching Hospital, Norman Bethune University of MedicalSciences, Changchun 130021, People’s Republic of China

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Materials and methods

Model establishment of ventricular fibrillation (VF) andcardiac arrest

In this study, 24 rabbits of either sex weighing 1.25 ± 0.25 kgwere randomly divided into three groups (8 each). Theanimals were killed by cervical dislocation. Through amidsternal thoracotomy, the heart was rapidly removed andattached to a Langendorff perfusion system. The aorta wascannulated to record left ventricular pressure. The heart wasperfused reversely through pulmonary artery for 10 minwith Tyrode’s solution warmed at 37°C, then perfused fromaorta for 10 min. Ventricular fibrillation (VF) and cardiacarrest were induced by electric stimuli under hypoxicconditions 95% N

2+5% CO

2 [4, 5]. The hearts of Group A and

B were resuscitated by a high-dose of epinephrine (180 µg/kg) (HDE, Group A) or a high-dose of epinephrine (180 µg/kg) consolidated with isoprenaline (25 µg/kg) (HDE+Iso,Group B) under normoxic conditions with 95% O

2+5%

CO2. The control underwent the process described above

except for electric stimulus and resuscitation. The ECG LVSPLVEDP ± dp/dt

max were recorded by 8-lead physiograph (RM-

6000, KOHDEN, Japan). The preload of isolated hearts was8.77 mmHg and the postload was 40.35 mmHg. Flow ratewas 10 ml/min. The composition of normal Tyrode’s solutionwas (in mmol/L): NaCl 136.9, KCl 5.4, CaCl

2 1.8, MgCl

2 0.5,

NaH2PO

4 0.25, glucose 5.5, HEPES 5, pH was adjusted to 7.3

with HCl.

Record of action potential

Suspended microelectrodes were used to record actionpotential in the isolated perfused hearts. Action potentialwas recorded with the electrodes implanted inside the cellsaccording to the technique of electrophysiology con-vention. The electrodes was filled with KCl (3 mol/L)internal solution. The parameters of membrane actionpotential including OS, APD

50, APD

90, V

max and APF were

analyzed by a computer.

Preparation of single ventricular cells

Single ventricular cells were isolated from the isolatedperfused rabbits hearts according to the procedure describedby Kazuto et al. [6]. Briefly, the heart was perfused for 2 minwith Ca2+-containing Tyrode’s solution and for 5 min Ca2+-free Tyrode’s solution, and then perfusion was switched in aCa2+-free Tyrode’s solution containing collagenase (12 mg/60ml, Yakult, Japan) for 10 min. The heart was then stored inthe storage solution at 4°C for at least 1 h. A small piece of

the ventricular tissue was dissected and gently agitated inthe recording chamber (0.5 ml in volume) filled with normalTyrode’s solution. The recording chamber was perfused at2–3 ml/min with normal Tyrode’s solution after the cellshad settled on the floor of the chamber. Experiments wereperformed at 36–37°C on rod-shaped quiescent single cellsthat had clear sarcomere striations.

Solution and membrane current measurements

Membrane currents were recorded using the patch-clamptechnique in the whole-cell configuration with a Dagan(model 8800) patch-clamp amplifier. Ventricular myocyteswere voltage-clamped with fire-polished patch pipettes (tipresistance, 1 ± 3 MΩ) filled with a solution containing (inmmol/L): Cs-aspartate110, CsCl 20, ATP 2, EGTA 5 andHEPES 5, the pH was adjusted to 7.2 with CsOH. The bathsolution contained the following composition (in mmol/L):CaCl

2 2, TEA 135, 4-AP 5, MgCl

2 1, glucose 10, HEPES 10,

TTX 0.02, pH 7.4 adjusted with CsOH. As a routine protocolfor I

Na recordings, Holing potential was –50 mV and test

voltages ranged from –50–60 mV in 10 mV increments andwere delivered at a frequency of 1 Hz in all experiments. Thetime constant of the capacitative transient was 296 ± 14 µSand the membrane capacitance was calculated to be 101 ± 6pF. Mean series resistance (Rs) was 5 ± 1 MΩ. All experimentswere conducted at room temperature of 22–24°C. Signalsobtained was filtered at 3 kHz, digitized at 1 KHz by usingPCLAMP software and Digidata 1200 A/B (Axon InstrumentsInc.) and stored in a computer. Original current recordingswere corrected for leaks either during an experiment using aP+P/n protocol or off-line using a scaled current recordingin which no membrane current was evident.

Statistics

Results are given as mean ± S.D. A paired t test was used forthe determination of significance, and a value of p < 0.05was considered statistically significant.

Results

The effects of epinephrine and isoprenaline on themyocardial contractibility

The myocardial contractibilities in Group A and Group Bwere determined (Table 1). The results show that parametersof hemodynamics of Group B is lower than control, butsignificantly enhanced than those of Group A (p < 0.01).

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The effects of epinephrine and isoprenaline on the actionpotential of ventricular myocytes

The effects of these drugs on the action potential of ven-tricular myocytes were determined (Table 2 and Fig. 1). TheOS and V

max of Group B increased 83.7 and 10.15% res-

pectively than that of Group A. APF of Group B was muchmore rapid than that of Group A but showed little differencebetween that of control. APD

50 and APD

90 of Group B

decreased 12.19% and 8.93% than that of Group A, but thevalues are larger than that of control. These results showedthat HDE+Iso could improve conductivity of ventricularmyocytes and significantly shorten AP compared to HDE.

The effect of epinephrine and isoprenaline on L-type Ca2+

channel of ventricular myocytes

The effect of these drugs on L-type Ca2+ channel of ven-tricular myocytes were determined (Table. 3). The I

Ca was

obtained in the cells that were dialyzed internally with theCs+-internal pipette solution and TTX through the patchelectrode and were superfused with Cs+-Tyrode’s solution.The cell was voltage clamped at –50 mV, and 200-msecdepolarizing test pulses from –50 to +60 mV were appliedevery 10 sec. The current through the L-type Ca2+ channelwas recorded. The result showed that amplitude current ofGroup B increased 23.26% compared to control, but wassignificantly lower than that of Group A (p < 0.05, Fig. 2).Active voltage and reverse voltage were not affected (Fig. 2).

A β-receptor blocker propranolol (Pro, 10–6 M) was shown tosignificantly depression the current through L-type Ca2+

channel, while the a-receptor blocker prazosin (Pra, 10–6 M)did not cause any depression. Our data indicate that the β-receptor played an important role on the AP of cardiomyocytesafter cardioversion of ventricular fibrillation in Group B(HDE+Iso).

Discussion

The activation of β- and a-adrenoceptor by low and high doseepinephrine respectively, and the β-adrenoceptor agonistnature of isoprenaline were well-documented [7]. It wasobserved in clinical studies that high-dose of epinephrinecould make cardiopulmonary resuscitation more successfullyby increase of coronary perfusion pressure [8], but the vitalbradycardia and atrioventricular block often made re-suscitation fail. In recent years, it was reported that high-doseof epinephrine combined with isoprenaline appeared to bebeneficial for resuscitation. The results of our study indicatethat it is likely that HDE+Iso directly affect the electro-physiology of cardiomyocytes, resulting in the improvementof hemodynamics in the isolated heart.

The control of our study underwent the process of O2-free

and O2-supplying. The result showed that in control, APD

50,

APD90

were shorter than routine results and amplitude of L-type Ca2+ channel was lower while OS and V

max were

significantly higher than Groups A and B (p < 0.05 and p <0.001, Table 2), APF was not different between that of GroupB (p > 0.05, Table 2). Although we did not assay for I

K (ATP),

we considered that the result maybe caused by increase of IK

(ATP) [9] and/or decrease of ICa

[10] during hypoxia, after O2-

supplying K+ channel return to normal while the L-type Ca2+

Table1. The effects of Group A (HDE) and Group B (HDE+Iso) onhemodynamics of isolated rabbit hearts (n = 8)

Control HDE HDE+Iso

LVSP (kPa) 10.21 ± 1.44 5.89 ± 1.098 8.38 ± 1.41***

LVEDP (kPa) 1.55 ± 0.073 1.13 ± 0.076 1.35 ± 0.15***

+dp/dtmax (kPa/s) 684.00 ± 191.55 353.00 ± 47.6 459.00 ± 60.2**

–dp/dtmax (kPa/s) 468.57 ± 104.79 221.70 ± 39.7 301.70 ± 35.6***

HR (beat/min) 175.00 ± 6.85 65.20 ± 6.25 136.14 ± 4.48***

**p < 0.01; ***p < 0.001 compared to HDE.

Table 2. The effects of Group A (HDE) and Group B (HDE+Iso) onaction potential of single ventricular myocyte of rabbit (n = 8)

Control HDE HDE+Iso

OS(mV) 28.68 ± 1.33 10.97 ± 2.74† 20.15 ± 3.460***†

APD50 (msec) 256.07 ± 17.45 345.65 ± 28.8† 303.52 ± 12.21***†

APD90 (msec) 307.43 ± 11.7 393.37 ± 13.3† 358.25 ± 20.29**†

Vmax (V/sec) 179.09 ± 22.68 124.69 ± 8.32† 137.35 ± 7.250***†

APF (min–1) 154.14 ± 19.20 55.85 ± 8.63† 138.38 ± 9.960***†

**p < 0.01, ***†p < 0.001 compared to HDE; †p < 0.001 compared tocontrol.

Fig. 1. The effects HDE and HDE+Iso on the action potential ofrabbit ventricular myocytes. The superimposition of action potentialrecorded control.

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channel does not. But the hemodynamics of control was notaffected (Table 1).

When Group A was compared to control, the APD50

andAPD

90 lengthened p < 0.001, OS and V

max were significantly

reduced (p < 0.001 respectively), and APF was decreased(55.83 ± 8.63 vs. 154.14 ± 19.20 min–1, p < 0.001). Thesefactors could cause a reduction of HR, which is not afavorable condition for the recovery of hemodynamics andcardiomyocyte injury after resuscitation of ventricularfibrillation.

The APD, OS and Vmax

in Group B were slightly lower thanthose in control, but higher than those in Group A, especiallywhen APD

50 and APD

90 were significantly shortened than

those in Group A (p < 0.01), so that the APD was closer to thatin control. The β-adrenergic effect of Group B such as rise of

Fig. 2. The effects of HDE and HDE+Iso on ICa of single isolatedrabbit ventricular myocytes.. The top panel shows current tracesinduced by 200 msec clamp pulses from a holding potential of –50 mVto +10 mV. The I–V relation are shown below. They were measuredat the peak ( control; HDE+Iso; +HDE).∆

APF and HR, improve of conductivity by increase of Vmax

and decrease of APD50

, may be factors which alleviate thebradycardia and atroventricular block after resuscitation byhigh-dose epinephrine.

The α-adrenergic effect of high-dose epinephrine caninhibit I

to of ventricular myocytes of rabbit [11], thus

prolonging the APD. The electrophysiological effect ofisoprenaline can be modulated by the concentration ofintracellular Ca2+. In our study, L-type Ca2+ channel wasassayed by patch clamp in normal solution. The amplitudein Group A was significantly higher than that of the control,at the same time the APD was prolonged, the amplitude inGroup B was also higher than that in control while lower thanthat in Group A. The α-receptor blocker parzosin had no effecton the amplitude of L-type Ca2+ channel but β-receptor cansignificantly inhibit it (Table 3, Fig. 2). We postulate thatHDE+Iso are effective for: (1) releasing the inhibition of L-type Ca2+ channel during normoxic and hypoxic conditions[10]; (2) showing that the β-adrenergic effect on increasingboth I

K and I

Ca [12] while I

K plays a more important role on

APD.In conclusion, the present study demonstrated that the

effect of combined administration of HDE+Iso is moreeffective than the single use of high-dose epinephrine. It isclear that the parameters of hemodynamics, action potentialof cardiomyocytes (APD, OS, V

max, APF) and HR are sig-

nificantly improved. The basis of its electrophysiology islikely interrelated to I

Ca and I

K, while mechanism of I

K requires

further studies.

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Table 3. The effects of Group A (HDE) and Group B (HDE+Iso) on L-type Ca2+ channel on cardiomyocyte of rabbit (n = 6)

Control HDE HDE+Iso HDE+Iso+Pro HDE+Iso+Pra

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***p < 0.001 compared to control; †p < 0.05 compared to HDE; ‡p < 0.001 compared to HDE+Iso

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