Br. J. Pharmacol. (1991), 102, 73-78

I'--,

Macmillan Press Ltd, 1991

Lack of correlation between the antiarrhythmic effect of L-propionylcarnitine on reoxygenation-induced arrhythmias and its electrophysiological properties M. Barbieri, tP.U. Carbonin, *E. Cerbai, tG. Gambassi, Jr, tfP. Lo Giudice, *1. Masini, 1A. Mugelli & tM. Pahor Institute of Pharmacology, University of Ferrara; tGerontology Division, Catholic University, Roma and *Department of Pharmacology, University of Firenze; Italy 1 The antiarrhythmic effect of L-propionylcarnitine (L-PC) was evaluated in the guinea-pig isolated heart; arrhythmias were induced with hypoxia followed by reoxygenation and by digitalis intoxication. 2 L-PC 1ipm, was found to be the minimal but effective antiarrhythmic concentration against reoxygenation-induced ventricular fibrillation. No antiarrhythmic effect was observed against digitalisinduced arrhythmias. D-Propionylcarnitine, L-carnitine and propionic acid did not exert antiarrhythmic effects. 3 During hypoxia and reoxygenation L-PC consistently prevented the rise of the diastolic left ventricular pressure, and significantly reduced the release of the cardiac enzymes creatine kinase (CK) and lactic dehydrogenase (LDH). 4 The electrophysiological effects of L-PC were then studied on either normal sheep cardiac Purkinje fibres or those manifesting oscillatory afterpotentials induced by barium or strophanthidin. 5 L-PC (1 and 10paM) did not significantly modify action potential characteristics and contractility of normal Purkinje fibres, or the amplitude of OAP induced by strophanthidin or barium. 6 It is concluded that the antiarrhythmic action of L-PC on reoxygenation-induced arrhythmias is not correlated with its direct electrophysiological effects studied on normoxic preparations.

Introduction Several studies have suggested that L-carnitine exerts protection of the ischaemic myocardium in both experimental animals and man (Folts et al., 1978; Liedtke & Nellis, 1979; Thomsen et al., 1979; Kamikawa et al., 1984). It has been reported that exogenous administration of L-carnitine may counteract the myocardial depletion of endogenous carnitine stores, resulting in myocardial protection (Liedtke & Nellis, 1979; 1981; Liedtke et al., 1982). L-Carnitine has also been reported to exert antiarrhythmic activity on ventricular arrhythmias caused by coronary ligation and/or reperfusion in the dog (Suzuki et al., 1981; Kobayashi et al., 1983; Imai et al., 1984). Recently, it has been shown that Lpropionylcarnitine (L-PC) protects the ischaemic myocardium more than L-carnitine or L-acetylcarnitine (Paulson et al., 1986). An antiarrhythmic effect of L-PC on reperfusioninduced arrhythmias in isolated hearts from spontaneously hypertensive rats (Carbonin et al., 1990) has also been observed. Reperfusion- and reoxygenation-induced arrhythmias may be caused by a common electrophysiological arrhythmogenic stimulus. Some evidence suggests that oscillatory afterpotentials (OAPs) could represent this electrophysiological mechanism (Corr & Witkowsky, 1983; Manning & Hearse, 1984; Amerini et al., 1985a; 1988). It is also well known that OAPs are responsible for digitalis-induced arrhythmias (Ferrier, 1977). We thought that it would be interesting to evaluate the antiarrhythmic properties of L-PC (in comparison with those of equimolar concentrations of D-propionylcarnitine, Lcarnitine, and propionic acid) on reoxygenation-induced arrhythmias. In an attempt to clarify the possible electrophysi' Author for correspondence at Institute of Pharmacology, Via Fossato di Mortara 64/b, 44100 Ferrara, Italy. 2 Present address: Institute for Research on Senescence, Sigma Tau,

Pomezia, Roma, Italy.

ological mechanism of its antiarrhythmic action, we evaluated the electrophysiological effects of antiarrhythmic concentrations of L-PC on the transmembrane potential characteristics of normal sheep Purkinje fibres and of preparations exposed to barium or strophanthidin and manifesting OAPs (Amerini et al., 1985a; 1988). This approach has been extremely helpful in the understanding of the mechanism of class I antiarrhythmic drugs on reoxygenation-, reperfusion- and digitalis-induced arrhythmias (Amerini et al., 1985a; 1988). Thus, the aim of this study was to evaluate the antiarrhythmic effect of L-PC on a well characterized model of arrhythmias in order to obtain information on its possible mechanisms of action.

Methods Perfusion of the isolated heart Guinea-pigs (body weight: 35400 g) were used. Thirty minutes after an intraperitoneal heparin injection (100iu) the animals were killed by a sharp blow at the base of the skull. The hearts were rapidly excised and placed in ice-cold Tyrode solution and utilized for Langendorff perfusion. The details of the technique have been described elsewhere (Carbonin et al., 1981). The hydrostatic aortic perfusion pressure was 8kPa. The control medium was equilibrated at 37°C with 95% °2 plus 5% CO2 gas mixture and contained (in mm): NaCl 117.0, NaHCO3 23.0, KCI 4.6, NaH2PO4 0.8, MgCl2 1.0, CaCl2 2.0 and glucose 5.5. The 02 and CO2 partial pressures and pH values of the perfusion fluid were periodically determined by means of a gas analyser (Instrumentation Laboratories model 213). Epicardial electrograms were recorded by means of an atraumatic electrode connected to an amplifier (E & M Instrument model V 1205). The left ventricular pressure was measured by inserting a 12cm polyethylene catheter (0.5mm diameter) into the left ventricle through the ventricular wall

74

M. BARBIERI et al.

and was recorded by means of a pressure transducer (Statham P23) connected to a pressure amplifier (E & M Instrument model V 2203). All data were recorded on paper with an E & M Instrument model VR 12 Simultrace recorder. The coronary flow rate was measured by collecting the effluent. Rhythm disturbances were subdivided into: conduction disturbances (sino-atrial and atrio-ventricular blocks) and ventricular tachyarrhythmias: (a) ventricular fibrillation (VF) and (b) ventricular arrhythmias (VA) including ventricular premature beats (VPB) and ventricular tachycardia (VT). A large and aberrant QRS complex and the absence of a preceding P wave identified VPB. More than 5 consecutive VPBs were considered VT. Complete morphological irregularity of at least 10 complexes was considered VF. VF and VA were quantified by counting the number of hearts that exhibited VF or VA over 1 min periods. As VPB or VT could obviously not be evaluated in the fibrillating hearts, the hearts manifesting VF were excluded from VA analysis. After 20min of control perfusion to obtain the stabilization of heart rate and ventricular function, the hearts in one group were exposed to hypoxia followed by reoxygenation and in a second group to perfusion with digitalis. Hypoxia was produced by gassing the medium with a mixture of 95% N2 plus 5% CO2 (02 partial pressure < 1.33 kPa). During the hypoxic period the hearts were perfused with a glucose-free medium. After 15 min of hypoxia, the perfusion with the oxygenated and glucosecontaining medium was rapidly restored and maintained for 15min (reoxygenation phase). Experiments of hypoxia and reoxygenation were performed in 130 hearts. They were exposed to 9 different treatment protocols as follows: controls (n = 20), 0.1lM L-PC (n = 15), 1 PM L-PC (n = 20), 10pUM L-PC (n = 15), 1,UM L-carnitine (n = 10), 1UM D-propionylcarnitine (n = 10) and 1 uM propionic acid (n = 10) added to the medium during the whole hypoxic and reperfusion periods, 1pM L-PC (n = 15) and 10pUM L-PC (n = 15) added to the medium only during reoxygenation. Digitalis intoxication was obtained by perfusing the heart with 1 uM digoxin for 30min. Four digitalis experiments were used as controls and in four experiments 1 M L-PC was added to the medium 10min before and during superfusion with

digoxin. Determination of creatine kinase and lactic dehydrogenase in the effluent Samples of cardiac effluent were collected periodically and assayed for creatine kinase (CK) and lactic dehydrogenase (LDH) content with kits obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.). All determinations were done on a Pye-Unicam spectrophotometer. The results were related to the ventricular wet mass.

Electrophysiological studies Sheep cardiac Purkinje fibres were excised from the ventricles and kept in oxygenated Tyrode solution until used. One strand was mounted in a tissue bath and superfused with Tyrode solution at a rate of 8 ml min 1. The composition of solution was (mM): NaCl 137.0, NaHCO3 11.9, KCl 4, NaH2PO4 0.42, MgCl2 0.5, CaCl2 2.7, and glucose 5. The Tyrode solution was equilibrated with 97% 02 and 3% CO2 (pH 7.4). One end of the preparation was fixed to the Sylgard floor of the tissue bath and the other was connected to an isometric force transducer (Mangoni GCO1). The preparations were stimulated with rectangular pulses (0.5 to 1 ms in duration and 1.5 times the threshold) through bipolar silver electrodes electrically insulated except for the tip. Action potentials were recorded by means of two 3 M KCIfilled glass microelectrodes, one of which was inserted intracellularly and the other placed in the solution close to the

preparation. Microelectrodes were coupled to two high-input impedance guard electrometer amplifiers (Bigongiari, Firenze). The action potential was displayed on a Tektronix (model 5113) dual beam storage oscilloscope and recorded on an FM tape recorder (Racal 14 DS). The records were played back into a chart recorder (Gould Brush 2400). An automated analysis of action potential was performed, as previously described (Fusi et al., 1984). Evaluation of the following parameters was carried out: action potential amplitude, overshoot, maximum diastolic potential, Vmax, action potential duration at -60mV (APD-60) and at 90% of repolarization

(APD9o).

OAPs were induced by exposing the preparations to low barium concentration or to strophanthidin, as described elsewhere (Mugelli et al., 1983; Amerini et al., 1985a,b; 1988). The drive stimulus was interrupted periodically (usually every minute for 30s) to assess the presence of OAPs. Drugs were superfused at increasing concentrations; the effect of each concentration was followed for at least 20 min. The drugs used in this study were chemically pure: Lcarnitine, L-propionylcarnitine, D-propionylcarnitine, propionic acid (Sigma Tau), digoxin (Boehringer Biochemia Robin), strophanthidin (Sigma).

Statistical analysis Results were expressed as means + standard error of the mean. Statistical analysis was performed by means of the Student's t test, the Fisher exact test, the chi-squared test and ANOVA corrected for multiple measures with Scheffes' procedure. To compare CK and LDH release during hypoxia and reoxygenation between control and treated hearts, ANOVA for multiple measures statistic was used (MANOVA procedure of the SPSS/PC + software). A P < 0.05 (two tailed) was considered statistically significant.

Results

Effect of L-propionylcarnitine on reoxygenation-induced arrhythmias Perfusion with a hypoxic glucose-free medium caused, as expected, a decrease in ventricular rate (Table 1) followed by conduction disturbances (Carbonin et al., 1981; Amerini et al., 1985a; 1988). Reoxygenation was associated with the rapid development of ventricular arrhythmias in all the control hearts and 55% of them developed ventricular fibrillation (VF) (Figure lab). The lowest but effective concentration of L-PC on reoxygenation-induced VF was 1 M when the drug was added to the medium during the hypoxic and reoxygenation phases (Figure 1). The effect was dose-dependent, with a higher concentration (10pM) being more effective on VF and also reducing significantly ventricular arrhythmias (VA). L-PC (0.1 uM) did not significantly modify the incidence of VA or VF (Figure lab). The rise of the diastolic left ventricular pressure which was consistently observed during hypoxia and Table 1 Effect of L-propionylCarnitine (L-PC) on heart rate (beats min -1) during hypoxia and reoxygenation L-PC

0.1 fIM

Time

Control

Baseline

0

254 + 14

255

Hypoxia

5

142 + 8

146 ± 11 108 + 10 78 + 9

15

Reoxygen.

104 + 14 85+ 14

+

12

2

187 + 16

191 + 15

4 6 10

205 + 10 227+6 250+7 249 + 10

218 + 16

15

234 +

11

253 + 9 254 + 8

1 M

10pM

258 + 10 146 + 9

259 + 9 135 + 11 101 + 8 78 + 11 191 + 16 214 + 16

104+11 80+ 11 194 + 17 212 + 18 225 + 8 247 + 6 253 + 6

233 + 9 249 + 8 251 + 9

75

ANTIARRHYTHMIC EFFECTS OF L-PROPIONYLCARNITINE

a

a

100

6

80 -C

60

4U

CU

F

U1)

-c U)

0.

40

C.2

0

20

F

Co Cn

4-5 min

9-10 min

14-15 min

Baseline

Co

Hypoxia

15 min min Reoxygenation

4

b

b 5'

4_

4 CU

CU

4-cco

CL3

0

T

2 a-

4-5 min

9-10 min

T

14-15 min

Figure 1 Guinea-pig isolated hearts: effect of increasing concentrations of L-propionylcarnitine (L-PC) (added during hypoxia and reoxygenation) on ventricular fibrillation (a) and on ventricular arrhythmias (b). The columns show the percentage of hearts in which ventricular fibrillation or ventricular arrhythmias were observed over 1 min periods of reoxygenation at the time indicated on the abscissa scale. (Control open columns; 0.1 PM L-PC hatched columns; 1 jfM L-PC cross-hatched columns; 10pM L-PC solid columns. * P < 0.05, ** P < 0.01 vs. control).

0

Baseline

Hypoxia

4 min

15 min Reoxygenation

Figure 2 Guinea-pig isolated hearts: variations of the left ventricular diastolic pressure (a) and of developed left ventricular pressure (DLVP) (b) after baseline perfusion (0 min), during hypoxia and reoxygenation of control hearts n = 20 (open columns), and hearts perfused with 0.1 PM L-propionylcarnitine (L-PC), n 15 (hatched columns); 1 ,M L-PC, n = 20 (cross-hatched columns) and 1OpM L-PC, n 15 (solid columns). Vertical bars show s.e.mean. L-PC was added during hypoxia and reoxygenation. * P < 0.05 vs. control. =

=

reoxygenation was also prevented by L-PC in a dosedependent manner (Figure 2a). Consequently, the recovery of the developed left ventricular pressure during reoxygenation was more pronounced in the groups of hearts perfused with L-PC (Figure 2b). The release of cardiac enzymes (CK and LDH) in the effluent was significantly reduced by 1 M L-PC during hypoxia and reoxygenation (Figure 3); however, L-PC did not significantly influence the heart rate and the coronary flow rate (Tables 1 and 2). The antiarrhythmic effect of L-PC is less pronounced when it is added to the medium only during the period of reoxygenation. In this case, in fact, the results obtained with 1 M L-PC were not significantly different from controls whereas 10pM L-PC still significantly inhibited VF (Figure 4). At a dose of IpM, L-carnitine, D-propionylcarnitine and propionic acid added to the medium during hypoxia and reoxygenation had no significant effects on reoxygenationinduced VA or VF compared to controls. In fact, after 15 min of reoxygenation, the percentage of VA and VF were, respectively, 67% and 40% (L-carnitine, n = 10), 67% and 50% (Dpropionylcarnitine, n = 10), 80% and 50% (propionic acid, n = 10), 80% and 50% (control, n = 20).

Electrophysiological effects ofL-propionylcarnitine in Purkinje fibres The electrophysiological and mechanical effects of L-PC (1 and to 10pM) on electrically driven sheep Purkinje fibres are shown in Table 3. It is apparent that L-PC did not modify the action potential characteristics and the contractile force. L-PC (1 and 10uM) did not significantly modify OAP amplitude in strophanthidin-treated preparations (n = 8) (5.6 + 3.6 and 5.8 + 1.8 mV, respectively, vs 5.2 + 2.2 mV of the control). In barium-treated preparations (n = 16), L-PC caused a reduction of OAP amplitude, from 7.8 + 2.2 in control to 6.2 + 1.4mV with 1p M L-PC and 3.9 + 1.OmV with 10pM

Table 2 Effect of L-propionylcarnitine (L-PC) flow (ml min 1)

Experiments with digoxin in the isolated heart Perfusion with 1 UM digoxin induced severe VA in all the isolated hearts (n = 4) within 4-7 min. This effect is consistent with our previous observations (Amerini et al., 1985a; 1988). Addition of 1 M L-PC (n 4) to the medium 10 min before and during perfusion with digoxin did not modify the incidence and severity of VA. In fact in both groups, 3 out of 4 hearts developed VF within 4-10min. Furthermore the time necessary for the appearance of VA was similar in control and L-PC-treated hearts (6 + 2 min and 7 + 3min respectively, NS).

on coronary

L-PC

Baseline

Hypoxia

=

Reoxygen.

Time

Control

0.1pM

1pM

1OpM

0 5 10 15 2 4 6 10 15

16.2 + 0.7 15.5 + 0.9 7.1 +0.7 4.8+0.6 8.2 + 1.0 10.7 + 0.8 13.3 +0.8 14.3 + 0.6 14.7 + 0.5

15.6 + 0.7 14.9 + 0.8 6.4 + 0.7 3.9 + 0.8 7.3 + 0.9 10.5 + 0.7 12.6 + 0.8 13.6 ± 0.6 14.1 + 0.6

15.8 + 0.6

15.4 + 0.9 14.7 + 1.0 6.7 + 0.8 4.5 + 0.6 9.0 + 1.3 11.1 + 1.0 13.3 + 0.9 14.4 + 0.9 14.4 + 0.6

15.1 + 0.9

6.7 + 0.5 3.9 + 0.7 7.5 + 0.8 9.8 + 0.7 12.7 + 0.7 13.7 ± 0.5 14.0 + 0.5

M. BARBIERI et al.

76

a

a 2000)i U-

CD

4-

1500 1

3.1

7

in

t

._C

co 0)

4-

*

1000 -

0

S-O

R

(n

CD

0)

0)

-s;

500 -

C)

4-5 min

9-10 min

14-15 min

4-5 min

9-10 min

14-15 min

b 100

5

0

10

15 17 20 Time (min)

25

30

< 80 .

60

t

b

Col

a)

40

-c

0

520 0)

600 F

I E

Figure 4 Guinea-pig isolated hearts: effect of increasing concentrations of L-propionylcarnitine (L-PC) added only during reoxygenation on ventricular fibrillation (VF) (a) and on ventricular arrhythmias

E 400 0) (A

Co 0 200

0

(VA) (b). The columns show the percentage of hearts in which VF or VA was observed over 1 min periods of reoxygenation. Control (open columns), 1 fUM L-PC (cross-hatched columns), 10p.M L-PC (solid

R

H

[

columns). * P < 0.05 vs control.

5

0

10

15 17

20

25

BaCI2

30

Time (min)

Figure 3 Guinea-pig isolated hearts: variations of creatine kinase (CK) (a) and of lactic dehydrogenase (LDH) (b) release in the effluent after baseline perfusion (Omin), during hypoxia (H) and during reoxygenation (R) in control hearts n = 10 (@) and in hearts perfused with 1 fM L-propionylcarnitine n 10 (L). Vertical lines show s.e.mean. * P < 0.05 vs. control refers to the curve analysed by ANOVA for multiple measures.

10

FM

I L-PC

1

FIM 31min

6 min

=

L-PC. The effect of L-PC, however, was not statistically significant due to its variability. In fact, while in 2 cases L-PC did not cause any change of the OAP amplitude, it decreased OAP amplitude in 50% of the remaining cases, and increased it in the others. A typical experiment in which L-PC caused a decrease of OAP amplitude is shown in Figure 5. It is apparent that superfusion with L-PC caused a reduction of the OAP amplitude despite the increase in contractile force. No clearcut correlation was observed between the effects of L-PC on OAP amplitude and contractility.

mvI I

mi

~

5

~

~

~

~

ms

7mgI U1t1

¶1L1tfL

Figure 5 Effect of 1pM L-propionylcarnitine (L-PC) on barium (lOMm)-induced oscillatory afterpotentials. Each panel shows the electrical (upper traces) and mechanical (lower traces) activity recorded during and after the interruption of the stimulation.

Table 3 Effect of L-propionylcarnitine (L-PC) on the transmembrane action potential and mechanical activity of driven (1 Hz) fibres of sheep

(n = 7)

Control L-PC L-PC

1pM

10p

M

Os (mV)

MDP

AP

(mV)

(mV)

APD_60 (Ms)

APDI90 (Ms)

(V/s)

40.0± 1.6 39.7 + 1.6 39.5 + 1.5

83.0 ± 0.8 83.7 ± 0.7 83.8 + 1.0

123.0 ± 1.6 123.5 ± 1.5 123.4 + 1.2

316 ± 27 315 ± 33 323 ± 33

352 ± 30 351 + 35 358 + 33

583 + 81 564 ± 80 570 + 89

..

Purkinje

Contraction (%)

100 103 ± 11 93 + 6

Data are presented as means ± s.e.mean. The number in parentheses indicates the number of experiments. OS = overshoot, MDP = maximum diastolic potential, AP action potential amplitude, APD-60 and APD9Q = action potential duration at - 60mV = maximum rate of upstroke. and 90% repolarization, respectively, =

k'.

ANTIARRHYTHMIC EFFECTS OF L-PROPIONYLCARNITINE

Discussion

The present results demonstrate that L-PC (and not Lcarnitine) exerts antiarrhythmic effects versus reoxygenationinduced arrhythmias. This effect appears to be associated with a protection of the hypoxic and reoxygenated myocardium: in fact the release of CK and LDH is significantly reduced by L-PC and the rise of diastolic left ventricular pressure is prevented. As a consequence, developed left ventricular pressure after reoxygenation is greater in the L-PC-treated hearts than in controls. This 'protection' does not appear to be connected to an effect on heart rate or coronary flow, both of which are unaffected by L-PC-treatment. We also evaluated the possibility that the antiarrhythmic action of L-PC could be due to a direct effect on the electrophysiological properties of the heart. However, concentrations of L-PC that are antiarrhythmic are devoid of any electrophysiological effect in normal Purkinje fibres. L-PC has recently been reported to affect action potential duration of canine Purkinje fibres but only at millimolar concentrations (Aomine et al., 1989). Similar behaviour has been described in guinea-pig ventricular muscle under acidic conditions (Aomine & Arita, 1987) and after addition of amphiphilic lipids (Aomine et al., 1988). Thus, it is unlikely that modifications of refractoriness could play a relevant role under our experimental conditions at micromolar concentrations of L-PC. L-PC was able to reduce the rise of diastolic left ventricular pressure observed during hypoxia and reoxygenation; the increase in diastolic left ventricular pressure is considered an expression of the intracellular calcium overload (Poole-Wilson et al., 1984). Calcium overload may result in OAPs and triggered activity (Manning & Hearse, 1984). One could consequently expect a reduction of the amplitude of OAPs as a result of the effect of L-PC. This is not the case, since L-PC is

77

not able to affect significantly OAP amplitude in two different experimental conditions, i.e. in barium- and strophanthidintreated preparations that we have described previously (Mugelli et al., 1983; Amerini et al., 1985b) and which are certainly suitable for the study of antiarrhythmic effects of drugs (Amerini et al., 1985a,b; 1988). However, since OAPs play a fundamental role in digitalis-induced arrhythmias (Ferrier, 1977) and L-PC was completely ineffective against digitalisinduced arrhythmias, it appears unlikely that L-PC can exert its protective effect on reoxygenation-induced arrhythmias through action of OAPs induced by ischaemia/reperfusion. However, we did not study the effects of L-PC on the electrophysiological properties of the guinea-pig heart under ischaemic or hypoxic conditions; thus the possibility cannot be excluded that L-PC might exert some direct electrophysiological effect under those circumstances. The effect of L-PC on diastolic tension and possibly on calcium overload appears to be operative only when the cause is an hypoxic (present results) or ischaemic (Paulson et al., 1986) insult, followed by reoxygenation or reperfusion, respectively. However, the mechanism by which these effects occur remains unsettled. Finally, these results confirm the data in the literature showing that L-PC is more effective than L-carnitine (Paulson et al., 1986; Siliprandi et al., 1987; Subramian et al., 1987) and demonstrate that the action we described is specific for L-PC. In conclusion, L-PC in micromolar concentrations is able to protect the heart from a period of hypoxia followed by reoxygenation, an antiarrhythmic effect which cannot be explained by its direct electrophysiological properties on normoxic preparations.

Partly supported by a grant from MPI 60%, University of Ferrara.

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(Received April 24, 1990 Revised September 4, 1990 Accepted September 5, 1990)