Molecular and Cellular Biochemistry 116: 147-153, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
L-propionylcarnitine and myocardial performance in stunned porcine myocardium L.M.A. Sassen, D.J. Duncker, A. Hogendoorn, E.O. McFalls, R. Krams, K. Bezstarosti, 1 J.M.J. Lamers 1 and P.D. Verdouw Laboratory for Experimental Cardiology (Thoraxcenter) and ~Department of Biochemistry, Erasmus University Rotterdam, Rotterdam, The Netherlands
Abstract Recently, we showed that L-propionylcarnitine did not affect recovery of regional contractile function of porcine myocardium subjected to 1 h of low-flow ischemia followed by 2 hr of reperfusion. In that study, ischemia may have been too severe and/or the duration of reperfusion too short to detect a beneficial effect of the compound. Therefore, in the present study we investigated the effects of saline (control group; n = 14) or pretreatment with L-propionylcarnitine (3 days of 50mg/kg p.o.b.i.d. + 50mg/kg i.v. prior to the experiment; n = 13) on recovery of regional contractile function of the myocardium in open-chest anesthetized pigs, subjected to two cycles of 10 rain of left anterior descending coronary artery (LADCA) occlusion, each followed by 30 min of reperfusion. In the control animals, at the end of the second reperfusion period, systemic vascular resistance had increased by 18%, which, however, was not observed in the L-propionylcarnitine-treated pigs. In the control group, during the first occlusion, systolic segment length shortening (SSLS) of the LADCA-perfused area decreased from 18.5 _+ 5.5% to - 3.7 = 3.2%. After 30rain of reperfusion, SSLS of the LADCA-perfused area had only partially recovered to 6.2 + 5.9%. During the second occlusion-reperfusion cycle similar values for SSLS were observed. In the treated animals, SSLS of the LADCA-perfused area was slightly improved after the second occlusion-reperfusion cycle (p -- 0.056). This effect did not result in an overall improvement in cardiac pump function. We conclude that in a model of myocardial stunning, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, possibly as a result of this effect, slightly ameliorates post-ischemic hypofunction. (Mol Cell Biochem 116: 147-153, 1992)
Key words: stunned myocardium, systemic hemodynamics, regional contractile function, L-propionylcarnitine, pig
Introduction L-propionylcarnitine, the naturally occurring derivate of L-carnitine, has been shown to enhance fatty acid utilization and to protect ischemic myocardium in the isolated working rat heart preparation [1, 2]. Additionally, a reduction in infarct size has been demonstrated in dogs by L-propionylcarnitine alone or in combination
with the calcium-antagonist tiapamil [3]. Moreover, Liedtke et al. [4] demonstrated reversal of myocardial stunning by L-propionylcarnitine in a porcine model of mild myocardial ischemia. Similar improvement in regional contractility was also found with propionylcarnitine taurine [5].
Address for offprints: P.D. Verdouw, Laboratory for Experimental CardioLogy, Thoraxcenter, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands
148 Recently, we investigated the effects of L-propionylcarnitine on changes in regional contractile function, high energy phosphates and calcium uptake by the sarcoplasmic reticulum after 60 min of low-flow myocardial ischemia in anesthetized swine but were unable to show a beneficial effect of the compound on these parameters, during the early phase (2hr) of reperfusion [6]. Since in that study ischemia may have been too severe to detect a beneficial effect, we now wished to investigate the effects of L-propionylcarnitine on recovery of regional contractile function, in an open-chest porcine model of myocardial stunning. In this model, two cycles of 10 rain of complete coronary artery occlusion each followed by 30 min of reperfusion result in approximately a 60% loss of myocardial contractile function, in the absence of myocardial necrosis [7]. One of the explanations for the beneficial effects of L-propionylcarnitine could be the repletion of intracellular carnitine. Therefore we also measured free carnitine and short chain acylcarnitine in transmural biopsies of the myocardium.
Materials and methods General Cross-bred Landrace × Yorkshire pigs (HVC, Hedel, The Netherlands) or either sex (25-45 kg, n = 27) were used to study the effects of no treatment (n = 14) or pretreatment with L-propionylcarnitine (n = 13) on changes in systemic hemodynamics and myocardial contractile function, induced by two sequences of myocardial ischemia (10 min) and reperfusion (30 rain). Lpropionylcarnitine pretreatment was started three days prior to the experiment and consisted of oral administration of 50 mg/kg L-propionylcarnitine twice a day (total dose: 100 mg/kg/day). On the day of the experiment the animals received 50 mg/kg/day orally in one dose two hours prior to the induction of anesthesia.
Preparation Animals were sedated with intramuscular ketamine (20mg/kg; AUV, Cuijk, The Netherlands), anesthetized with intravenous metomidate (5 mg/kg; Janssen Pharmaceutica, Beerse, Belgium), intubated and connected to a ventilator for intermittent positive pressure ventilation with a mixture of 02 and N 2 (1:2). Respira-
tory rate and tidal volume were set to keep arterial blood gases, measured with an ABL3 (Radiometer, Copenhagen, Denmark) within the normal range: 7.35 < p H o9
ii i
A
*
I
E E
2
W
> 0
BL
01
R1
0 2
R2
BL
0 1 R1
02
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Fig. 1. Systemic h e m o d y n a m i c s in non-treated (open bars) and L-propionylcarnitine-treated (hatched bars) anesthetized pigs before (BL) and after two cycles of 10 rain occlusion (O) of the left anterior descending coronary artery each followed by 30 min of reperfusion (R). M A P = m e a n arterial pressure, L V dP/dtmax = m a x i m u m rate of rise in left ventricular pressure, C O = cardiac output, H R = heart rate, SVR = systemic vascular resistance, L V E D P = left ventricular end diastolic pressure. 4t p < 0.05 versus baseline, • L-propionylcarnitine-induced changes versus baseline are significantly different from changes versus baseline in control swine.
from 90+ 14mmHg to 85 + 13mmHg (NS), while cardiac output fell from 2.9 + 0.2 L/min to 2.4 + 0.2L/ min (p < 0.05). Consequently, calculated systemic vascular resistance increased by approximately 10%. Heart rate did not change from its baseline value of 106 + 15 beats/min, implying that the reduction in cardiac output was solely due to the attenuation of stroke volume. Finally, LV dP/dtmax decreased by 20%, while left ventricular end-diastolic pressure was elevated from 8 + 1 mmHg to 14 +_ 2 mmHg (p < 0.05). During the first
reperfusion period, all ischemia-induced changes in hemodynamic parameters were maintained, except for left ventricular end-diastolic pressure which fully recovered. During the second cycle of coronary artery occlusion and reperfusion, changes in a similar direction as in the first cycle were observed although they were in general of a smaller dimension. L-propionylcarnitine had no effect on systemic hemodynamics under baseline conditions (Fig. 1). Pretreatment with the drug, however, resulted in a drop in
151 mean arterial blood pressure of 15% during the 2 cycles of occlusion and reperfusion, as an increase in systemic vascular resistance was prevented.
T
Regional myocardial segment length changes (Fig. 2)
10 09
_J
The non-LADCA perfused segment showed a tendency towards enhanced shortening during occlusion from 14.9 + 1.2% to 16.9 + 1.5% (p > 0.05). During the first occlusion, the segment perfused by the LADCA became dyskinetic as SSLS decreased from 18.5 + 1.5% to - 3 . 7 + 0.9% and recovered to 6.2 + 1.6% after 30 min of reperfusion. During the second cycle of occlusion and reperfusion similar values for SSLS were observed. Baseline value of SSLS of the non-LADCA area was significantly lower in the L-propionylcarnitine-treated animals (Fig. 2). L-propionylcarnitine induced a slight improvement in regional SSLS of the area perfused by the LADCA after 2 cycles of occlusion and reperfusion (p-- 0.056).
O9 09 BL
01
02
R2
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.
.
.
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Carnitine levels in plasma and myocardium (Tables 1 and 2) In the control group plasma levels of carnitine and short chain acyl carnitine remained unchanged during the experimental period (Table 1). Two cycles of occlusion and reperfusion caused free carnitine of the stunned myocardium to decrease by 20%, while the level of short chain acyl carnitine was not affected (Table 2). Pretreatment with L-propionylcarnitine caused marked increases in plasmalevels of carnitine and short chain acylcarnitine (Table 1). After infusion of the bolus these
R1
.
O1
.
.
R1
.
.
.
.
02
R2
Fig. 2. Systolic segment length shortening (SSLS) of the area remote from that perfused by the left anterior descending coronary artery (non-LADCA, panel a) and of that perfused by the L A D C A (panel b) in non-treated (open bars) and L-propionylcarnitine-treated (hatched bars) anesthetized pigs before (BL) and after two cycles of 10 min occlusion (O) of the left anterior descending coronary artery and 30min of reperfusion (R). ° p < 0.05 versus control animals, * p < 0.05 versus baseline, + p = 0.056 (L-propionylcarnitine-induced change from baseline versus change from baseline in control swine).
Table 1. Plasma levels of carnitine and short-chain acylcarnitine in non-treated and L-propionylcarnitine-pretreated anesthetized pigs before and at the end of two sequences of 10 min occlusion of the left anterior descending coronary artery and 30 min of reperfusion
Carnitine
Short chain acyl carnitine
Group
n
Pre-baseline (before i.v. bolus)
Baseline (after i.v. bolus)
30 rain reperfusion-2
C LPC
6 14
23.4 + 1.7"
8.8 + 0.9 87.4 + 4.0 °+
8.3 + 0.9 75.8 + 4.5 *~
C LPC
6 14
5.4 + 0.9"
0.9 + 0.2 163.3 + 27.4 "+
0.6 + 0.2 49.4 + 7.5 *#
C = control; LPC = L-propionylcarnitine. Date are in/zM and have been expressed as mean + SEM; "p < 0.05 versus Baseline in control swine; +p < 0.05 versus Pre-baseline in L-propionylcarnitine treated swine; *p < 0.05 versus Baseline; ~ L-propionylcarntine-induced changes versus Baseline after bolus are significantly different from those in the control animals.
152 levels increased even more noticeable. In the pretreated animals the myocardial level of carnitine at baseline was higher than in the control animals (Table 2). After two cycles of ischemia-reperfusion this difference was maintained in the control but not in the ischemic area. The myocardial level of short chain acyl carnitine was not affected by treatment. A decrease, as observed in the LADCA-perfused myocardium of the control animals after occlusion-reperfusion, was, however, prevented by L-propionylcarnitine treatment.
Discussion The present study demonstrates that in a model of stunned myocardium L-propionylcarnitine is capable of modestly improving regional contractile function. Recently, we investigated the effects of L-propionylcarnitine in a model in which 60 min of low-flow ischemia was followed by 2hr of reperfusion [6]. In that model no improvement of function by L-propionylcarnitine was observed, because the ischemic episode was too severe. It has been reported that in the model we used in the present study a loss of myocardial contractile function of approximately 60% occurs in the absence of myocardial necrosis [7]. We observed a 72% loss in the control group while in the treated animals the loss was 61%, which was borderline significantly different. A number of possible explanations can be forwarded. Firstly, regional myocardial systolic segment shortening is a load-dependent index of myocardial contractile function. Therefore, the prevention by L-propionylcarnitine of peripheral systemic vasoconstriction, and the consequently lower arterial blood pressure (afterload) may have been responsible for the improvement in systolic segment shortening. Indeed, a vasodila-
tory action of L-propionylcarnitine has also been observed in anesthetized dogs, where L-propionylcarnitine (bolus: 20-200 mg/kg) dose-dependently decreased total peripheral resistance by up to 10% [13]. In parallel, we have found that in pigs after one hour of lowflow ischemia followed by 2 hr of reperfusion, L-propionylcarnitine caused vasodilation in the non-ischemic myocardium as well as in some peripheral organs, also revealing a vasodilatory effect [6]. However, we must keep in mind that in the latter study the higher myocardial work in the L-propionylcarnitine-treated animals could also have resulted in metabolic autoregulation secondary to the higher myocardial oxygen demand. Secondly, L-propionylcarnitine may have caused partial reversal of myocardial stunning, similar to that found by Liedtke et al. [4] in a porcine model of mild myocardial ischemia. The latter group suggested that a positive inotropic action of L-propionylcarnitine might be responsible for this beneficial effect. A positive inotropic action of the compound has also been found in rabbits [14] as well as in man [15] although this was not confirmed by others in dogs [13]. In the present study we found in the pretreated animals a lower segment length shortening of the non-LADCA area at baseline (P = 0.034), which, however, was not noticed in the area perfused by the LADCA. We therefore believe that with this dose the positive inotropic effect on the stunned myocardium, if present, is only of minor importance. The beneficial effect on regional contractile function of the stunned myocardium did not result in an overall improvement in cardiac pump function. This is in accordance with the results of the study by Liedtke et al. [4], where left ventricular developed pressure, heart rate and LV d P / d t m a x w e r e also not affected by L-propionylcarnitine.
Table 2. Myocardial tissue levels of carnitine and short-chain acylcarnitine in non-treated and L-propionylcarnitine-pretreated anesthetized pigs before and at the end of two sequences of 10 min occlusion of the left anterior descending coronary artery and 30 min of reperfusion Group
n
Baseline
30 min reperfusion-2 Control area
Ischernic area
Carnitine
C LPC
8 6
4 . 0 ± 0.5 5 . 7 ± 0.6 °
4 . 3 ± 0.4 5 . 8 ± 0.4 °
3 . 2 ± 0.3 + 4 . 4 ± 0.6
Short chain acyl carnitine
C LPC
8 6
1 . 3 ± 0.1 1 . 3 ± 0.2
1 . 2 ± 0.2 1 . 2 ± 0.2
1 . 7 ± 0.2 1 . 1 ± 0.2 ~
C = control; L P C = L-propionylcarnitine. D a t a are in txmol/g protein and have been expressed as m e a n +_ SEM; ° p < 0.05 versus control animals; +p < 0.05 versus control area; # p < 0.05 L-propionylcarnitine-induced changes from Baseline versus changes from Baseline in the control pigs.
153 The present study was not designed to unravel the mechanism by which L-propionylcarnitine exerts its protective effect. However, one possibility deserved attention. L-propionylcarnitine could simply act by repleting carnitine, lost during ischemia. The decrease after ischemia-reperfusion was, however, not affected by L-propionylcarnitine. Similarly, in a former study on L-propionylcarnitine of our laboratory [6] we showed that ischemia followed by reperfusion indeed depleted the intracellular carnitine pool which, however, was not prevented by L-propionylcarnitine. On the other hand, baseline levels of myocardial free carnitine were higher in the L-propionylcarnitine-treated animals which might be beneficial by preventing a fall below critical levels. The increased plasma free carnitine after intravenous administration of L-propionylcarnitine has been reported before [6] and is most likely due to hydrolysation from L-propionylcarnitine to free carnitine in tissues or exchange for free carnitine. In conclusion, in a model of myocardial stunning, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, possibly as a result of this effect, slightly ameliorates postischemic hypofunction. However, a protective effect in combination with a positive inotropic effect can not be excluded.
Acknowledgements L-propionylcarnitine was generously supplied by Sigma Tau Pharmaceutical of Rome, Italy.
References 1. Paulson D J, Traxler J, Schmidt M, Noonan J, Shug AL: Protection of the ischaemic myocarium by L-propionylcarnitine: effects on the recovery of cardiac output after ischaemia and reperfusion, carnitine transport, and fatty acid oxidation. Cardiovasc Res 20: 536-541, 1986 2. Shug A, Paulson D, Subraimanian R, Regitz V: Protective effects of propionyl-L-carnitine during ischemia and reperfusion. Cardiovasc Drugs Ther 5 (suppl 1): 7%83, 1991
3. Leasure JE, Kordenat K: Effect of propionyl-L-carnitine on experimental myocardial infarction in dogs. Cardiovasc Drugs Ther 5 (suppl 1): 85-95, 1991 4. Liedtke AJ, DeMaison A, Nellis DSH: Effect of L-propionylcarnitine on mechanical recovery during reflow in intact hearts. Am J Physiol 255 (Heart Circ Physiol 24): H169-H176, 1988 5. Molaparast-Saless F, Nellis SH, Liedtke AJ: The effects of propionylcarnitine taurine on cardiac performance in aerobic and ischemic myocardium. J Moll Cell Cardiol 20: 63-74, 1988 6. Sasscn LMA, Bezstarosti K, van der Giessen W J, Lamers JMJ, Verdouw PD: L-propionylcarnitine increases post-ischemic blood flow but does not affect recovery of the energy charge. Am J Physiol 261 (Heart Circ Physiol 30): H172-H180, 1991 7. Schott RJ, Rohmann S, Braun ER, Schaper W: Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res 66: 913931, 1990 8. Guth BD, Schultz R, Heusch G: Evaluation of parameters for the assessment of regional myocardial contractile function during asynchronous left ventricular contraction. Basic Res Cardiol 84: 22%246, 1989 9. Barth PG, Scholte HR, Berden JA, van der Klei-van Moorsel JM, Luyt-Houwen IEM, van het Veer-Korthof ETh, van der Harten J J, Sobotka-Plojhar MA: An X-linked mitochondrial disease affecting cardiac muscle skeletal muscle and neutrophil leukocytes. J Neurol Sci 62: 327-355, 1983 10. Lamers JMJ, De Jonge-Stinis JT, Verdouw PD, Htilsmann WC: On the possible role of long chain fatty acycarnitine accumulation in producing functional and calcium permeability changes in membranes during myocardial ischaemia. Cardiovasc Res 21: 313-322, 1987 11. Lowry OH, Rosenbrough NJ, Fare AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265-275, 1951 12. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976 13. Cevese A, Schena F, Cerutti G: Short-term hemodynamic effects of intravenous propionyl-L-carnitine in anesthetized dogs. Cardiovasc Drugs Ther 5 (suppl 1): 45-56, 1991 14. Ferrari R, Pasini E, Condorelli E, Boraso A, Lisciani R, Marzo A, Visioli O: Effect of propionyl-L-carnitine on mechanical function of isolated rabbit heart. Cardiovasc Drugs Ther 5 (suppl 1): 1%23, 1991 15. Chiddo A, Gaglione A, Musci S, Troito G, Grimaldi N, Locuratolo N, Rizzon P: Hemodynamic study of intravenous propionyl-L-carnitine in patients with ischaemic heart disease and normal left ventricular function. Cardiovasc Drugs Ther 5 (suppl 1): 10%11l, 1991
L-propionylcarnitine and myocardial performance in stunned porcine myocardium L.M.A. Sassen, D.J. Duncker, A. Hogendoorn, E.O. McFalls, R. Krams, K. Bezstarosti, 1 J.M.J. Lamers 1 and P.D. Verdouw Laboratory for Experimental Cardiology (Thoraxcenter) and ~Department of Biochemistry, Erasmus University Rotterdam, Rotterdam, The Netherlands
Abstract Recently, we showed that L-propionylcarnitine did not affect recovery of regional contractile function of porcine myocardium subjected to 1 h of low-flow ischemia followed by 2 hr of reperfusion. In that study, ischemia may have been too severe and/or the duration of reperfusion too short to detect a beneficial effect of the compound. Therefore, in the present study we investigated the effects of saline (control group; n = 14) or pretreatment with L-propionylcarnitine (3 days of 50mg/kg p.o.b.i.d. + 50mg/kg i.v. prior to the experiment; n = 13) on recovery of regional contractile function of the myocardium in open-chest anesthetized pigs, subjected to two cycles of 10 rain of left anterior descending coronary artery (LADCA) occlusion, each followed by 30 min of reperfusion. In the control animals, at the end of the second reperfusion period, systemic vascular resistance had increased by 18%, which, however, was not observed in the L-propionylcarnitine-treated pigs. In the control group, during the first occlusion, systolic segment length shortening (SSLS) of the LADCA-perfused area decreased from 18.5 _+ 5.5% to - 3.7 = 3.2%. After 30rain of reperfusion, SSLS of the LADCA-perfused area had only partially recovered to 6.2 + 5.9%. During the second occlusion-reperfusion cycle similar values for SSLS were observed. In the treated animals, SSLS of the LADCA-perfused area was slightly improved after the second occlusion-reperfusion cycle (p -- 0.056). This effect did not result in an overall improvement in cardiac pump function. We conclude that in a model of myocardial stunning, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, possibly as a result of this effect, slightly ameliorates post-ischemic hypofunction. (Mol Cell Biochem 116: 147-153, 1992)
Key words: stunned myocardium, systemic hemodynamics, regional contractile function, L-propionylcarnitine, pig
Introduction L-propionylcarnitine, the naturally occurring derivate of L-carnitine, has been shown to enhance fatty acid utilization and to protect ischemic myocardium in the isolated working rat heart preparation [1, 2]. Additionally, a reduction in infarct size has been demonstrated in dogs by L-propionylcarnitine alone or in combination
with the calcium-antagonist tiapamil [3]. Moreover, Liedtke et al. [4] demonstrated reversal of myocardial stunning by L-propionylcarnitine in a porcine model of mild myocardial ischemia. Similar improvement in regional contractility was also found with propionylcarnitine taurine [5].
Address for offprints: P.D. Verdouw, Laboratory for Experimental CardioLogy, Thoraxcenter, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands
148 Recently, we investigated the effects of L-propionylcarnitine on changes in regional contractile function, high energy phosphates and calcium uptake by the sarcoplasmic reticulum after 60 min of low-flow myocardial ischemia in anesthetized swine but were unable to show a beneficial effect of the compound on these parameters, during the early phase (2hr) of reperfusion [6]. Since in that study ischemia may have been too severe to detect a beneficial effect, we now wished to investigate the effects of L-propionylcarnitine on recovery of regional contractile function, in an open-chest porcine model of myocardial stunning. In this model, two cycles of 10 rain of complete coronary artery occlusion each followed by 30 min of reperfusion result in approximately a 60% loss of myocardial contractile function, in the absence of myocardial necrosis [7]. One of the explanations for the beneficial effects of L-propionylcarnitine could be the repletion of intracellular carnitine. Therefore we also measured free carnitine and short chain acylcarnitine in transmural biopsies of the myocardium.
Materials and methods General Cross-bred Landrace × Yorkshire pigs (HVC, Hedel, The Netherlands) or either sex (25-45 kg, n = 27) were used to study the effects of no treatment (n = 14) or pretreatment with L-propionylcarnitine (n = 13) on changes in systemic hemodynamics and myocardial contractile function, induced by two sequences of myocardial ischemia (10 min) and reperfusion (30 rain). Lpropionylcarnitine pretreatment was started three days prior to the experiment and consisted of oral administration of 50 mg/kg L-propionylcarnitine twice a day (total dose: 100 mg/kg/day). On the day of the experiment the animals received 50 mg/kg/day orally in one dose two hours prior to the induction of anesthesia.
Preparation Animals were sedated with intramuscular ketamine (20mg/kg; AUV, Cuijk, The Netherlands), anesthetized with intravenous metomidate (5 mg/kg; Janssen Pharmaceutica, Beerse, Belgium), intubated and connected to a ventilator for intermittent positive pressure ventilation with a mixture of 02 and N 2 (1:2). Respira-
tory rate and tidal volume were set to keep arterial blood gases, measured with an ABL3 (Radiometer, Copenhagen, Denmark) within the normal range: 7.35 < p H o9
ii i
A
*
I
E E
2
W
> 0
BL
01
R1
0 2
R2
BL
0 1 R1
02
R2
Fig. 1. Systemic h e m o d y n a m i c s in non-treated (open bars) and L-propionylcarnitine-treated (hatched bars) anesthetized pigs before (BL) and after two cycles of 10 rain occlusion (O) of the left anterior descending coronary artery each followed by 30 min of reperfusion (R). M A P = m e a n arterial pressure, L V dP/dtmax = m a x i m u m rate of rise in left ventricular pressure, C O = cardiac output, H R = heart rate, SVR = systemic vascular resistance, L V E D P = left ventricular end diastolic pressure. 4t p < 0.05 versus baseline, • L-propionylcarnitine-induced changes versus baseline are significantly different from changes versus baseline in control swine.
from 90+ 14mmHg to 85 + 13mmHg (NS), while cardiac output fell from 2.9 + 0.2 L/min to 2.4 + 0.2L/ min (p < 0.05). Consequently, calculated systemic vascular resistance increased by approximately 10%. Heart rate did not change from its baseline value of 106 + 15 beats/min, implying that the reduction in cardiac output was solely due to the attenuation of stroke volume. Finally, LV dP/dtmax decreased by 20%, while left ventricular end-diastolic pressure was elevated from 8 + 1 mmHg to 14 +_ 2 mmHg (p < 0.05). During the first
reperfusion period, all ischemia-induced changes in hemodynamic parameters were maintained, except for left ventricular end-diastolic pressure which fully recovered. During the second cycle of coronary artery occlusion and reperfusion, changes in a similar direction as in the first cycle were observed although they were in general of a smaller dimension. L-propionylcarnitine had no effect on systemic hemodynamics under baseline conditions (Fig. 1). Pretreatment with the drug, however, resulted in a drop in
151 mean arterial blood pressure of 15% during the 2 cycles of occlusion and reperfusion, as an increase in systemic vascular resistance was prevented.
T
Regional myocardial segment length changes (Fig. 2)
10 09
_J
The non-LADCA perfused segment showed a tendency towards enhanced shortening during occlusion from 14.9 + 1.2% to 16.9 + 1.5% (p > 0.05). During the first occlusion, the segment perfused by the LADCA became dyskinetic as SSLS decreased from 18.5 + 1.5% to - 3 . 7 + 0.9% and recovered to 6.2 + 1.6% after 30 min of reperfusion. During the second cycle of occlusion and reperfusion similar values for SSLS were observed. Baseline value of SSLS of the non-LADCA area was significantly lower in the L-propionylcarnitine-treated animals (Fig. 2). L-propionylcarnitine induced a slight improvement in regional SSLS of the area perfused by the LADCA after 2 cycles of occlusion and reperfusion (p-- 0.056).
O9 09 BL
01
02
R2
b 20
o~
10
o9 _1 o9 o9
_10 ~ .
.
.
.
BL
Carnitine levels in plasma and myocardium (Tables 1 and 2) In the control group plasma levels of carnitine and short chain acyl carnitine remained unchanged during the experimental period (Table 1). Two cycles of occlusion and reperfusion caused free carnitine of the stunned myocardium to decrease by 20%, while the level of short chain acyl carnitine was not affected (Table 2). Pretreatment with L-propionylcarnitine caused marked increases in plasmalevels of carnitine and short chain acylcarnitine (Table 1). After infusion of the bolus these
R1
.
O1
.
.
R1
.
.
.
.
02
R2
Fig. 2. Systolic segment length shortening (SSLS) of the area remote from that perfused by the left anterior descending coronary artery (non-LADCA, panel a) and of that perfused by the L A D C A (panel b) in non-treated (open bars) and L-propionylcarnitine-treated (hatched bars) anesthetized pigs before (BL) and after two cycles of 10 min occlusion (O) of the left anterior descending coronary artery and 30min of reperfusion (R). ° p < 0.05 versus control animals, * p < 0.05 versus baseline, + p = 0.056 (L-propionylcarnitine-induced change from baseline versus change from baseline in control swine).
Table 1. Plasma levels of carnitine and short-chain acylcarnitine in non-treated and L-propionylcarnitine-pretreated anesthetized pigs before and at the end of two sequences of 10 min occlusion of the left anterior descending coronary artery and 30 min of reperfusion
Carnitine
Short chain acyl carnitine
Group
n
Pre-baseline (before i.v. bolus)
Baseline (after i.v. bolus)
30 rain reperfusion-2
C LPC
6 14
23.4 + 1.7"
8.8 + 0.9 87.4 + 4.0 °+
8.3 + 0.9 75.8 + 4.5 *~
C LPC
6 14
5.4 + 0.9"
0.9 + 0.2 163.3 + 27.4 "+
0.6 + 0.2 49.4 + 7.5 *#
C = control; LPC = L-propionylcarnitine. Date are in/zM and have been expressed as mean + SEM; "p < 0.05 versus Baseline in control swine; +p < 0.05 versus Pre-baseline in L-propionylcarnitine treated swine; *p < 0.05 versus Baseline; ~ L-propionylcarntine-induced changes versus Baseline after bolus are significantly different from those in the control animals.
152 levels increased even more noticeable. In the pretreated animals the myocardial level of carnitine at baseline was higher than in the control animals (Table 2). After two cycles of ischemia-reperfusion this difference was maintained in the control but not in the ischemic area. The myocardial level of short chain acyl carnitine was not affected by treatment. A decrease, as observed in the LADCA-perfused myocardium of the control animals after occlusion-reperfusion, was, however, prevented by L-propionylcarnitine treatment.
Discussion The present study demonstrates that in a model of stunned myocardium L-propionylcarnitine is capable of modestly improving regional contractile function. Recently, we investigated the effects of L-propionylcarnitine in a model in which 60 min of low-flow ischemia was followed by 2hr of reperfusion [6]. In that model no improvement of function by L-propionylcarnitine was observed, because the ischemic episode was too severe. It has been reported that in the model we used in the present study a loss of myocardial contractile function of approximately 60% occurs in the absence of myocardial necrosis [7]. We observed a 72% loss in the control group while in the treated animals the loss was 61%, which was borderline significantly different. A number of possible explanations can be forwarded. Firstly, regional myocardial systolic segment shortening is a load-dependent index of myocardial contractile function. Therefore, the prevention by L-propionylcarnitine of peripheral systemic vasoconstriction, and the consequently lower arterial blood pressure (afterload) may have been responsible for the improvement in systolic segment shortening. Indeed, a vasodila-
tory action of L-propionylcarnitine has also been observed in anesthetized dogs, where L-propionylcarnitine (bolus: 20-200 mg/kg) dose-dependently decreased total peripheral resistance by up to 10% [13]. In parallel, we have found that in pigs after one hour of lowflow ischemia followed by 2 hr of reperfusion, L-propionylcarnitine caused vasodilation in the non-ischemic myocardium as well as in some peripheral organs, also revealing a vasodilatory effect [6]. However, we must keep in mind that in the latter study the higher myocardial work in the L-propionylcarnitine-treated animals could also have resulted in metabolic autoregulation secondary to the higher myocardial oxygen demand. Secondly, L-propionylcarnitine may have caused partial reversal of myocardial stunning, similar to that found by Liedtke et al. [4] in a porcine model of mild myocardial ischemia. The latter group suggested that a positive inotropic action of L-propionylcarnitine might be responsible for this beneficial effect. A positive inotropic action of the compound has also been found in rabbits [14] as well as in man [15] although this was not confirmed by others in dogs [13]. In the present study we found in the pretreated animals a lower segment length shortening of the non-LADCA area at baseline (P = 0.034), which, however, was not noticed in the area perfused by the LADCA. We therefore believe that with this dose the positive inotropic effect on the stunned myocardium, if present, is only of minor importance. The beneficial effect on regional contractile function of the stunned myocardium did not result in an overall improvement in cardiac pump function. This is in accordance with the results of the study by Liedtke et al. [4], where left ventricular developed pressure, heart rate and LV d P / d t m a x w e r e also not affected by L-propionylcarnitine.
Table 2. Myocardial tissue levels of carnitine and short-chain acylcarnitine in non-treated and L-propionylcarnitine-pretreated anesthetized pigs before and at the end of two sequences of 10 min occlusion of the left anterior descending coronary artery and 30 min of reperfusion Group
n
Baseline
30 min reperfusion-2 Control area
Ischernic area
Carnitine
C LPC
8 6
4 . 0 ± 0.5 5 . 7 ± 0.6 °
4 . 3 ± 0.4 5 . 8 ± 0.4 °
3 . 2 ± 0.3 + 4 . 4 ± 0.6
Short chain acyl carnitine
C LPC
8 6
1 . 3 ± 0.1 1 . 3 ± 0.2
1 . 2 ± 0.2 1 . 2 ± 0.2
1 . 7 ± 0.2 1 . 1 ± 0.2 ~
C = control; L P C = L-propionylcarnitine. D a t a are in txmol/g protein and have been expressed as m e a n +_ SEM; ° p < 0.05 versus control animals; +p < 0.05 versus control area; # p < 0.05 L-propionylcarnitine-induced changes from Baseline versus changes from Baseline in the control pigs.
153 The present study was not designed to unravel the mechanism by which L-propionylcarnitine exerts its protective effect. However, one possibility deserved attention. L-propionylcarnitine could simply act by repleting carnitine, lost during ischemia. The decrease after ischemia-reperfusion was, however, not affected by L-propionylcarnitine. Similarly, in a former study on L-propionylcarnitine of our laboratory [6] we showed that ischemia followed by reperfusion indeed depleted the intracellular carnitine pool which, however, was not prevented by L-propionylcarnitine. On the other hand, baseline levels of myocardial free carnitine were higher in the L-propionylcarnitine-treated animals which might be beneficial by preventing a fall below critical levels. The increased plasma free carnitine after intravenous administration of L-propionylcarnitine has been reported before [6] and is most likely due to hydrolysation from L-propionylcarnitine to free carnitine in tissues or exchange for free carnitine. In conclusion, in a model of myocardial stunning, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, possibly as a result of this effect, slightly ameliorates postischemic hypofunction. However, a protective effect in combination with a positive inotropic effect can not be excluded.
Acknowledgements L-propionylcarnitine was generously supplied by Sigma Tau Pharmaceutical of Rome, Italy.
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