ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 288, No. 2, August 1, pp. 533-537, 1991
Free Radical Scavenging Is Involved in the Protective Effect of L-Propionyl-carnitine against IschemiaReperfusion Injury of the Heart L. Packer,’ M. Valenza, E. Serbinova, P. Starke-Reed,*
K. Frost, and V. Kagan
Department of Molecular and Cell Biology, University of California, Berkeley, of Experimental Medicine, George Washington University School of Medicine,
California 94720; and *Department Washington, D.C. 20037
Received October 19, 1990, and in revised form April 8, 1991
L-Propionyl-carnitine is known to improve the recovery of myocardial function and metabolic parameters reduced in the course of ischemia-reperfusion of the heart. The mechanism of this protective effect of L-propionylcarnitine is not fully understood. The purpose of this study was to elucidate the effects of L-propionyl-carnitine in Langendorff perfused rat hearts subjected to 40 min of ischemia followed by 20 min of reperfusion. We tested the hypothesis that L-propionyl-carnitine suppresses generation of oxygen radicals and subsequent oxidative modification of myocardial proteins during reperfusion. Our data show that the protective effect of L-propionylcarnitine in the course of ischemia-reperfusion is highly significant in terms both of mechanical properties of the heart (developed pressure) and of high-energy phosphates (ATP, creatine phosphate). Myocardial creatine phosphokinase (CPK) activity decreased in the course of the reperfusion period. The loss of CPK activity was partially prevented by L-propionyl-carnitine. Two other effects were observed when L-propionyl-carnitine was present in the perfusion solution: (i) the reperfusion-induced sharp increase in oxidative protein modification was completely prevented as detected by the formation of protein carbonyls, and (ii) generation of hydroxyl radicals was significantly inhibited as detected by the formation of the adducts with the spin trap 5,5-dimethyl1-pyrrolinel-oxide. We conclude that the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart is at least due in part to its ability to suppress the development of oxidative stress and free 0 1991 Academic Press, Inc. radical damage.
The hypothesis that free radical species of oxygen and subsequently formed peroxyl radicals of lipids and proteins are responsible for myocardial injury (in particular, ’ To whom correspondence
should be addressed.
0003-9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
ischemic and postischemic injury, anthracycline antibiotics-induced injury, chelated and nonchelated iron-dependent injury, etc.) has obtained much support over the past few years (l-3). The actual generators of oxygenderived free radicals in ischemic and reperfused heart tissue are still not known, but several sources have been postulated, viz. mitochondrial electron transport, activated polymorphonuclear cells, xanthine oxidase, autooxidation of catecholamines, peroxisomal fatty acid oxidation, biosynthesis of prostaglandins, and endothelial relaxing factor (4-7). Evidence in support of this free radical hypothesis is based mainly on: (i) the detection of radical intermediates arising in the course of ischemia-reperfusion (ESR-detectable spin adducts of oxygen free radicals) (6,8-10) and specific oxidation products of chemical probes-e.g., salicylate-(11,12), and (ii) the protection of ischemic-reperfused cardiac tissue by antioxidant enzymes, chelators of transition metals, or free radical scavengers (spin traps, antioxidants) (13-19). A large multinational epidemiological study showed that higher dietary intake and plasma levels of the antioxidant vitamins and antioxidants were associated with reduced risk of mortality from ischemia heart disease (20). Recently it was reported that L-propionyl-carnitine has a pronounced protective effect against ischemia-reperfusion damage (21-24). Although L-propionyl-carnitine does not possess direct radical scavenging activity (25,26), its protective effects under conditions where enhanced production of free radicals might be primarily responsible for the damage were documented (21,26). In the present investigation our purpose was to study the effects of L-propionyl-carnitine in Langendorff perfused rat hearts subjected to 40 min of global ischemia to clarify its role in protection of the heart. We tested the hypothesis that L-propionyl-carnitine decreases the production of free radicals and oxidative damage during reperfusion. 533
Inc. reserved.
534 MATERIALS
PACKER
AND
METHODS
Animals and perfusion experiments. Male Sprague-Dawley rata (280300 g) were used for the study. The animals were anesthetized with diethyl ether and injected with 400 units of heparin intravenously before excision of the heart. The perfusion apparatus was constructed essentially as described earlier (27) and thermostated to 38°C. A retrograde aortic perfusion system (28) without recirculation was employed. The perfusion solution was an oxygenated modified Krebs-bicarbonate buffer (1.2 mM MgClz. 6H,O), 5.9 mM KCl, 17 mM d-glucose, 25 mM NaHC03, 2.0 mM CaCl,, 117 mM NaCl), pH 7.4, and was continuously bubbled with 95% Ox and 5% COr. The medium used in the L-propionyl-camitine experiments contained 10 mM L-propionyl-carnitine hydrochloride. When L-propionyl-carnitine was added, the pH of the perfusate was adjusted to 7.4 and the sodium chloride concentration lowered respectively to retain constant osmolarity. After excision of the heart, the aorta was immediately cannulated and Langendorff perfusion (left ventricle perfusion) was initiated for 10 min (preperfusion period). During this period a cannula was attached to the pressure transducer and inserted into the left ventricle through the right atrium. Hearts were then subjected to a period of global ischemia for 40 min. Ischemia was achieved by clamping the aortic cannula. The hearts were reoxygenated (reperfusion period) for 20 min after ischemia. Control hearts were subjected to 60 min of perfusion (no ischemia) both with and without L-propionylcarnitine. Left ventricular pressure was measured with a Gould/Statham P23 pressure transducer. Recovery of the heart was measured before and after ischemia by calculating the developed pressure from the trace obtained from the pressure transducer (Gilson Duograph). At the end of the perfusion experiments, all hearts were freeze-clamped and stored in liquid nitrogen. Extraction. The frozen hearts were crushed with a chilled mortar and pestle and the powder thus formed added to 2 ml of 6% perchloric acid in 10 mM EDTA at 0°C. The mixture was homogenized and then centrifuged at 3600g for 10 min. The solids were again homogenized in 2 ml of ice-cold 6% perchloric acid in 10 mM EDTA and centrifuged. The pooled supernatants were neutralized to pH 7.0 with the addition of ice-cold 1 M KOH. The precipitate of KClO, was removed by centrifugation and filtration. The extracts were then lyophilized and redissolved in 2.0 ml H,O and 0.5 ml D,O (24). 31P NMR spectroscopy was performed at 121.5 NMR spectroscopy. MHz on a Bruker AM 300 spectrometer. D20 was used as a field lock and chemical shifts were assigned relative to the creatine phosphate (CP)‘peak (-2.5 ppm relative to 85% phosphoric acid). A 60-s excitation pulse and a 2.1-s repetition time were used to acquire 3072 scans over 108 min. A ~-HZ line broadening was applied to reduce spectral noise. Quantification of the spectra was achieved by acquiring a second spectrum of each sample after addition of CP and ATP standards. The integrals of the spectral peaks from the two spectra were compared to determine the amount of phosphate present in the original sample. The reproducibility of the measurements of 31P metabolites was found to be 5% for identical samples.
ET AL. The resulting supernatant was used for the protein nations as previously described (30). Administration of 5,5-dimethyl-l-Wrroline-l-oxide (0.1 M) was infused via a side arm of aortic cannula rate of 1 ml/min. Coronary flow was approximately the first 3 min of Langendorff perfusion. DMPO was the reperfusion period. DMPO was initially purified a charcoal column before use.
2 Ahbreviations used: CP, creatine phosphate; phokinase; DMPO, 5,5-dimethyl-1-pyrroline-l-oxide; tricular developed pressure.
CPK, creatine phosLVDP, left ven-
determi-
(DMPO). DMPO into the heart at a 10 ml/min during infused only during by passing through
ESR measurements. ESR spectra were recorded in glass accupette pipets at room temperature using a Bruker ER 200 D-SRC electron spin resonance spectrometer (X-band). Instrument settings: modulation amplitude 2.5, G; scan range, 100 G; central field, 3380 G; microwave power, 10 mW. The DMPO concentration was measured spectrophotometrically (e = 8 X lo3 M-’ cm-’ at 227 nm). To prevent spin adduct decay the perfusate was frozen in liquid nitrogen immediately after the sample was taken. No significant loss of ESR signal occurred during the storage of DMPO adducts. Hyperfine coupling constants were measured directly from the field scan using Mn*+ as a marker for calibration. As a model system generating hydroxyl radicals, the Fenton reagent consisting of 50 nM FeSO, * 6H,O and 80 mM H,Oz was used in the presence of 100 mM DMPO. Reagents. The authors thank Dr. C. Trevisani (Sigma Tau Co., Pomezia, Italy) for a generous gift of L-propionyl-carnitine hydrochloride used in this study. Heparin was from Upjohn Co. (Kalamazoo, MI). The other chemicals were from Sigma Chemical Co. (St. Louis, MO).
RESULTS
Left ventricular developed pressure (LVDP) and mechanical recovery. L-propionyl-carnitine did not affect the preischemic LVDP. The postischemic LVDP was significantly higher in the L-propionyl-carnitine-treated group, compared to the postischemic LVDP in the hearts without L-propionyl-carnitine (Table I). After 40 min of ischemia the mechanical recovery of the hearts treated with 10 mM L-propionyl-carnitine was 80% of the control value, while in the hearts without L-propionyl-carnitine it was only 26% of the control level (Table I). High-energy phosphates. The results of the 31P NMR determinations of ATP and CP contents in the heart extracts are shown in Table II. Global ischemia for 40 min followed by 20 min of reperfusion caused a 75% loss of myocardial ATP and an 84% loss of CP. L-Propionylcarnitine (10 mM) protected the hearts from the loss of ATP and phosphocreatine: the ATP and CP concentrations of the hearts exposed to ischemia-reperfusion in the presence of 10 mM L-propionyl-carnitine did not differ
Creatine phosphokinase assay. Creatine phosphokinase (CPK) activity was assayed using a standard kit obtained from Sigma Chemical Co. Carbonyl assay. Following treatment the hearts were minced and placed in a 60-mm cell culture plate. Added to the dish was 2 ml of the Hepes buffer plus a protease inhibitor cocktail as described in (29) with the addition of 0.1% digitonin. The plates were placed on a rotating apparatus and the tissue gently swirled at room temperature for 15 min. The resulting fluid was removed and further centrifuged at a top speed in a table-top clinical centrifuge for 5 min to precipitate the digitonin.
oxidation
TABLE
I
Effect of L-Propionyl-carnitine on Recovery of Left Ventricular Pressure (LDVP) in Isolated Perfused Rat Hearts after 40 min of Global Ischemia LVDP
Control +lO mM L-propionyl-carnitine
(mmHg)
Before ischemia
After ischemia
78 t 18 80 rt_ 17
20 f 18 64 f 20
Note. Mean values -+ SD are given; n = 3.
L-PROPIONYL-CARNITINE TABLE
AND
ISCHEMIA-REPERFUSION
535
INJURY
MODEL
II
SYSTEM
Effect of L-Propionyl-carnitine on the Content of the HighEnergy Phosphates and Creatine Phosphokinase Activity (CPK) in Isolated Perfused Rat Hearts 31PMetabolites (%) ATP 60
min perfusion
Modified Krebs +lO mM L-propionyl-carnitine 10 min perfusion
100
100
86.7 2 10.7
100
100
79.9f 2.9
25 + 14 90 + 15
16 + 10 89 + 14
32.3 f 70.4 f
as=a,
I
+ 40 min ischemia + 20 min reperfusion
Modified Krebs +lO mM L-propionyl-carnitine Note.
CP
CPK activity (units/mg protein)
4.4 1.7
HEART
I
PERFUSATE
Mean values f SD are given; n = 3. BEFORE
significantly from those of control hearts perfused without ischemia. CPK activity in proteins solubilized from cardiac homogenates. The specific activity of CPK was determined in the same extracts as the ones used for the determination of the accumulation of carbonyl groups. Values of CPK activity in the hearts after 10 min of perfusion + 40 min of ischemia had a tendency to be slightly elevated, but changes in activity were not significant (data not shown). However, after 10 min perfusion + 40 min of ischemia + 20 min reperfusion, the activity of CPK was markedly lower. L-Propionyl-carnitine partially prevented this loss of activity (Table II). Content of carbonyls. There was no significant difference in the protein carbonyl content in the control hearts perfused 60 min with or without 10 mM L-propionyl-carnitine. Perfusion for 10 min + ischemia for 40 min did not change significantly the concentration of carbonyls in the proteins both in the hearts perfused with 10 mM L-propionyl-carnitine and in the hearts perfused without L-propionyl-carnitine. In control hearts 20 min of reperfusion after 40 min of global ischemia resulted in a drastic decrease in the protein carbonyl content. This was completely prevented in 10 mM L-propionyl-carnitine-treated hearts (Table III). DMPO spin adducts. ESR spectra consisting of a 1:2:2:1 quartet with splittings of aN = aH = 14.9 G, where aN and aH denote the hyperfine splittings of the nitroxyl nitrogen and a-hydrogen, respectively, were observed in a model system (HzOz + Fe(I1)) generating hydroxyl radicals and in perfusates (Fig. 1). Based on these splittings and the 1:2:2:1 lineshape the spectra were assigned to the DMPO-OH adduct (31). Only small indications of the DMPO-OH radical adduct signal were observed in ESR spectra of perfusates before ischemia. After 40 min of global no-flow ischemia followed by 20 min of reperfusion
AFTER
ISCHEMIA
IS
H’
’
FIG. 1. ESR spectra of DMPO spin adducts in the model system and in the heart per&sate. The model system contained 100 mM DMPO, 80 mM H,Oz, 50 j&M ferrous ammonium sulfate, in 50 pl of Krebs-Ringer solution, pH 7.4, at 25°C. The spectrum was recorded immediately after the mixing of the reagents. Scan time was 4 min. Conditions for perfusate measurements are described under Materials and Methods.
a prominent ESR signal of the DMPO-OH spin adducts was found in the perfusate (Fig. 1). On examination of the time course for the appearance of the spin-trapped signals, maximum signal intensity was observed during
TABLE
III
Effect of 10 mM L-Propionyl-carnitine on Carbonyl Content of Proteins Extracted from Isolated Perfused Rat Hearts Carbonyl content (nmol/mg protein)
Control +lO mM L-propionylcarnitine
10 min perfusion
10 min perfusion + 40 min ischemia
1.83 f 0.25
1.80 f 0.25
1.80
+
0.25
1.63
t
Note. Mean values + SD are given; n = 3.
0.25
10 min perfusion + 40 min ischemia + 20 min reperfusion 4.90 *
0.20
?
0.40
1.63
PACKER
-
+ 10 mH L-proplonyl-carnltine I
0
100
200
300
TIME, set
FIG. 2. Effect of 10 mM L-propionyl-carnitine on the time course of ESR signal of DMPO spin adducts in the heart perfusate after 40 min of global ischemia. Conditions are described under Materials and Methods.
the 1.5 to 2.5 min of reperfusion. In the samples from the the time hearts perfused with 10 mM L-propionyl-carnitine course of DMPO-OH adduct was the same but the magnitude of the signal was markedly suppressed (Fig. 2). In a pure chemical system containing HzOz and DMPO in the same Krebs-Ringer solution as the one used for the heart reperfusion with no exogenous iron added (but with adventitions iron present) a typical ESR signal of DMPO-OH spin adducts which slowly decayed over time was observed (Fig. 3A). Addition of exogenous iron resulted in an increase of the magnitude of the ESR signals (Fig. 1) and accelerated their decay (data not shown). LPropionyl-carnitine had a dual effect on DMPO-OH adducts: it decreased the magnitude of the initial signals and caused a monotonous increase in the magnitude of the signal over time (Fig. 3B). DISCUSSION
There are several reports in the literature showing that L-propionyl-carnitine improves the recovery of myocardial function and various metabolic parameters after ischemia-reperfusion (21-24). Our experiments confirm these findings and indicate that in Langendorff perfused rat hearts subjected to 40 min of global nonflow ischemia the protective effect of L-propionyl-carnitine is highly significant both in terms of mechanical properties of the heart (developed pressure) and in sparing high-energy phosphates (ATP, CP). L-Propionyl-carnitine also prevented the loss of creatine phosphokinase activity in the myocardial soluble protein fraction induced by ischemiareperfusion. However, the mechanism of L-propionylcarnitine protective action against ischemia-reperfusion damage is not fully understood. If the hypothesis that free radicals are implicated in the genesis of ischemia-reperfusion-induced functional disturbances of the heart is valid, then agents which effectively prevent the formation of free radicals or trap them should also possess protective properties and vice versa-compounds exerting pronounced cardioprotection
ET AL.
against ischemia-reperfusion injury should suppress generation of radicals and subsequent oxidation of intracellular targets. In this study we used two different approaches to follow the development of oxidative stress in postischemia reperfused hearts: (i) assay of carbonyls indicating oxidative modifications in proteins (29,30) and (ii) ESR DMPO spin trapping to document generation of short-lived radicals. The amount of oxidatively modified proteins was remarkably increased during reperfusion but did not differ from the control values if L-propionyl-carnitine was present in the perfusion solution. The importance of the free radical scavenging pathway in the L-propionyl-carnitine protection of the heart against ischemia-reperfusion injury is further substantiated by our data on the formation of DMPO spin adducts. In accordance with the other data (6,8-12) our results show a pronounced transient increase in the steady-state concentrations of DMPO-OH spin adduct formation in the early reperfusion period. This burst of generation of oxygen radicals in the course of reperfusion was significantly quenched by L-propionylcarnitine. The specific mechanism responsible for the suppression of oxidative stress during ischemia-reperfusion of the heart by L-propionyl-carnitine is unclear. Since L-propionyl-carnitine does not have any direct free radical scavenging activity (25,26) its antioxidant effects should be indirect. We suggest that the transient accumulation of DMPO-OH spin adducts in the perfusate after ischemia-reperfusion is due to a release of reactive oxygen species and probably iron in a form which may be active in OH’ generation. We further hypothesize that the Lpropionyl-carnitine protective effect on myocardial cell function, protein oxidation, and accumulation of DMPOOH spin adducts is due to the iron-binding and the formation of complexes which are not active in the production of OH’ radicals. The inhibitory effect of L-propionylcarnitine on ischemia-preperfusion-generated DMPO-
A
B 2 min 4 min 6 min 6 mln 10 min 12 min
FIG. 3. ESR spectra of DMPO spin adducts generated in the model system containing H202 and adventitious iron. (A) The model system contained 100 mM DMPO, 80 mM H202, in 50 ~1 of Krebs-Ringer solution, pH 7.4, at 25°C. (B) Effect of 10 mML-propionyl-carnitine added to the same system.
L-PROPIONYL-CARNITINE
AND
OH adducts in the perfusate corresponded well in time with the initial inhibitory effect of L-propionyl-carnitine on DMPO-OH adducts generated in a model system containing H202 and no exogenous iron added. However, the nature of the subsequent growth of DMPO-OH adduct ESR signal in model systems remains to be clarified. The experiments with exogenously added iron showed that the duration of the initial inhibitory phase and the magnitudes of the DMPO-OH signals were strongly dependent on the values of the L-propionyl-carnitine:iron ratio (data not shown). One possible explanation suggests that oxidation of Fe(II)-L-propionyl-carnitine complex may produce a species that over time forms OH’ at a rate which counterbalances the decay of the ESR-detectable DMPOOH adducts, as was recently reported for an iron-bleomycin complex (32). The formation of such an OH’-generating iron-L-propionyl-carnitine complex may potentially be detrimental. However, L-propionyl-carnitine gave only an inhibitory effect on DMPO-OH adducts generated in the ischemia-reperfused heart perfusates. Iron chelation (e.g., by 1,2-dimethyl-3-hydroxy-4-pyridone) has been earlier reported to be beneficial for the protection of the heart against ischemia-reperfusion injury (33). L-Propionyl-carnitine has been recently shown to partially inhibit iron-induced lipid peroxidation in liposomes (34). The experiments carried out in this laboratory demonstrated the formation of complexes of Lpropionyl-carnitine with free iron. Addition of an iron chelator, deferoxamine, to the perfusion fluid protected the heart against ischemia-reperfusion injury (unpublished data). In conclusion our data show that the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart is at least partly due to its ability to prevent generation of oxygen radicals and subsequent oxidative modification of proteins in myocardial cells. The evaluation of the specific contribution of the free radical scavenging pathway versus the nonradical metabolic pathway to overall L-propionyl-carnitine protection against ischemia-reperfusion injury may be accomplished by comparing its effect with the effect of its stereoisomer, L-propionyl-carnitine, which may not participate in the enzymatic metabolic turnover. Such experiments are now under way to compare the protective effects of propionylcarnitine stereoisomers.
ISCHEMIA-REPERFUSION
3. Bolli, R., Jeroudi, M. O., Patel, B. S., DuBose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1989) Proc. N&l. Acad. Sci. USA,
86,4695-4699. 4. Flaherty, J. T., and Weisfeldt, M. L. (1988) Free Radicals Biol. Med., 5,409-419. 5. Rowe, G. T., Eaton, L. R., and Hess, M. L. (1984) J. Mol. Cell. Curdiol. 16,1075-1082. 6. Zweier, L. J. (1988) J. Biol. Chem. 263, 1353-1357. 7. Crescimanno, M., Armata, M. G., Rausa, L., Gueli, M. C., Nicotra, C., and D’Alessandro,
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N. (1989) Free Radicals Res. Commun. 7,67-
72. 8. Hearse, D. J., and Tosaki, A. (1987) J. Cardiovasc. Pharmacol. 9, 641-650. 9. Arroyo, C. M., Kramer, J. H., Dickens, B. F., and Weglicki, W. B. (1987) FEBS Lett. 22,101-104.
10. Garlick, P. B., Davies, M. J., Hearse, D. J., and Slater, T. F. (1987) Circ. Res. 61, 757-760. 11. Das, D. K., George, A., Lin, X. K., and Rae, P. S. (1989) Biochem. Biophys. Res. Commun. 165, 1004-1009. 12. Powell, S. R., and Hall, D. (1990) Free Radicals Biol. Med. 9, 133141. 13. Hearse, D. J., and Tosaki, A. (1987) Circ. Res. 60, 375-383. 14. Tosaki, A., Blasig, I. E., Pali, T., and Ebert, B. (1990) Free Radical Biol. Med. 8, 363-372. 15. van der Kraaij, A. M. M., van Eijk, H. G., and Koster, J. F. (1989) Circulation 80, 158-164. 16. Shlafer, M., Kane, P. F., and Kirsh, M. M. (1982) J. Z’horac. Cardiovasc. Surg. 83, 830-839. 17. Jolly, S. R., Kane, W. J., Bailie, M. B., Abrams, G. D., and Lucchesi, B. R. (1984) Circ. Rex 54, 277-285. 18. Przyklenk, K., and Kloner, R. A. (1986) Circ. Res. 58, 148-156. 19. Kehrer, J. P. (1989) Free Radicals Res. Commun. 5, 305-314.
20. Gey, K. F., and Puska, P. (1989) Ann. N. Y. Acad. Sci. 570,26%282. 21. Subramanian, R., Plehn, S., Noonan, J., Schmidt, M., and Shug, A. L. (1987) 2. Kurdiol.
76, (Suppl. 5), 41-45.
22. Di Lisa, F., Menabo, R., and Siliprandi, N. (1989) Mol. Cell. Biochem. 88, 1699173.
23. Ferrari, R., Ceconi, C., Curello, S., Pasini, E., and Visioli, 0. (1989) Mol. Cell. Biochem. 88, 161-168.
24. Leipala, J., Bhatnagar, R., Pineda, E., Najibi, S., Massoumi, K., and Packer, L. J. Appl. Physiol., submitted
for publication.
25. De Lange, R. J., and Glazer, A. (1989) Anal. Biochem. 177,300-306. 26. Ferrari, R., Ciampalini, G., Agnoletti, G., Cargnoni, A., Ceconi, C., and Visioli,
0. (1988) Pharmacol.
Res. Commun. 20, 125-132.
21. Takala, T. (1981) Basic Res. Cardiol. 76, 44-61. 28. Langendorff, 0. (1895) P/lugers Arch. 61, 291-332. 29. Starke-Reed, P. E., and Oliver, C. N. (1989) Arch. B&hem. Biophys. 257.559-567. 30. Rodney, L. L., Garland, D., Oliver, C. N., and Amici, A., Climent,
ACKNOWLEDGMENTS The authors thank Drs. Norris Siliprandi, Fabio Di Lisa, Carlo Trevisani, and Abraham Reznick for valuable discussions.
537
INJURY
31. 32. 33. 34.
I., Lens, A., Ahn, B., Shaaltiel, S., and Stadtman, E. (1990) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 186, pp. 464-478, Academic Press, San Diego. Buettner, G. R. (1987) Free Radicals Biol. Med. 3, 259-303. Antholine, W. E., Kalyanaraman, B., Templin, J. A., Byrnes, R. W., and Petering, D. H. (1991) Free Radicals Btil. Med. 10, 119-123. van der Kraaij, A. M. M., Mostert, L. J., Eijk, H. G., and Koster, J. F. (1988) Circulation, 78, 442-449. Arduini, A., Fernandez, E., Pallini, R., Mancinelli, G., Sanita di Toppi, G., Belfiglio, M., Scurti, R., and Federici, G. (1990) Free Radicals Biol. Med. 10, 325-332.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 288, No. 2, August 1, pp. 533-537, 1991
Free Radical Scavenging Is Involved in the Protective Effect of L-Propionyl-carnitine against IschemiaReperfusion Injury of the Heart L. Packer,’ M. Valenza, E. Serbinova, P. Starke-Reed,*
K. Frost, and V. Kagan
Department of Molecular and Cell Biology, University of California, Berkeley, of Experimental Medicine, George Washington University School of Medicine,
California 94720; and *Department Washington, D.C. 20037
Received October 19, 1990, and in revised form April 8, 1991
L-Propionyl-carnitine is known to improve the recovery of myocardial function and metabolic parameters reduced in the course of ischemia-reperfusion of the heart. The mechanism of this protective effect of L-propionylcarnitine is not fully understood. The purpose of this study was to elucidate the effects of L-propionyl-carnitine in Langendorff perfused rat hearts subjected to 40 min of ischemia followed by 20 min of reperfusion. We tested the hypothesis that L-propionyl-carnitine suppresses generation of oxygen radicals and subsequent oxidative modification of myocardial proteins during reperfusion. Our data show that the protective effect of L-propionylcarnitine in the course of ischemia-reperfusion is highly significant in terms both of mechanical properties of the heart (developed pressure) and of high-energy phosphates (ATP, creatine phosphate). Myocardial creatine phosphokinase (CPK) activity decreased in the course of the reperfusion period. The loss of CPK activity was partially prevented by L-propionyl-carnitine. Two other effects were observed when L-propionyl-carnitine was present in the perfusion solution: (i) the reperfusion-induced sharp increase in oxidative protein modification was completely prevented as detected by the formation of protein carbonyls, and (ii) generation of hydroxyl radicals was significantly inhibited as detected by the formation of the adducts with the spin trap 5,5-dimethyl1-pyrrolinel-oxide. We conclude that the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart is at least due in part to its ability to suppress the development of oxidative stress and free 0 1991 Academic Press, Inc. radical damage.
The hypothesis that free radical species of oxygen and subsequently formed peroxyl radicals of lipids and proteins are responsible for myocardial injury (in particular, ’ To whom correspondence
should be addressed.
0003-9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
ischemic and postischemic injury, anthracycline antibiotics-induced injury, chelated and nonchelated iron-dependent injury, etc.) has obtained much support over the past few years (l-3). The actual generators of oxygenderived free radicals in ischemic and reperfused heart tissue are still not known, but several sources have been postulated, viz. mitochondrial electron transport, activated polymorphonuclear cells, xanthine oxidase, autooxidation of catecholamines, peroxisomal fatty acid oxidation, biosynthesis of prostaglandins, and endothelial relaxing factor (4-7). Evidence in support of this free radical hypothesis is based mainly on: (i) the detection of radical intermediates arising in the course of ischemia-reperfusion (ESR-detectable spin adducts of oxygen free radicals) (6,8-10) and specific oxidation products of chemical probes-e.g., salicylate-(11,12), and (ii) the protection of ischemic-reperfused cardiac tissue by antioxidant enzymes, chelators of transition metals, or free radical scavengers (spin traps, antioxidants) (13-19). A large multinational epidemiological study showed that higher dietary intake and plasma levels of the antioxidant vitamins and antioxidants were associated with reduced risk of mortality from ischemia heart disease (20). Recently it was reported that L-propionyl-carnitine has a pronounced protective effect against ischemia-reperfusion damage (21-24). Although L-propionyl-carnitine does not possess direct radical scavenging activity (25,26), its protective effects under conditions where enhanced production of free radicals might be primarily responsible for the damage were documented (21,26). In the present investigation our purpose was to study the effects of L-propionyl-carnitine in Langendorff perfused rat hearts subjected to 40 min of global ischemia to clarify its role in protection of the heart. We tested the hypothesis that L-propionyl-carnitine decreases the production of free radicals and oxidative damage during reperfusion. 533
Inc. reserved.
534 MATERIALS
PACKER
AND
METHODS
Animals and perfusion experiments. Male Sprague-Dawley rata (280300 g) were used for the study. The animals were anesthetized with diethyl ether and injected with 400 units of heparin intravenously before excision of the heart. The perfusion apparatus was constructed essentially as described earlier (27) and thermostated to 38°C. A retrograde aortic perfusion system (28) without recirculation was employed. The perfusion solution was an oxygenated modified Krebs-bicarbonate buffer (1.2 mM MgClz. 6H,O), 5.9 mM KCl, 17 mM d-glucose, 25 mM NaHC03, 2.0 mM CaCl,, 117 mM NaCl), pH 7.4, and was continuously bubbled with 95% Ox and 5% COr. The medium used in the L-propionyl-camitine experiments contained 10 mM L-propionyl-carnitine hydrochloride. When L-propionyl-carnitine was added, the pH of the perfusate was adjusted to 7.4 and the sodium chloride concentration lowered respectively to retain constant osmolarity. After excision of the heart, the aorta was immediately cannulated and Langendorff perfusion (left ventricle perfusion) was initiated for 10 min (preperfusion period). During this period a cannula was attached to the pressure transducer and inserted into the left ventricle through the right atrium. Hearts were then subjected to a period of global ischemia for 40 min. Ischemia was achieved by clamping the aortic cannula. The hearts were reoxygenated (reperfusion period) for 20 min after ischemia. Control hearts were subjected to 60 min of perfusion (no ischemia) both with and without L-propionylcarnitine. Left ventricular pressure was measured with a Gould/Statham P23 pressure transducer. Recovery of the heart was measured before and after ischemia by calculating the developed pressure from the trace obtained from the pressure transducer (Gilson Duograph). At the end of the perfusion experiments, all hearts were freeze-clamped and stored in liquid nitrogen. Extraction. The frozen hearts were crushed with a chilled mortar and pestle and the powder thus formed added to 2 ml of 6% perchloric acid in 10 mM EDTA at 0°C. The mixture was homogenized and then centrifuged at 3600g for 10 min. The solids were again homogenized in 2 ml of ice-cold 6% perchloric acid in 10 mM EDTA and centrifuged. The pooled supernatants were neutralized to pH 7.0 with the addition of ice-cold 1 M KOH. The precipitate of KClO, was removed by centrifugation and filtration. The extracts were then lyophilized and redissolved in 2.0 ml H,O and 0.5 ml D,O (24). 31P NMR spectroscopy was performed at 121.5 NMR spectroscopy. MHz on a Bruker AM 300 spectrometer. D20 was used as a field lock and chemical shifts were assigned relative to the creatine phosphate (CP)‘peak (-2.5 ppm relative to 85% phosphoric acid). A 60-s excitation pulse and a 2.1-s repetition time were used to acquire 3072 scans over 108 min. A ~-HZ line broadening was applied to reduce spectral noise. Quantification of the spectra was achieved by acquiring a second spectrum of each sample after addition of CP and ATP standards. The integrals of the spectral peaks from the two spectra were compared to determine the amount of phosphate present in the original sample. The reproducibility of the measurements of 31P metabolites was found to be 5% for identical samples.
ET AL. The resulting supernatant was used for the protein nations as previously described (30). Administration of 5,5-dimethyl-l-Wrroline-l-oxide (0.1 M) was infused via a side arm of aortic cannula rate of 1 ml/min. Coronary flow was approximately the first 3 min of Langendorff perfusion. DMPO was the reperfusion period. DMPO was initially purified a charcoal column before use.
2 Ahbreviations used: CP, creatine phosphate; phokinase; DMPO, 5,5-dimethyl-1-pyrroline-l-oxide; tricular developed pressure.
CPK, creatine phosLVDP, left ven-
determi-
(DMPO). DMPO into the heart at a 10 ml/min during infused only during by passing through
ESR measurements. ESR spectra were recorded in glass accupette pipets at room temperature using a Bruker ER 200 D-SRC electron spin resonance spectrometer (X-band). Instrument settings: modulation amplitude 2.5, G; scan range, 100 G; central field, 3380 G; microwave power, 10 mW. The DMPO concentration was measured spectrophotometrically (e = 8 X lo3 M-’ cm-’ at 227 nm). To prevent spin adduct decay the perfusate was frozen in liquid nitrogen immediately after the sample was taken. No significant loss of ESR signal occurred during the storage of DMPO adducts. Hyperfine coupling constants were measured directly from the field scan using Mn*+ as a marker for calibration. As a model system generating hydroxyl radicals, the Fenton reagent consisting of 50 nM FeSO, * 6H,O and 80 mM H,Oz was used in the presence of 100 mM DMPO. Reagents. The authors thank Dr. C. Trevisani (Sigma Tau Co., Pomezia, Italy) for a generous gift of L-propionyl-carnitine hydrochloride used in this study. Heparin was from Upjohn Co. (Kalamazoo, MI). The other chemicals were from Sigma Chemical Co. (St. Louis, MO).
RESULTS
Left ventricular developed pressure (LVDP) and mechanical recovery. L-propionyl-carnitine did not affect the preischemic LVDP. The postischemic LVDP was significantly higher in the L-propionyl-carnitine-treated group, compared to the postischemic LVDP in the hearts without L-propionyl-carnitine (Table I). After 40 min of ischemia the mechanical recovery of the hearts treated with 10 mM L-propionyl-carnitine was 80% of the control value, while in the hearts without L-propionyl-carnitine it was only 26% of the control level (Table I). High-energy phosphates. The results of the 31P NMR determinations of ATP and CP contents in the heart extracts are shown in Table II. Global ischemia for 40 min followed by 20 min of reperfusion caused a 75% loss of myocardial ATP and an 84% loss of CP. L-Propionylcarnitine (10 mM) protected the hearts from the loss of ATP and phosphocreatine: the ATP and CP concentrations of the hearts exposed to ischemia-reperfusion in the presence of 10 mM L-propionyl-carnitine did not differ
Creatine phosphokinase assay. Creatine phosphokinase (CPK) activity was assayed using a standard kit obtained from Sigma Chemical Co. Carbonyl assay. Following treatment the hearts were minced and placed in a 60-mm cell culture plate. Added to the dish was 2 ml of the Hepes buffer plus a protease inhibitor cocktail as described in (29) with the addition of 0.1% digitonin. The plates were placed on a rotating apparatus and the tissue gently swirled at room temperature for 15 min. The resulting fluid was removed and further centrifuged at a top speed in a table-top clinical centrifuge for 5 min to precipitate the digitonin.
oxidation
TABLE
I
Effect of L-Propionyl-carnitine on Recovery of Left Ventricular Pressure (LDVP) in Isolated Perfused Rat Hearts after 40 min of Global Ischemia LVDP
Control +lO mM L-propionyl-carnitine
(mmHg)
Before ischemia
After ischemia
78 t 18 80 rt_ 17
20 f 18 64 f 20
Note. Mean values -+ SD are given; n = 3.
L-PROPIONYL-CARNITINE TABLE
AND
ISCHEMIA-REPERFUSION
535
INJURY
MODEL
II
SYSTEM
Effect of L-Propionyl-carnitine on the Content of the HighEnergy Phosphates and Creatine Phosphokinase Activity (CPK) in Isolated Perfused Rat Hearts 31PMetabolites (%) ATP 60
min perfusion
Modified Krebs +lO mM L-propionyl-carnitine 10 min perfusion
100
100
86.7 2 10.7
100
100
79.9f 2.9
25 + 14 90 + 15
16 + 10 89 + 14
32.3 f 70.4 f
as=a,
I
+ 40 min ischemia + 20 min reperfusion
Modified Krebs +lO mM L-propionyl-carnitine Note.
CP
CPK activity (units/mg protein)
4.4 1.7
HEART
I
PERFUSATE
Mean values f SD are given; n = 3. BEFORE
significantly from those of control hearts perfused without ischemia. CPK activity in proteins solubilized from cardiac homogenates. The specific activity of CPK was determined in the same extracts as the ones used for the determination of the accumulation of carbonyl groups. Values of CPK activity in the hearts after 10 min of perfusion + 40 min of ischemia had a tendency to be slightly elevated, but changes in activity were not significant (data not shown). However, after 10 min perfusion + 40 min of ischemia + 20 min reperfusion, the activity of CPK was markedly lower. L-Propionyl-carnitine partially prevented this loss of activity (Table II). Content of carbonyls. There was no significant difference in the protein carbonyl content in the control hearts perfused 60 min with or without 10 mM L-propionyl-carnitine. Perfusion for 10 min + ischemia for 40 min did not change significantly the concentration of carbonyls in the proteins both in the hearts perfused with 10 mM L-propionyl-carnitine and in the hearts perfused without L-propionyl-carnitine. In control hearts 20 min of reperfusion after 40 min of global ischemia resulted in a drastic decrease in the protein carbonyl content. This was completely prevented in 10 mM L-propionyl-carnitine-treated hearts (Table III). DMPO spin adducts. ESR spectra consisting of a 1:2:2:1 quartet with splittings of aN = aH = 14.9 G, where aN and aH denote the hyperfine splittings of the nitroxyl nitrogen and a-hydrogen, respectively, were observed in a model system (HzOz + Fe(I1)) generating hydroxyl radicals and in perfusates (Fig. 1). Based on these splittings and the 1:2:2:1 lineshape the spectra were assigned to the DMPO-OH adduct (31). Only small indications of the DMPO-OH radical adduct signal were observed in ESR spectra of perfusates before ischemia. After 40 min of global no-flow ischemia followed by 20 min of reperfusion
AFTER
ISCHEMIA
IS
H’
’
FIG. 1. ESR spectra of DMPO spin adducts in the model system and in the heart per&sate. The model system contained 100 mM DMPO, 80 mM H,Oz, 50 j&M ferrous ammonium sulfate, in 50 pl of Krebs-Ringer solution, pH 7.4, at 25°C. The spectrum was recorded immediately after the mixing of the reagents. Scan time was 4 min. Conditions for perfusate measurements are described under Materials and Methods.
a prominent ESR signal of the DMPO-OH spin adducts was found in the perfusate (Fig. 1). On examination of the time course for the appearance of the spin-trapped signals, maximum signal intensity was observed during
TABLE
III
Effect of 10 mM L-Propionyl-carnitine on Carbonyl Content of Proteins Extracted from Isolated Perfused Rat Hearts Carbonyl content (nmol/mg protein)
Control +lO mM L-propionylcarnitine
10 min perfusion
10 min perfusion + 40 min ischemia
1.83 f 0.25
1.80 f 0.25
1.80
+
0.25
1.63
t
Note. Mean values + SD are given; n = 3.
0.25
10 min perfusion + 40 min ischemia + 20 min reperfusion 4.90 *
0.20
?
0.40
1.63
PACKER
-
+ 10 mH L-proplonyl-carnltine I
0
100
200
300
TIME, set
FIG. 2. Effect of 10 mM L-propionyl-carnitine on the time course of ESR signal of DMPO spin adducts in the heart perfusate after 40 min of global ischemia. Conditions are described under Materials and Methods.
the 1.5 to 2.5 min of reperfusion. In the samples from the the time hearts perfused with 10 mM L-propionyl-carnitine course of DMPO-OH adduct was the same but the magnitude of the signal was markedly suppressed (Fig. 2). In a pure chemical system containing HzOz and DMPO in the same Krebs-Ringer solution as the one used for the heart reperfusion with no exogenous iron added (but with adventitions iron present) a typical ESR signal of DMPO-OH spin adducts which slowly decayed over time was observed (Fig. 3A). Addition of exogenous iron resulted in an increase of the magnitude of the ESR signals (Fig. 1) and accelerated their decay (data not shown). LPropionyl-carnitine had a dual effect on DMPO-OH adducts: it decreased the magnitude of the initial signals and caused a monotonous increase in the magnitude of the signal over time (Fig. 3B). DISCUSSION
There are several reports in the literature showing that L-propionyl-carnitine improves the recovery of myocardial function and various metabolic parameters after ischemia-reperfusion (21-24). Our experiments confirm these findings and indicate that in Langendorff perfused rat hearts subjected to 40 min of global nonflow ischemia the protective effect of L-propionyl-carnitine is highly significant both in terms of mechanical properties of the heart (developed pressure) and in sparing high-energy phosphates (ATP, CP). L-Propionyl-carnitine also prevented the loss of creatine phosphokinase activity in the myocardial soluble protein fraction induced by ischemiareperfusion. However, the mechanism of L-propionylcarnitine protective action against ischemia-reperfusion damage is not fully understood. If the hypothesis that free radicals are implicated in the genesis of ischemia-reperfusion-induced functional disturbances of the heart is valid, then agents which effectively prevent the formation of free radicals or trap them should also possess protective properties and vice versa-compounds exerting pronounced cardioprotection
ET AL.
against ischemia-reperfusion injury should suppress generation of radicals and subsequent oxidation of intracellular targets. In this study we used two different approaches to follow the development of oxidative stress in postischemia reperfused hearts: (i) assay of carbonyls indicating oxidative modifications in proteins (29,30) and (ii) ESR DMPO spin trapping to document generation of short-lived radicals. The amount of oxidatively modified proteins was remarkably increased during reperfusion but did not differ from the control values if L-propionyl-carnitine was present in the perfusion solution. The importance of the free radical scavenging pathway in the L-propionyl-carnitine protection of the heart against ischemia-reperfusion injury is further substantiated by our data on the formation of DMPO spin adducts. In accordance with the other data (6,8-12) our results show a pronounced transient increase in the steady-state concentrations of DMPO-OH spin adduct formation in the early reperfusion period. This burst of generation of oxygen radicals in the course of reperfusion was significantly quenched by L-propionylcarnitine. The specific mechanism responsible for the suppression of oxidative stress during ischemia-reperfusion of the heart by L-propionyl-carnitine is unclear. Since L-propionyl-carnitine does not have any direct free radical scavenging activity (25,26) its antioxidant effects should be indirect. We suggest that the transient accumulation of DMPO-OH spin adducts in the perfusate after ischemia-reperfusion is due to a release of reactive oxygen species and probably iron in a form which may be active in OH’ generation. We further hypothesize that the Lpropionyl-carnitine protective effect on myocardial cell function, protein oxidation, and accumulation of DMPOOH spin adducts is due to the iron-binding and the formation of complexes which are not active in the production of OH’ radicals. The inhibitory effect of L-propionylcarnitine on ischemia-preperfusion-generated DMPO-
A
B 2 min 4 min 6 min 6 mln 10 min 12 min
FIG. 3. ESR spectra of DMPO spin adducts generated in the model system containing H202 and adventitious iron. (A) The model system contained 100 mM DMPO, 80 mM H202, in 50 ~1 of Krebs-Ringer solution, pH 7.4, at 25°C. (B) Effect of 10 mML-propionyl-carnitine added to the same system.
L-PROPIONYL-CARNITINE
AND
OH adducts in the perfusate corresponded well in time with the initial inhibitory effect of L-propionyl-carnitine on DMPO-OH adducts generated in a model system containing H202 and no exogenous iron added. However, the nature of the subsequent growth of DMPO-OH adduct ESR signal in model systems remains to be clarified. The experiments with exogenously added iron showed that the duration of the initial inhibitory phase and the magnitudes of the DMPO-OH signals were strongly dependent on the values of the L-propionyl-carnitine:iron ratio (data not shown). One possible explanation suggests that oxidation of Fe(II)-L-propionyl-carnitine complex may produce a species that over time forms OH’ at a rate which counterbalances the decay of the ESR-detectable DMPOOH adducts, as was recently reported for an iron-bleomycin complex (32). The formation of such an OH’-generating iron-L-propionyl-carnitine complex may potentially be detrimental. However, L-propionyl-carnitine gave only an inhibitory effect on DMPO-OH adducts generated in the ischemia-reperfused heart perfusates. Iron chelation (e.g., by 1,2-dimethyl-3-hydroxy-4-pyridone) has been earlier reported to be beneficial for the protection of the heart against ischemia-reperfusion injury (33). L-Propionyl-carnitine has been recently shown to partially inhibit iron-induced lipid peroxidation in liposomes (34). The experiments carried out in this laboratory demonstrated the formation of complexes of Lpropionyl-carnitine with free iron. Addition of an iron chelator, deferoxamine, to the perfusion fluid protected the heart against ischemia-reperfusion injury (unpublished data). In conclusion our data show that the protective effect of L-propionyl-carnitine against ischemia-reperfusion injury of the heart is at least partly due to its ability to prevent generation of oxygen radicals and subsequent oxidative modification of proteins in myocardial cells. The evaluation of the specific contribution of the free radical scavenging pathway versus the nonradical metabolic pathway to overall L-propionyl-carnitine protection against ischemia-reperfusion injury may be accomplished by comparing its effect with the effect of its stereoisomer, L-propionyl-carnitine, which may not participate in the enzymatic metabolic turnover. Such experiments are now under way to compare the protective effects of propionylcarnitine stereoisomers.
ISCHEMIA-REPERFUSION
3. Bolli, R., Jeroudi, M. O., Patel, B. S., DuBose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1989) Proc. N&l. Acad. Sci. USA,
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