protective effect of propionyl-l-carnitine against ischaemia and reperfusion-damage

Molecular and Cellular Biochemistry 88: 161-168, 1989. © 1989 Kluwer Academic Publishers. Printed in the Netherlands.

Invited Paper

Protective effect of propionyl-L-carnitine against ischaemia and reperfusion-damage

Roberto Ferrari, Claudio Ceconi, Salvatore Curello, Evasio Pasini and Odoardo Visioli Cattedra di Cardiologia dell' Universitd di Brescia, Brescia, Italy

Accepted 28 December 1988

Key words." propionyl-L-carnitine, myocardial-protection, post-ischaemic reperfusion, oxidative damage

Summary

Reperfusion of isolated rabbit heart after 60min of ischaemia resulted in poor recovery of mechanical function, release of reduced (GSH) and oxidized glutathione (GSSG), reduction of tissue GSH/GSSG ratio and shift of cellular thiol redox state toward oxidation, suggesting the occurrence of oxidative stress. Pretreatment of the isolated heart with propionyl-L-carnitine (10 .7 M) improved the functional recovery of the myocardium, reduced GSH and GSSG release and attenuated the accumulation of tissue GSSG. This effect was specific for propionyl-L-carnitine as L-carnitine and propionic acid did not modify myocardial damage.

Introduction

Carnitine is a natural occurring and essential cofac- tot in the mitochondrial transfer of activated fatty acids in mammalian heart muscle [1]. Depletion of this cofactor has been reported in severa ! patholog- ical conditions, including myocardial ischaemia_ [2-4]. There is also an increasing number of reports supporting that carnitine and carnitine derivatives have a pIotective effect against myocardial ischaemia in experimental animals [5-11] and humans [12, 13]; propionyl-L-carnitine being the most ef- fective derivative [10]. Despite considerable efforts to find explanation for this beneficial effect, a com- monly accepted basic mechanism is still lacking.

Recently it has been described that propionyl-L- carnitine reduces the age-dependent accumulation of lipo fuscines [14] and protects heart mitochondria membranes from the damage induced by lipid peroxidation [15]. We now describe the effect of propionyl-L-carnitine on the oxidative stress which

occurs in the isolated and perfused rabbit heart during post-ischaemic reperfusion [16, 17]. Oxida- tive stress was determined by measuring rate of release and tissue changes of reduced (GSH) and oxidized (GSSG) glutathione. As index of myocardial function, the pressure developed by isolated heart was also determined.

Experimental procedure

Perfusion of the hearts

Adult, New Zealand white rabbits (2.5 to 3.0 Kg) were stunned by a blow on the head. The hearts were rapidly excised and perfused with a modified Krebs-Henseleit buffer solution by the non recircu- lating Langendorff technique as previously described [16]. All hearts were paced at 180 beats/rain using suprathreshold rectangular pulses of 1.0ms duration. A period of 30 min equilibration was al-

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lowed before any experimental intervention. Then an infusion of either L-carnitine (10 7 M), propionic acid (10 -7 M) or propionyl-L-carnitine (10 -7 M) was started into the aortic inflow tract and maintained until the end of the experiments. Ischaemia was induced by reducing coronary flow to 1 ml/min.

Left ventricular pressure measurements

To obtain an isovolumetrically beating prepara- tion, a fluid-filled balloon was inserted into the left ventricular cavity via the atrium. The intraventric- ular balloon was then connected by a fluid-filled polyethylene catheter to a Statham pressure trans- ducer (P 23 D6) for the determination of left ventricular pressure.

Coronary effluent analysis

During each perfusion the coronary effluent was collected in chilled glass vials and assayed, on the same day, for GSH and GSSG following the method described later in this section.

Reduced (GSH) and oxidized glutathione (GSSG) determination

The tissue ( - 100 mg) was deproteinized with 3 M HCLO4 and the supernatant obtained after centrif- ugation at 6000 g for 15 min was neutralized with 2M K2CO3. A sample of the neutralized extract was analysed for total glutathione by the method of Tietze [18] modified by us [17]. The reaction mixture (1.0ml) contained: 0.05M potassium phosphate buffer pH7.4, l m M EDTA, 0.1mM 5-5'- dithiobis-(2 nitrobenzoic acid) (DTNB), 0.15 mM NADPH and an appropriate volume of sample. After 2 min of preincubation, the reaction was initi- ated by the addition of i U of glutathione reductase and the rate of reduction of the DTNB was contin- uously monitored at 412 nm; the slope was propor- tional to glutathione concentration over the range of 0-1/xM. Oxidized glutathione was measured as described above after the preliminary reaction of

GSH with 20 mM N-ethyl maleimide followed by complete removal of unreacted sulphydryl reagent with diethylether.

Determination of protein sulphydryl groups

To determine the content of tissue thiol (SH) groups a portion of the left ventricle was homogenized as described by Sedlack and Lindsay [19], with 20 mM EDTA pH 4.7, and filtered on nylon. Samples of homogenated were mixed with 0.2 M Tris HC1 pH8.2, 0.01 M DTNB and methanol to a final volume of 10ml. A reagent blank (without sample) and a sample blank (without DTNB) were prepared in a similar manner. The mixture was incubated for 30 min at room temperature. The absorbance was read at 412 nm and a molar extinc- tion coefficient of 13600 M -1 cm -1 was used.

The acid-soluble SH group content (as an ex- pression of non-protein sulphydryl groups) was determined, in the same manner, in the supernatant fluid obtained after denaturation of the homogenate with ice-cold 50% TCA.

Protein SH groups were determined by subtract- ing the acid-soluble from the total SH-group content.

Determination of glutathione peroxidase (GPD) and glutathione reductase ( GRD)

Frozen tissues were homogenized in 10 vol. of ice- cold 0.05M potassium phosphate buffer, pH7.2, centrifuged at 3000 g for 10 min and the superna- tants were used for enzymatic determinations.

Glutathione peroxidase activity was assayed by a modification of the method of Paglia and Valentine [20]. The reaction mixture consisted, in a final volume of lml, of 50mM Tris-HC1 buffer pH7.3, 0.1 mM EDTA, 0.12 mM NADPH, 0.25 mM GSH, 1 unit/ml of glutathione reductase (1 unit/ml corre- sponds to 1 ~tmol of NADPH oxidized/min) and an appropriate amount of supernatant. After 5 rain of incubation, the assay mixture was made 0.2 mM in cumene hydroperoxide and the rate of disappear- ance of NADPH was monitored at 340nm. The

activity of GPD was calculated as nmoles of NADPH oxidized/min/mg protein.

Glutathione reductase activity was assayed in a medium containing in a final volume of 1 ml:0.1 M Tris buffer pH8.0, 0.94ram EDTA, 4.6mM GSSG, 0.16mM NADPH and an appropriate amount of supernatant. The activity of GRD was calculated as nmoles of NADPH oxidized/min/mg protein.

Protein determination

Protein determination was carried out according to Bradford [21], using bovine serum albumin as stan- dard.

Experimental compounds and statistical evaluation

The reagents were analytic reagent grade quality. All enzymes used for the biochemical determinations were obtained from the Sigma Company (U.S.A.). L-carnitine and propiony!-L-carnitine were kindly supplied by Sigma-Tau S.p.A., Rome (Italy).

The data are expressed as mean + S.E. of at least 6 experiments, where each experimental is an individual perfusion. A one-way analysis of varia- nce was first carried out to test for any differences between all groups. If a difference was established, each of the groups was compared with the control group using the unpaired 't'-test, considering significant only results given p values below 0.05.

Results

In these experiments propionyl-L-carnitine was used at 10 7 M because in a previous dose-response study the best protective effect was found at this dosage [22]. From Fig. 1 it appears that infusion of L-carnitine, propionic acid or propionyl-L-carnitine did not cause significant changes in cardiac performance during the 30 min of aerobic perfusion. Ischaemia resulted in a rapid decline of developed pressure to zero in all groups and the period

163

of still detectable contractile activity was equal (2.5 to 3.5 min). The ischaemic and reperfusion induced contracture, as indicated by the increase in diastolic pressure, was significantly less pronounced in heart receiving propionyl-L-carnitine, whilst it was not affected by L-carnitine or propionic acid. Fur- thermore, the percentage of recovery of developed pressure after reperfusion was significantly improved in the heart treated with propionyl-L-carnitine but not in those receiving L-carnitine or propionic acid.

The data on release and cellular content of GSH and GSSG are shown in Fig. 2 and Table 1, respec- tively. In control hearts there was a small release of GSH and almost no release of GSSG during aerobic perfusion (Fig. 2). Ischaemia did not alter the rate of GSH and GSSG release (Fig. 2), whilst tissue contents of GSH and of protein-SH were significantly reduced. Tissue GSSG after ischaemia was unchanged (Table 1). Reperfusion of these hearts resulted in a marked and sustained increase of the rate of GSH and of GSSG release with further reduction of cellular GSH content (Fig. 2, Table 1). Furthermore, in control hearts, tissue content of GSSG was significantly increased after the readmission of coronary flow, despite the reperfusion-induced release of GSSG. Tissue concentration of protein-SH after reperfusion re- mained low (Table 1).

Administration of L-carnitine, propionic acid or propionyl-L-carnitine did not affect tissue content or rate of GSH and GSSG release under aerobic condition (Fig. 2, Table 1). L-carnitine and propionic acid failed to modify the alteration induced by ischaemia and reperfusion on glutathione status. On the contrary, in the hearts receiving propionyl-L-carnitine the decline of tissue GSH after ischaemia was not prevented, but there was a recovery on reperfusion. In addition, the reperfusion induced increase of GSSG was reduced, leading to almost a normal GSH/GSSG ratio (Table 1). In addition, in the hearts treated with propionyl-L- carnitine, the reperfusion induced release of GSH and GSSG was significantly reduced and protein- SH content after reperfusion was ameliorated (Fig. 2, Table 1).

Table i also shows that GPD and GRD activities

164

-, ,,; ~" 75 . - , ~

+ ¢n ,-- 60 IJJ IZ ¢ o. E 45

UJ 30

~ o

A E R O B I A I S C H A E M I A REPERFUSION

i • I,~ ~__..;---;

J i l ii/ , i ,

-60 -30 0 30 60 70 80 90

" ' " ; 75

== ~ 60

0 30

~ 0

A E R O B I A I S C H A E M I A REPERFUSION

-,,- ,., C , f _ , ,~ " -~ • "*-

• #,.B 4~ ,,~

. . . . . e5 . . . . . ~ ' ~ 2.~---" ~ - -

-60 - 3 0 0 30 60 70 80 90

e - - - e C O N T R O L , , - - - , , PROPIONIC ACID

o---o L -CARNIT INE o - - - o L -PROPIOYL-L -CARNIT INE

, p < 0 . 0 5 * * p<0 .01

Fig. l. Effect of L-carnitine, propionic acid and propionyl-L-carnitine on the ischaemia and reperfusion induced alterations of left ventricular pressure. Under aerobic and reperfusion conditions the hearts were perfused at a mean coronary flow of 25 ml/min. lschaemia was induced (at time 0) by reducing coronary flow to 1 ml/min. The administration of the different substances was started 30 minutes before the onset of ischaemia. Each point is the mean of at least six separate experiments. P relates to the significance of the

difference between the controls and the relative treated groups.

165

c +1

c

10

1.0

ua ~ 0.8 m

uJ o~ u.i .a ' - ( 6 0.6 U J " ~

E +l ,~ o.a

m E 0.2 ¢~ ,.-

0

/ / ~ / / / / / / / / / / / / / / / / / / ~

AEROBIA ISCHAEMIA

8 I

6 I i

4

2

o - ~ . . . . ~ . . . . ~ - - - - ~ . . . .

I I [ I

min. -60 -30 0 30

...t-

~11 , , ,

60 70 80 90

: : : : : : : : : : : : : : : : : : : : : : : : . . . . ~ . . . . ~, . . . . ~ . . . .

l I I I

U--K.~!~. :.~;~h~,~y~ AEROBIA ISCHAEMIA REPERFUSION

} !

d? 10 , I I

min. -60 -30 0 30 60 70 80 90

e- . - -e CONTROL ,,___A PROPIONIC ACID

o - - - o L-CARNITINE n - - - n L-PROPIONYL-L-CARNITINE

* p<0.05 * * p<0.01

Fig. 2. Effect of L-carmtine, propionic acid and propionyl-L-carmtine on the ischaemia and reperfusion induced alterations in the rate of

GSH and GSSG release. Under aerobic and reperfusion conditions the hearts were perfused at a mean coronary flow of 25 ml/min.

Ischaemia was induced (at t ime 0) by reducing coronary flow to 1 ml/min. The administration of the different substances was started 30

minutes before the onset of ischaemia. Each point is the mean of at least six separate experiments. P relates to the significance of the

difference between the controls and the relative treated groups.

166

were not al tered by ischaemia and reperfusion and that L-carnit ine, propionic acid or propionyl-L-

carnit ine had no effects on these enzymes.

D i s c u s s i o n

Our results indicate that in control hearts ischae-

mia and part icularly reperfusion resulted in a

marked reduct ion of G S H / G S S G ratio and in a

shift of cellular thiol redox state towards oxidation.

These alterations are known to cause disturbances

in calcium transport , al terations of m e m b r a n e per-

meabil i ty and impai rment of muscle contract ion

[16] which could in par t account for the poor recov-

ery of funct ion observed after reperfusion. Unde r these condit ions the glutathione system, which rep-

resents an impor tan t mechanism of defence against

oxygen toxicity, is under stress, the reduced avail-

ability of cellular G S H becoming a rate limiting

factor for detoxification of oxygen metabol i tes [16,

17].

F rom these data it also appears that propionyl-L- carnit ine is able to provide protect ion. The in-

crease of diastolic pressure during ischaemia was

at tenuated. On reperfusion there was a bet ter recovery of mechanical function, a reduced rate of

G S H and G S S G release and a less evident accumu-

lation of GSSG, which is considered a reliable in-

dex of oxidative stress [16, 17]. This effect is quite

specific for propionyl-L-carni t ine as L-carnit ine

and propionic acid were not active. The lack of a

protect ive effect of L-carnit ine on an isolated prep-

arat ion is consistent with our earlier findings in rabbits [23]and with previous studies in rats [24--26]

but not with studies on intact animals using either

dogs [7] or swine [5, 6]. The explanat ion for this

discrepancy is unknown, but it may relate to the

slow rate of carnitine t ranspor t in perfused heart

[10] or to indirect peripheral effects of L-carnitine.

It has been recently suggested [10] that propionyl-

L-carni t ine is t ranspor ted with greater affinity than

L-carnit ine into the isolated cardiac myocytes and

this could explain why in our experiments only

Table I. Effect of L-carnitine, propionic acid and propionyl-L-carnitine on tissue content of reduced (GSH) and oxidized (GSSG) glutathione, protein SH groups and on the activity of glutathione peroxidase (GPD) and glutathione reductase (GRD). All the determinations and the enzyme activities were measured in the homogenate as described in the text. Under aerobic and reperfusion conditions the hearts were perfused at a mean coronary flow of 25 ml/min. Ischaemia was induced by reducing coronary flow to 1 ml/min. Results are given as mean _+ S.E. The number of experiments is reported in brackets. P relates to the significance of the difference between control and the different groups. Tissue GSH, GSSG and protein SH are expressed as nmoles/mg prot., whilst GPD and GRD activities are expressed as nmoles NADPH oxidized/min/mg prot

GSH GSSG Protem-SH GPD GRD

Aerobia (150 rain) Control 13.0 _+ 0.7 (6) L-carnitine (10-VM) 12.1 + 0.9 (5) Propionic acid (10 7M) 12.7 _+ 0.7 (5) Propionyl-L-carnitine (10-7M) 12.6 + 0.6 (6) lschaemia (60 rain) Control L-carnitine (10-7M) Propionic acid (10 VM) Propionyl-L-carnitine (10-7M) Reperfusion (30 rain) Control L-carnitine (10-7M) Propionic acid (10 7M) Propionyl-L-carnitine (10-7M)

5.9 _+ 0.3 (6) 6.3 _+ 0.7 (5) 6.0 _+ 0.4 (5) 7.2 _+ 0.5 (6)*

4.0 _+ 0.3 (6) 4.6 _+ 0.8 (5) 3.9 _+ 0.7 (5) 7.3 _+ 0.4 (6)**

0.24_+0.05 (6) 337+10.7 (6) 21.1_+1.7 (5) 45.6_+2.1 (5) 0.20 _+ 0.07 (5) 303 _+ 12.1 (5) 19.3 +_ 1.6 (4) 43.2 _+ 1.9 (4) 0.23+0.06 (5) 323_+13.1 (5) 22.0_+1.3 (4) 44.6_+1.9 (4) 0.26_+0.05 (6) 309+10.3 (6) 23.4_+0.9 (6) 47.1_+1.5 (6)

0.20+0,04 (6) 107_+11.3 (6) 21.2_+1.3 (5) 46.1_+2.9 (5) 0.21 _+ 0.03 (5) 126 _+ 12.3 (5) 22.3 _+ 1.9 (4) 45.2 _+ 1.8 (4) 0.23_+0,06 (5) 111+13.1 (5) 20.3_+1.6 (4) 43.1_+2.2 (4) 0.24 _+ 0.07 (6) 141 _+ 14.3 (6) 21.3 _+ 0.9 (6) 47.1 _+ 1.6 (6)

0.53_+0.08 (6) 193_+13.1 (6) 20.6_+1.3 (5) 43.1+2.3 (5) 0.49_+0.07 (5) 212_+14.3 (5) 19.9_+1.6 (4) 48.3+2.2 (4) 0.51 -+ 0.06 (5) 207 _+ 13.3 (5) 19.7 _+ 1.0 (4) 44.5 _+ 1.9 (4) 0.29_+0.06 (6)** 276_+12.1 (6)* 21.3_+1.0 (6) 46.1_+2.0 (6)

* P<0.05. ** P<0.01.

propionyl-L-carnitine exerted protective effects. The mechanism by which propionyl-L-carnitine

is protective is unclear, although a number of fac- tors can be eliminated. A lowered myocardial oxygen demand at the time of ischaemia is an unlikely factor as propionyl-L-carnitine did not modify peak of developed pressure before ischaemia or the onset of quiescence during ischaemia. In addition, heart rate and afterload were maintained constant. As coronary flow was also kept constant, the beneficial effect of propionyl-L-carnitine is unlikely to be due to an enhanced delivery of oxygen during ischaemia. Pretreatment with propionyl-L-carni- fine did not increase tissue GSH or protein-SH content after aerobic and ischaemic reperfusion and had no effect on the activity of GRD and GPD. Thus, it is unlikely that its protection is secondary to the maintenance of reduced thiol pool or to a direct effect on the enzymes regulating cellular glutathione status.

We have previously demonstrated [15] that propionyl-L-carnitine is able to protect isolated heart mitochondria against the ferrous ions-induced lipid peroxidation damage. This protective effect of propionyl-L-carnitine could not be explained in terms of a reduction of lipid peroxidation, as malondial- dehyde formation was not modified [15]. We, therefore, concluded that the most likely explanation was an interaction of the compound with the lipid bilayer, leading to a stabilizing effect.

A similar mechanism of action may be involved also in the present experimental model. In support of this hypothesis is the reduced rate of GSH and of CPK (data not shown) release which we found in the hearts receiving propionyl-L-carnitine. The consequent greater availability of cellular GSH could, in turn, explain why reperfusion in these hearts did not lead to an oxidative stress. In conclu- sion, propionyl-L-carnitine ameliorates post-ischaemic dysfunction in isolated heart. These effects are not mediated by changes in classic oxygen supply-demand mechanisms. Instead, our in vitro

experiments, suggest that the mechanism by which propionyl-L-carnitine improves recovery of myocardial function may be mediated by a reduction of oxidative stress due to oxygen derived free rad- icals.

167

Acknowledgements

This work was supported by the Italian C.N.R. grant 087432. We thank Miss Afra Benini for secre- tarial assistance in preparing the manuscript.

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Address for offprints: R. Ferrari, Cattedra di Cardiologia, Uni- versit~ degli Studi di Brescia, c/o Spedali Civili, P. le Spedali Civili, 1 25100 Brescia, Italy

protective effect of propionyl-l-carnitine against ischaemia and reperfusion-damage

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