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Effects of nicotinic acid and mepacrine on fatty acid accumulation and myocardial damage during ischemia and reperfusion
J Mol
Cell
Cardiol
22, 1555163
(1990)
Effects of Nicotinic Acid and Mepacrine Accumulation and Myocardial Damage and Reperfusion Marc van Bilsen, Will A. Coumans,
Ger J. van der Vusse, Theo H. M. Roemen
on Fatty Acid During Ischemia
Peter H. M. Willemsen, and Robert S. Reneman
Department of Physiology, University of Limburg, P.O. Box 616, 6200 MD, The Netherlands
Maastricht,
(Received 15 February 1989, accepted in reuisedform 10 October 1989) M. VAN BILSEN, G. J. VAN DER VUSSE, P. H. M. WILLEMSEN, W. A. COUMANS, T. H. M. ROEMEN AND R. S. RENEMAN. Effects of Nicotinic Acid and Mepacrine on Fatty Acid Accumulation and Myocardial Damage During Ischemia and Reperfusion, Journal of Molecular and Cellular Cardiology (1990) 22, 1555163. To assess the nature of &herniaand reperfusion-induced lipid changes and their consequences for myocardial function and integrity, Krebs-Henseleit perfused, isolated, working rat hearts were treated with nicotinic acid or mepacrine, putative inhibitors of triacylglycerol and phospholipid hydrolysis, respectively. In non-treated hearts 60 min ischemia resulted in a marked rise in myocardial fatty acid (FA) content. The FA content sharply increased further during 30 min reperfusion. Seven out of 16 (44%) hearts fibrillated continuously during reperfusion. Post-ischemic recovery of cardiac output (CO) of the non-fibrillating hearts amounted to 68 2 15% of the preischemic value. Nicotinic acid (10 PM) significantly reduced FA accumulation during ischemia (P < 0.05), but not during reperfusion (0.05 < P < 0.10). Post-ischemic recovery of CO was improved (87 f 12%). This was neither associated with preservation of myocardial adenine nucleotide content, nor significant reduction of enzyme release. Mepacrine (1 PM) completely abolished reperfusion arrhythmias and improved recovery of CO (88 f 7% of pre-ischemic value). The reduction of FA content in ischemic and reperfused hearts did not reach the level of significance. Enzyme release was not attenuated. At 10 P(M, mepacrine completely prevented accumulation of FAs during ischemia and reperfusion, abolished reperfusion-arrhythmias, and reduced enzyme release. No concomitant preservation of adenine nucleotides was observed. In conclusion, nicotinic acid and mepacrine are able to reduce ischemia- and reperfusion-induced changes in myocardial lipid metabolism, In addition, both drugs improve post-ischemic functional recovery. It remains to be established whether these effects are causally related. KEY WORDS: Fatty heart.
acids; Nicotinic
acid; Glycerol;
Mepacrine;
Introduction Derangements in myocardial lipid metabolism are believed to play an important, if not crucial, role in the sequence of events leading to irreversible damage during ischemia (reviewed by Farber el’ al., 1981; Katz and Messineo, 198 1; Corr et al., 1984). Previous studies from our laboratory showed that triacyglycerol and phospholipid homeostasis are disturbed during ischemia and reperfusion (Van B&en et al., 1989). Accordingly, specific interventions aiming at the reduction of these disturbances in lipid metabolism should be beneficial to the jeopardized myocardium. It should be noted, however, that lipid degradation may also reflect autolysis of irreversibly 0022-2828/90/020155
+ 09 $03.00/O
Adenine
nucleotides;
Ischemia;
Reperfusion;
Rat
damaged cells and, hence, represents an epiphenomenon (Van Bilsen et al., 1989; Va.n der Vusse et al., 1989). Inhibitors of phospholipid hydrolysis, like chlorpromazine, U26,384 and mepacrine have been shown to protect cardiac tissue (Chien et al., 1979; Das et al., 1986; Chiariello et al., 1987; Sen et al., 1988). Nicotinic acid was foupd to limit ischemia-induced ST-segment elevation and to reduce infarct size (Kjekshus and Mjas, 1973; Kjekshus, 1981; Vik-Mo et al., 1979, 1986). The protective effect of nicotinic acid has been attributed mainly to the reduced supply of fatty acids (FAs) to the jeopardized myocardium through inhibition of lipolysis in adipose tissue (Kjekshus, 198 1; 0 1990 Academic
Press Limited
156
M. van Bilsen
Lamping et al., 1984; Vik-Mo et al., 1979, 1986). Several investigators have discussed the possibility that this drug might also inhibit lipolysis in ischemic cardiac tissue (Vik-Mo et al., 1979; Otani et al., 1988). The present study was designed to delineate the relative importance of derangements in phospholipid and triacylglycerol homeostasis to myocardial damage during ischemia and reperfusion. To this end isolated, working rat hearts, rendered transiently ischemic, were perfused in the presence of either mepacrine or nicotinic acid, putative inhibitors of phospholipid and triacylglycerol hydrolysis, respectively. The effect of the drugs on myocardial lipid metabolism was assessed by monitoring the tissue content of FAs, a sensitive marker for derangements of myocardial lipid metabolism, of ischemic and reperfused hearts (Van der Vusse et al., 1982; Chien et al., 1984; Van Bilsen et al., 1989). Myocardial protection, if any, was assessed from (a) the incidence and duration of ventricular fibrillation after restoration of flow, (b) postischemic hemodynamic recovery, (c) the preservation of the myocardial high-energy phosphate content during ischemia and reperfusion and (d) enzyme release, a marker for loss of cell membrane integrity, during reperfusion. Methods Perfusion of the hearts Hearts from male Lewis rats were isolated and cannulated as described in detail previously (Snoeckx et al., 1986). The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer, supplemented with glucose ( 11 mM) and pyruvate (5 mM) as substrates. Nicotinic acid and mepacrine were purchased from Sigma (St. Louis, USA). Following 10 min of perfusion as Langendorff hearts (stabilization period), the hearts were perfused via the left atrium as assisted, working hearts. Left atria1 filling pressure was set at 1.0 kPa and diastolic aortic pressure at 8.0 kPa. In the control situation the aortic input impedance used resulted in an aortic pressure pulse of about 4.5 kPa. After 30 min of perfusion as working hearts, the hearts were rendered globally ischemic for 60 min (no-flow ischemia), which was followed by 30 min reperfusion. The hearts were
et al.
retrogradely perfused at a perfusion pressure of 8.0 kPa during the first 5 min of reperfusion and antegradely perfused thereafter. Hemodynamic variables, like aortic pressure, left ventricular pressure and aortic flow were measured as previously described (Snoeckx et al., 1986). Platinum electrodes were attached to the surface of the left ventricle for the continuous recording of the electrogram. Coronary flow was assessed by timed collection of the coronary eflluent in a graded cylinder. Cardiac output was calculated by adding aortic llow and coronary flow. Left ventricular developed pressure was defined as the difference between systolic and enddiastolic left ventricular pressure. Nicotinic acid (10 ,UM final concentration) or mepacrine (1 PM or 10 PM final concentration) was added to the perfusion medium when indicated. The drugs were present during the pre-ischemic as well as post-ischemic phase. Hearts were freeze-clamped at the end of the pre-ischemic control period (nontreated, n = 9), at the end of ischemia (nontreated, n = 8; nicotinic acid treated, n = 6; mepacrine treated, n = 6 and n = 3 in the 1 ,UM and 10 ,DM group, respectively), or at the end of reperfusion (non-treated, n = 16; nicotinic acid treated, n = 7; mepacrine treated, n=7andn=4inthel,uMandlO/&igroup, respectively). To assess possible effects of the drugs on normoxic myocardial function, hearts were perfused antegradely for 120 min in the absence (n = 6) or in the presence of either nicotinic acid (10 PM, n = 3) or mepacrine (1 PM, n = 4) and subsequently freezeclamped. Biochemical
analysis
Perchloric acid extraction of freeze-dried tissue samples was performed as described before (Snoeckx et al., 1986). ATP, ADP and AMP in the neutralized perchloric acid extract were determined by reversed phase high performance liquid chromatography (HPLC) according to the method of Wynants and Van Belle ( 1985). The neutralized extract was also used for the fluorometric determination of glycerol-3-phosphate and glycerol (modified after Laurel1 and Tibbling, 1966) and lactate (according to Passonneau, 1974). Myocardial lipids were extracted using the Folch proce-
Myocardial
Fatty Acid Accumulation
dure as described in detail elsewhere (Van der Vusse et al., 1982). Neutral and polar lipids were separated by silica gel column chromatography (Roemen and Van der Vusse, 1985). In the polar lipid fraction the phosphorus content of phospholipids was estimated according to Bartlett (1959). The neutral lipid fraction was further separated by thin layer chromatography. After elution from the silica gel powder, FAs and triacylglycerols were (trans)methylated and determined by gasliquid chromatography (Van der Vusse et al., 1982). Samples of the coronary effluent were used to determine lactate dehydrogenase (Bergmeyer and Bernt, 1974). Statistical analysis Results are expressed as mean values and standard deviations. Differences between groups were analyzed for significance using Dunn’s multiple comparison procedure, based on the Kruskal-Wallis one-way analysis of variance (Hollander and Wolfe, 1973). P values less than 0.05 were considered to be statistically significant.
157
centration of 10 PM, mepacrine had a depressant effect on myocardial performance. In this experimental group left ventricular developed pressure was insufficient (less than 5 kPa) to generate cardiac output. At a concentration of 1 PM the negative inotropic effect was limited.. At this dose level left ventricular developed pressure and cardiac output amounted to 10.8 &- 0.9 kPa and 63 f 5 ml/min, respectively, a small reduction as compared to the values in the control group (12.5 ) 0.8 kPa and 68 + 7 ml/min, respectively). During 120 min of normoxic perfusion the decline in cardiac output of the control hearts as well as the hearts treated with nicotinic acid (10 PM) or mepacrine (1 FM) did not exceed 5%. Following 120 min of antegrade perfusion the myolcardial triacyglycerol content (about 25 pmol fatty acid equivalents/g, dry wt) was comparable for all experimental groups. In nicotinic acid treated hearts the average FA content was somewhat higher (0.46 pmol/g dry wt) as compared with non-treated hearts (0.30 pmol/g dry wt), but this difference diid the level of significance not reach (0.05 < P < 0.10). The FA content of mepa.crine treated hearts was similar to that of nontreated hearts.
Results Normoxic perfusion
Ischemia and reperfusion
Nicotinic acid (10 ,UM) did not affect the hemodynamics of the working heart, normoxically perfused for 120 min. In contrast, at a con-
As shown in Table 1, hemodynamic recovery of non-treated hearts was severely depressed after 60 min of no-flow ischemia. In fact, seven
TABLE
1. Recovery of hemodynamic treated hearts and hearts
function during reperfusion following treated with nicotinic acid or mepacrine
60 min
of ischemia
of non-
LVDP E
(%I
Non-treated (102.;.: LNicotinic
acid
( 10
,UM)
(104.2 Mepacrine
(1
PM)
4.4) n.c. F 21.1)
103.9 (103.9
+ 7.8 + 7.8)
:;
49.5 (88.0
+ 45.7 f 9.9)
38.2 + 36.5 (68.0 * 15.1)
83.7 (97.6
f 37.3" f 6.1)
74.9 * (87.3 f
96.7 (96.7
IfI 9.3” + 9.3)
87.7 (87.7
34.ga 12.2)b
+ 6.9" + 6.9)b
Data presented as means f S.D. n refers to the number of hearts. Heart rate (HR), left ventricular developed pressure (LVDP) and cardiac output (CO) are presented as percentages of the pre-ischemic values. n.c. = not calculated because of the presence of fibrillating hearts. Data in parentheses refer to the subgroups of hearts that resumed regular rhythm within 30 min of reperfusion. “Significantly different (P < 0.05) from the total group of non-treated hearts. bSignificantly different from the subgroup of non-treated hearts, which resumed regular rhythm within 30 min of reperfusion.
158
M.
van
B&en
out of 16 hearts fibrillated during the entire reperfusion phase. The other nine hearts librillated initially, but were able to resume spontaneous beating within a few minutes. In this subgroup cardiac output recovered to 68% of its pre-ischemic value after 30 min of reperfusior (Table 1). When nicotinic acid was administered, only one out of seven hearts fibrillated during the reperfusion phase (not ,;tatistically different from non-treated reperfused hearts). In the non-fibrillating hearts contractile activity was restored immediately upon reperfusion. Post-ischemic recovery of left ventricular developed pressure and cardiac output was significantly better in nicotinic acid treated hearts than in non-treated hearts. Mepacrine (1 PM) completely prevented the occurrence of fibrillation during reperfusion. Hemodynamic recovery was ameliorated and comparable to that of the nonfibrillating nicotinic acid treated hearts (Table 1). Because of the substantial negative inotropic effects of mepacrine at the higher dose level ( 10 PM), hemodynamic recovery was not analyzed in this experimental group. Adenine nucleotides Table 2 shows that the presence nicotinic acid nor mepacrine (1 TABLE
2. Myocardial reperfused
content of adenine hearts, treated with
ATP Non-treated Pre-ischemia Ischemia Reperfusion
Mepacrine (1 Ischemia Reperfusion
PM)
Mepacrine (10 Ischemia Reperfusion
and
Triacyglycerol
and phospholipids
The myocardial triacylglycerol and total phospholipid contents of non-treated hearts were not significantly affected during ischemia or reperfusion and averaged about 25 pmol fatty acid equivalents/g dry wt and 170 pmol lipid phosphorus/g dry wt, respectively. Administration of nicotinic acid (10 /AM) or mepacrine (1 or 10 PM) did not influence the
nucleotides and energy charge nicotinic acid or mepacrine
of pre-ischemic,
ischemic
and
Energy charge
Total
AMP
5.4 + 1.2
1.0 f 0.5
25.4 f
2.6
0.85
f 0.05
5.5 * 3.7 *
1.3 0.5
3.6 + 1.7 1.4 f 0.7
14.8 f 3.2 13.5 + 2.7
0.55 0.75
5 0.14 f 0.11
5.6 + 1.2 10.4 + 2.1
6.1 + 0.5 3.9 & 0.5
1.7 1 0.3” 0.9 f 0.5
13.3 + 1.1 15.2 f 2.0
0.64 0.81
f 0.04 + 0.06
6.2 + 1.5 10.2 f 1.9
5.6 f 1.5 3.2 + 0.4b
2.0 + 1.4” 0.4 f O.Ob
13.8 + 1.8 13.8 k 2.4
0.60 0.86
+ 0.13 f 0.02b
7.9 * 3.9 10.7 &- 2.1
4.5 + 0.2 2.7 + 0.7b
1.3 2 0.6” 0.6 f- 0.2
13.6 + 3.3 13.7 + 1.6
0.73 0.86
+ 0.12 * 0.05b
3.1
5.7 ‘I 3.1 8.4 + 3.1 (10
cantly preserved the total adenine nucleotide content during ischemia or reperfusion. However, during ischemia the rise in myocardial content of AMP was less pronounced when either of the drugs was administered. During reperfusion ATP content was partially restored, whereas the contents of ADP and AMP declined in each of the experimental groups. In comparison with non-treated hearts nicotinic acid did not significantly improve the energy charge of reperfused hearts. The energy charge restored to pre-ischemic levels when mepacrine (1 PM) was present. Despite the marked negative inotropic effect, 10 /AM mepacrine did not attenuate the depletion of the total adenine nucleotide pool during ischemia and reperfiision. At this dose the energy charge of reperfused hearts was similar to hearts treated with 1 pM mepacrine.
ADP
18.9 f
Nicotinic acid Ischemia Reperfusion
of neither signifi-
,UM)
et al.
PM)
PM)
Data presented as means + SD. Adenine nucleotides are expressed in pmol/g dry wt. Total AMP. Energy charge is defined as (ATP + 0.5 ADP)/(ATP + ADP + AMP). “Significantly different (P < 0.05) from non-treated ischemic hearts. bSignificantly different from non-treated reperfused hearts.
refers
to sum ofATP,
ADP
Myocardial
triacylglycerol ischemic and shown).
and phospholipid reperfused hearts
Fatty
content of (data not
Glycerol-3-phosphate, glycerol and lactate In pre-ischemic, non-treated hearts the tissue content of glycerol-3-phosphate and glycerol amounted to 0.6 k 0.3 and 0.5 ) 0.1 pmol/g dry wt, respectively, As shown in Figure 1 the accumulation of glycerol-3-phosphate was less pronounced in ischemic hearts treated with nicotinic acid or mepacrine, especially at the At the higher dose level (10 PM) of mepacrine. end of ischemia the average glycerol content of nicotinic acid treated hearts tended to be lower as compared with non-treated hearts (0.05 < P < 0.10). At a concentration of 10 PM mepacrine reduced the rise of glycerol during ischemia. Reperfusion resulted in a normalization of the tissue levels of glycerol-3phosphate and glycerol in all experimental groups. In non-treated hearts the lactate content rose from 10.5 + 6.9 to 213.7 + 26.3 hmol/g dry wt during 60 min of ischemia. Nicotinic acid and mepacrine (1 and 10 ,UM) significantly reduced the ischemia-induced accumulation of lactate (149.0 * 17.1, 157.5 -+_ 28.3 and 129.8 +_ 22.1 pmoljg dry wt, respectively).
Acid
Accumulation
159
acid (10 ,UM) reduced FA accumulation during ischemia. In the presence of 1 PM mepacrine the ischemic rise in FAs was intermediate. The tissue FA content was neither statistically different from the pre-ischemic value nor from the ischemic value of non-treated hearts. At a concentration of IO PM, mepacrine completely blocked the ischemia-induced rise in FAs. Restoration of flow was associated with enhanced accumulation of FAs in non-treated hearts (Fig. 2). The increase of tissue FA content of hearts that resumed regular rhythm during reperfusion and hearts that continued to fibrillate was found to be similar (3.80 k 0.94 and 4.55 + 0.99 pmol FA/g dry wt, respectively). At the end of reperfusion the accumulation of FAs was about halvled when nicotinic acid (10 PM) or mepacrine were administered. However, these (1 PM) reduced values were statistically not significant from corresponding values in non-treated hearts, due to the large inter-individual variation in the FA content of reperfused hearts. At the higher dose level of mepacrine (10 PM) the FA content of reperfused hearts averaged about 0.39 pmol/g ‘dry wt (n = 4) only, a value not different from the pre-ischemic value. As a result of ischemia and reperfusion not only the total FA content, but also the contribution of individual FAs to total FA accumulation changed (Table 3). In non-treated
Myocardial fat9 acid content FAs accumulated in non-treated hearts rendered ischemic for 60 min (Fig. 2). Nicotinic 5 6
3000
-
)
P -1; 40002000c 2
II
-^
y2
1 tI
IOOO&ss
O
P
/ 1
I
R
I
R
I
R
0
-
I
R
in-71
Non-treated
Nmtmc Mepocrcne acid (IO PM) (I PM)
Mepacme (IO p.Ml
II
Non-
treated
h&
acid (IO
/IM)
Mepacrine (I pl)
Mepocrine (IO pd
FIGURE 1. Tissue content of glycerol-3-phosphate (0) and glycerol (a) of non-treated, nicotinic acid treated, and mepacrine treated hearts subjected to 60 min ofischemia. Data presented as means + S.D. Star indicates significantly different (P < 0.05) from non-treated ischemic hearts.
FIGURE 2. Myocardial content of FAs of pre-ischemic hearts (P), hearts subjected to 60 min ofischemia (I), and reperfused hearts (R), perfused in the absence or presence of either nicotinic acid (10 PM) or mepacrine (1 or 10 PM). Data presented as means rt S.D. *, significantly different (P < 0.05) from pre-ischemic non-treated hearts; h, significant difference between drug-treated and non-treated ischemic hearts; 0, significant difference between drugtreated and non-treated reperfiised hearts.
160
M. van Bilsen
TABLE
3. Myocardial total FA content and percentage contribution ofindividual of non-treated pre-ischemic, ischemic and reperfused hearts
Total FA content Percentage composition C16:O C18:O C18: 1 C18:2 C20:4 C22:6 Data presented their chemical are shown. aSignificantly
as means notation and different
60 min ischemia
60 min ischemia + 30 min reperfusion
0.25 + 0.05
1.06 + 0.39”
4.13 + 2.11”
12.8 19.4 4.9 11.5 12.7 33.9
23.9 20.6 15.5 21.0 9.7 6.5
18.4 21.3 15.6 22.5 11.4 7.3
+ S.D. Total are expressed (P < 0.05)
from
-t + f f + &-
4.7 4.2 4.3 2.8 2.3 11.2
FA content is expressed as pmol/g as percentages of the total. Only pre-ischemic
cumulative release of LDH during 30 min reduced reperfusion tended to be < P < 0.10) when nicotinic acid (10 ,UM) administered (Fig. 3). Mepacrine at a
1 C
4 NA
M
M
(IO p.4
(I pd
(IO p4)
f + IfI * + +
1.8” 1.9 1.9” 3.3” 0.7 1.4”
dry wt. The the quantitatively
+ f f + * rt
1.5” 3.7 1.3” 3.4” 2.1 2.3”
individual FAs are denoted by most important FA species
value.
Enzyme release
0
FAs to total FA content
Pre-ischemic
ischemic and reperfused hearts the percentage contribution of Cl6:0, C18: 1 and Cl8:2 increased, whereas that of C22 : 6 substantially declined. Except for a small, but significant, rise in the percentage contribution of C22 : 6 to the FA pool, nicotinic acid (10 ,UM) and mepacrine (1 and 10 PM) did not affect the contribution of individual fatty acids to the total FA content of reperfused hearts (data not shown).
The of (0.05 was
et al.
_
FIGURE 3. Cumulative release of lactate dehydrogenase (LDH) during 30 min of reperfusion following 60 min of ischemia of non-treated hearts (C) and hearts treated with nicotinic acid (NA) or mepacrine (M). Star indicates significantly different (P < 0.05) from non-treated group.
concentration of 1 pM did not affect LDH release. However, at a concentration of 10 pM of mepacrine LDH release was markedly reduced.
Discussion Effects of nicotinic acid The findings of the present study indicate that nicotinic acid directly effects myocardial metabolism. Nicotinic acid reduces the accumulation of FAs during ischemia, suggesting that at least part of the FAs accumulating during ischemia are formed by net hydrolysis of triacyglycerols. Since cardiac tissue is unable to metabolize glycerol to any significant extent (Scheuer and Olson, 1967), glycerol production is considered to be an index of triacylglycerol hydrolysis in the heart. In non-treated ischemic hearts (i.e. 4.6 the accumulation of glycerol ,umol/g dry wt, corresponding to 13.8 ,umol fatty acid equivalents) outnumbers the rise in FAs (i.e. 0.8 pmol/g dry wt) manyfold. This indicates reesterification of FAs and, hence, the existence of an operative triacylglycerol-FA cycle, as suggested by Trach and coworkers (1986). As a result, a considerable part of the anaerobically produced ATP will be converted to AMP in this so-called “futile cycle”. The present findings show that nicotinic acid tends to lower the tissue content of glycerol in ischemic hearts
Myocardial
Fatty
indicating that the activity of the triacylglycerol-FA cycle is partly inhibited. Consequently, nicotinic acid will diminish ATP usage of the ischemic tissue, and a positive effect on the energy status may be anticipated. The attenuation of the ischemic rise in AMP and the preservation of the energy charge are in concert with this notion. It is interesting to note that nicotinic acid also inhibits the formation of glycerol-3phosphate and lactate during ischemia, indicating that this drug somehow influences anaerobic glycolysis. The true nature of the interplay between anaerobic glycolysis and lipolysis (Schoonderwoerd et al., 1987), and the effect of nicotinic acid on these processes, remains to be established in more detail. After restoration of flow nicotinic acid markedly improves functional recovery, without preserving the tissue content of adenine nucleotides. As compared to non-treated reperfused hearts the FA content and release of LDH only tends to be reduced. Efects of mepacrine At a concentration of 1 PM, mepacrine exerts only a weak negative inotropic effect. During reperfusion arrhythmias are abolished and functional recovery is ameliorated, indicating that at this dose level mepacrine affords protection to the heart. Notably, the protective effect is not associated with a better preservation of the adenine nucleotide content or with substantial reduction of FA accumulation and LDH release from reperfused hearts. The higher dose level of mepacrine (10 PM) prevents the accumulation of FAs during ischemia and reperfusion. At this dose level enzyme release during reperfusion is markedly reduced. It cannot be excluded that the observed reduction of enzyme release and PA accumulation are related to the negative inotropic effects of mepacrine. However, during no-flow ischemia mechanical activity rapidly ceases and, hence, negative inotropic effects are likely to be less relevant under these circumstances. The observation that during ischemia the depletion of the total adenine nucleotide pool is not mitigated is in favor of this notion. In pig hearts high doses of mepacrine prevented the loss of phospholipids during reper-
Acid
Accumulation
16 1
fusion and preserved myocardial ATP levels, but did not improve post-ischemic hemodynamic function. Mepacrine prevented the activation of microsomal phospholipase A;!, whereas it did not affect acyl-CoA synthetase and lysophospholipid acyltransferase activity, suggesting that mepacrine merely affects the rate of phospholipid hydrolysis (Das et al., 1986; Otani et al., 1986). effects The present fi n d ings that mepacrine glycerol levels in ischemic hearts (see Fig. 1) , suggests that mepacrine also influences triacylglycerol metabolism. In addition, mepacrine reduces the ischemic rise of glycerol-3phosphate and lactate. In this respect the mechanism of action of mepacrine and nicotinic acid shows common features. Several studies indicate that mepacrine acts through mechanisms other than the inhibition of phospholipase AZ activity alone. For instance, mepacrine has been found to inhibit the activity of lysophospholipase and monoacylglycerol lipase (Kunze et al., 1982) and to reduce lipid peroxidation (Jackson et al., 1984). Besides, mepacrine is likely to have effects on myocardial calcium homeostasis (Philipson et al., 1985).
Fatty acid accumulation, jibrillation and myocardial damage The present findings indicate that reperfusion results in a marked release of FAs from endogenous lipid pools. It should be stressed that the measurement of tissue levels of FAs possibly underestimates the degradation of esterified lipid pools. Parts of the FAs released may be further metabolized (formation of acylCoA and acyl-carnitine; p-oxidation). Furthermore, preferential oxidation of the more saturated FAs might effect the contribution of individual FAs to total FA accumulation. In the absence of albumin substantial release of FAs into the coronary effluent is not very likely to occur. The post-ischemic rise in arachidonic acid, a fatty acid almost exclusively present in the phospholipid pool, in both non-treated and drug-treated hearts, strongly suggests that the FAs released originate from phospholipids for a large part. Future experiments with labeled FAs might provide additional information with respect to the exact source and
162
M. van Bilsen
destination of FAs released from endogenous lipid pools. It is shown that nicotinic acid and mepacrine, putative inhibitors of hormonestimulated triacylglycerol hydrolysis and phospholipase activity, respectively, improve hemodynamic recovery, without preserving the ATP and total adenine nucleotide content after reinstallation of flow. Besides, both drugs are found to improve post-ischemic electrical stability. A relation between a rise in the tissue content of amphiphilic lipid-intermediates and arrhythmias has been frequently proposed (Corr et al., 1984). At first sight the ability to resume regular rhythm during reperfusion of nicotinic acid and mepacrine treated hearts might be attributed to a reduction of the myocardial FA content. However, for the non-treated reperfused hearts it is shown that the FA levels of fibrillating and non-fibrillating hearts are comparable. This observation suggests a complex relation, if any, between tissue FA levels and reperfusioninduced fibrillation. It is generally believed that disturbances in lipid metabolism are able to induce cell damage (Katz and Messineo, 1981). Accordingly, a positive relationship between tissue FA accumulation (largely reflecting phospholipid hydrolysis) and enzyme release (reflecting cell damage) during reperfusion might be anticipated (Van Bilsen et al., 1989). In addition, interventions aiming at the reduction of these disturbances should lessen cell damage. Plotting the paired data of individual hearts (Fig. 4) points out that interventions with nicotinic acid and mepacrine result in a weak, albeit significant, relation between FA accumulation and LDH release (rS = 0.53). Comparable r,-values were obtained for the relation between the accumulation of individual FAs and enzyme release. For instance, rSvalues of 0.46 and 0.53 were calculated for oleic acid (relatively abundant in triacylglyerols) and arachidonic acid (almost exclusively in phospholipids), respectively. Obvi-
et al.
oh 0
2000
4000
FA hmol/g
6000
8000
dry wt)
FIGURE 4. Relation between FA content of reperfused hearts and the cumulative release of LDH during 30 min of reperfusion. Non-treated (0); 10 ELM nicotinic acid ( n ); 1 pM mepacrine (A); 10 pM mepacrine (a). r,=O.53 (Spearman correlation coefficient) at P
ously, such weak correlations do not allow a firm conclusion as to the nature of the relation between derangements in myocardial lipid metabolism and tissue damage. To summarize, both nicotinic acid and mepacrine ameliorate post-ischemic functional recovery, indicating a protective effect of putative inhibitors of triacylglycerol and phospholipid hydrolysis. It is tempting to speculate that the reduction of the tissue FA level is responsible for this phenomenon. Further investigation is required to decide whether derangements in lipid metabolism are causally related to irreversible cell damage (Van der Vusse et al., 1989). Application of new, highly specific antilipolytic drugs might be a useful approach for solving this intriguing issue. Acknowledgements This work was supported by a grant from Medigon/NWO (nr. 900-516-091). The authors are grateful to Miss L. de Boer and Mrs E. van Roosmalen for secretarial assistance.
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chromatography. J Biol Chem 234: 46-68. Lactate Dehydrogenase with Pyruvate and NADH. Weinheim, Verlag Chemie GmbH, pp 574-579.
In:
Methods
of
Myocardial
Fatty
Acid
Accumulation
163
CHIARIELLO M, AM~ROSIO G, CAPELLA-BIGAZZI M, NEVOLA E, PERRONE-FILARDI P, MARONE G, CONDORELLI M (1987) Inhibition of ischemia-induced phospholipase activation by quinacrine protects jeopardized myocardium in rats with coronary artery occlusion. J Pharmacol Exp Ther 241: 560-568. CHIEN KR, HAN A, SEN A, BUJA LM, WILLERSON JT (1984) Accumulation of unesterified arachidonic acid in ischemic canine myocardium. Circ Res 54: 3 13-322. CHIEN KR, PFAU RG, FARBER JL (1979) Ischemic myocardial cell injury. Prevention by chlorpromazine of an accelerated phospholipid degradation and associated membrane dysfunction. Am J Pathol97: 5055530. CORR PB, GROSS RW, SOBEL BE (1984) Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ Res 55: 135-154. DAS DK, ENGELMAN RM, Rousou JA, BREYER RH, OTANI H, LEMESHOW S (1986) Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am J Physiol251: H71-H79. FARBER JL, CHIEN KR, MITTNACHT S (1981) The pathogenesis ofirreversible cell injury in ischemia.. Am J Pathol102: 271-281. HOLLANDER W, WOLFE DA (1973) Nonparametric Statistical Methods. New York, John Wiley. JACKSON MJ, JONES DA, HARRIS EJ (1984) Inhibition oflipid peroxidation in muscle homogenates by phospholipase & inhibitors. Biosci Rep 4: 581-587. KATZ AM, MESSINEO FC (1981) Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium. Circ Res 48: 1-16. KJEKSHUS JK (1981) Effect of inhibition oflipolysis on acute myocardial injury during coronary occlusion in normal and reserpinized dogs. Acta Med Stand 645: 85-91. KJEKSHUS JK, MJSS OD (1973) Effect of inhibition of lipolysis on infarct size after experimental coronary artery occlusion. J Clin Invest 52: 1770-l 778. KUNZE H, HESSE B, BOHN E (1982) Effects of antimalarial drugs on several rat-liver lysosomal enzymes involved in phosphatidylethanolamine catabolism. Biochim Biophys Acta 713: 112-l 17. LAMPINC KA, MENAHAN LA, GROSS GJ (1984) Nicotinic acid, free fatty acids and myocardial function during coronary occlusion and reperfusion in the dog. J Pharmacol Exp Ther 231: 532-538. LAURELL S, TIBBLINC G (1966) An enzymatic fluorometric micromethod for the determination of glycerol. Clin Chim Acta 13: 3 17-322. OTANI H, ENGELMAN RM, BREYER RH, Rousou JA, LEMESHOW S, DAS DK (1986) Mepacrine, a phospholipase inhibitor. A potential tool for modifying myocardial reperfusion injury, J Thor Cardiovasc Surg 92: 247-254. OTANI H, ENCELMAN RM, DATTA S, JONES RM, CORDIS GA, Rousou JA, BREYER RH, DAS DK (1988) Enhanced myocardial preservation by nicotinic acid, an antilipolytic compound. J Thorac Cardiovasc Surg 96: 81-87. PASSONNEAU J (1974) Fluorometric Method for the Determination of Lactate with LDH. In: Methods of Engmatic Ana&, Vol3, edited by HU Bergmeyer. Weinheim, Verlag Chemie GmbH, pp 1468-1472. PHILIPSON KD, LANCER GA, RICH TL (1985) Charged amphiphiles regulate heart contractility and sarcolemma-Ca2+ interactions. Am I Phvsiol248: H147-H150. ROEMEN THM, VAN DER VUSSE GJ (1985) Application of silicagel column chromatography in the assessment of nonesterified fatty acids and phosphoglycerides in myocardial tissue. J Chromatogr 344: 304-308. SCHEUER J, OLSON RE (1967) Metabolism of exogenous triglyceride by the isolated perfused rat heart. Am J Physiol 212: 301-307. SCHOONDERWOERD K, BROEKHOVEN-SCHOKKER S, H~~LSMANN WC, STAM H (1987) Stimulation of myocardial neutral triglyceride lipase activity by adenosine-3’:5’-monophosphate: involvement of glycogenolysis. Bas Res Cardiol 82 [Suppl 11: 29-35. SEN A, MILLER JC, REYNOLDS R, WILLERSON JT, BUJA M, CHIEN KR (1988) Inhibition of the release of arachidonic acid prevents the development of sarcolemmal membrane defects in cultured rat myocardial cells during adenosine triphosphate depletion. J Clin Invest 82: 1333-1338. SNOECKX LHEH, VAN DER VUSSE GJ, COUMANS WA, WILLEMSEN PHM, VAN DER NAGEL T, RENEMAN RS (1986) Myocardial function in normal and spontaneously hypertensive rats during reperfusion after a period of global ischemia. Cardiovasc Res 20: 67-75 i 1986) TRACH V, BUSCHMANS-DENKEL E, SCHAPER W (1986) Relation between lipolysis and glycolysis during ischemia in the isolated rat heart. Basic Res Cardio181: 454-464. VAN BILSEN M, VAN DER VUSSE GJ, WILLEMSEN PHM, COUMANS WA, ROEMEN THM, RENEMAN RS (1989) Lipid alterations in isolated, working rat hearts during &hernia and reperfusion: its relation to myocardial damage. Circ Res 64: 304-314. VAN DER VUSSE GJ, ROEMEN THM, PRINZEN FW, COUMANS WA, RENEMAN RS (1982) Uptake and tissue content offatty acids in dog myocardium under normoxic and ischemic conditions. Circ Res 50: 538-546. VAN DER VUSSE GJ, VAN BILSEN M, RENEMAN RS (1989) Is phospholipid degradation a critical event in ischemia and reperfusion induced damage. NIPS 4: 49-53. VIK-MOHH, RIEMERSMA RA, MJ~S OD, OLIVER MF (1979) Effect of myocardial ischaemia and antilipolytic agents on lipolysis and fatty acid metabolism in the in situ dog heart. Stand J Clin Lab Invest 39: 559-568. VIK-MO H, MJ~S OD, NEF.=Y JR, RIBEIRO LGT (1986) Limitation ofmyocardial infarct size by metabolic interventions that reduce accumulation of fatty acid metabolites in ischemic myocardium. Am Heart J 111: 1048-1054. WYNANTS J, VAN BELLE H (1985) Single-run high performance liquid chromatography of nucleotides, nucleosides and major purine bases and its application to different tissue extracts. Anal Biochem 144: 25&266.
Cell
Cardiol
22, 1555163
(1990)
Effects of Nicotinic Acid and Mepacrine Accumulation and Myocardial Damage and Reperfusion Marc van Bilsen, Will A. Coumans,
Ger J. van der Vusse, Theo H. M. Roemen
on Fatty Acid During Ischemia
Peter H. M. Willemsen, and Robert S. Reneman
Department of Physiology, University of Limburg, P.O. Box 616, 6200 MD, The Netherlands
Maastricht,
(Received 15 February 1989, accepted in reuisedform 10 October 1989) M. VAN BILSEN, G. J. VAN DER VUSSE, P. H. M. WILLEMSEN, W. A. COUMANS, T. H. M. ROEMEN AND R. S. RENEMAN. Effects of Nicotinic Acid and Mepacrine on Fatty Acid Accumulation and Myocardial Damage During Ischemia and Reperfusion, Journal of Molecular and Cellular Cardiology (1990) 22, 1555163. To assess the nature of &herniaand reperfusion-induced lipid changes and their consequences for myocardial function and integrity, Krebs-Henseleit perfused, isolated, working rat hearts were treated with nicotinic acid or mepacrine, putative inhibitors of triacylglycerol and phospholipid hydrolysis, respectively. In non-treated hearts 60 min ischemia resulted in a marked rise in myocardial fatty acid (FA) content. The FA content sharply increased further during 30 min reperfusion. Seven out of 16 (44%) hearts fibrillated continuously during reperfusion. Post-ischemic recovery of cardiac output (CO) of the non-fibrillating hearts amounted to 68 2 15% of the preischemic value. Nicotinic acid (10 PM) significantly reduced FA accumulation during ischemia (P < 0.05), but not during reperfusion (0.05 < P < 0.10). Post-ischemic recovery of CO was improved (87 f 12%). This was neither associated with preservation of myocardial adenine nucleotide content, nor significant reduction of enzyme release. Mepacrine (1 PM) completely abolished reperfusion arrhythmias and improved recovery of CO (88 f 7% of pre-ischemic value). The reduction of FA content in ischemic and reperfused hearts did not reach the level of significance. Enzyme release was not attenuated. At 10 P(M, mepacrine completely prevented accumulation of FAs during ischemia and reperfusion, abolished reperfusion-arrhythmias, and reduced enzyme release. No concomitant preservation of adenine nucleotides was observed. In conclusion, nicotinic acid and mepacrine are able to reduce ischemia- and reperfusion-induced changes in myocardial lipid metabolism, In addition, both drugs improve post-ischemic functional recovery. It remains to be established whether these effects are causally related. KEY WORDS: Fatty heart.
acids; Nicotinic
acid; Glycerol;
Mepacrine;
Introduction Derangements in myocardial lipid metabolism are believed to play an important, if not crucial, role in the sequence of events leading to irreversible damage during ischemia (reviewed by Farber el’ al., 1981; Katz and Messineo, 198 1; Corr et al., 1984). Previous studies from our laboratory showed that triacyglycerol and phospholipid homeostasis are disturbed during ischemia and reperfusion (Van B&en et al., 1989). Accordingly, specific interventions aiming at the reduction of these disturbances in lipid metabolism should be beneficial to the jeopardized myocardium. It should be noted, however, that lipid degradation may also reflect autolysis of irreversibly 0022-2828/90/020155
+ 09 $03.00/O
Adenine
nucleotides;
Ischemia;
Reperfusion;
Rat
damaged cells and, hence, represents an epiphenomenon (Van Bilsen et al., 1989; Va.n der Vusse et al., 1989). Inhibitors of phospholipid hydrolysis, like chlorpromazine, U26,384 and mepacrine have been shown to protect cardiac tissue (Chien et al., 1979; Das et al., 1986; Chiariello et al., 1987; Sen et al., 1988). Nicotinic acid was foupd to limit ischemia-induced ST-segment elevation and to reduce infarct size (Kjekshus and Mjas, 1973; Kjekshus, 1981; Vik-Mo et al., 1979, 1986). The protective effect of nicotinic acid has been attributed mainly to the reduced supply of fatty acids (FAs) to the jeopardized myocardium through inhibition of lipolysis in adipose tissue (Kjekshus, 198 1; 0 1990 Academic
Press Limited
156
M. van Bilsen
Lamping et al., 1984; Vik-Mo et al., 1979, 1986). Several investigators have discussed the possibility that this drug might also inhibit lipolysis in ischemic cardiac tissue (Vik-Mo et al., 1979; Otani et al., 1988). The present study was designed to delineate the relative importance of derangements in phospholipid and triacylglycerol homeostasis to myocardial damage during ischemia and reperfusion. To this end isolated, working rat hearts, rendered transiently ischemic, were perfused in the presence of either mepacrine or nicotinic acid, putative inhibitors of phospholipid and triacylglycerol hydrolysis, respectively. The effect of the drugs on myocardial lipid metabolism was assessed by monitoring the tissue content of FAs, a sensitive marker for derangements of myocardial lipid metabolism, of ischemic and reperfused hearts (Van der Vusse et al., 1982; Chien et al., 1984; Van Bilsen et al., 1989). Myocardial protection, if any, was assessed from (a) the incidence and duration of ventricular fibrillation after restoration of flow, (b) postischemic hemodynamic recovery, (c) the preservation of the myocardial high-energy phosphate content during ischemia and reperfusion and (d) enzyme release, a marker for loss of cell membrane integrity, during reperfusion. Methods Perfusion of the hearts Hearts from male Lewis rats were isolated and cannulated as described in detail previously (Snoeckx et al., 1986). The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer, supplemented with glucose ( 11 mM) and pyruvate (5 mM) as substrates. Nicotinic acid and mepacrine were purchased from Sigma (St. Louis, USA). Following 10 min of perfusion as Langendorff hearts (stabilization period), the hearts were perfused via the left atrium as assisted, working hearts. Left atria1 filling pressure was set at 1.0 kPa and diastolic aortic pressure at 8.0 kPa. In the control situation the aortic input impedance used resulted in an aortic pressure pulse of about 4.5 kPa. After 30 min of perfusion as working hearts, the hearts were rendered globally ischemic for 60 min (no-flow ischemia), which was followed by 30 min reperfusion. The hearts were
et al.
retrogradely perfused at a perfusion pressure of 8.0 kPa during the first 5 min of reperfusion and antegradely perfused thereafter. Hemodynamic variables, like aortic pressure, left ventricular pressure and aortic flow were measured as previously described (Snoeckx et al., 1986). Platinum electrodes were attached to the surface of the left ventricle for the continuous recording of the electrogram. Coronary flow was assessed by timed collection of the coronary eflluent in a graded cylinder. Cardiac output was calculated by adding aortic llow and coronary flow. Left ventricular developed pressure was defined as the difference between systolic and enddiastolic left ventricular pressure. Nicotinic acid (10 ,UM final concentration) or mepacrine (1 PM or 10 PM final concentration) was added to the perfusion medium when indicated. The drugs were present during the pre-ischemic as well as post-ischemic phase. Hearts were freeze-clamped at the end of the pre-ischemic control period (nontreated, n = 9), at the end of ischemia (nontreated, n = 8; nicotinic acid treated, n = 6; mepacrine treated, n = 6 and n = 3 in the 1 ,UM and 10 ,DM group, respectively), or at the end of reperfusion (non-treated, n = 16; nicotinic acid treated, n = 7; mepacrine treated, n=7andn=4inthel,uMandlO/&igroup, respectively). To assess possible effects of the drugs on normoxic myocardial function, hearts were perfused antegradely for 120 min in the absence (n = 6) or in the presence of either nicotinic acid (10 PM, n = 3) or mepacrine (1 PM, n = 4) and subsequently freezeclamped. Biochemical
analysis
Perchloric acid extraction of freeze-dried tissue samples was performed as described before (Snoeckx et al., 1986). ATP, ADP and AMP in the neutralized perchloric acid extract were determined by reversed phase high performance liquid chromatography (HPLC) according to the method of Wynants and Van Belle ( 1985). The neutralized extract was also used for the fluorometric determination of glycerol-3-phosphate and glycerol (modified after Laurel1 and Tibbling, 1966) and lactate (according to Passonneau, 1974). Myocardial lipids were extracted using the Folch proce-
Myocardial
Fatty Acid Accumulation
dure as described in detail elsewhere (Van der Vusse et al., 1982). Neutral and polar lipids were separated by silica gel column chromatography (Roemen and Van der Vusse, 1985). In the polar lipid fraction the phosphorus content of phospholipids was estimated according to Bartlett (1959). The neutral lipid fraction was further separated by thin layer chromatography. After elution from the silica gel powder, FAs and triacylglycerols were (trans)methylated and determined by gasliquid chromatography (Van der Vusse et al., 1982). Samples of the coronary effluent were used to determine lactate dehydrogenase (Bergmeyer and Bernt, 1974). Statistical analysis Results are expressed as mean values and standard deviations. Differences between groups were analyzed for significance using Dunn’s multiple comparison procedure, based on the Kruskal-Wallis one-way analysis of variance (Hollander and Wolfe, 1973). P values less than 0.05 were considered to be statistically significant.
157
centration of 10 PM, mepacrine had a depressant effect on myocardial performance. In this experimental group left ventricular developed pressure was insufficient (less than 5 kPa) to generate cardiac output. At a concentration of 1 PM the negative inotropic effect was limited.. At this dose level left ventricular developed pressure and cardiac output amounted to 10.8 &- 0.9 kPa and 63 f 5 ml/min, respectively, a small reduction as compared to the values in the control group (12.5 ) 0.8 kPa and 68 + 7 ml/min, respectively). During 120 min of normoxic perfusion the decline in cardiac output of the control hearts as well as the hearts treated with nicotinic acid (10 PM) or mepacrine (1 FM) did not exceed 5%. Following 120 min of antegrade perfusion the myolcardial triacyglycerol content (about 25 pmol fatty acid equivalents/g, dry wt) was comparable for all experimental groups. In nicotinic acid treated hearts the average FA content was somewhat higher (0.46 pmol/g dry wt) as compared with non-treated hearts (0.30 pmol/g dry wt), but this difference diid the level of significance not reach (0.05 < P < 0.10). The FA content of mepa.crine treated hearts was similar to that of nontreated hearts.
Results Normoxic perfusion
Ischemia and reperfusion
Nicotinic acid (10 ,UM) did not affect the hemodynamics of the working heart, normoxically perfused for 120 min. In contrast, at a con-
As shown in Table 1, hemodynamic recovery of non-treated hearts was severely depressed after 60 min of no-flow ischemia. In fact, seven
TABLE
1. Recovery of hemodynamic treated hearts and hearts
function during reperfusion following treated with nicotinic acid or mepacrine
60 min
of ischemia
of non-
LVDP E
(%I
Non-treated (102.;.: LNicotinic
acid
( 10
,UM)
(104.2 Mepacrine
(1
PM)
4.4) n.c. F 21.1)
103.9 (103.9
+ 7.8 + 7.8)
:;
49.5 (88.0
+ 45.7 f 9.9)
38.2 + 36.5 (68.0 * 15.1)
83.7 (97.6
f 37.3" f 6.1)
74.9 * (87.3 f
96.7 (96.7
IfI 9.3” + 9.3)
87.7 (87.7
34.ga 12.2)b
+ 6.9" + 6.9)b
Data presented as means f S.D. n refers to the number of hearts. Heart rate (HR), left ventricular developed pressure (LVDP) and cardiac output (CO) are presented as percentages of the pre-ischemic values. n.c. = not calculated because of the presence of fibrillating hearts. Data in parentheses refer to the subgroups of hearts that resumed regular rhythm within 30 min of reperfusion. “Significantly different (P < 0.05) from the total group of non-treated hearts. bSignificantly different from the subgroup of non-treated hearts, which resumed regular rhythm within 30 min of reperfusion.
158
M.
van
B&en
out of 16 hearts fibrillated during the entire reperfusion phase. The other nine hearts librillated initially, but were able to resume spontaneous beating within a few minutes. In this subgroup cardiac output recovered to 68% of its pre-ischemic value after 30 min of reperfusior (Table 1). When nicotinic acid was administered, only one out of seven hearts fibrillated during the reperfusion phase (not ,;tatistically different from non-treated reperfused hearts). In the non-fibrillating hearts contractile activity was restored immediately upon reperfusion. Post-ischemic recovery of left ventricular developed pressure and cardiac output was significantly better in nicotinic acid treated hearts than in non-treated hearts. Mepacrine (1 PM) completely prevented the occurrence of fibrillation during reperfusion. Hemodynamic recovery was ameliorated and comparable to that of the nonfibrillating nicotinic acid treated hearts (Table 1). Because of the substantial negative inotropic effects of mepacrine at the higher dose level ( 10 PM), hemodynamic recovery was not analyzed in this experimental group. Adenine nucleotides Table 2 shows that the presence nicotinic acid nor mepacrine (1 TABLE
2. Myocardial reperfused
content of adenine hearts, treated with
ATP Non-treated Pre-ischemia Ischemia Reperfusion
Mepacrine (1 Ischemia Reperfusion
PM)
Mepacrine (10 Ischemia Reperfusion
and
Triacyglycerol
and phospholipids
The myocardial triacylglycerol and total phospholipid contents of non-treated hearts were not significantly affected during ischemia or reperfusion and averaged about 25 pmol fatty acid equivalents/g dry wt and 170 pmol lipid phosphorus/g dry wt, respectively. Administration of nicotinic acid (10 /AM) or mepacrine (1 or 10 PM) did not influence the
nucleotides and energy charge nicotinic acid or mepacrine
of pre-ischemic,
ischemic
and
Energy charge
Total
AMP
5.4 + 1.2
1.0 f 0.5
25.4 f
2.6
0.85
f 0.05
5.5 * 3.7 *
1.3 0.5
3.6 + 1.7 1.4 f 0.7
14.8 f 3.2 13.5 + 2.7
0.55 0.75
5 0.14 f 0.11
5.6 + 1.2 10.4 + 2.1
6.1 + 0.5 3.9 & 0.5
1.7 1 0.3” 0.9 f 0.5
13.3 + 1.1 15.2 f 2.0
0.64 0.81
f 0.04 + 0.06
6.2 + 1.5 10.2 f 1.9
5.6 f 1.5 3.2 + 0.4b
2.0 + 1.4” 0.4 f O.Ob
13.8 + 1.8 13.8 k 2.4
0.60 0.86
+ 0.13 f 0.02b
7.9 * 3.9 10.7 &- 2.1
4.5 + 0.2 2.7 + 0.7b
1.3 2 0.6” 0.6 f- 0.2
13.6 + 3.3 13.7 + 1.6
0.73 0.86
+ 0.12 * 0.05b
3.1
5.7 ‘I 3.1 8.4 + 3.1 (10
cantly preserved the total adenine nucleotide content during ischemia or reperfusion. However, during ischemia the rise in myocardial content of AMP was less pronounced when either of the drugs was administered. During reperfusion ATP content was partially restored, whereas the contents of ADP and AMP declined in each of the experimental groups. In comparison with non-treated hearts nicotinic acid did not significantly improve the energy charge of reperfused hearts. The energy charge restored to pre-ischemic levels when mepacrine (1 PM) was present. Despite the marked negative inotropic effect, 10 /AM mepacrine did not attenuate the depletion of the total adenine nucleotide pool during ischemia and reperfiision. At this dose the energy charge of reperfused hearts was similar to hearts treated with 1 pM mepacrine.
ADP
18.9 f
Nicotinic acid Ischemia Reperfusion
of neither signifi-
,UM)
et al.
PM)
PM)
Data presented as means + SD. Adenine nucleotides are expressed in pmol/g dry wt. Total AMP. Energy charge is defined as (ATP + 0.5 ADP)/(ATP + ADP + AMP). “Significantly different (P < 0.05) from non-treated ischemic hearts. bSignificantly different from non-treated reperfused hearts.
refers
to sum ofATP,
ADP
Myocardial
triacylglycerol ischemic and shown).
and phospholipid reperfused hearts
Fatty
content of (data not
Glycerol-3-phosphate, glycerol and lactate In pre-ischemic, non-treated hearts the tissue content of glycerol-3-phosphate and glycerol amounted to 0.6 k 0.3 and 0.5 ) 0.1 pmol/g dry wt, respectively, As shown in Figure 1 the accumulation of glycerol-3-phosphate was less pronounced in ischemic hearts treated with nicotinic acid or mepacrine, especially at the At the higher dose level (10 PM) of mepacrine. end of ischemia the average glycerol content of nicotinic acid treated hearts tended to be lower as compared with non-treated hearts (0.05 < P < 0.10). At a concentration of 10 PM mepacrine reduced the rise of glycerol during ischemia. Reperfusion resulted in a normalization of the tissue levels of glycerol-3phosphate and glycerol in all experimental groups. In non-treated hearts the lactate content rose from 10.5 + 6.9 to 213.7 + 26.3 hmol/g dry wt during 60 min of ischemia. Nicotinic acid and mepacrine (1 and 10 ,UM) significantly reduced the ischemia-induced accumulation of lactate (149.0 * 17.1, 157.5 -+_ 28.3 and 129.8 +_ 22.1 pmoljg dry wt, respectively).
Acid
Accumulation
159
acid (10 ,UM) reduced FA accumulation during ischemia. In the presence of 1 PM mepacrine the ischemic rise in FAs was intermediate. The tissue FA content was neither statistically different from the pre-ischemic value nor from the ischemic value of non-treated hearts. At a concentration of IO PM, mepacrine completely blocked the ischemia-induced rise in FAs. Restoration of flow was associated with enhanced accumulation of FAs in non-treated hearts (Fig. 2). The increase of tissue FA content of hearts that resumed regular rhythm during reperfusion and hearts that continued to fibrillate was found to be similar (3.80 k 0.94 and 4.55 + 0.99 pmol FA/g dry wt, respectively). At the end of reperfusion the accumulation of FAs was about halvled when nicotinic acid (10 PM) or mepacrine were administered. However, these (1 PM) reduced values were statistically not significant from corresponding values in non-treated hearts, due to the large inter-individual variation in the FA content of reperfused hearts. At the higher dose level of mepacrine (10 PM) the FA content of reperfused hearts averaged about 0.39 pmol/g ‘dry wt (n = 4) only, a value not different from the pre-ischemic value. As a result of ischemia and reperfusion not only the total FA content, but also the contribution of individual FAs to total FA accumulation changed (Table 3). In non-treated
Myocardial fat9 acid content FAs accumulated in non-treated hearts rendered ischemic for 60 min (Fig. 2). Nicotinic 5 6
3000
-
)
P -1; 40002000c 2
II
-^
y2
1 tI
IOOO&ss
O
P
/ 1
I
R
I
R
I
R
0
-
I
R
in-71
Non-treated
Nmtmc Mepocrcne acid (IO PM) (I PM)
Mepacme (IO p.Ml
II
Non-
treated
h&
acid (IO
/IM)
Mepacrine (I pl)
Mepocrine (IO pd
FIGURE 1. Tissue content of glycerol-3-phosphate (0) and glycerol (a) of non-treated, nicotinic acid treated, and mepacrine treated hearts subjected to 60 min ofischemia. Data presented as means + S.D. Star indicates significantly different (P < 0.05) from non-treated ischemic hearts.
FIGURE 2. Myocardial content of FAs of pre-ischemic hearts (P), hearts subjected to 60 min ofischemia (I), and reperfused hearts (R), perfused in the absence or presence of either nicotinic acid (10 PM) or mepacrine (1 or 10 PM). Data presented as means rt S.D. *, significantly different (P < 0.05) from pre-ischemic non-treated hearts; h, significant difference between drug-treated and non-treated ischemic hearts; 0, significant difference between drugtreated and non-treated reperfiised hearts.
160
M. van Bilsen
TABLE
3. Myocardial total FA content and percentage contribution ofindividual of non-treated pre-ischemic, ischemic and reperfused hearts
Total FA content Percentage composition C16:O C18:O C18: 1 C18:2 C20:4 C22:6 Data presented their chemical are shown. aSignificantly
as means notation and different
60 min ischemia
60 min ischemia + 30 min reperfusion
0.25 + 0.05
1.06 + 0.39”
4.13 + 2.11”
12.8 19.4 4.9 11.5 12.7 33.9
23.9 20.6 15.5 21.0 9.7 6.5
18.4 21.3 15.6 22.5 11.4 7.3
+ S.D. Total are expressed (P < 0.05)
from
-t + f f + &-
4.7 4.2 4.3 2.8 2.3 11.2
FA content is expressed as pmol/g as percentages of the total. Only pre-ischemic
cumulative release of LDH during 30 min reduced reperfusion tended to be < P < 0.10) when nicotinic acid (10 ,UM) administered (Fig. 3). Mepacrine at a
1 C
4 NA
M
M
(IO p.4
(I pd
(IO p4)
f + IfI * + +
1.8” 1.9 1.9” 3.3” 0.7 1.4”
dry wt. The the quantitatively
+ f f + * rt
1.5” 3.7 1.3” 3.4” 2.1 2.3”
individual FAs are denoted by most important FA species
value.
Enzyme release
0
FAs to total FA content
Pre-ischemic
ischemic and reperfused hearts the percentage contribution of Cl6:0, C18: 1 and Cl8:2 increased, whereas that of C22 : 6 substantially declined. Except for a small, but significant, rise in the percentage contribution of C22 : 6 to the FA pool, nicotinic acid (10 ,UM) and mepacrine (1 and 10 PM) did not affect the contribution of individual fatty acids to the total FA content of reperfused hearts (data not shown).
The of (0.05 was
et al.
_
FIGURE 3. Cumulative release of lactate dehydrogenase (LDH) during 30 min of reperfusion following 60 min of ischemia of non-treated hearts (C) and hearts treated with nicotinic acid (NA) or mepacrine (M). Star indicates significantly different (P < 0.05) from non-treated group.
concentration of 1 pM did not affect LDH release. However, at a concentration of 10 pM of mepacrine LDH release was markedly reduced.
Discussion Effects of nicotinic acid The findings of the present study indicate that nicotinic acid directly effects myocardial metabolism. Nicotinic acid reduces the accumulation of FAs during ischemia, suggesting that at least part of the FAs accumulating during ischemia are formed by net hydrolysis of triacyglycerols. Since cardiac tissue is unable to metabolize glycerol to any significant extent (Scheuer and Olson, 1967), glycerol production is considered to be an index of triacylglycerol hydrolysis in the heart. In non-treated ischemic hearts (i.e. 4.6 the accumulation of glycerol ,umol/g dry wt, corresponding to 13.8 ,umol fatty acid equivalents) outnumbers the rise in FAs (i.e. 0.8 pmol/g dry wt) manyfold. This indicates reesterification of FAs and, hence, the existence of an operative triacylglycerol-FA cycle, as suggested by Trach and coworkers (1986). As a result, a considerable part of the anaerobically produced ATP will be converted to AMP in this so-called “futile cycle”. The present findings show that nicotinic acid tends to lower the tissue content of glycerol in ischemic hearts
Myocardial
Fatty
indicating that the activity of the triacylglycerol-FA cycle is partly inhibited. Consequently, nicotinic acid will diminish ATP usage of the ischemic tissue, and a positive effect on the energy status may be anticipated. The attenuation of the ischemic rise in AMP and the preservation of the energy charge are in concert with this notion. It is interesting to note that nicotinic acid also inhibits the formation of glycerol-3phosphate and lactate during ischemia, indicating that this drug somehow influences anaerobic glycolysis. The true nature of the interplay between anaerobic glycolysis and lipolysis (Schoonderwoerd et al., 1987), and the effect of nicotinic acid on these processes, remains to be established in more detail. After restoration of flow nicotinic acid markedly improves functional recovery, without preserving the tissue content of adenine nucleotides. As compared to non-treated reperfused hearts the FA content and release of LDH only tends to be reduced. Efects of mepacrine At a concentration of 1 PM, mepacrine exerts only a weak negative inotropic effect. During reperfusion arrhythmias are abolished and functional recovery is ameliorated, indicating that at this dose level mepacrine affords protection to the heart. Notably, the protective effect is not associated with a better preservation of the adenine nucleotide content or with substantial reduction of FA accumulation and LDH release from reperfused hearts. The higher dose level of mepacrine (10 PM) prevents the accumulation of FAs during ischemia and reperfusion. At this dose level enzyme release during reperfusion is markedly reduced. It cannot be excluded that the observed reduction of enzyme release and PA accumulation are related to the negative inotropic effects of mepacrine. However, during no-flow ischemia mechanical activity rapidly ceases and, hence, negative inotropic effects are likely to be less relevant under these circumstances. The observation that during ischemia the depletion of the total adenine nucleotide pool is not mitigated is in favor of this notion. In pig hearts high doses of mepacrine prevented the loss of phospholipids during reper-
Acid
Accumulation
16 1
fusion and preserved myocardial ATP levels, but did not improve post-ischemic hemodynamic function. Mepacrine prevented the activation of microsomal phospholipase A;!, whereas it did not affect acyl-CoA synthetase and lysophospholipid acyltransferase activity, suggesting that mepacrine merely affects the rate of phospholipid hydrolysis (Das et al., 1986; Otani et al., 1986). effects The present fi n d ings that mepacrine glycerol levels in ischemic hearts (see Fig. 1) , suggests that mepacrine also influences triacylglycerol metabolism. In addition, mepacrine reduces the ischemic rise of glycerol-3phosphate and lactate. In this respect the mechanism of action of mepacrine and nicotinic acid shows common features. Several studies indicate that mepacrine acts through mechanisms other than the inhibition of phospholipase AZ activity alone. For instance, mepacrine has been found to inhibit the activity of lysophospholipase and monoacylglycerol lipase (Kunze et al., 1982) and to reduce lipid peroxidation (Jackson et al., 1984). Besides, mepacrine is likely to have effects on myocardial calcium homeostasis (Philipson et al., 1985).
Fatty acid accumulation, jibrillation and myocardial damage The present findings indicate that reperfusion results in a marked release of FAs from endogenous lipid pools. It should be stressed that the measurement of tissue levels of FAs possibly underestimates the degradation of esterified lipid pools. Parts of the FAs released may be further metabolized (formation of acylCoA and acyl-carnitine; p-oxidation). Furthermore, preferential oxidation of the more saturated FAs might effect the contribution of individual FAs to total FA accumulation. In the absence of albumin substantial release of FAs into the coronary effluent is not very likely to occur. The post-ischemic rise in arachidonic acid, a fatty acid almost exclusively present in the phospholipid pool, in both non-treated and drug-treated hearts, strongly suggests that the FAs released originate from phospholipids for a large part. Future experiments with labeled FAs might provide additional information with respect to the exact source and
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destination of FAs released from endogenous lipid pools. It is shown that nicotinic acid and mepacrine, putative inhibitors of hormonestimulated triacylglycerol hydrolysis and phospholipase activity, respectively, improve hemodynamic recovery, without preserving the ATP and total adenine nucleotide content after reinstallation of flow. Besides, both drugs are found to improve post-ischemic electrical stability. A relation between a rise in the tissue content of amphiphilic lipid-intermediates and arrhythmias has been frequently proposed (Corr et al., 1984). At first sight the ability to resume regular rhythm during reperfusion of nicotinic acid and mepacrine treated hearts might be attributed to a reduction of the myocardial FA content. However, for the non-treated reperfused hearts it is shown that the FA levels of fibrillating and non-fibrillating hearts are comparable. This observation suggests a complex relation, if any, between tissue FA levels and reperfusioninduced fibrillation. It is generally believed that disturbances in lipid metabolism are able to induce cell damage (Katz and Messineo, 1981). Accordingly, a positive relationship between tissue FA accumulation (largely reflecting phospholipid hydrolysis) and enzyme release (reflecting cell damage) during reperfusion might be anticipated (Van Bilsen et al., 1989). In addition, interventions aiming at the reduction of these disturbances should lessen cell damage. Plotting the paired data of individual hearts (Fig. 4) points out that interventions with nicotinic acid and mepacrine result in a weak, albeit significant, relation between FA accumulation and LDH release (rS = 0.53). Comparable r,-values were obtained for the relation between the accumulation of individual FAs and enzyme release. For instance, rSvalues of 0.46 and 0.53 were calculated for oleic acid (relatively abundant in triacylglyerols) and arachidonic acid (almost exclusively in phospholipids), respectively. Obvi-
et al.
oh 0
2000
4000
FA hmol/g
6000
8000
dry wt)
FIGURE 4. Relation between FA content of reperfused hearts and the cumulative release of LDH during 30 min of reperfusion. Non-treated (0); 10 ELM nicotinic acid ( n ); 1 pM mepacrine (A); 10 pM mepacrine (a). r,=O.53 (Spearman correlation coefficient) at P
ously, such weak correlations do not allow a firm conclusion as to the nature of the relation between derangements in myocardial lipid metabolism and tissue damage. To summarize, both nicotinic acid and mepacrine ameliorate post-ischemic functional recovery, indicating a protective effect of putative inhibitors of triacylglycerol and phospholipid hydrolysis. It is tempting to speculate that the reduction of the tissue FA level is responsible for this phenomenon. Further investigation is required to decide whether derangements in lipid metabolism are causally related to irreversible cell damage (Van der Vusse et al., 1989). Application of new, highly specific antilipolytic drugs might be a useful approach for solving this intriguing issue. Acknowledgements This work was supported by a grant from Medigon/NWO (nr. 900-516-091). The authors are grateful to Miss L. de Boer and Mrs E. van Roosmalen for secretarial assistance.
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