Effect of exogenous fatty acids on reperfusion arrhythmias in isolated working perfused hearts NORMAN J. DAVIES,? RAYMOND E. LOVLIN, AND GARY D. LOPASCHUK Division of Cardiology, Department of Medicine, Departments of Pediatrics and Pharmacology, and Heritage Cardiovascular Research Group, University of Alberta, Edmonton, Alberta T6G 2S2, Canada Davies, Lopaschuk.
Norman
J., Raymond
E. Lovlin,
and Gary
D.
Effect of exogenous fatty acids on reperfusion arrhythmias in isolated working perfused hearts. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): Hl796-Hl801,1992.-Exogenous fatty acids may promote arrhythmias during ischemia and reperfusion, perhaps by increasing myocardial concentrations of long-chain acylcarnitines. We therefore studied the effects of high concentrations of fatty acids on reperfusion arrythmias and acylcarnitine accumulation in isolated working rat hearts subjected to regional ischemia and reperfusion. Hearts were perfused with buffer containing 3% albumin, 5.9 mM K+, and either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate. After 15 min of aerobic work, the left anterior descending artery was reversibly ligated for 10 min and released, and the hearts were subsequently reperfused for 3 min. Although ischemic zone acylcarnitine accumulation after reperfusion was significantly greater in glucose plus palmitate-perfused hearts (247 & 149 vs. 717 t 176 nmol/g d ry wt in glucose- vs. palmitate-perfused hearts, respectively), no significant differences in the incidence (67 vs. 44%) or duration (95 t 17 vs. 56 t 17 s) of ventricular fibrillation (VF) were seen in glucose or glucose plus palmitate hearts, respectively. Because low K+ concentration ([K’]) has been reported to increase reperfusion arrythmias in similar models, we reduced perfusate [K+] to 4.0 mM. This significantly increased the incidence and duration of VF in hearts perfused with glucose alone but had no effect in palmitate-perfused hearts. We conclude that acylcarnitine accumulation is not arrhythmogenic in this model and that fatty acids may actually have antiarrhythmic effects if exogenous [K+] is low. acylcarnitine; potassium; ventricular fibrillation HIGH CONCENTRATIONS of exogenous fatty acids have been associated with worsened prognosis in the setting of acute myocardial infarction, perhaps through an increase in arrhythmogenesis (2 1, 22). Amphipathic fatty acid intermediates, such as acylcarnitine and acylcoenzyme A, accumulate during reperfusion after noflow ischemia (3,16,18) and have been implicated in the pathogenesis of both mechanical stunning and malignant arrythmias after myocardial ischemia. These molecules produce in isolated cell systems a number of biochemical and electrophysiological .derangements that have been thought to account for these phenomena (3). However, we have recently provided evidence that acylcarnitine accumulation, although increased when concentrations of exogenous fatty acids are high, may not be important in the pathogenesis of mechanical stunning in isolated ischemic-reperfused rat hearts (18). An arrhythmogenic effect of fatty acid has been demonstrated inconsistently in vivo (14, 22, 23). These inconsistencies have been ascribed to differences in exogenous K+ concentration ([K+]) in different canine models, implying that an interaction exists between TDeceased H1796
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fatty acids and extracellular [K+] (14, 22). Recently, additional evidence has arisen to support an interaction between fatty acids and K+ channels (13). This effect is similar to that seen for the sulfonylurea glyburide (29), which was antiarrythmic properties in isolated perfused hearts (12, 30). In this study, we attempted to clarify the role of exogenous fatty acids in arrythmogenesis using the isolated working rat heart subjected to transient regional ischemia and reperfusion. Our data suggest that acylcarnitine accumulation, although increased by exogenous fatty acids, is not arrythmogenic. Our data further suggest that when exogenous [K+] is decreased, exogenous fatty acids may have antiarrythmic effects during reperfusion. METHODS Perfusion conditions. Hearts were excised from pentobarbital sodium-anesthetized Sprague-Dawley male rats (250-300 g), placed in ice-cold buffer, and cannulated initially as Langendorff hearts. As indicated in the individual experiments, a number of hearts were subsequently cannulated and perfused as working hearts as previously described (17, 18). Hearts were perfused with Krebs-Henseleit buffer (pH 7.4), gassed with 95% 02-5% COz, containing 2.5 mM free Ca2+ and either 5.9 or 4.0 mM K+. As indicated, hearts were perfused with 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate. All perfusates contained 3% bovine serum albumin previously dialyzed against 10 vol Krebs-Henseleit buffer. Palmitate, when used, was prebound to the albumin. In this study, we chose to use palmitate as a representative fatty acid, since it makes up -40% of total free fatty acids in the plasma and under conditions of physiological stress palmitate shows the greatest relative increase in plasma concentration of any fatty acid (27). Furthermore, we have had considerable previous experience using palmitate in the isolated perfused heart model. Working hearts were perfused with recirculated buffer (100 ml) at a 60-mmHg hydrostatic afterload and (in working hearts) a 11.5-mmHg left atria1 preload. Coronary flow was determined by cannulating the pulmonary artery and was measured at 5-min intervals as the sum of pulmonary flow and perfusate “drip out” from the heart as previously described (17,18). Previous workers (4) have demonstrated that coronary flow almost exclusively represents ventricular flow in this model, since the atria receive their nutrient supply through superfusion from the perfusate. Protocol and method of occlusion. All hearts underwent 15 min of aerobic perfusion during which heart rate and peak systolic pressure were recorded every 5 min. After 5 min of aerobic perfusion, coronary arterial occlusion was prepared by placing a suture (6-O silk) under the proximal left anterior descending coronary artery (LAD), and aerobic perfusion was then continued for an additional 10 min. Regional ischemia was produced by tying a short (cl.0 cm) piece of polyethylene tubing (1.0 mm diam) against the proximal LAD. After 10 min of ischemia, the suture was cut with a
0 1992 the
American
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scalpel blade and the ischemic segment was reperfused for an additional 3 min. Approximately 100 mg of myocardial tissue from the epicardial surfaces of the ischemic and nonischemic segments were then quickly frozen using small bulldog clamps cooled to the temperature of liquid nitrogen and then excised, weighed, and stored in liquid nitrogen for metabolite determinations. A number of hearts were also frozen in this manner at the end of ischemia before reperfusion. A lo-min period of ischemia was chosen because initial experiments in our fatty acid-perfused hearts showed that the incidence and duration of ventricular fibrillation (VF) was lower when the ischemic interval was shortened to 5 min or increased to 20 min. This parallels what has previously been shown in glucose-perfused Langendorff hearts (4, 5). Ischemic zone size was determined in a number of hearts by the injection of 1 ml 0.05% Amido Black dye into the preload line immediately after ischemia to demarcate the ischemic and nonischemic zones. The heart was cut from the perfusion apparatus, and after the atria and excess mediastinal tissue were excised, the ischemic zone was cut along its borders as delineated by the dye. The tissue was then dried for determination of the dry weight of the ischemic and nonischemic zones. Tissue analysis. Frozen ventricular tissue from ischemic and nonischemic zones of hearts frozen at the end of the ischemia or at the end of reperfusion was weighed and powdered in a mortar and pestle cooled to the temperature of liquid nitrogen. A portion of the powdered tissues was used in the calculation of the final concentration of metabolite determinations. Lactate and long-chain acylcarnitine were extracted from frozen tissue with perchloric acid as described previously (17, 18). Extracted longchain acylcarnitine was hydrolyzed to free carnitine and measured radiometrically (17, 18). Lactate levels were determined spectrophotometrically using standard enzymatic assays (18). Measurement of cardiac rhythm disturbances. Throughout each experiment, cardiac rhythm was recorded using a Grass physiological recorder via needle electrodes attached to the apex of the heart and the aortic cannula. VF was defined according to the criteria of Walker et al. (28). Recordings were analyzed visually to determine the incidence and duration of VF as previously described (4, 5). Statistical analysis. Statistical analysis was performed using analysis of variance followed by comparison of group means using group tests. Statistical significance was set at P < 0.05. RESULTS
Effects of LAD ligation on coronary flow and myocardial perfusion. The effect of LAD ligation on heart rate, cor-
onary flow, and (in working hearts) peak systolic pressure development and rate pressure product is presented in Table 1. In both Langendorff and working hearts, LAD ligation led to a significant reduction in coronary flow but no significant effect on heart rate. In working hearts,
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significant reductions in peak systolic pressure development and rate pressure product were also seen. No significant difference in coronary flow reduction was seen in working hearts perfused in the presence or absence of fatty acids. The effect of LAD ligation on ischemic zone size is depicted in Table 2. In Langendorff hearts, ischemic zone dry weight averaged 35.1 t 1.6% of total myocardial dry weight; no significant difference in ischemic zone size was observed between Langendorff and working glucose-perfused hearts. Effects of work on reperfusion arrhythmias. Previous studies (1, 5, 6) examining the incidence and duration of reperfusion arrhythmias have been performed in glucoseperfused Langendorff hearts. We therefore initially performed a series of perfusions in which hearts were perfused with 11 mM glucose alone (perfusate [K+] = 5.9 mM) and subjected to 15 min of Langendorff perfusion, 10 min of LAD ligation, and 3 min of reperfusion. In these hearts, the incidence and duration of reperfusion VF paralleled that observed in the previous studies (Fig. 1). These Langendorff hearts were compared with another series of glucose-perfused working hearts subjected to the same aerobic perfusion-ischemia-reperfusion protocol (Fig. 1). Compared with Langendorff hearts, working hearts displayed a significantly greater incidence (67 vs. 20%, P < 0.05) and duration (15.6 t 7.5 vs. 53.0 t 10.6% of 3-min reperfusion period for working vs. Langendorff hearts, respectively, P < 0.05) of reperfusion VF (Fig. 1, A and B). Effects of exogenous fatty acids on reperfusion arrhythmias. To determine the effects of fatty acids on
arrhythmia formation, a further series of working hearts (n = 6) were perfused with 11 mM glucose or 11 mM glucose and 1.2 mM palmitate (perfusate [K+] = 5.9 mM) and were subjected to the same ischemia-reperfusion protocol. The incidence and duration of VF in glucose plus palmitate-perfused hearts was not significantly different from that seen in hearts perfused with glucose alone (67 vs. 45% of 3-min reperfusion period in glucose vs. glucose plus palmitate hearts respectively, Fig. 2, A and B). Tissue levels of lactate and acylcarnitine in the ischemic-reperfused and nonischemic zones were determined in these hearts and are presented in Table 3. Levels of these metabolites were also measured in a series of hearts perfused under identical conditions except that they were frozen at the end of ischemia. Accumulation of acylcarnitine was seen in both the ischemic and nonischemic
t-1 Table 1. Effect of left anterior descending artery ligation on coronary JLOW and heart function parameters
in Langendorff
and working hearts
Langendorff
Coronary flow, ml/min HR, beats/min PSP, mmHg HR x PSP x 10-3, beats mmHg min-l l
l
Glucose
Working
FA Working
Aerobic
Ischemia
Aerobic
Ischemia
Aerobic
Ischemia
12.4t0.8 238t9.7
7.5kO.6” 208t6.6
17.0t1.9 221t9.4 98t3.3 22.7kO.8
13.0t1.5” 203k9.7 78.2t5.0 15.8+1.0*t
16.Ok1.4 220t9.7 99.6k2.3 21.9t0.9
lO.Ot0.9 190t10.4 83.0+3.2*t 14.0+1.3*t
x 10v3
Values are means t SE. Hearts (n = 12-19/group) were perfused as described in METHODS. FA, Fatty acid; HR, heart rate; PSP, peak systolic pressure. Because pressure measurements were not recorded in Langendorff hearts, values for PSP and HR x PSP are not given. * P < 0.05 vs. Langendorff aerobic. t P < 0.05 vs. working aerobic.
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Table 2. Ischemic zone size in hearts subjected to left anterior descending artery ligation Nonischemic Zone
Total
A
Ischemic Zone
0.062t0.003 Tissue wt, g dry wt 0.17~Joo.008 0.114t0.007 64.9k2.0 35.1t1.6 % Total tissue wt Values are means t SE. Hearts (n = 5) were perfused as Langendorff hearts as described in METHODS.
A c .-0
m-m o-o
loo-
ARRHYTHMIAS
.-uE 0
-r 13 .-G
1001
80-
60-
-:
Working Langendorff 20
10
min.
80-
-z .y x.3 .-
B loo
L+Oy--Y 0 8
E .-w
40-
30
0
Glucose
m
Fatty
Acid
80
0
.-= .-2 LL
6 .--0 -2
25
Time (minutes)
lschemia
zo-
60
55 40
00
.-6 4-J 15
20
25
30
Time (minutes)
2
20
0
B loo
1
; .-w 0 .-=
80-
_ts .LLn Q-v x
60-
El .-
40 -
0
Working
m
Langendorff
Fig. 2. Incidence (A) and duration (B) of VF in working hearts perfused with 5.9 mM K+ and either 11 mM glucose or 11 mM glucose + 1.2 mM palmitate. Preload and afterload were as described in METHODS. Duration of VF is expressed as mean & SE of percentage of 3-min reperfusion period for each group. n = 7 each of 2 groups.
* I *
decreased the incidence and duration of VF compared with that in glucose-perfused hearts (Fig. 3).
0
5 2
DISCUSSION 20-
0-
Fig. 1. Incidence (A) and duration (B) of ventricular fibrillation (VF) in glucose-perfused Langendorff and working hearts. Hearts were perfused with 5.9 mM K+and 11 mM glucose. Langendorff hearts were perfused at a 60-mmHg pressure, whereas working hearts perfused at 11.5-mmHg left atria1 preload and 60-mmHg aortic afterload as described in METHODS. Duration of VF is expressed as mean t SE of percentage of 3-min reperfusion period for each group. n = 6 in each group. * P < 0.05 vs. Langendorff hearts
zones of hearts perfused with palmitate; this increase was significantly different in glucose-perfused hearts in the ischemic zone during both ischemia and reperfusion. Significant ischemic zone accumulation of lactate was also seen during ischemia in both substrate groups in keeping with the severe ischemia produced by LAD ligation. To detect a possible interaction between fatty acids and exogenous [K+] , the ischemia-reperfusion experiments were repeated in working hearts perfused in the presence or absence of fatty acids using a perfusate [K+] of 4.0 mM. As depicted in Fig. 3, both the incidence (A) and duration (B) of reperfusion VF increased in glucoseperfused hearts compared with hearts perfused with 5.9 mM K+ (Fig. 2). In contrast, an increase in the incidence and duration of VF did not occur in fatty acid-perfused hearts (Figs. 2 and 3). As a result, in the presence of 4.0 mM K+, addition of exogenous fatty acid significantly
Exogenous fatty acids have been implicated in the production of depressed contractility and electrophysiological derangements seen during ischemia and reperfusion. The possibility that fatty acids may potentiate arrhythmias during acute myocardial ischemia and infarction was first suggested by Oliver and colleagues (21). Some investigators (9, 22) have also reported such an association, but others (23, 25) failed to find a consistent relationship between levels of circulating free fatty acids and the presence of ventricular or atria1 arrhythmias. The arrythmogenic effect of exogenous fatty acids may be related to increases in concentrations of fatty acid intermediates such as long chain acylcarnitine. Previous studies have demonstrated that acylcarnitine does accumulate during ischemia and reperfusion (3,11,18). These amphiphilic molecules may have a number of deleterious and potentially arrhythmogenic effects, including concentration-dependent alterations of Ca2+ transport in sarcoplasmic reticulum, inhibition of Ca2+-adenosinetriphosphatase (ATPase) and Na+-K+-ATPase in the sarcolemma, inhibition of ATP translocase in the mitochondrial membrane, concentration-dependent decreases in maximum diastolic potential, amplitude, maximal shortening velocity of phase 0, and action potential duration of Purkinje cells, decreased excitability, postrepolarization refractoriness, electrical alternans phenomena, and enhanced a-receptor expression (3, 7, 8, 10, 15, 24, 26). These effects are partially dependent on the type of
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Table 3. Effect of exogenous fatty acids on tissue LeveLsof lactate and acylcarnitine Perfusion Substate
Ischemic Nonischemic Glucose + fatty acid Ischemic Nonischemic Values are means & SE expressed in nmol/g dry wt. Hearts nonischemic. t P < 0.05 vs. glucose. n
-
10
min.
lschemia
\
80
Fatty
Lactate
Acylcarnitine
54t13* 25t7
227k64 183k25 700&121*‘f 433t62
34k5 36k8 87t19* 43k7
247H49 32Ok249 717k176”t 500t137
94t5* 33t5
(n = 7 in each group) were perfused as described in
Acid
* * M-D
30
25
20 Time (minutes)
B loo
f
0
Glucose
m
Fatty
Acid
.-5 80 -w -.=0 h.- 60 LL y-2-4 O5 .c, E)
40
2
20
Postreperfusion Acylcarnitine
,, 15
hearts
Lactate
m Glucose
0-O
c 0
in ischemic-reperfused
Postischemia
Myocardial Zone
Glucose
.-eu -0
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0
Fig. 3. Incident (A) and duration (B) of VF in working hearts perfused with 4.0 mM K+ and either 11 mM glucose or 11 mM + 1.2 mM palmitate (fatty acid) as described in METHODS. Duration of VF is expressed as in Figs. 1 and 2. n = 7 for each of 2 groups. * P < 0.05 vs. fatty acid-perfused hearts.
cardiac tissue being studied, the species, and pH (3). Enhancement of free radical-induced lipid peroxidative injury to sarcolemmal membranes by lipid amphiphiles has also been reported (19). Phospholipid degradation due to phospholipase activation and defective reacylation of unesterified fatty acid may be potentiated by fatty acid intermediates. This may also cause arrhythmias, enzyme inhibition, and membrane dysfunction. Recent studies in our laboratory have suggested, however, that acylcarnitine accumulation may not be important in the production of mechanical stunning in the postischemic heart (18). We demonstrated that the impairment in reperfusion recovery seen in isolated hearts perfused with high concentrations of fatty acids could be overcome through the stimulation of glucose oxidation by the carnitine acyltransferase I inhibitor, etomoxir, independent of changes in tissue levels of acylcarnitine. This suggested that postischemic accumulation of fatty acids intermediates, although enhanced by the presence of high concentrations of exogenous fatty acids, does not account for fatty acid-induced depression
METHODS.
*
P < 0.05 vs.
of function seen during reperfusion. In this study we also did not see an association between the levels of longchain acylcarnitine in the heart and the incidence of reperfusion-induced arrythmias. As shown in Table 3, addition of fatty acids resulted in a rise in long chain acylcarnitine levels in the ischemic zone from 247 to 717 pmol/g dry wt and in the nonischemic zone from 320 to 500 pmol/g dry wt. Despite this increase, a greater incidence of reperfusion-induced arrythmias did not occur in these hearts. This does not preclude the fact, however, that larger increases in long-chain acylcarnitine may have been arrythmogenic. In addition, the involvement of acylcarnitine in other types of arrythmias cannot be excluded. It does suggest, however, that the role of long-chain acylcarnitines in the genesis of arrythmias requires further study. In this study we did not attempt to correlate changes in incidence of VF after reperfusion with changes in level of acylcarnitine. However, in previous work (18) we demonstrated that long-chain acylcarnitine levels 30 min after reperfusion of hearts with high concentrations of fatty acids are much higher than immediately postischemia in isolated fatty acid-perfused hearts subjected to transient global ischemia (Table 3). This suggests that acylcarnitine levels increase progressively during reperfusion rather than after the bimodal concentration curve which might be expected from the known differences in incidence of reperfusion VF. To assess arrythmias in this study, we used a simple adaptation of the isolated regionally ischemic-reperfused Langendorff heart model of Hearse and colleagues (1, 4), which allowed a direct comparison of reperfusion arrhythmias in working hearts under physiological substrate conditions with high concentrations of exogenous fatty acids. Hearts perfused in working mode had a higher incidence and duration of reperfusion VF that Langendorff hearts; the mechanism of this effect is not clear from our data. However, high concentrations of exogenous fatty acids prevented the increased incidence and duration of reperfusion VF in glucose-perfused working hearts seen when exogenous [K+] was reduced. In this study, isolated working hearts subjected to a transient period of regional ischemia showed a higher incidence of arrythmias than Langendorff hearts (Fig. 1). The reason that this occurred is not certain, but possible mechanisms include an increase in wall stress and myocardial oxygen consumption during reperfusion, which could lead to a prolongation of ischemia at the level of the cardiac myocyte and a resultant increase in levels of lactate, H+, and Ca 2+. This phenomenon is even more interesting in light of the frequent observation by clinicians that malignant ventricular arrhythmias often become
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easier to control when concurrent congestive heart failure is adequately treated; treatment in this circumstance lowers left ventricular filling pressures, reduces wall stress, and reduces myocardial oxygen consumption. The increase in VF in the setting of low [K+] is consistent with the findings of other investigators. Using a Langendorff rat heart model, Curtis and Hearse (4) showed that the incidence and duration of VF in Langendorff rat hearts during ischemia correlated inversely with log,, [K+] . During ischemia, K+ loss from myocardial cells occurs, leading to accumulation of extracellular K+. Mechanisms proposed to explain K+ accumulation include inhibition of the Na+-K+ pump, K+ efflux coupled to intracellular ions such as lactate, modification of K+ channels, and opening of ATP-sensitive K+ channels. Regardless of the mechanism by which low [K+] increases arrhythmogenesis, our data demonstrate that fatty acid actually decreased arrhythmia formation in hearts perfused with low [K+]. It is not clear why palmitate had an antiarrythmic effect at lower [K+]. One possible explanation for this effect is the potential effect of fatty acids on the ATPsensitive K+ channel. Kim and Duff (13) have reported that the ATP-sensitive K+ channel, which mediates K+ efflux during ischemia when ATP concentrations fall to very low levels, is inhibited by unesterified free fatty acids such as arachidonic acid. Kantor and colleagues (12) reported that the inhibition of this channel by the sulfonylurea glyburide reduced reperfusion arrhythmias in glucose-perfused nonworking hearts subjected to global lowflow ischemia. Wilde et al. (29) used glyburide in patchclamp investigations of isolated guinea pig cardiomyocyte membranes. These studies showed that this drug slowed the rate of extracellular K+ accumulation during ischemia. In our study, lowering perfusate [K+] would be expected to increase the transmembrane K+ concentration gradient, producing increased K+ efflux during ischemia. Wolleben et al. (30) have shown that stimulation of the ATP-dependent K+ channel contributes to the development of fibrillation and that glyburide, a blocker of the ATP-dependent K+ channel, has an antiarrhythmic effect. In guinea pig myocytes, Naykaya et al. (20) demonstrated that glyburide inhibits action potential shortening, which would be expected to have antiarrythmic effects. In humans, Cacciapuoti et al. (2) showed that glyburide reduces the frequency of premature complexes and tachycardias during transient myocardial ischemia. Whether palmitate at lower [K+] has an antiarrhythmic effect similar to that of glyburide has not been determined. It is therefore possible that exogenous fatty acid may have exerted an antiarrhythmic effect through an inhibitory effect on the ATP-sensitive K+ channel. It has yet to be determined how glyburide interacts with fatty acid on the ATP-dependent K+ channel. In summary, our results indicate that long-chain acylcarnitine accumulation during reperfusion after transient regional ischemia does not promote reperfusion arrhythmias in working hearts subjected to reversible LAD occlusion. Furthermore, our data are consistent with fatty acid-mediated inhibition of K+ efflux during ischemia. Studies to verify the mechanism of arrhythmogenesis in
ARRHYTHMIAS
this model and to determine the relevance of these finding to clinically observed arrhythmias are indicated. This study was supported by a grant from the Medical Research Council of Canada. G. D. Lopaschuk is a scholar of the Alberta Heritage Foundation for Medical Research. Address for reprint requests: G. D. Lopaschuk, Dept. of Pediatrics and Pharmacology, University of Alberta, 423 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Received 30 January 1991; accepted in final form 8 January 1992. REFERENCES M., D. J. Hearse, and A. S. Manning. Reperfusioninduced arrhythmias and oxygen-derived free radicals: studies with “anti-free radical” interventions and a free radical-generating system in the isolated perfused rat heart. Circ. Res. 58: 331-340,
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Trauma metabolism and the heart: uptake of substrates and effects of insulin early after cardiac operations. J. Thoruc. Cardiouasc. Surg. 99: Nilsson,
1063-1073,
1990. J. A., M. J. Curtis, D. J. Hearse, R. W. F. M. J. Janne, D. J. Yellon, S. M. Cobbe, S. J. J. B. Harness, D. W. Harron, A. J. Higgins, D. J. M. J. Lab, A. S. Manning, B. J. Northover, J. R. R. A. Riemersma, E. Riva, D. C. Russell, D. J. E. Winslow, and B. Woodward. The Lambeth
Walker, 28* Campbell,
M.
Coker, Julian, Parratt, Sheridan,
S.
Failure of high concentrations of circulating free fatty acids to provoke arrhythmias in experimental myocardial infraction. Lancet 1: 818-822, 1971. Pitts, Wood,
D.
electrophysiological of early ventricular
1153-1157,
and T. W. Greenwood. Relation between serum-free fatty acids and arrhythmias and death after acute myocardial infraction. Lancet 1: 710-715, 1968. 22. Opie, L. H. Metabolism of free fatty acids, glucose and catecholamines in acute myocardial infarction: relation to mycocardial ischemia and infarct size. Am. J. CardioL. 36: 938-953, 1975.
24.
26. Russell, 686-688,
1986. Nakaya,
103: 1019-1026,1991. 21. Oliver, M. F., V. A. Kurien,
23.
ARRHYTHMIAS
Conventions: guidelines for the study of arrhythmias in myocardial ischemias, infarction and reperfusion. Curdiouasc. Res. 22: 29.
447-455, Wilde, ringer,
1988. A. A. M., M. Mestre,
sium accumulation 30.
Circ. Res. Wolleben
D.
Escande, C. A. Schumacker, D. J. W. T. Fiolet, and M. J. Janse.
in the globally ischemic mammalian
67: 835-843, C. D., M.
1990. C. Sanquinetti,
Influence of ATP-sensitive ischemia-induced fibrillation Curdiol.
and
P.
K.
Thu-
Potasheart.
S. Siegl.
potassium channel modulators on in isolated rat hearts. J. Mol. Cell.
21: 783-788,1989.
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Norman
J., Raymond
E. Lovlin,
and Gary
D.
Effect of exogenous fatty acids on reperfusion arrhythmias in isolated working perfused hearts. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): Hl796-Hl801,1992.-Exogenous fatty acids may promote arrhythmias during ischemia and reperfusion, perhaps by increasing myocardial concentrations of long-chain acylcarnitines. We therefore studied the effects of high concentrations of fatty acids on reperfusion arrythmias and acylcarnitine accumulation in isolated working rat hearts subjected to regional ischemia and reperfusion. Hearts were perfused with buffer containing 3% albumin, 5.9 mM K+, and either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate. After 15 min of aerobic work, the left anterior descending artery was reversibly ligated for 10 min and released, and the hearts were subsequently reperfused for 3 min. Although ischemic zone acylcarnitine accumulation after reperfusion was significantly greater in glucose plus palmitate-perfused hearts (247 & 149 vs. 717 t 176 nmol/g d ry wt in glucose- vs. palmitate-perfused hearts, respectively), no significant differences in the incidence (67 vs. 44%) or duration (95 t 17 vs. 56 t 17 s) of ventricular fibrillation (VF) were seen in glucose or glucose plus palmitate hearts, respectively. Because low K+ concentration ([K’]) has been reported to increase reperfusion arrythmias in similar models, we reduced perfusate [K+] to 4.0 mM. This significantly increased the incidence and duration of VF in hearts perfused with glucose alone but had no effect in palmitate-perfused hearts. We conclude that acylcarnitine accumulation is not arrhythmogenic in this model and that fatty acids may actually have antiarrhythmic effects if exogenous [K+] is low. acylcarnitine; potassium; ventricular fibrillation HIGH CONCENTRATIONS of exogenous fatty acids have been associated with worsened prognosis in the setting of acute myocardial infarction, perhaps through an increase in arrhythmogenesis (2 1, 22). Amphipathic fatty acid intermediates, such as acylcarnitine and acylcoenzyme A, accumulate during reperfusion after noflow ischemia (3,16,18) and have been implicated in the pathogenesis of both mechanical stunning and malignant arrythmias after myocardial ischemia. These molecules produce in isolated cell systems a number of biochemical and electrophysiological .derangements that have been thought to account for these phenomena (3). However, we have recently provided evidence that acylcarnitine accumulation, although increased when concentrations of exogenous fatty acids are high, may not be important in the pathogenesis of mechanical stunning in isolated ischemic-reperfused rat hearts (18). An arrhythmogenic effect of fatty acid has been demonstrated inconsistently in vivo (14, 22, 23). These inconsistencies have been ascribed to differences in exogenous K+ concentration ([K+]) in different canine models, implying that an interaction exists between TDeceased H1796
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fatty acids and extracellular [K+] (14, 22). Recently, additional evidence has arisen to support an interaction between fatty acids and K+ channels (13). This effect is similar to that seen for the sulfonylurea glyburide (29), which was antiarrythmic properties in isolated perfused hearts (12, 30). In this study, we attempted to clarify the role of exogenous fatty acids in arrythmogenesis using the isolated working rat heart subjected to transient regional ischemia and reperfusion. Our data suggest that acylcarnitine accumulation, although increased by exogenous fatty acids, is not arrythmogenic. Our data further suggest that when exogenous [K+] is decreased, exogenous fatty acids may have antiarrythmic effects during reperfusion. METHODS Perfusion conditions. Hearts were excised from pentobarbital sodium-anesthetized Sprague-Dawley male rats (250-300 g), placed in ice-cold buffer, and cannulated initially as Langendorff hearts. As indicated in the individual experiments, a number of hearts were subsequently cannulated and perfused as working hearts as previously described (17, 18). Hearts were perfused with Krebs-Henseleit buffer (pH 7.4), gassed with 95% 02-5% COz, containing 2.5 mM free Ca2+ and either 5.9 or 4.0 mM K+. As indicated, hearts were perfused with 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate. All perfusates contained 3% bovine serum albumin previously dialyzed against 10 vol Krebs-Henseleit buffer. Palmitate, when used, was prebound to the albumin. In this study, we chose to use palmitate as a representative fatty acid, since it makes up -40% of total free fatty acids in the plasma and under conditions of physiological stress palmitate shows the greatest relative increase in plasma concentration of any fatty acid (27). Furthermore, we have had considerable previous experience using palmitate in the isolated perfused heart model. Working hearts were perfused with recirculated buffer (100 ml) at a 60-mmHg hydrostatic afterload and (in working hearts) a 11.5-mmHg left atria1 preload. Coronary flow was determined by cannulating the pulmonary artery and was measured at 5-min intervals as the sum of pulmonary flow and perfusate “drip out” from the heart as previously described (17,18). Previous workers (4) have demonstrated that coronary flow almost exclusively represents ventricular flow in this model, since the atria receive their nutrient supply through superfusion from the perfusate. Protocol and method of occlusion. All hearts underwent 15 min of aerobic perfusion during which heart rate and peak systolic pressure were recorded every 5 min. After 5 min of aerobic perfusion, coronary arterial occlusion was prepared by placing a suture (6-O silk) under the proximal left anterior descending coronary artery (LAD), and aerobic perfusion was then continued for an additional 10 min. Regional ischemia was produced by tying a short (cl.0 cm) piece of polyethylene tubing (1.0 mm diam) against the proximal LAD. After 10 min of ischemia, the suture was cut with a
0 1992 the
American
Physiological
Society
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scalpel blade and the ischemic segment was reperfused for an additional 3 min. Approximately 100 mg of myocardial tissue from the epicardial surfaces of the ischemic and nonischemic segments were then quickly frozen using small bulldog clamps cooled to the temperature of liquid nitrogen and then excised, weighed, and stored in liquid nitrogen for metabolite determinations. A number of hearts were also frozen in this manner at the end of ischemia before reperfusion. A lo-min period of ischemia was chosen because initial experiments in our fatty acid-perfused hearts showed that the incidence and duration of ventricular fibrillation (VF) was lower when the ischemic interval was shortened to 5 min or increased to 20 min. This parallels what has previously been shown in glucose-perfused Langendorff hearts (4, 5). Ischemic zone size was determined in a number of hearts by the injection of 1 ml 0.05% Amido Black dye into the preload line immediately after ischemia to demarcate the ischemic and nonischemic zones. The heart was cut from the perfusion apparatus, and after the atria and excess mediastinal tissue were excised, the ischemic zone was cut along its borders as delineated by the dye. The tissue was then dried for determination of the dry weight of the ischemic and nonischemic zones. Tissue analysis. Frozen ventricular tissue from ischemic and nonischemic zones of hearts frozen at the end of the ischemia or at the end of reperfusion was weighed and powdered in a mortar and pestle cooled to the temperature of liquid nitrogen. A portion of the powdered tissues was used in the calculation of the final concentration of metabolite determinations. Lactate and long-chain acylcarnitine were extracted from frozen tissue with perchloric acid as described previously (17, 18). Extracted longchain acylcarnitine was hydrolyzed to free carnitine and measured radiometrically (17, 18). Lactate levels were determined spectrophotometrically using standard enzymatic assays (18). Measurement of cardiac rhythm disturbances. Throughout each experiment, cardiac rhythm was recorded using a Grass physiological recorder via needle electrodes attached to the apex of the heart and the aortic cannula. VF was defined according to the criteria of Walker et al. (28). Recordings were analyzed visually to determine the incidence and duration of VF as previously described (4, 5). Statistical analysis. Statistical analysis was performed using analysis of variance followed by comparison of group means using group tests. Statistical significance was set at P < 0.05. RESULTS
Effects of LAD ligation on coronary flow and myocardial perfusion. The effect of LAD ligation on heart rate, cor-
onary flow, and (in working hearts) peak systolic pressure development and rate pressure product is presented in Table 1. In both Langendorff and working hearts, LAD ligation led to a significant reduction in coronary flow but no significant effect on heart rate. In working hearts,
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significant reductions in peak systolic pressure development and rate pressure product were also seen. No significant difference in coronary flow reduction was seen in working hearts perfused in the presence or absence of fatty acids. The effect of LAD ligation on ischemic zone size is depicted in Table 2. In Langendorff hearts, ischemic zone dry weight averaged 35.1 t 1.6% of total myocardial dry weight; no significant difference in ischemic zone size was observed between Langendorff and working glucose-perfused hearts. Effects of work on reperfusion arrhythmias. Previous studies (1, 5, 6) examining the incidence and duration of reperfusion arrhythmias have been performed in glucoseperfused Langendorff hearts. We therefore initially performed a series of perfusions in which hearts were perfused with 11 mM glucose alone (perfusate [K+] = 5.9 mM) and subjected to 15 min of Langendorff perfusion, 10 min of LAD ligation, and 3 min of reperfusion. In these hearts, the incidence and duration of reperfusion VF paralleled that observed in the previous studies (Fig. 1). These Langendorff hearts were compared with another series of glucose-perfused working hearts subjected to the same aerobic perfusion-ischemia-reperfusion protocol (Fig. 1). Compared with Langendorff hearts, working hearts displayed a significantly greater incidence (67 vs. 20%, P < 0.05) and duration (15.6 t 7.5 vs. 53.0 t 10.6% of 3-min reperfusion period for working vs. Langendorff hearts, respectively, P < 0.05) of reperfusion VF (Fig. 1, A and B). Effects of exogenous fatty acids on reperfusion arrhythmias. To determine the effects of fatty acids on
arrhythmia formation, a further series of working hearts (n = 6) were perfused with 11 mM glucose or 11 mM glucose and 1.2 mM palmitate (perfusate [K+] = 5.9 mM) and were subjected to the same ischemia-reperfusion protocol. The incidence and duration of VF in glucose plus palmitate-perfused hearts was not significantly different from that seen in hearts perfused with glucose alone (67 vs. 45% of 3-min reperfusion period in glucose vs. glucose plus palmitate hearts respectively, Fig. 2, A and B). Tissue levels of lactate and acylcarnitine in the ischemic-reperfused and nonischemic zones were determined in these hearts and are presented in Table 3. Levels of these metabolites were also measured in a series of hearts perfused under identical conditions except that they were frozen at the end of ischemia. Accumulation of acylcarnitine was seen in both the ischemic and nonischemic
t-1 Table 1. Effect of left anterior descending artery ligation on coronary JLOW and heart function parameters
in Langendorff
and working hearts
Langendorff
Coronary flow, ml/min HR, beats/min PSP, mmHg HR x PSP x 10-3, beats mmHg min-l l
l
Glucose
Working
FA Working
Aerobic
Ischemia
Aerobic
Ischemia
Aerobic
Ischemia
12.4t0.8 238t9.7
7.5kO.6” 208t6.6
17.0t1.9 221t9.4 98t3.3 22.7kO.8
13.0t1.5” 203k9.7 78.2t5.0 15.8+1.0*t
16.Ok1.4 220t9.7 99.6k2.3 21.9t0.9
lO.Ot0.9 190t10.4 83.0+3.2*t 14.0+1.3*t
x 10v3
Values are means t SE. Hearts (n = 12-19/group) were perfused as described in METHODS. FA, Fatty acid; HR, heart rate; PSP, peak systolic pressure. Because pressure measurements were not recorded in Langendorff hearts, values for PSP and HR x PSP are not given. * P < 0.05 vs. Langendorff aerobic. t P < 0.05 vs. working aerobic.
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Table 2. Ischemic zone size in hearts subjected to left anterior descending artery ligation Nonischemic Zone
Total
A
Ischemic Zone
0.062t0.003 Tissue wt, g dry wt 0.17~Joo.008 0.114t0.007 64.9k2.0 35.1t1.6 % Total tissue wt Values are means t SE. Hearts (n = 5) were perfused as Langendorff hearts as described in METHODS.
A c .-0
m-m o-o
loo-
ARRHYTHMIAS
.-uE 0
-r 13 .-G
1001
80-
60-
-:
Working Langendorff 20
10
min.
80-
-z .y x.3 .-
B loo
L+Oy--Y 0 8
E .-w
40-
30
0
Glucose
m
Fatty
Acid
80
0
.-= .-2 LL
6 .--0 -2
25
Time (minutes)
lschemia
zo-
60
55 40
00
.-6 4-J 15
20
25
30
Time (minutes)
2
20
0
B loo
1
; .-w 0 .-=
80-
_ts .LLn Q-v x
60-
El .-
40 -
0
Working
m
Langendorff
Fig. 2. Incidence (A) and duration (B) of VF in working hearts perfused with 5.9 mM K+ and either 11 mM glucose or 11 mM glucose + 1.2 mM palmitate. Preload and afterload were as described in METHODS. Duration of VF is expressed as mean & SE of percentage of 3-min reperfusion period for each group. n = 7 each of 2 groups.
* I *
decreased the incidence and duration of VF compared with that in glucose-perfused hearts (Fig. 3).
0
5 2
DISCUSSION 20-
0-
Fig. 1. Incidence (A) and duration (B) of ventricular fibrillation (VF) in glucose-perfused Langendorff and working hearts. Hearts were perfused with 5.9 mM K+and 11 mM glucose. Langendorff hearts were perfused at a 60-mmHg pressure, whereas working hearts perfused at 11.5-mmHg left atria1 preload and 60-mmHg aortic afterload as described in METHODS. Duration of VF is expressed as mean t SE of percentage of 3-min reperfusion period for each group. n = 6 in each group. * P < 0.05 vs. Langendorff hearts
zones of hearts perfused with palmitate; this increase was significantly different in glucose-perfused hearts in the ischemic zone during both ischemia and reperfusion. Significant ischemic zone accumulation of lactate was also seen during ischemia in both substrate groups in keeping with the severe ischemia produced by LAD ligation. To detect a possible interaction between fatty acids and exogenous [K+] , the ischemia-reperfusion experiments were repeated in working hearts perfused in the presence or absence of fatty acids using a perfusate [K+] of 4.0 mM. As depicted in Fig. 3, both the incidence (A) and duration (B) of reperfusion VF increased in glucoseperfused hearts compared with hearts perfused with 5.9 mM K+ (Fig. 2). In contrast, an increase in the incidence and duration of VF did not occur in fatty acid-perfused hearts (Figs. 2 and 3). As a result, in the presence of 4.0 mM K+, addition of exogenous fatty acid significantly
Exogenous fatty acids have been implicated in the production of depressed contractility and electrophysiological derangements seen during ischemia and reperfusion. The possibility that fatty acids may potentiate arrhythmias during acute myocardial ischemia and infarction was first suggested by Oliver and colleagues (21). Some investigators (9, 22) have also reported such an association, but others (23, 25) failed to find a consistent relationship between levels of circulating free fatty acids and the presence of ventricular or atria1 arrhythmias. The arrythmogenic effect of exogenous fatty acids may be related to increases in concentrations of fatty acid intermediates such as long chain acylcarnitine. Previous studies have demonstrated that acylcarnitine does accumulate during ischemia and reperfusion (3,11,18). These amphiphilic molecules may have a number of deleterious and potentially arrhythmogenic effects, including concentration-dependent alterations of Ca2+ transport in sarcoplasmic reticulum, inhibition of Ca2+-adenosinetriphosphatase (ATPase) and Na+-K+-ATPase in the sarcolemma, inhibition of ATP translocase in the mitochondrial membrane, concentration-dependent decreases in maximum diastolic potential, amplitude, maximal shortening velocity of phase 0, and action potential duration of Purkinje cells, decreased excitability, postrepolarization refractoriness, electrical alternans phenomena, and enhanced a-receptor expression (3, 7, 8, 10, 15, 24, 26). These effects are partially dependent on the type of
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Table 3. Effect of exogenous fatty acids on tissue LeveLsof lactate and acylcarnitine Perfusion Substate
Ischemic Nonischemic Glucose + fatty acid Ischemic Nonischemic Values are means & SE expressed in nmol/g dry wt. Hearts nonischemic. t P < 0.05 vs. glucose. n
-
10
min.
lschemia
\
80
Fatty
Lactate
Acylcarnitine
54t13* 25t7
227k64 183k25 700&121*‘f 433t62
34k5 36k8 87t19* 43k7
247H49 32Ok249 717k176”t 500t137
94t5* 33t5
(n = 7 in each group) were perfused as described in
Acid
* * M-D
30
25
20 Time (minutes)
B loo
f
0
Glucose
m
Fatty
Acid
.-5 80 -w -.=0 h.- 60 LL y-2-4 O5 .c, E)
40
2
20
Postreperfusion Acylcarnitine
,, 15
hearts
Lactate
m Glucose
0-O
c 0
in ischemic-reperfused
Postischemia
Myocardial Zone
Glucose
.-eu -0
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0
Fig. 3. Incident (A) and duration (B) of VF in working hearts perfused with 4.0 mM K+ and either 11 mM glucose or 11 mM + 1.2 mM palmitate (fatty acid) as described in METHODS. Duration of VF is expressed as in Figs. 1 and 2. n = 7 for each of 2 groups. * P < 0.05 vs. fatty acid-perfused hearts.
cardiac tissue being studied, the species, and pH (3). Enhancement of free radical-induced lipid peroxidative injury to sarcolemmal membranes by lipid amphiphiles has also been reported (19). Phospholipid degradation due to phospholipase activation and defective reacylation of unesterified fatty acid may be potentiated by fatty acid intermediates. This may also cause arrhythmias, enzyme inhibition, and membrane dysfunction. Recent studies in our laboratory have suggested, however, that acylcarnitine accumulation may not be important in the production of mechanical stunning in the postischemic heart (18). We demonstrated that the impairment in reperfusion recovery seen in isolated hearts perfused with high concentrations of fatty acids could be overcome through the stimulation of glucose oxidation by the carnitine acyltransferase I inhibitor, etomoxir, independent of changes in tissue levels of acylcarnitine. This suggested that postischemic accumulation of fatty acids intermediates, although enhanced by the presence of high concentrations of exogenous fatty acids, does not account for fatty acid-induced depression
METHODS.
*
P < 0.05 vs.
of function seen during reperfusion. In this study we also did not see an association between the levels of longchain acylcarnitine in the heart and the incidence of reperfusion-induced arrythmias. As shown in Table 3, addition of fatty acids resulted in a rise in long chain acylcarnitine levels in the ischemic zone from 247 to 717 pmol/g dry wt and in the nonischemic zone from 320 to 500 pmol/g dry wt. Despite this increase, a greater incidence of reperfusion-induced arrythmias did not occur in these hearts. This does not preclude the fact, however, that larger increases in long-chain acylcarnitine may have been arrythmogenic. In addition, the involvement of acylcarnitine in other types of arrythmias cannot be excluded. It does suggest, however, that the role of long-chain acylcarnitines in the genesis of arrythmias requires further study. In this study we did not attempt to correlate changes in incidence of VF after reperfusion with changes in level of acylcarnitine. However, in previous work (18) we demonstrated that long-chain acylcarnitine levels 30 min after reperfusion of hearts with high concentrations of fatty acids are much higher than immediately postischemia in isolated fatty acid-perfused hearts subjected to transient global ischemia (Table 3). This suggests that acylcarnitine levels increase progressively during reperfusion rather than after the bimodal concentration curve which might be expected from the known differences in incidence of reperfusion VF. To assess arrythmias in this study, we used a simple adaptation of the isolated regionally ischemic-reperfused Langendorff heart model of Hearse and colleagues (1, 4), which allowed a direct comparison of reperfusion arrhythmias in working hearts under physiological substrate conditions with high concentrations of exogenous fatty acids. Hearts perfused in working mode had a higher incidence and duration of reperfusion VF that Langendorff hearts; the mechanism of this effect is not clear from our data. However, high concentrations of exogenous fatty acids prevented the increased incidence and duration of reperfusion VF in glucose-perfused working hearts seen when exogenous [K+] was reduced. In this study, isolated working hearts subjected to a transient period of regional ischemia showed a higher incidence of arrythmias than Langendorff hearts (Fig. 1). The reason that this occurred is not certain, but possible mechanisms include an increase in wall stress and myocardial oxygen consumption during reperfusion, which could lead to a prolongation of ischemia at the level of the cardiac myocyte and a resultant increase in levels of lactate, H+, and Ca 2+. This phenomenon is even more interesting in light of the frequent observation by clinicians that malignant ventricular arrhythmias often become
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easier to control when concurrent congestive heart failure is adequately treated; treatment in this circumstance lowers left ventricular filling pressures, reduces wall stress, and reduces myocardial oxygen consumption. The increase in VF in the setting of low [K+] is consistent with the findings of other investigators. Using a Langendorff rat heart model, Curtis and Hearse (4) showed that the incidence and duration of VF in Langendorff rat hearts during ischemia correlated inversely with log,, [K+] . During ischemia, K+ loss from myocardial cells occurs, leading to accumulation of extracellular K+. Mechanisms proposed to explain K+ accumulation include inhibition of the Na+-K+ pump, K+ efflux coupled to intracellular ions such as lactate, modification of K+ channels, and opening of ATP-sensitive K+ channels. Regardless of the mechanism by which low [K+] increases arrhythmogenesis, our data demonstrate that fatty acid actually decreased arrhythmia formation in hearts perfused with low [K+]. It is not clear why palmitate had an antiarrythmic effect at lower [K+]. One possible explanation for this effect is the potential effect of fatty acids on the ATPsensitive K+ channel. Kim and Duff (13) have reported that the ATP-sensitive K+ channel, which mediates K+ efflux during ischemia when ATP concentrations fall to very low levels, is inhibited by unesterified free fatty acids such as arachidonic acid. Kantor and colleagues (12) reported that the inhibition of this channel by the sulfonylurea glyburide reduced reperfusion arrhythmias in glucose-perfused nonworking hearts subjected to global lowflow ischemia. Wilde et al. (29) used glyburide in patchclamp investigations of isolated guinea pig cardiomyocyte membranes. These studies showed that this drug slowed the rate of extracellular K+ accumulation during ischemia. In our study, lowering perfusate [K+] would be expected to increase the transmembrane K+ concentration gradient, producing increased K+ efflux during ischemia. Wolleben et al. (30) have shown that stimulation of the ATP-dependent K+ channel contributes to the development of fibrillation and that glyburide, a blocker of the ATP-dependent K+ channel, has an antiarrhythmic effect. In guinea pig myocytes, Naykaya et al. (20) demonstrated that glyburide inhibits action potential shortening, which would be expected to have antiarrythmic effects. In humans, Cacciapuoti et al. (2) showed that glyburide reduces the frequency of premature complexes and tachycardias during transient myocardial ischemia. Whether palmitate at lower [K+] has an antiarrhythmic effect similar to that of glyburide has not been determined. It is therefore possible that exogenous fatty acid may have exerted an antiarrhythmic effect through an inhibitory effect on the ATP-sensitive K+ channel. It has yet to be determined how glyburide interacts with fatty acid on the ATP-dependent K+ channel. In summary, our results indicate that long-chain acylcarnitine accumulation during reperfusion after transient regional ischemia does not promote reperfusion arrhythmias in working hearts subjected to reversible LAD occlusion. Furthermore, our data are consistent with fatty acid-mediated inhibition of K+ efflux during ischemia. Studies to verify the mechanism of arrhythmogenesis in
ARRHYTHMIAS
this model and to determine the relevance of these finding to clinically observed arrhythmias are indicated. This study was supported by a grant from the Medical Research Council of Canada. G. D. Lopaschuk is a scholar of the Alberta Heritage Foundation for Medical Research. Address for reprint requests: G. D. Lopaschuk, Dept. of Pediatrics and Pharmacology, University of Alberta, 423 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada. Received 30 January 1991; accepted in final form 8 January 1992. REFERENCES M., D. J. Hearse, and A. S. Manning. Reperfusioninduced arrhythmias and oxygen-derived free radicals: studies with “anti-free radical” interventions and a free radical-generating system in the isolated perfused rat heart. Circ. Res. 58: 331-340,
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Effectiveness of glibenclamide on myocardial ischemic ventricular arrythmias in non-insulin-dependent diabetes mellitus. Am. J. Curdiol. 67: 843-847, 1991. 3. Corr, P. B., R. W. Gross, and B. E. Sobel. Amphipathic metabolites and membrane dysfunction in the ischemic myocardium. Circ. Res. 55: 135-154, 1984. 4. Curtis, M. J., and D. J. Hearse. Ischemia-induced and reperfusion-induced arrhythmias differ in their sensitivity to potassium: implications for mechanisms of initiation and maintenance of ventricular fibrillation. J. MOL. Cell. CardioZ. 21: 21-40, 1989. 5. Curtis, M. J., and D. J. Hearse. Reperfusion-induced arrhythmias are critically dependent upon occluded zone size: relevance to the mechanism of arrhythmogenesis. J. Mol. Cell. Cardiol. 21: and
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increase in alphal-adrenergic Circ. Res. 61: 735-746, 1987. 11. Idell-Wenger,
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Coenzyme A and carnitine distribution in normal and ischemic hearts. J. Biol. Chem. 253: 4310-4318, 1978. 12. Kantor, P. F., W. A. Coetzee, and L. H. Opie. Reduction of 13. 14.
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