Arrhythmogenic Action of Thrombin During Myocardial Reperfusion via Release of Inositol 1,4,5-Triphosphate
Background Cardiac reperfusion initiates release of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and arrhythmogenesis via norepinephrine stimulation of α1-adrenergic receptors. The present study examines arrhythmogenic effects of thrombin-stimulated Ins(1,4,5)P3 release under these conditions.
Methods and Results [3H]Ins(1,4,5)P3 release was measured in [3H]inositol-labeled rat hearts by high-performance liquid chromatography. Arrhythmia studies were performed in buffer-perfused rat hearts. Two-minute reperfusion after 20 minutes of global ischemia increased [3H]Ins(1,4,5)P3 from 1123±77 to 2238±44 cpm/mg tissue. No increase was observed in catecholamine-depleted hearts (755±89 cpm/mg). The addition of thrombin (5 IU/mL) or thrombin receptor agonist peptide (TRAP1-6, 50 μmol/L) restored the reperfusion Ins(1,4,5)P3 response (thrombin, 1518±68 cpm/mg and TRAP1-6, 1755±128 cpm/mg). Ins(1,4,5)P3 release initiated by norepinephrine or thrombin was inhibited by gentamicin (150 μmol/L; 986±52 and 868±125 cpm/mg, respectively). The thrombin response was inhibited by the phospholipase C inhibitor U-73122 (5 μmol/L; 394±59 cpm/mg) but not by its inactive isomer U-73343. The norepinephrine response was not inhibited by U-73122 (2126±74 cpm/mg). Ventricular tachycardia and ventricular fibrillation were observed in intact hearts but not in hearts from catecholamine-depleted rats (ventricular fibrillation duration, 110±19 versus 0±0 seconds). The addition of thrombin or TRAP1-6 increased arrhythmias in catecholamine-depleted hearts (112±32 and 89±28 seconds, respectively). Gentamicin and U-73122 but not U-73343 prevented thrombin-induced arrhythmias. Gentamicin inhibited norepinephrine-initiated arrhythmias, but U-73122 was ineffective.
Conclusions This study demonstrates that the development of reperfusion arrhythmias under these conditions depends on the release of Ins(1,4,5)P3.
Reperfusion after regional myocardial ischemia in isolated, perfused rat hearts causes release of Ins(1,4,5)P31 and development of arrhythmias.2 Both responses require the release of norepinephrine and activation of α1-adrenergic receptors. The Ins(1,4,5)P3 response to norepinephrine stimulation under reperfusion conditions was quantitatively greater than the response to norepinephrine observed during normoxia.1 Furthermore, a number of agents that bind PtdIns(4,5)P2 to inhibit the release of Ins(1,4,5)P33 4 have been shown also to inhibit reperfusion arrhythmias.1 The close correlation between Ins(1,4,5)P3 release and the incidence of reperfusion arrhythmias suggested the possibility that Ins(1,4,5)P3 release initiates reperfusion arrhythmias in this model.
Thrombin, in addition to its role in coagulation, exerts a range of stimulatory effects in many cell types5 6 (including cardiomyocytes7 ) by binding specific cell surface receptors coupled via G proteins to pathways such as inositol phosphate release. Cardiac thrombin receptors activate a PLC, which is sensitive to inhibition by the PLC inhibitor U-73122.8 In contrast, the norepinephrine-stimulated release of Ins(1,4,5)P3 is insensitive to U-73122.8 This difference in the specificities of Ins(1,4,5)P3 release initiated by norepinephrine and thrombin provides a means to establish the critical role of Ins(1,4,5)P3 in the origin of reperfusion arrhythmias in this model.
Adult male Sprague-Dawley rats (weight, 250 to 350 g) were used. All experiments were approved by the Alfred Hospital and Baker Institute Animal Ethics Committee.
Inositol Phosphate Studies
Where indicated, rats were treated with reserpine (5 mg/kg IP, 18 hours before the experiment) to deplete endogenous norepinephrine. All rats were given heparin (1 IU/g IP) 30 minutes before decapitation. The hearts were excised, placed in ice-cold normal saline, and cannulated via the ascending aorta to initiate Langendorff perfusion with HEPES-buffered Krebs-Henseleit solution (37°C) equilibrated with 5% CO2/95% O2 at 5 mL/min. After a 15-minute equilibration period, hearts were labeled with myo-[3H]inositol (2 μCi/mL) for 2 hours. Labeled medium was then removed and replaced with medium containing propranolol (1 μmol/L) and lithium chloride (10 mmol/L) to block β-adrenergic receptors and to inhibit inositol phosphate metabolism, respectively.9 Gentamicin, U-73122, and U-73343 were also added at this point. Normothermic global ischemia was initiated by cessation of perfusion for 20 minutes, and reperfusion was initiated by reinitiating flow at 5 mL/min. Inositol phosphate accumulation was terminated by freezing the hearts in liquid nitrogen. The frozen ventricles were weighed, and inositol phosphates were extracted by use of trichloroacetic acid and quantitated by anion exchange high-performance liquid chromatography exactly as described previously.1 10
Rats were anesthetized with pentobarbital (60 mg/kg IP) and given heparin (200 IU IV). Hearts were cannulated in situ via the ascending aorta and perfused at 5 mL/min with Krebs-Henseleit medium constantly gassed with 95% O2/5% CO2 at 37°C. A 10-minute period was allowed to stabilize the preparation before the experiment, then the left main coronary artery was ligated to produce regional ischemia. After 20 minutes, the ligature was released to initiate reperfusion. Effective coronary artery ligation was confirmed by an increase in coronary perfusion pressure. The perfusion flow rate was adjusted coincidentally with coronary occlusion and reperfusion to maintain a constant perfusion pressure. During the 20 minutes of ischemia and 5 minutes of reperfusion, the epicardial ECG was monitored. Ventricular arrhythmias that occurred during monitoring were quantified according to the Lambeth convention guidelines.11 The perfusate included propranolol (1 μmol/L) to block β-adrenergic receptors and lithium chloride (10 mmol/L) to replicate the conditions used in studies of inositol phosphate release. We have previously demonstrated that the incidence of ischemic VT and VF was significantly reduced by propranolol to 40% and 30%, respectively, compared with 80% and 75% in the control group (both P<.05).2 Ischemic ventricular arrhythmias were unaffected by lithium chloride. The incidence of reperfusion arrhythmias was not changed by propranolol or lithium, alone or in combination.
Reserpine, propranolol, lithium chloride, and thrombin were obtained from Sigma. TRAP1-6 was obtained from Peninsula Laboratories. U-73122 (1-6((17β-3-methoxyestra-1,3,5-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) and U-73343 [(1-6((17β-3-methoxyestra-1,3,5-trien-17-yl)amino)hexyl)-1H-pyrrolidine-2,5-dione] were obtained from Sapphire Bioscience. Gentamicin was obtained from Delta West, and myo-[3H]inositol was from Amersham.
Group differences for nonparametric data (incidence of VT and VF) were examined by Kruskal-Wallis one-way ANOVA, followed by the Mann-Whitney test if significant differences between the groups were detected. Comparison was made with the respective control data. Parametric data (arrhythmia duration and Ins(1,4,5)P3 response) were examined by ANOVA followed by Student’s t tests if significant group differences were found. A value of P<.05 was considered significant.
Inositol Phosphate Studies
Isolated perfused rat hearts were labeled with [3H]inositol and subjected to 20 minutes of global ischemia or 20 minutes of ischemia followed by 2 minutes of reperfusion. Ischemia for 20 minutes did not result in release of inositol phosphates, and there was no observable effect of catecholamine depletion either on [3H]Ins(1,4,5)P3 content (948±134 cpm/mg for reserpine-treated hearts compared with 1123±76 cpm/mg for hearts not treated with reserpine; mean±SEM, n=5) or on total inositol phosphates (10 167±2596 cpm/mg compared with 6040±2161 cpm/mg). Reperfusion of intact hearts for 2 minutes caused a release of Ins(1,4,5)P3, but no such release was observed after treatment with reserpine (Figure⇓).
Experiments were performed to examine whether thrombin could replace norepinephrine as the activator of Ins(1,4,5)P3 release under reperfusion conditions. [3H]Inositol-labeled norepinephrine-depleted hearts were subjected to 20 minutes of ischemia followed by 2 minutes of reperfusion in the presence of thrombin (5 IU/mL). As is shown in the Figure⇑, thrombin caused significant increases in [3H]Ins(1,4,5)P3 over the 2-minute reperfusion period. In contrast, thrombin stimulation of normoxic hearts for 2 minutes did not cause detectable increases in [3H]Ins(1,4,5)P3 (from 1214±251 to 964±100 cpm/mg, mean±SEM, n=5) or in total inositol phosphates (from 10 191±2184 to 6535±1316 cpm/mg), although increases could be detected at later time points. Thus, thrombin-stimulated release of Ins(1,4,5)P3 is enhanced under reperfusion conditions, as described previously for norepinephrine. Similar experiments were performed with TRAP1-6 (SFLLRN). TRAP1-6 (50 μmol/L) caused a release of Ins(1,4,5)P3 under reperfusion conditions similar to that observed with thrombin (Figure⇑).
The effects of the PLC inhibitor U-73122 on inositol phosphate release were tested under reperfusion conditions in the presence of norepinephrine (intact hearts) or thrombin (norepinephrine-depleted hearts). U-73122 (5 μmol/L) was added to the perfusate before ischemia and was maintained throughout the ischemia/reperfusion protocol. As shown in the Figure⇑, U-73122 inhibited the Ins(1,4,5)P3 response to thrombin but had no effect on the response mediated by norepinephrine. Similar experiments were performed with U-73343, an inactive isomer of U-73122. U-73343 (5 μmol/L) did not inhibit the thrombin-stimulated Ins(1,4,5)P3 response during reperfusion (Figure⇑).
Aminoglycosides inhibit release of Ins(1,4,5)P3 by binding to its precursor PtdIns(4,5)P2. Effects of gentamicin on the reperfusion Ins(1,4,5)P3 responses initiated by thrombin and norepinephrine were investigated. Gentamicin was added to the perfusate before ischemia and was maintained throughout the experiment. Gentamicin (150 μmol/L) inhibited Ins(1,4,5)P3 responses both to endogenous norepinephrine and to exogenous thrombin (5 IU/mL) (Figure⇑).
Reperfusion arrhythmias were measured over a 5-minute period after 20 minutes of regional ischemia in perfused rat hearts. There was no significant difference in the incidence of reperfusion arrhythmias between control groups from different experiments in the case of non–catecholamine-depleted rats, and therefore combined data from the non–catecholamine-depleted control rats were pooled together in one group. Depletion of catecholamines caused a reduction in reperfusion-induced VT and VF from 100% in the control group to 0% in animals treated with reserpine. However, the addition of thrombin or TRAP1-6 to catecholamine-depleted hearts restored the arrhythmogenic responses (Figure⇑ and Table⇓).
Gentamicin (150 μmol/L), which was added to the perfusate throughout the experiment, reduced VT and VF in both intact (catecholamine-replete) and thrombin-stimulated (catecholamine-depleted) hearts (Figure⇑ and Table⇑). Addition of U-73122 (5 μmol/L) to the perfusate inhibited the arrhythmogenic response initiated by thrombin but not that initiated by norepinephrine, in parallel with findings in inositol phosphate studies (Figure⇑ and Table⇑). To confirm that the antiarrhythmic actions of U-73122 were specific to its activity as a PLC inhibitor, experiments were performed with its inactive isomer U-73343. U-73343 (5 μmol/L) was ineffective in inhibiting thrombin-induced arrhythmias.
Previous studies in our laboratory1 have shown that reperfusion after acute ischemia causes a rapid, transient release of Ins(1,4,5)P3 (mass and [3H]-labeled) dependent on local release of norepinephrine and mediated by α1-adrenergic receptors. Subsequent studies2 demonstrated a close correlation between Ins(1,4,5)P3 release and the incidence of reperfusion arrhythmias. However, more specific evidence for an association between Ins(1,4,5)P3 and reperfusion arrhythmias was required to establish a pivotal role for Ins(1,4,5)P3 release.
We and others7 12 have previously demonstrated that thrombin can directly activate inositol phosphate release in cardiac tissue, and thrombin is known to be directly proarrhythmic. The current study shows that, like norepinephrine, thrombin caused both release of Ins(1,4,5)P3 and arrhythmias during early reperfusion. Like the norepinephrine response, thrombin-induced Ins(1,4,5)P3 release under reperfusion conditions was greater than that observed in normoxic tissue. The TRAP1-6 peptide was similarly effective, both in studies of inositol phosphate release and arrhythmogenesis, demonstrating that both actions are mediated by thrombin receptors.13 Thus, thrombin receptors appear to function similarly to α1-adrenergic receptors under these conditions. However, the inositol phosphate response to thrombin differs from the norepinephrine response in its sensitivity to the PLC inhibitor U-73122. Inhibition of inositol phosphate release was observed under both normoxic conditions8 and conditions of postischemic reperfusion. Furthermore, U-73122 prevented development of thrombin-induced arrhythmias under reperfusion conditions. A closely related compound, U-73343, was ineffective in inhibiting either inositol phosphate release or arrhythmogenesis. This compound differs chemically from U-73122 only in one double bond, and the only functional difference lies in their efficacy for inhibition of PtdIns-specific PLC.14 Thus, the effectiveness of U-73122 but not of U-73343 in preventing thrombin-induced arrhythmias indicates involvement of a PLC enzyme.
In contrast to thrombin-induced responses, U-73122 did not inhibit norepinephrine-stimulated inositol phosphate release in intact heart tissue under normoxic or reperfusion conditions, demonstrating that the two receptor classes involved couple to different PLC enzymes. In parallel, U-73122 did not prevent reperfusion arrhythmias initiated by norepinephrine. The other inhibitor of PtdIns-PLC that we investigated, gentamicin, inhibits Ins(1,4,5)P3 release by binding to its precursor PtdIns(4,5)P23 and thus does not differentiate between different PLC enzymes. Gentamicin inhibited both the Ins(1,4,5)P3 response and the ventricular arrhythmias caused by activation of either α1-adrenergic receptors or thrombin receptors. The specificity of the antiarrhythmic effect of U-73122 for thrombin-induced arrhythmias, together with the ineffectiveness of U-73343, precludes the possibility of an indirect effect of this agent. Thus, the data demonstrate that release of Ins(1,4,5)P3 is an essential component of the cascade that causes ventricular arrhythmias under reperfusion conditions.
A direct proarrhythmic effect of thrombin has been reported previously,7 15 and this has been associated with inositol phosphate release7 and with activation of Na+/H+ exchange and release of lysophosphatidylcholine.15 Such increases in Na+/H+ exchange could be secondary to protein kinase C activation1 16 initiated by release of sn-1,2-diacylglycerol in parallel with release of inositol phosphates.17 However, in previous studies,2 we have shown that inhibition of protein kinase C did not prevent arrhythmias in our model, indicating that it was the inositol phosphates rather than the sn-1,2-diacylglycerol that were proarrhythmic. It is likely that a number of events are required for the generation of arrhythmias in our reperfusion model, including increased cytosolic pH and Ca2+ overload1 in addition to release of Ins(1,4,5)P3. Ins(1,4,5)P3 has been reported to enhance Ca2+ oscillations in the heart,18 19 and this is a likely mechanism for its proarrhythmic action, especially in the presence of Ca2+ overload.
In conclusion, the present study demonstrates that reperfusion arrhythmias can be initiated either by thrombin or by norepinephrine, and studies with specific inhibitors of PtdIns-PLC enzymes indicate that the development of these arrhythmias requires the release of Ins(1,4,5)P3. Thus, inhibition of release of Ins(1,4,5)P3 provides a potential target for the development of antiarrhythmic drugs.
Selected Abbreviations and Acronyms
|TRAP1-6||=||thrombin receptor agonist peptide|
This work was supported by the Australian National Health and Medical Research Council and by grants-in-aid from the National Heart Foundation of Australia and the Alfred Health Care Group. Dr Jacobsen is the recipient of a medical postgraduate scholarship from the National Heart Foundation.
- Received August 3, 1995.
- Revision received September 25, 1995.
- Accepted October 19, 1995.
- Copyright © 1996 by American Heart Association
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