- Research Article
- Open Access
Thrombin and Its Receptor Enhance ST-Segment Elevation in Acute Myocardial Infarction by Activating the KATP Channel
- Ming Long†1,
- Lei Yang†1,
- Genya Huang1,
- Liping Liu2,
- Yugang Dong1,
- Zhimin Du1,
- Anli Tang1,
- Chenghen Hu1,
- Ruimin Gu2,
- Xiuren Gao1 and
- Lilong Tang1Email author
© The Feinstein Institute for Medical Research 2010
- Received: 28 January 2010
- Accepted: 8 April 2010
- Published: 9 April 2010
ST-segment elevation is the major clinical criterion for committing patients with chest pain to have emergent coronary revascularizations; however, the mechanism responsible for ST-segment elevation is unknown. In a guinea pig model of ST-segment elevation acute myocardial infarction (AMI), local application of hirudin, a thrombin antagonist, significantly decreased AMI-induced ST-segment elevation in a dose-dependent manner. Hirudin-induced (5 antithrombin units [ATU]) decrease in ST elevation was reversed by 250 nmol/L thrombin receptor activator peptide (TRAP). TRAP (250 nmol/L [100 µL]) significantly induced ST-segment elevation in hearts without AMI. The TRAP effect was blocked by 4 mg/kg glibenclamide and 4 mg/kg HMR1098 and partially blocked by 3 mg/kg 5HD. Pinacidil (0.45 mg/kg) simulated the effect of TRAP (250 nmol/L [100 µL]) on hearts without AMI. Moreover, single-channel recordings showed that TRAP induced ATP-sensitive K+ channel (KATP channel) activity, and this effect was blocked by HMR1098 but not 5HD. Finally, TRAP significantly shortened the monophasic action potential (MAP) at 90% repolarization (MAP90) and epicardial MAP (EpiMAP) duration. These effects of TRAP were completely reversed by HMR1098 and partially reversed by 5HD. Thrombin and its receptor activation enhanced ST-segment elevation in an AMI model by activating the sarcolemmal KATP channel.
ST-segment elevation is a typical hallmark to diagnose AMI and is the major clinical criterion for committing patients with chest pain to emergent coronary revascularizations. In spite of this knowledge, the mechanism of ST-segment elevation remains unclear. For decades, two mechanistic theories, the “injury current” theory and the “transmural potential gradient” theory, have aimed to explain ST-segment elevation. However, neither theory can explain how the transmural voltage gradient that leads to ST-segment elevation is generated during ventricular repolarization or explain its ionic basis. Evidence indicates that the potassium channel may be critical for this process. For example, potassium current activation is sufficient to cause ST elevation during acute ischemia (1). Activation of the ATP-sensitive K+ channel (KATP channel) owing to hypoxia is generally hypothesized to result in ST-segment elevation in AMI (2). Additionally, Kir6.2 was shown to be responsible for ST-segment elevation in Kir6.2-knockout mice (3).
Thrombin, a serine protease, is generated at sites of vascular injury and is a key molecule in the pathogenesis of AMI because it is involved in thrombus formation. The generation of thrombin during AMI is a long-lasting process, which extends beyond the acute phase of myocardial infarction (4). Thrombin binds to its receptor and cleaves it in the NH2-terminal portion. The exposed new NH2-terminus has been proposed to function as a “tethered peptide ligand” binding to this receptor in hirudinlike regions to cause receptor activation. Hirudin could combine the hirudinlike region of the receptor to compete with tethered peptide ligand binding to this region. Thrombin receptor activating peptide (TRAP), the synthetic peptide of 5-14 amino acids, correspond to the tethered ligand sequence. TRAPs have been found to be agonists for receptor activation (5,6). Thrombin and the thrombin receptor have been shown to play important roles in diverse biological processes, such as platelet aggregation, inflammation and cell proliferation, in a wide variety of tissues (7). Because ST-segment elevation is the major criterion for thrombolytic therapy, we aimed to examine the role of thrombin and its receptor activation in ST-segment elevation during AMI.
All experimental protocols complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the regulations of the Animal Care and Use Committees at Sun Yat-sen University. Albino male guinea pigs (Experimental Animal Center, Guangdong, China) weighing 350–400 g were used in our experiments. The animal AMI model was created as described (8). Briefly, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (40–50 mg/kg). The guinea pigs were then restrained in a supine position, and a tracheotomy was performed. The animals were ventilated with oxygen-enriched room air (90 breaths/min, 5 mL/kg). Electrocardiogram (ECG) limb electrodes were positioned to record in the standard lead II configuration, and each needle electrode was placed under the skin. A left thoracotomy was performed, and then the proximal left coronary artery (LCA) between the pulmonary artery outflow tract and the left atrium was occluded with a silk suture. Myocardial ischemia was confirmed by the presence of regional cyanosis and ST-segment elevation in the ECG and was further confirmed by Evans blue perfusion after every experiment. Only the data from animals with infarct indices between 15% and 25% were analyzed. In the sham control animals, a silk suture was placed under the LCA without ligation. The ECG was recorded at least 5min after opening the chest for acclimatization and immediately after the LCA occlusion to allow continuous monitoring of changes in the ST segment during ischemia. In some experiments, reagents with restricted volumes of 100 µL were injected into the midmyocardial wall of infarcted or uninfarcted left ventricles. ST-segment elevation in animal models can be variable. For instance, ST-segment elevation can be caused by a vector change that results from ischemia, a mechanical alteration of the heart geometry or other factors. Therefore, we analyzed only stable ST segment changes recorded after surgery.
Ventricular Myocyte Preparation and Patch-Clamp Experiments
Action Potential Recordings in Langendorff-Perfused Hearts
Isolated guinea pig hearts were perfused as described (10). Briefly, guinea pigs were anesthetized with sodium pentobarbital (40–50 mg/kg) and given 200 IU heparin. The heart was quickly excised and submerged in ice-cold Krebs-Henseleit solution and equilibrated with 95% O2/5% CO2 (pH 7.4, 37°C). For endocardial recordings, a small access window was created in the interventricular septum to gain access to the left ventricular endocardium. Each heart was perfused retrogradely using the Langendorff method and was provided a constant flow rate of 15 mL/min modified Krebs-Henseleit solution before the right ventricular free wall was partly removed and a small access window in the septum was created. Thereafter, the perfused heart was stabilized and monophasic action potentials (MAPs) were recorded.
The MAP recording protocols used were described by Killeen et al. (11). Briefly, two custom-made MAP electrodes were constructed from two spring-shaped, twisted strands of Teflon-coated silver wire (1-mm diameter, 99.99% purity). The electrodes were positioned onto the left ventricular free wall under a stable contact pressure until MAP signals were reproducibly recorded on the BL-420E+ Biological Function Analysis System (Taimeng, Chengdu, China). MAPs were amplified, band-pass filtered and digitized before they were extracted and analyzed. Recordings were considered reproducible if they had a stable baseline, a rapid upstroke phase with a consistent amplitude, a smooth contoured repolarization phase and a stable duration.
MAPs were simultaneously recorded from epicardial (EpiMAP) and endocardial (EndoMAP) sites in the center of left ventricular free walls (10). A transmural ECG was recorded with two silver chloride electrodes (2-mm internal diameter with fishhook-shaped tip) placed at the epicardial (+) and endocardial (−) surfaces of the left ventricular free wall along approximately the same axis as the transmural recordings. The duration of the MAP was measured at 90% repolarization (MAP90).
All values are presented as means ± SEM. Pearson correlation analysis was used to evaluate the relationship between variables. Differences were evaluated using analysis of variance followed by the Student t test (paired or unpaired). Differences were considered to be statistically significant at P < 0.05.
All supplementary materials are available online at www.molmed.org .
Animal Model for ST-Segment Elevation
Studies were conducted on an animal model of ST-segment elevation after LCA ligation. As shown in Figure 1A, the nonperfused zones were clearly identified. ST-segment elevation following AMI gradually returned to baseline in a time-dependent manner. ST-segment elevation had no relationship to the infarct index (Figure 1B). In addition, thrombin activity in the infarcted left ventricular wall significantly increased 5 min after LCA ligation in the model (Figure, online Data Supplement).
Effect of Hirudin and TRAP on the ST Segment after AMI
To further examine the effect of the thrombin on the ST segment after AMI, the thrombin receptor was activated with a local injection of exogenous TRAP (250 nmol/L [100 µL]) into the infarcted left ventricular as shown in Figure 3A. Interestingly, as shown in Figure 3B, C, TRAP (250 nmol/L [100 µL]) can significantly reverse the effect of 5 ATU hirudin, which decreased ST/QRS+. The infarct index in the 5 ATU hirudin group (18.6% ± 2.33%) and 5 ATU hirudin + TRAP group (19.5% ± 2.33%) showed no significant differences.
Effect of the KATP Channel on TRAP-Induced ST-Segment Elevation
Effect of TRAP on KATP Channels
ST-Segment Elevation in ECG, EpiMAP and EndoMAP in Left Ventricle Walls
EpiMAP90 and EndoMAP90 duration.
A. After TRAP and vehicle (TFA) perfusion to the left ventricular walls
10 min after perfusion
10 min after wash
153 ± 4
154 ± 2
135 ± 3a#
156 ± 4
154 ± 2
159 ± 5
151 ± 2
152 ± 3
159 ± 2
156 ± 5
154 ± 4
153 ± 5
B. Before and after perfusion with HMR1098 + TRAP or 5-HD + TRAP
Perfusion of K-H solution
149 ± 2
152 ± 3
150 ± 2
151 ± 3
10 min after HMR1098/5HD + K-H solution perfusion
162 ± 3
164 ± 5
171 ± 4
173 ± 4
10 min after TRAP + HMR1098/5HD perfusion
152 ± 3a,c
168 ± 3
165 ± 5
170 ± 3
5 min after washed by HMR1098/5HD
+ K-H solution perfusion
162 ± 4
165 ± 5
168 ± 2
171 ± 3
10 min after washed by K-H solution
151 ± 5
150 ± 1
156 ± 3
156 ± 4
Our studies demonstrated that eliminating endogenous thrombin with hirudin significantly decreased ST-segment elevation, which was induced by AMI in our guinea pig model. Some previous studies demonstrated that tying a single vessel off for a few days to weeks did not cause myocardial infarction in guinea pig hearts (14,15). However, other researchers showed that LCA ligation in guinea pigs resulted in infarction within 3 days of surgery (16–18). Our data indicate that LCA ligation resulted in a nonperfused zone within 1 hour in nearly all the animals. Possible reasons for the differences among these studies include the following: (a) Ligation of LCA does not ligate only a single vessel. The area between pulmonary artery outflow tract and left atrium was ligated, which may include the left descending artery and left circumflex artery. (b) Establishing collateral vessels takes time. Collateral vessels usually do not open in a normal situation, but open only when the main artery is narrowed or blocked, and this process is very slow. We can see such phenomena very commonly in our clinical activities. Slowly developing, high-grade coronary artery stenoses may progress to complete occlusion without precipitating an AMI because of the development of a rich collateral network over time. However, if plaque fissuring and rupturing occur during the natural evolution of lipidladen atherosclerotic plaques, this can result in thrombus formation and lead to AMI (19).
We observed no relationship between ST-segment elevation and the infarct index in our study. Previously, infarct size was reported to have a low correlation with ST-segment elevation (20). We used the ratio of ST-segment elevation to positive QRS amplitude rather than absolute ST-segment elevation in our study because absolute ST-segment elevation is dramatically variable in animals with different weights.
Our study showed that thrombin receptor activation plays a very important role in ST-segment elevation in our AMI model. TRAP perfusion was shown to induce ST-segment elevation in uninfarcted hearts (7), but this effect was attributed to TRAP-induced coronary artery constriction and heart dysfunction (21). To exclude the disturbances caused by ATP depletion and TRAP-induced vessel constriction, we locally injected TRAP in left ventricles without AMI, and we observed that TRAP directly affected ST-segment elevation by sarcKATP channels, although the duration of TRAP-induced ST-segment elevation is much shorter than that of AMI-induced ST-segment elevation and the concentration of TRAP is also much higher than the upper limitation of physiological thrombin concentration. We also applied pinacidil, a KATP channel opener that relaxes coronary arteries (22), and observed that the effect of TRAP was nearly replicated by pinacidil. Therefore, TRAP-induced ST-segment elevation was not related to coronary artery constriction. Furthermore, our singlechannel recordings indicated that TRAP increased KATP channel activity, and this effect could be blocked by HMR1098 but not by the mitochondrial KATP channel blocker 5HD (10 µM), as reported in other cell types (23). 5HD is usually considered a mitochondrial KATP channel blocker. Notsu et al. reported that 5HD at concentrations >1 µM had an effect similar to that of glibenclamide (24). Therefore, sarcKATP channels play an important role in TRAP-induced ST-segment elevation. The mechanism of increased KATP channel activity by TRAP is unclear. TRAP not only stimulates accumulation of IP2 and IP3 in cardiac myocytes but also modulates cardiac contractile function by the protein kinase C (PKC) pathway (24). Increased PKC in cardiac cells enhances KATP activity (25).
Our work clearly demonstrates that TRAP induced MAP duration change by activating sarcKATP channels, which resulted in ST-segment elevation. MAP recorded in cardiac ventricular walls in our experiment is different from transmembrane action potential recorded in single cells. MAP is measured from a group of cells with extracellular electrodes that have a diameter of 1 to 2 mm (26). Therefore, a number of dissimilarities exist between the MAP and transmembrane action potential from single cells. For instance, MAP has less diastolic and action potential amplitude than transmembrane action potential from single cells. The maximum upstroke velocity of MAP is much smaller than that measured in a single cell (26). A notch, the remnant of the “intrinsic deflection,” may appear in the MAP plateau phase, mimicking “phase I repolarization.” This should not represent predominant KIto (a transient outward current) channel activity of epicardial myocardium (27). TRAP induced different electrophysiological responses, including a shortened MAP duration and EpiMAP duration but not EndoMAP duration, by activating sarcKATP channels and causing ST-segment elevation. This indicates that the KATP channels in the endocardial and epicardial cells are different, as suggested by previous studies (28–30). Studies have reported that ST-segment elevation was always accompanied by the shortening of action potential after AMI (31,32). There is a general consensus, though not universal, that IKATP is the major current responsible for MAP duration shortening during acute ischemia (33). In the ECG, a ST-segment elevation is related to shortened EpiMAP duration but not EndoMAP duration (34).
Thrombin can be produced only during thrombus formation during AMI. Therefore, thrombin-induced ST-segment elevation during AMI indicates fresh thrombus forming in the coronary arteries. In addition, thrombin is released during revascularization treatments, and antithrombin therapy with bivalirudin has begun to be used as an important adjuvant therapy during revascularization treatments (35). Decrease in ST-segment elevation following coronary artery recanalization is a major clinical criterion used to judge the effect of revascularization treatments. Therefore, our work indicates that decreases in ST-segment elevation following revascularization treatments with strong antithrombin therapies should be carefully evaluated for their effectiveness.
In conclusion, our study demonstrates that thrombin and its receptor activation significantly enhances ST-segment elevation during AMI. The activation of the thrombin receptor opens sarcKATP channels, which shortens the MAP duration and EpiMAP duration.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank Dai Gang for his technical assistance with the animal model and MAP recordings. This work was supported by National Natural Science Foundation of China (no. 30770897/C03030201 for L Tang).
- Litovsky SH, Antzelevitch C. (1989) Rate dependence of action potential duration and refractoriness in canine ventricular endocardium differs from that of epicardium: the role of the transient outward current. J. Am. Coll. Cardiol. 14:1053.View ArticleGoogle Scholar
- Kubota I, et al. (1993) Role of ATP-sensitive K+ channel on ECG ST segment elevation during a bout of myocardial ischemia: a study on epicardial mapping in dogs. Circulation 88:1845–1851.View ArticleGoogle Scholar
- Li RA, Leppo M, Miki T, Seino S, Marbán E. (2000) Molecular basis of electrocardiography ST segment elevation. Circ. Res. 87:837–839.View ArticleGoogle Scholar
- Szczeklik A, Dropinski J, Radwan J, Krzanowski M. (1992) Persistent generation of thrombin after acute myocardial infarction. Arterioscler. Thromb. Vasc. Biol. 12:548–553.View ArticleGoogle Scholar
- Vu TKH, Hung DT, Wheaton VI, Coughlin SR. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991;64:1057–68.View ArticleGoogle Scholar
- Nanevicz T, et al. (1995) Mechanism of thrombin receptor agonist specificity: chimeric receptors and complementary mutations identify an Nanagonist recognition site. J. Biol. Chem. 270:21619–25.View ArticleGoogle Scholar
- Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, Plevin R. (2001) Proteinase-activated receptors. Pharmacol. Rev. 53:245–282.PubMedGoogle Scholar
- Tang L, et al. (2008) Thrombin receptor and ventricular arrhythmias after acute myocardial infarction. Mol. Med. 14:131–140.View ArticleGoogle Scholar
- Redel A, et al. (2008) Impact of ischemia and reperfusion times on myocardial infarct size in mice in vivo. Exp. Biol. Med. 233:84–93.View ArticleGoogle Scholar
- Bai C-X, Kurokawa J, Tamagawa M, Nakaya H, Furukawa T. (2005) Nontranscriptional regulation of cardiac repolarization currents by testosterone. Circulation 112:1701–1710.View ArticleGoogle Scholar
- Killeen MJ, et al. (2008) Effects of potassium channel openers in the isolated perfused hypokalaemic murine heart. Acta Physiol. 193:25–36.View ArticleGoogle Scholar
- Kleber AG. (1984) Extracellular potassium accumulation in acute myocardial ischemia. J. Mol. Cell Cardiol. 16:389–394.View ArticleGoogle Scholar
- Wilde AAM, et al. (1990) Potassium accumulation in the globally ischemic mammalian heart: a role for the ATP-sensitive potassium channel. Circ. Res. 67:835–843.View ArticleGoogle Scholar
- Hearse DJ. (2000) The elusive coypu: the importance of collateral flow and the search for an alternative to the dog. Cardiovasc. Res. 45:215–219.View ArticleGoogle Scholar
- Rösen R, Marsen A, Klaus W. (1984) Local myocardial perfusion and epicardial NADH-fluorescence after coronary artery ligation in the isolated guinea pig heart. Basic Res. Cardiol. 79:59–67.View ArticleGoogle Scholar
- Johns TNP, Olson BJ. (1954) Experimental myocardial infarction: I. A method of coronary occlusion in small animals. Ann. Surg. 140:675–682.View ArticleGoogle Scholar
- Dawson TA, et al. (2008) Cardiac cholinergic NOcGMP signaling following acute myocardial infarction and nNOS gene transfer. Am. J. Physiol. Heart Circ. Physiol. 295:H990–H998.View ArticleGoogle Scholar
- du Toit EF, Genis A, Opie LH, Pollesello P, Lochner A. (2008) A role for the RISK pathway and KATP channels in pre- and post-conditioning induced by levosimendan in the isolated guinea pig heart. Br. J. Pharmacol. 154:41–50.View ArticleGoogle Scholar
- Falk E. (1983) Plaque rupture with severe preexisting stenosis precipitating coronary thrombosis: characteristics of coronary atherosclerotic plaque underlying fatal occlusive thrombi. Br. Heart J. 50:127–131.View ArticleGoogle Scholar
- Sciagra R, et al. (2006) ST-segment analysis to predict infarct size and functional outcome in acute myocardial infarction treated with primary coronary intervention and adjunctive abciximab therapy. Am. J. Cardiol. 97:48–54.View ArticleGoogle Scholar
- Damiano BP, Cheng WM, Mitchell JA, Falotico R. (1996) Cardiovascular actions of thrombin receptor activation in vivo. J. Pharmacol. Exp. Ther. 279:1365–1378.PubMedGoogle Scholar
- Deka DK, Raviprakash V, Mishra SK. (1998) Basal nitric oxide release differentially modulates vasodilations by pinacidil and levcromakalim in goat coronary artery. Eur. J. Pharmacol. 348:11–23.View ArticleGoogle Scholar
- Moritani K, et al. (1994) Blockade of ATP-sensitive potassium channels by 5-hydroxydecanoate suppresses monophasic action potential shortening during regional myocardial ischemia. Cardiovasc. Drugs Ther. 8:749–756.View ArticleGoogle Scholar
- Notsu T, Tanaka I Takano M, Noma A. (1992) Blockade of the ATP-sensitive K+ channel by 5-hydroxydecanoate in guinea pig ventricular myocytes. J. Pharmacol. Exp. Ther. 260:702–708.PubMedGoogle Scholar
- Jiang T, et al. (1996) Thrombin receptor actions in neonatal rat ventricular myocytes. Circ. Res. 78:553–563.View ArticleGoogle Scholar
- Franz MR. (1999) Current status of monophasic action potential recording: theories, measurements and interpretations. Cardiovasc. Res. 41:25–40.View ArticleGoogle Scholar
- Tande PM, Mortensen E, Refsum H. (1991) Rate-dependent differences in dog epi- and endocardial monophasic action potential configuration in vivo. Am. J. Physiol. 261:H1387–H1391.PubMedGoogle Scholar
- Furukawa T, Kimura S, Furukawa N, Bassett AL, Myerburg RJ. (1991) Role of cardiac ATP-regulated potassium channels in differential responses of endocardial and epicardial cells to ischemia. Circ. Res. 68:1693–1702.View ArticleGoogle Scholar
- Gilmour RF Jr, Zipes DP. (1980) Different electro-physiological responses of canine endocardium and epicardium to combined hyperkalemia, hypoxia, and acidosis. Circ. Res. 46:814–825.View ArticleGoogle Scholar
- Kimura S, Bassett AL, Kohya T, Kozlovskis PL, Myerburg RJ. (1986) Simultaneous recording of action potentials from endocardium and epicardium during ischemia in the isolated cat ventricle: relation of temporal electrophysiologic heterogeneities to arrhythmias. Circulation 74:401–409.View ArticleGoogle Scholar
- Samson WE, Scher AM. (1960) Mechanism of ST-segment alteration during acute myocardial injury. Circ. Res. 8:780–787.View ArticleGoogle Scholar
- Franz MR, Flaherty JT, Platia EV, Bulkley BH, Weisfeldt ML. (1984) Localization of regional myocardial ischemia by recording of monophasic action potentials. Circulation 69:593–604.View ArticleGoogle Scholar
- Shaw RM, Rudy Y. (1997) Electrophysiologic effects of acute myocardial ischemia: a theoretical study of altered cell excitability and action potential duration. Cardiovasc. Res. 35:256–272.View ArticleGoogle Scholar
- Kingaby RO, Lab MJ, Cole AWG, Palmer NT. (1986) Relation between monophasic action potential duration, ST segment elevation, and regional myocardial blood flow after coronary occlusion in the pig. Cardiovasc. Res. 20:740–751.View ArticleGoogle Scholar
- Stone GW, et al. (2008) Bivalirudin during primary PCI in acute myocardial infarction. N. Engl. J. Med. 358:2218–2230.View ArticleGoogle Scholar