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Endothelin(ET)-1 (ET-1) increases after myocardial infarction and may have effects on myocardial function. ET-1 has also been shown to affect the action potential (AP) which may be arrhythmogenic and predispose to ventricular fibrillation (VF). The effects of ET-2 and ET-3 are uncertain. We hypothesized that the ETs increase during acute ischemia and that plasma levels are predictive of ischemically induced VF. Thirty-four domestic swine underwent balloon occlusion of the proximal LAD coronary artery. Occlusion was confirmed angiographically. Venous samples were collected from the right atrium at baseline and at 5 min intervals for 30 min or until VF induction. ET-1, ET-2, and ET-3 were measured using ELISA. Changes in plasma concentrations were assessed using repeated measures ANOVA with Dunnett's. A p<0.05 was considered statistically significant. All animals had angiographic evidence of successful proximal LAD occlusion. ET-1 levels were significantly increased from a baseline at 20 min and remained elevated during 30 min of occlusion. ET-2 and ET-3 levels did not change from baseline values (figure, mean±SE). VF occurred in 60% of animals. Peak ET-1 values were not significantly different between VF and non-VF animals (6.2±2.2 vs. 4.8±2.3 pg/mL). No single ET-1 value had a VF predictive value >50%. There is a significant increase in ET-1 level within 20 min of acute myocardial ischemia. Despite known effects of ET-1 on the AP, this increase did not correlate with the occurrence of VF.
Endothelins (ETs) are 21-amino-acid peptides produced by the endothelium and play an important role in cardiovascular physiology during acute myocardial ischemia (AMI) (Myauchi and others 1989). Endothelin-1 (ET-1) may increase during AMI, resulting in electrical instability, myocardial dysfunction, and adverse clinical outcomes (Ruo and others 2005).
Elevated plasma levels of ET-1 have been reported in animal models of AMI (Stewart and others 1991). Exogenous administration of ET-1 increases the incidence of ventricular fibrillation (VF) in dogs (Vago and others 2004) and has been implicated in the etiology of reperfusion arrhythmias occurring during AMI (Isaka and others 2007).
The objective of this study was to determine if physiologic elevations of ET-1 during AMI are related to arrhythmogenesis or to the occurrence of VF.
This investigation was approved by our institution's Animal Care and Utilization Review Committee. Yorkshire swine (n=34) of both sexes were premedicated with ketamine and xylazine. General anesthesia was induced via nasal isoflurane and maintained with isoflurane and oxygen/nitrous oxide after intubation.
Under fluoroscopy and continuous ECG monitoring, micromanometer tipped catheters (Millar Instruments, Houston, TX, USA) were positioned in the ascending aorta and right atrium for pressure monitoring. The left anterior descending coronary artery (LAD) was occluded distal to the first septal perforator using a standard PTCA balloon catheter with angiographic confirmation. Animals were observed until VF or for 30 min. No further samples were collected after 30 min and the study protocol did not occlude any animals that had VF due to reperfusion arrhythmias after balloon deflation. ECG and hemodynamic data were recorded and stored on a laptop computer using PowerLab Chart v. 5.2 (ADInstruments, Castle Hill, Australia).
Venous blood was sampled from the right atrium prior to occlusion and at 5 min intervals for 30 min or until VF. Samples were placed in sterile, chilled (0°C), EDTA tubes, and centrifuged at 5,000 rpm for 10 min. Plasma was immediately separated and stored at −80°C until analysis. Endothelin levels (ET-1, ET-2, and ET-3) were determined by using a quantitative sandwich ELISA customized for porcine plasma (R&D Systems, Minneapolis, MN, USA).
The number of premature ventricular contractions (PVC) was determined using Chart Pro (ADInstruments, Castle Hill, Australia) and confirmed visually.
Data was entered on a Sigma Stat spreadsheet (Statistical Solutions v3.0, Cork, Ireland). Statistical comparisons were assessed using the student's t-test and associations determined using Pearson Product Moment with p<0.05 considered statistically significant.
Twenty-seven of the thirty-four animals fibrillated within 30 min after LAD occlusion. The mean time to fibrillation was 21.5±8.7 min. Compared to baseline, ET-1 levels were increased at 30 min in all animals without increases in ET-2 and ET-3 (p<0.05, Fig. 1). At 30 min and at 5 min intervals, there was no significant difference in ET-1 levels between the animals that fibrillated (n=27) and those that did not (n=7). While PVCs were common after occlusion of the LAD, regression analysis failed to show that levels of ET-1 were an independent predictor of the number of PVCs (p=0.12). The peak ET-1 levels in the animals that fibrillated was 3.9±0.8 pg/mL and 4.1±2.1 pg/mL in the animals that did not fibrillate (95% CI for difference of means −4.1 to 3.8, p=0.93).
The pathophysiology of ischemia-induced ventricular arrhythmias is complex and may be due to metabolic and ionic changes in the ischemic myocardium resulting in a dispersion of conduction and refractoriness predisposing the myocardium to reentrant arrhythmias (Watanabe and others 1991).
Animal studies have reported contradictory findings with regard to the arrhythmogenic role of ET-1, including whether there is a direct arrhythmogenic effect of ET-1 or an indirect effect mediated by coronary vasoconstriction resulting in worsened myocardial ischemia (Szabo and others 2000). Studies of selective ET antagonists in animal ischemia models have reported mixed results with some studies reporting fewer ectopic beats (Raschack and others 1998; Clozel and others 2002; Baltogiannis and others 2005) but not reduced infarct size (Takahashi and others 2001; Baltogiannis and others 2005) and with variation in different species (Vago and others 2002).
Our study shows that ET-1 significantly increases within 15 min of occlusion in a juvenile swine model. This increase in ET-1 was not associated with an increase in PVCs or an increased likelihood of VF. This observation is at odds with previous studies suggesting that ET receptor blockade decreased the frequency of ventricular ectopy and incidence of VF. Many previous studies used open thoracotomy and ligation of the LAD to induce ischemia, which may have caused a hyper-catabolic stress state resulting in excess ET release (Hiramatsu and others 1997).
In this study, ischemia was induced via percutaneous balloon occlusion of the LAD and resulted in endogenous ET-1 increase, which was unrelated to VF or PVCs. In the complex inflammatory milieu surrounding acute LAD occlusion, other cytokines such as hepatocyte growth factor (Yumoto and others 2005), interleukin-8 (IL-8), metalloproteinase-1 (TIMP-1) (Elmas and others 2007), or activation of the phosphatidylinositol-3 kinase pathway (Takahama and others 2006), may have a more causal effect on development of VF in AMI. A lessened role of ET in the pathogenesis of VF may account for the disparate results of ET receptor blockade in the literature.
There were several limitations to this study. The first was that we did not assess ET levels following reperfusion. Secondly, we did not observe differences over time between the group of animals that fibrillated and the group that did not. However, the number of animals in the non-VF group was small.
In conclusion, while there was a significant increase of ET-1 over baseline during acute myocardial ischemia, increased ET-1 levels did not predict onset of VF or increased incidence of PVCs. Further research is needed to elucidate the relationship of ET-1 within the complex inflammatory milieu that may be responsible for the development of PVCs and VF in the setting of acute myocardial ischemia.
Supported, in part, by a grant from the National Institutes of Health, NHLBI R01 HL076671.