Sevoflurane Alleviates Reperfusion Arrhythmia by Ameliorating TDR and MAPD90 in Isolated Rat Hearts after Ischemia-Reperfusion

Objective To investigate the effect of sevoflurane on the monophasic action potentials (MAPs) in isolated rat hearts after ischemia-reperfusion. Methods Twenty-four healthy SD male rats, weighing 280–320 g, were randomly divided into three groups after successful preparation of a Langendorff isolated heart perfusion model with a stabilization period perfusion of 15 min with Krebs–Henseleit (K–H) fluid (n = 8): the control group (group A, continuously perfused with K–H fluid for 105 min), the ischemia-reperfusion group (group B, continuously perfused with K–H fluid for 15 min, and then exposed to 60 min of global ischemia induced by Thomas solution followed by 30 min of reperfusion), and the sevoflurane group (group C, K–H fluid contained 1.0 MAC sevoflurane, and other procedures were same as in group B). Heart rate (HR) and MAPs including time course (MAPD50 or MAPD90) of the epicardium (Epi) and endocardium (Endo) were recorded at the time of balance perfusion for 15 min (T0), continuous perfusion for 15 min (T1), reperfusion for 15 min/continuous perfusion for 105 min (T2), and reperfusion for 30 min/continuous perfusion for 120 min (T3), and the transmural dispersion of repolarization (TDR) was calculated. The incidence of arrhythmia, time for restoration of spontaneous heart beat, and duration of arrhythmia were recorded during the period of reperfusion. Results HR in group B and group C was lower at T2 and T3 than that in group A, while that in group B was significantly lower than that in group A at T2 and T3, and HR in group C was higher than that in group B at T2 and T3 (P < 0.05). There was no difference of TDR in each group at T0 and T1 (P > 0.05), while TDR in group B was increased at T2 and T3 compared with that in group C and group A (P < 0.05). TDR in group C was decreased compared with that in group B at T2 and T3 (P < 0.05), while there was no such difference between group C and group A (P > 0.05). The time for restoration of spontaneous heart beat and duration of arrhythmia in group C were shorter than those in group B (P < 0.05), while cardiac arrhythmia scores in group B were higher than those in group C (P < 0.05). There was no difference of MAPD50 in each group (P > 0.05). The MAPD90 in group B was much longer than that in other groups at T2 and T3 (P < 0.05), while there was no such difference between group C and group A (P > 0.05). The prolonged MAPD90 at T2 and T3 in group B strikingly differed from that at T0 and T1 (P < 0.05). Nevertheless, there was no such difference in other groups at different time points (P > 0.05). Conclusion Sevoflurane alleviates reperfusion arrhythmia induced by myocardial ischemia-reperfusion through the shortening of MPAD90 in isolated rat hearts.


Introduction
Reperfusion arrhythmias (RA) are the main complications of cardiopulmonary bypass (CPB) in heart surgery. Arrhythmias, such as ventricular tachycardia and ventricular fibrillation, can induce hemodynamic disorders and sudden cardiac death [1,2]. In the study of arrhythmias, monophasic action potentials (MAPs) is an indicator for changes of cardiac vectors of cardiac arrhythmias, which can describe the repolarization phase of cardiac myocytes intuitively and steadily, and MAPs is an important means to observe the electrical activity of cardiac myocytes [3,4]. e action potential duration recorded by the single-phase action potential synchronous recording technique is closely related to arrhythmia.
Studies have shown that sevoflurane can reduce the incidence of RA [5][6][7], but it is not clear whether sevoflurane can affect MAPs in ischemia-reperfusion myocardium. is study is aimed to explore the effects of sevoflurane on the MAPs in an isolated rat heart of ischemia-reperfusion by synchronous recording in the endocardium and epicardium of cardiac myocytes.

Materials and Methods
2.1. Animals. Healthy male SD rats weighing 280 to 320 g were kept in standard transparent polyethylene cages in a light cycle (darkness of 12 h-light of 12 h) in an animal room at 22°C. ey had free access to water and food until the time of test. All experiments were approved by the Institutional Animal Care and Use Committee of Guizhou Medical University, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals.
omas solution (g/l) contained sodium chloride (6.44), potassium chloride (1.2), magnesium chloride (1.52), sodium bicarbonate (0.83), and calcium chloride (0.14). All of these components were dissolved in distilled water, but calcium chloride was added after all the ingredients to prevent precipitation.

Surgical Procedure for Isolated Heart Preparation.
e animals were pretreated with intraperitoneal injection of 300 IU heparin and then anaesthetized by sodium pentobarbital (50-60 mg/kg). After sacrifice of the animals, their hearts were excised rapidly and mounted on a nonrecirculating Langendorff apparatus under a constant pressure of 80 mmHg at 37°C and perfused with K-H solution that previously equilibrated with 95% O 2 and 5% CO 2 (pH � 7.4).

Study Protocols.
In preparation of the hearts as well as the Langendorff setup, we followed the methods of Haghi et al. [8]. e rat hearts were randomly divided into three groups after successful preparation of the Langendorff isolated heart perfusion model and 15 min balanced perfusion of K-H fluid. In the control group (group A), K-H fluid was under continuous perfusion for 105 min. In the ischemia-reperfusion group (group B), K-H fluid was stopped after continuous balanced perfusion for 15 min and cardiac arrest of global ischemia was induced for 60 min with the perfusion of omas solution (4°C, 20 ml/kg), while the heart was protected by the low temperature omas solution (4°C) around it. Reperfusion of omas solution (4°C, 10 ml/kg) was performed for 30 min, and the heart was resuscitated by the perfusion of K-H fluid for 30 min. In the sevoflurane group (group C), K-H fluid contained 1.0 MAC sevoflurane, and other procedures were same as in group IR. e K-H fluid was equilibrated with sevoflurane using Vapor2000 with an air bubbler. e concentration of sevoflurane was also measured in the buffer before entering the aortic cannula using a volatile anesthetic gas monitor.

MAPs
Recording. MAPs was recorded by a custommade electrode, which could record the MAPs of the epicardium (Epi) and endocardium (Endo) simultaneously. e MAPs signals were amplified and recorded with a biological signal collecting and processing system. HR and MAPs including time course (MAPD 50 or MAPD 90 ) of endocardium and epicardium were recorded at the time of balance perfusion for 15 min (T 0 ), continuous perfusion for 15 min (T 1 ), reperfusion for 15 min/continuous perfusion for 105 min (T 2 ), and reperfusion for 30 min/continuous perfusion for 120 min (T 3 ), and transmural dispersion of repolarization (TDR) was calculated. e incidence of arrhythmia, time for restoration of spontaneous heart beat, and duration of arrhythmia were recorded during the period of reperfusion.

Arrhythmias Analysis.
Based on the Lambeth conventions [9], the MAPs were analyzed to determine the number of single ectopic beats, salvos (couplets and triplets), ventricular tachycardia (VT), the total number of ventricular ectopic beats (VEBs), incidence, and duration of VT during the 30 min of reperfusion time.

Statistical Analysis.
Statistical analyses were performed using the SPSS18.0 software. Except for the arrhythmia scores, the other data are expressed as mean ± SD after test for normality. e comparison between groups was carried out by using one-way analysis of variance. Pairwise comparison was carried out using the LSD method. e X 2 -test was used for enumeration data, and values of P < 0.05 were considered statistically significant.

Effects of Sevoflurane on Heart Rate (HR) and Transmural
Dispersion of Repolarization (TDR) in I/R Rats. HR in group B and group C was lower at T 2 and T 3 than that in group A, while it in group B was significantly lower than that in group A at T 2 and T 3 , and HR in group C was higher than that in group B at T 2 and T 3 (P < 0.05, Table 1). ere was no difference of TDR in each group at T 0 and T 1 (P > 0.05), while TDR in group B was increased at T 2 and T 3 compared with that in group C and group A (P < 0.05). TDR in group C was decreased compared with that in group B at T 2 and T 3 (P < 0.05), while there was no such difference between group C and group A (P > 0.05, Table 1).

Effects of Sevoflurane on Reperfusion Arrhythmia (RA) in I/R Rats.
Arrhythmia scores in group B was higher than that in group C, while the time for restoration of spontaneous heart beat and duration of arrhythmia in group C were shorter than that in group B (P < 0.05, Table 2).

Effects of Sevoflurane on MAPs in I/R Rats.
ere was no difference of MAPD 50 in each group (P > 0.05, Table 3). e MAPD 90 in group B was much longer than that in other groups at T 2 and T 3 (P < 0.05), while there was no such difference between group C and group A (P > 0.05). e prolonged MAPD 90 at T 2 and T 3 in group B strikingly differed from that at T 0 and T 1 (P < 0.05). Nevertheless, there was no such difference in other groups at different time points (P > 0.05, Table 3).

Discussion
Langendorff isolated perfused heart model, which may reveal pathophysiological changes in the heart more accurately compared to tissue and cells and eliminate the negatives of neurohumoral factors and the preload and after load of heart [10][11][12], was used in this study for a favorable assessment for the MAPs on ischemia-reperfusion isolated heart by sevoflurane.
Our results showed that sevoflurane could shorten MAPD 90 of ischemia-reperfusion myocardium and decrease TDR and the incidence of reperfusion arrhythmias and the time for restoration of spontaneous heart beat and duration of arrhythmia. Lee showed sevoflurane can prolong the QT interval and QTc interval [13], and Gong found that sevoflurane can lengthen APD of single cardiomyocyte under normal physiological conditions [14]. While our study revealed that MAPD 90 prolonged after ischemia-reperfusion and sevoflurane could shorten it, these all suggested that sevoflurane directly played a role in ameliorating the myocardial repolarization in isolated rat hearts after ischemia-reperfusion.
In the study of arrhythmia, MAPs, an indicator of vector cardiograph for a population of cells, could record myocardial repolarization intuitively, especially after myocardial depolarization [15,16]. erefore, it has been regarded as an effective method for the detection of the electrical activity of cardiomyocytes and pharmacologically electrophysiological Table 1: Effects of sevoflurane on heart rate (HR) and transmural dispersion of repolarization (TDR) in I/R rats (n � 8, x ± s).

Parameters
Group Note: compared with the T 0 , a P < 0.05; compared with the T 1 , b P < 0.05; compared with group B, c P < 0.05.   [17,18]. Monophasic action potentials could avoid the heterogeneity from different origins. We ensured that MAPs were derived from the same transmural origin. In this way, the sevoflurane effects on each myocardium layer were investigated apart from the evaluation of myocardial repolarization.
Various ion channel currents are involved in cardiac action potential, but the APD is determined by outward and inward currents [19,20]. MAPD 50, where ICa-L, Ikr, and Iks are involved, largely reflects plateau of action potential [21]. APD could be prolonged when the Ca 2+ inward current increases, which is liable to cause arrhythmia and early after depolarization [22]. e action potential repolarization could be prolonged when Ikr and Iks are inhibited [23]. Iks, a ventricular myocardium repolarization reserve and a remarkable prolonged APD, will present in appearance of suppressed Iks [24]. MAPD 90 is a representative of phase 3 where mainly mediated by Ikr and Ik1 [25,26]. ese two are the currents which are responsible for the early stage and late stage of phase 3, respectively [27,28]. e prolonged MAPD 90 shows that when inhibited, Ikr and Ik1 slow down K + current rate. TDR reflects the nonuniformity of ventricular myocyte repolarization of myocardial layers. e increased TDR is the basic factor of a variety of malignant arrhythmia, especially polymorphic ventricular tachycardia and torsades de pointes ventricular tachycardia. erefore, sevoflurane may alter MAPs through influencing icon channels.

Conclusion
In summary, we found sevoflurane could alleviate reperfusion arrhythmia by ameliorating TDR and MAPD 90 in isolated rat hearts after ischemia-reperfusion.

Data Availability
e data used to support the findings of this study have been deposited at http://www.anesthscape.com/Home/Content? Id�1728.

Conflicts of Interest
e authors declare that they have no conflicts of interest.