Rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist has been used to treat type II diabetes. Despite debates on its cardioprotection, the effects of rosiglitazone on cardiac electrophysiology are still unclear. We determined the effect of rosiglitazone on ventricular fibrillation (VF) incidence, VF threshold (VFT), defibrillation threshold (DFT), and mitochondrial function during ischemic and reperfusion periods in swine. Methods: Twenty-six domestic pigs were used. In each pig, either rosiglitazone (1mg/kg) or normal saline solution was administered intravenously within 60 minutes. Then, the left anterior descending coronary artery was ligated for 60 minutes and released to promote reperfusion for 120 minutes. The cardiac electrophysiologic parameters were determined at the beginning of the study and during ischemic and reperfusion period. The heart was removed and the area at risk and the infarct size in each heart were determined. Cardiac mitochondria isolated from remote and ischemic areas were used for mitochondrial function analysis. Results: Rosiglitazone did not alter the DFT and VFT during ischemic/reperfusion periods. In rosiglitazone group, VF incidence was increased (58.33 vs. 10 %) and time to the first occurrence of VF was decreased (3.17 ± 2.40 vs. 18.5±.13), compared to vehicle group (P<0.05). However, infarct size related to area at risk in rosiglitazone group was significantly decreased (P<0.05). In cardiac mitochondria and isolated cardiac mitochondrial function, rosiglitazone did not alter level of reactive oxygen species production and could not prevent mitochondrial membrane potential change and mitochondrial swelling. Conclusion: Rosiglitazone increased propensity for VF, and could neither increase defibrillation efficacy nor improve cardiac mitochondrial function.
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Keywords: Rosiglitazone, VF, Defibrillation, Mitochondria, Ischemic, Reperfusion
Rosiglitazone is a synthetic agonist of the peroxisome proliferator-activated receptor gamma (PPARγ) which has been used to improve insulin resistance in the treatment of type 2 diabetes (1). Rosiglitazone exerts its potent insulin sensitizer by improving glycemic control(2). Data from various studies in the past few years suggested that therapeutic effects of rosiglitazone reach far beyond their use as insulin sensitizers since rosiglitazone also exerts their other therapeutic effects such as the improvement of cardiac contractile dysfunction(3), the inhibition of the inflammatory response by reducing neutrophils and macrophage accumulation(4) and the protection of myocardial injury during ischemic/reperfusion (I/R) periods in different animals models(4-8).
Despite its beneficial effect, the effects of rosiglitazone on cardiovascular diseases remain controversy. Recent studies in porcine models have shown that treatment with rosiglitazone increased propensity for ventricular fibrillation (VF) during ischemia by blocking cardiac KATP channels that cause the shortening of cardiac action potential(9). Moreover, the discrepancy in findings regarding the effects of rosiglitazone on infarct size reduction has been reported (3, 5, 10, 11). Growing evidence on clinical trial also indicates the possible cardiac adverse effect in patient treated with rosiglitazone (12-15). Graham et al. reported that rosiglitazone was associated with increased risk of stroke, heart failure and the mortality (12). Regardless of these contraindicated finding, the effect of rosiglitazone on VF inducibility and defibrillation efficacy during I/R periods is not known.
In the present study, we aimed to determine the effect of rosiglitazone on cardiac electrophysiology during I/R periods in swine. The VF threshold (VFT), the incidence of VF occurrence and the defibrillation threshold (DFT) during I/R periods were determined. We tested the hypothesis that rosiglitazone attenuates the occurrence of VF by increasing the VFT, improves the defibrillation efficacy decreasing the DFT, and reduces the infarct size during I/R periods in swine hearts. We also determined the effect of rosiglitazone on cardiac mitochondria isolated from ischemic and remotes areas of each heart. We tested the hypothesis that rosiglitazone prevents mitochondrial damage in I/R swine heart. Moreover, we tested the effect of rosiglitazone on isolated cardiac mitochondrial function which exposed to oxidative stress induced by H2O2.
MATERIALS AND METHODS
Twenty-six domestic pigs (20-25 kg) of either sex were used in this study. All animals were cared for according to the Institutional Animal Care and Use Committees of the Faculty of Medicine, Chiang Mai University. Pigs were anesthetized by a combination of atropine (0.04 mg/kg), zolitil (5 mg/kg), and xylazine (2.2 mg/kg), and maintained by 1.5- 3.0% isoflurane delivered in 100% oxygen. After cuffed-endotracheal intubation, mechanical ventilation (volume controlled, tidal volume=12 ml/kg, respiratory rate=10-15 cycles/min) was started with pigs in a restrained dorsally recumbent position. Pavulon (2 mg/kg loading, 0.5 mg/kg/hr maintenance) was administered intravenously to minimize skeletal muscle contraction. The vital signs including the surface electrocardiogram (lead II), femoral arterial blood pressure (BP), heart rates (HR), respiratory rate and core temperature were continuously monitored. Blood gases and electrolytes were also monitored every 30 minutes and maintained within acceptable physiologic ranges (ABP=70-125 mmHg, pCO2=35-60 mmHg and pH=7.35-7.45) by giving intravenous sodium bicarbonate or by adjusting ventilation parameters as needed(16). Under fluoroscopic guidance, catheters with a 34- mm and 6-8 platinum coated titanium coil electrodes (Guidant Corp.) were inserted into the right ventricular apex respectively, at the junction between right atrium and superior vena cava, to deliver shocks (16, 17). The chest was opened trough a median sternotomy and the heart were suspended in a pericardial cradle. The pacing electrodes were affixed to the epicardium at the RV outflow tract and LV apex for effective refractory period (ERP) and diastolic pacing threshold (DPT) determination. The left anterior descending coronary artery (LAD) was identified and dissected from surrounding tissues.
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In the I/R study, pigs were divided into 2 groups. Group I (n=12) received rosiglitazone and group II (n=10) received normal saline solution (NSS). In each pig, the cardiac electrophysiological parameters, including DPT, ERP, QTc, VFT and DFT were determined at the beginning of the study (i.e. control parameter). Rosiglitazone potassium salt (Cayman Chemical, Ann Arbor, MI, USA) 1 mg/kg of body weight dissolved in NSS 30 ml and NSS as a vehicle in the same volume were administered intravenously over 60 minutes in groups I and II, respectively. After drug or vehicle was administered, the LAD was ligated at 5 centimeters above a distal branch to perform a regional occlusion. During the first 20 minutes of occlusion, if spontaneous VF occurred, the defibrillation shock was delivered to determine the DFT. If VF did not occur within 20 minutes, the DFT and VFT were determined using a three-reversal up/down protocol. The ischemic period was sustained for 60 minutes and then the LAD ligation was released to promote reperfusion for 120 minutes. All studied parameters were determined again beginning at 30 minutes after reperfusion. In this study, another group of 4 pigs without LAD occlusion were also studied for the electrophysiological effects of rosiglitazone in normal hearts. In this group, hemodynamic parameters as well as the DPT, ERP and DFT were determined before and after rosiglitazone administration.
Diastolic pacing threshold (DPT) protocol
The DPT testing was performed by delivered a train of 10 stimuli of a 5-ms square pulse (S1) via the electrode at the tip of RV catheter at 500-ms interval. The current was started from 0.1 mA and was increased in 0.1-mA steps until all drive trains elicit a ventricular response (capture). The DPT was defined as the minimum current strength necessary to capture the ventricle (16, 18).
Effective refractory period (ERP) protocol
In each train of 10 S1 stimuli, an S2 stimulus (5x DPT) was introduced in late diastole of the last S1 paced beat (350 ms after the R wave) to elicit a capture. The basic S1-S2 coupling interval was decreased in 10-ms steps until S2 failed to elicit a capture. The ERP was defined as the longest S1-S2 interval at which an S2 stimulus fails to elicit a ventricular response(16).
Ventricular fibrillation threshold (VFT) protocol
The heart was paced 3 times. The interval between the last S1 and the mid T-wave was determined each time and the average of S1-mid T wave interval was used as a coupling interval between the last S1 and S2 shock. The VFT testing was performed by delivering S2 shock started at 100 V. If this shock induced VF, the decrement of 10-V step was used for each successive shock until VF was no longer induced. If the 100-V did not induce VF, the increment of 10-V step was used for each successive shock until VF was induced. The lowest shock strength that could induce VF was the VFT (16).
Defibrillation threshold (DFT) protocol
In this protocol, the defibrillation shock was delivered after 10 seconds of VF to determine the DFT by using a three-reversal up/down protocol. Briefly, the initial shock strength was chosen at 400 V. In the event of successful defibrillation, the leading edge voltage was decreased in 80 V steps per defibrillation attempt until a first reversal from successful defibrillation to failure was achieved. If the initial shock was unsuccessful, the voltage was increased in 80 V steps per defibrillation attempt until a reversal from failed to successful defibrillation is achieved. At each reversal point, the algorithm was iterated in the opposite direction except that after the first reversal the voltage step size was diminished to 40 V and 20V for a total of three reversals. The DFT was defined as the lowest energy required for successful defibrillation of VF after three reversal points, when the next lower setting failed to defibrillate the heart. During the VF event, if the shock failed to defibrillate, a rescue shock (600-700 Volts) was delivered to successfully defibrillate the heart. A period of 4 minutes will be allowed between each VF induction so that the heart could return to physiologic condition (16).
Isolated cardiac mitochondrial function study
Cardiac mitochondrial isolation(19)
In this protocol, cardiac mitochondrial were isolated from male Wistar rats (weight 350 g). Rats were induced anesthetized and myocardium will rapidly excised and placed in ice cold mitochondrial isolation buffer (MIB), pH 7.2 (4°C), containing 300 mM sucrose, 0.2 mM EGTA, 5 mM TES. Myocardium will minced and homogenized in the same buffer (MIB). Homogenates will be centrifuged at 800xg, 4 °C for 5 min. The supernatant will be collected and centrifuged at 8800xg, 4 °C for 5 min. Then, resuspend mitochondrial pellet in MIB and at 8800xg, 4 °C for 5 min. The mitochondria will be collected and the protein concentration will be determined by the bicinchonic acid method.
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The Bicinchoninic Acid (BCA) assay for protein quantitation (standard assay)(20)
Reagent A (sodium bicinchoninate (0.1 g), Na2CO3 . H2O (2.0 g), sodium tartrate (dehydrate) 0.16 g, NaOH (0.4 g), NaHCO3 (0.95), made up to 100 ml. If necessary, adjust pH to 11.25 with NaHCO3 or NaOH) and Reagent B (CuSo4. H2O (0.4g) in 10 ml of water) will be prepare. Then, standard working reagent (SWR) will be made from 100 vol of reagent A with 2 vol of reagent B (the solution is apple green in color and is stable at room temperature for 1 week). SWR (1 ml) will be added to mitochondria protein and incubated at 60°C for 30 min. Cool the sample to room temperature, then will measure the absorbance at 562 nm using a spectrophotometer. A calibration curve will be constructed using dilution of stock 1 mg/ml solution of bovine serum albumin (BSA)
Reactive oxygen species (ROS) measurement in isolated cardiac mitochondria(21)
The production of superoxide will be detected by fluorescent probe, dichlorohydro-fluorescein diacetate (DCFDA). Cardiac mitochondrial proteins 0.4 mg/ml will be incubated with 2 μM DCFDA at 25°C for 25 minutes. ROS will be determined as fluorescence intensity using a fluorescent microplate reader by excited wavelength at 485 nm (bandwidth 5 nm) and emission wavelength at 530 nm (bandwidth 10 nm).
The mitochondrial membrane potential (âˆ†Ψm) change measurement in isolated cardiac mitochondria(22)
Mitochondrial membrane potential change (âˆ†Ψm) will be measured with the dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Mitochondrial proteins 0.4 mg/ml will be stained with JC-1 (5μM) at 37°C for 30 minutes. Mitochondrial membrane potential will be determined as fluorescence intensity by using a fluorescent microplate reader. JC-1 monomer (green) fluorescence is excited wavelength at 485 nm and detected the emission wavelength at 530 nm. JC-1 aggregate (red) fluorescence is excited wavelength at 585 nm and recorded the emission wavelength at 590nm. The change in mitochondrial membrane potential will be calculated the ratio of red to green fluorescence.
Isolated cardiac mitochondrial were confirmed morphology using electron microscopy. Isolated cardiac mitochondria was divide into 8 groups (n = 5 in each group): 1) control group, 2) H2O2 treated group, 3) rosiglitazone 10 µM pretreated group for 15 minutes, 4) rosiglitazone 25 µM pretreated group for 15 minutes, 5) rosiglitazone 50 µM pretreated group for 15 minutes, 6) rosiglitazone 10 µM pretreated group for 15 minutes then follow by H2O2 application and incubation for another 5 minutes, 7) rosiglitazone 25 µM pretreated group for 15 minutes then follow by H2O2 application and incubation for another 5 minutes, 8) rosiglitazone 50 µM pretreated group for 15 minutes then follow by H2O2 application and incubation for another 5 minutes. At the end of experiment, isolated cardiac mitochondria in each group were determined ROS production and âˆ†Ψm.
The basic electrophysiological and hemodynamic parameters in intact normal heart are shown in Table 1. Acute intravenous administration of rosiglitazone (1.0mg/kg) altered neither hemodynamics nor cardiac electrophysiologic parameters.
For the I/R experiment, the basic electrophysiology and hemodynamic parameters prior to and after I/R are shown in Table 2. Both saline and rosiglitazone did not alter any of those hemodynamic parameters during I/R period.
In rosiglitazone group, both the VFT and DFT during I/R period was not different from the baseline values (Table 3). The impedance and pulse width during I/R period were similar to these at baseline (Table 3). Similar to the rosiglitazone group, the DFT determined during I/R period was not different than that at baseline in the saline group (Table 3).
For the occurrence of VF, acute intravenous administration of rosiglitazone significantly (p<0.05) reduced the time interval from LAD ligation to the onset of the first spontaneous VF during I/R, compared to that in saline group (Figure 1A). The occurrence of spontaneous VF incidence was also significantly increased in rosiglitazone group (7/12 pigs), compared to the saline (1/10 pigs) group (Figure 1B). The areas of myocardial infarction (i.e. the ratio of infarct size to AAR) in rosiglitazone group were decreased (p<0.05), compared to the normal saline group (Figure 2).
For isolated cardiac mitochondria, rosiglitazone could neither prevent cardiac mitochondrial swelling (Figure 3A) nor alter the level of reactive oxygen species (ROS) production (Figure 3B) in both ischemic and remote areas. Moreover, rosiglitazone did not improve mitochondrial membrane potential changes (Figure 3C).
Similarly in normal cardiac mitochondria, treated with rosiglitazone 10, 25 and 50 µM did not alter mitochondrial function. However, rosiglitazone could not improve mitochondrial function in isolated cardiac mitochondrial which mimics I/R injury by exposed to oxidative stress environment induced by H2O2. Rosiglitazone 10, 25 and 50 µM could not reduced mitochondrial ROS production when compared with mitochondrial group which treated with H2O2 only. In addition all dose of rosiglitazone could not prevent mitochondrial membrane depolarization represent by âˆ†Ψm in rosiglitazone treated with H2O2 did not different from treated with H2O2 only.
The major findings of the present study are as follows: (1) acute intravenous administration of rosiglitazone did not alter basic electrophysiology and hemodynamic parameters in both intact and ischemic/reperfusion heart; (2) in I/R heart, acute intravenous administration of rosiglitazone could not improve defibrillation efficacy, but caused higher occurrence of ventricular fibrillation than the vehicle group; (3) acute intravenous administration of rosiglitazone reduced myocardial infarction area (4) rosiglitazone did not prevent cardiac mitochondrial dysfunction against I/R injury.
According to the effect of rosiglitazone on defibrillation efficacy, the present study was the first study that demonstrated that intravenous administration of rosiglitazone cannot improve defibrillation efficacy in I/R injury heart. Acute administration of rosiglitazone did not alter hemodynamic parameters this finding consistent with previous reports that administration of rosiglitazone in absent of ischemia, they found that rosiglitazone did not alter blood pressure(9).
During ischemic period, when pigs develop ventricular fibrillation, we found that treatment with rosiglitazone significantly shortened the time to onset of the first spontaneous VF that can indicate that VF inducibility was facilitated by rosiglitazone. A similar pro-fibrillatory effect was consistent with reports from the previous study(9) in the porcine model that demonstrated during completed coronary occlusion, intravenous administration of rosiglitazone in the same dose with present study (1.0 mg/kg) significantly decreased time to onset VF. This effect also found after treatment with the selective sarcolemmal KATP blocker, HMR-1098, suggesting that the mechanism of pro-arrhythmia of rosiglitazone was via KATP channels blockade. The previous study also demonstrated that rosiglitazone markedly attenuated degree of action potential duration shortening during ischemic period when compared with the vehicle group(9). This similarly result was found in our study that the ERP during ischemic period tended to increase when treated with rosiglitazone. Furthermore, the previous study demonstrated that rats treated with rosiglitazone increased late mortality after MI that presumed due to an increased risk of sudden cardiac death (23).
The present study demonstrated that acute intravenous administration of rosiglitazone significantly reduced the areas of myocardial infarction. Supporting the previous in vivo study that chronic treated mice with rosiglitazone reduced the ratio of infarct size to area at risk(24). From these results indicate that rosiglitazone have anti-apoptotic effect and the cardioprotection in ischemic/reperfusion injury.
In this present study, isolated cardiac mitochondria treated with rosiglitazone neither reduced ROS production nor prevent mitochondrial membrane potential depolarization. These results suggested that acute intravenous administration of rosiglitazone cannot improve cardiac mitochondrial function during ischemic and reperfusion period. However, the result of the infarction size demonstrated that the ratio of infarct size to area at risk was reducing when treated with rosiglitazone. From these results can suggest that anti-apoptotic effect of rosiglitazone did not directly act with cardiac mitochondria. Rosiglitazone may be involved in the extrinsic apoptosis signaling pathway. The extrinsic apoptosis signaling pathway is mitochondrial-independent apoptosis pathway that mediated cellular apoptosis by activated death receptors and transmits apoptotic signals after ligation with specific ligands (25, 26).
According to the pro-fibrillatory effect of rosiglitazone in our pre-clinical study, in isolated cardiac mitochondrial function study also demonstrated the similarly effect. Mitochondrial membrane potential change in isolated cardiac mitochondrial treated with rosiglitazone 25 µM which equally to dose of rosiglitazone 1.0 mg/kg could not prevent mitochondrial membrane potential depolarization against I/R injury. From this results, supported the rosiglitazone increase VF incidence and time to onset first VF occurrence in swine heart with I/R injury model. Suggesting that rosiglitazone may cause of cardiac arrhythmia and may not be used as the cardioprotective agent.
Future studies with different injured heart model as well as defibrillation studies at the cellular level in normal and diseased hearts are needed to determine.
This work was supported by the Faculty of Medicine Endowment Fund and grants from the Thailand Research Fund and RTA 52060008 (N.C.) and RMU 510700xx (S.C.).