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Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats

Abstract Microtubule integrity is important in cardio-protection, and microtubule disruption has been implicated in the response to ischemia in cardiac myocytes. However, the effects of Taxol, a common microtubule stabilizer, are still unknown in ischemic ventricular arrhythmias. The arrhythmia model was established in isolated rat hearts by regional ischemia, and myocardial infarction model by ischemia/reperfusion. Microtubule structure was immunohistochemically measured. The potential mechanisms were studied by measuring reactive oxygen species (ROS), activities of oxidative enzymes, intracellular calcium concentration ([Ca2+]i) and Ca2+ transients by using fluorometric determination, spectrophotometric assays and Fura-2-AM and Fluo-3-AM, respectively. The expression and activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) was also examined using real-time polymerase chain reaction, Western blot and pyruvate/Nicotinamide adenine dinucleotide-coupled reaction. Our data showed that Taxol (0.1, 0.3 and 1 μM) effectively reduced the number of ventricular premature beats and the incidence and duration of ventricular tachycardia. The infarct size was also significantly reduced by Taxol (1 μM). At the same time, Taxol preserved the microtubule structure, increased the activity of mitochondrial electron transport chain complexes I and III, reduced ROS levels, decreased the rise in [Ca2+]i and preserved the amplitude and decay times of Ca2+ transients during ischemia. In addition, SERCA2a activity was preserved by Taxol during ischemia. In summary, Taxol prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study presents evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.


Introduction
Ventricular arrhythmias are a major public health problem and are common events leading to sudden death [1]. Most traditional pharmacological therapies for this condition have yielded disap-ing [9,10]. In cardiac cells from newborn rats, disruption of microtubules has been found to stimulate the rate of spontaneous contraction [11]. Tubulin-binding agents that effectively stabilize microtubules have been reported to reverse adriamycin-induced arrhythmias and to affect the probability of eliciting stretchinduced arrhythmia in rabbits [11,12].
Ischemia is the most common cause of ventricular arrhythmias [13,14]. Microtubule disruption has been implicated in the response to ischemia in cardiac myocytes [15]. Taxol is a common microtubule stabilizer, and its main mechanism of action is to promote tubulin polymerization and to stabilize existing microtubules [7]. However, the effects of Taxol on ischemic ventricular arrhythmias remain unknown.

Establishment of the ischemic ventricular arrhythmia model
The ischemic ventricular arrhythmia model was established as previously described [16,17]. Adult male Sprague-Dawley rats weighing 250-280 g were killed by stunning and cervical dislocation. Hearts were rapidly removed and placed in ice-cold Krebs-Henseleit (K-H) buffer containing the following (mM): NaCl 118.0, KCl 4.7, KH2PO4 1.2, NaHCO3 25, MgSO4 1.2, CaCl2 2.5, glucose 11.0. The hearts were then mounted onto a Langendorff apparatus and perfused retrogradely at a constant pressure of 75 mmHg with K-H buffer gassed with 95% O2/5% CO2 equilibrated at pH 7. 35-7.45 and maintained at 37ЊC for 20 min. The left main coronary artery was ligated within 2 mm of where it emerged adjacent to the left atrium. With this procedure, coronary flow was reduced by at least 25%. The duration of coronary artery ligation was 30 min. No necrosis was found in this model, as determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (Fig. 1B). The electrogram and coronary flow rate were monitored before and during coronary artery ligation. Animals used in this study were maintained in accordance with the Guide for Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1996) and the Policy of the Animal Care and Use Committee of Tongji University.

Assessment of arrhythmias and pharmacological intervention
Arrhythmias were determined and diagnosed in accordance with the Lambeth Convention criteria [18]. Experiments were terminated or excluded from the final data analysis if any of the following conditions occurred: absence of signs of successful coronary artery occlusion, severe arrhythmias prior to coronary artery occlusion or severe atrioventricular block during the first 5 min. of ischemia. The pharmacological intervention protocol is shown in Figure 1A. Taxol was dissolved in dimethyl sulfoxide (DMSO). The exact concentration of DMSO in the perfusate as well as in the medium used in the later experiments on isolated cells was 1:1000. This concentration does not increase the susceptibility to arrhythmias. The heart rate and coronary flow among all groups in this study were comparable (data not shown).

Measurement of microtubule disruption
Myocardial tissues were perfused with a periodate-lysine-paraformaldehyde fixative at the end of the ischemia protocol. After that, the left ventricles were rinsed successively in phosphate-buffered saline (PBS) solutions containing 10%, 15% and 20% sucrose (2 hrs each). Frozen sections of 2-3 m in thickness were generated using a cryostat (CM 3050; Leica, Benshiem, Germany) and then mounted on gelatine-coated glass slides, rinsed three times in PBS (30 min. each) [19].

Measurement of infarct size
The myocardial infarction model was established as previously described [20]. The pharmacological intervention protocol is shown in Figure 1C. Briefly, the hearts of adult male Sprague-Dawley rats were mounted onto a Langendorff apparatus and perfused with K-H buffer. For hearts subjected to regional ischemia, the left main coronary artery was ligated. Reperfusion was achieved by releasing the ligation. In the present study, the isolated heart was subjected to 30 min. ischemia followed by 120 min. of reperfusion, which caused myocardial infarction. To determine the infarct size, the left main coronary artery was relegated at the end of reperfusion, and 2.5% Evans blue was perfused to delineate the area of risk. The blue staining region was removed and the rest, regarded as the risk area, was frozen and cut into slices, which were then incubated in a sodium phosphate buffer containing 1% (w/v) TTC for 15 min. to visualize the unstained infarcted region. The infarcted region was weighed, and the infarct size was expressed as a percentage of the risk zone.

Measurement of intracellular calcium concentration ([Ca 2؉ ] i) and Ca 2؉ transients
Ventricular myocytes were isolated as previously described [21].

Measurement of expression and activities of sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA2a)
To measure the expression and activities of SERCA2a, the left ventricles from hearts subjected to the ischemia protocol were used. SERCA2a mRNA expression was measured by real-time polymerase chain reaction with the green fluorescence dye SYBR Green (Roche, Indianapolis, IN, USA), which was validated with respect to reproducibility and linearity within the measurement range. The assay was performed in quadruplicate using Stratagen 3500 (Stratagen, La Jolla, CA, USA). RNA was extracted and reverse transcribed after complete DNA digestion. RNA yields were comparable in all groups (data not shown). To correct for potential variances between samples in mRNA extraction or in reverse transcribed efficiency, the mRNA content of each gene was normalized to the expression of the stably expressed reference gene GAPDH within the same sample. cDNA sequences were obtained from the public GenBank sequence database of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), and primers were designed with Primer3 software (http://fokker.wi.mit. edu/primer3/input.htm). Sequences for all polymerase chain reaction primers were as follows: forward 5Ј-TCC CTC AAG ATT GTC AGC AA-3Ј and reverse 5Ј-AGA TCC ACA ACG GAT ACA TT-3Ј for GAPDH; forward 5Ј-GGA ATT CCT AGA GAT GTG CAA TGC CCT C-3Ј and reverse 5Ј-CGG GAT CCC GTA ACA ACG CAC ATG CAC GCA-3Ј for SERCA2a.
SERCA2a protein expression was measured by western blot as previously described [22].    [22]. The sarcoplasmic reticulum membrane was prepared as previously described [22]. Protein content was determined using the BCA Protein Assay Kit (Merck, Darmstadt, Germany). A photometer adjusted to a wavelength of 340 nm was used to assess NADH oxidation (which is coupled to SERCA2a) at 37ЊC in the membrane preparations using the difference of the total absorbance and basal absorbance. The reaction was carried out in a volume of 1 ml. The SERCA2a activity was calculated as ⌬ absorbency/ (6.22 ϫ protein ϫ time) in nmol ATP/(mg protein ϫ min.).

Measurement of reactive oxygen species (ROS) and activities of oxidative enzymes
To evaluate ROS qualitatively, the left ventricles from hearts subjected to the ischemia protocol were placed in 20% sucrose for 12 hrs and embedded in optimal cutting temperature compound. Frozen sections of 10 m in thickness were generated using a cryostat ( Some homogenized solutions were centrifuged at 5000 ϫ g (4ЊC) for 10 min., and the supernatant was used for various assays. Xanthine oxidase activity was assayed through H2O2 production using p-hydroxybenzoic acid, 4-aminoantipyrine and horseradish peroxidase according to the manufacturer's protocol (Genmed Scientifics, Inc.), using a plate reader at 510 nm. Where indicated, the samples were incubated with hypoxanthine for 15 min. at 37ЊC before reading. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity was analysed using a tissue NADPH oxidase assay kit (Genmed Scientifics, Inc.). Briefly, tissue extract (100 g total protein) was incubated with NADPH. NADPH consumption was monitored by the decrease in absorbance at 340 nm for 5 min. Specific oxidase activity was measured by monitoring the rate of consumption of NADPH inhibited by the addition of DPI. The superoxide dismutase (SOD) assay was performed with an SOD Assay Kit (Genmed Scientifics, Inc.), which is based on the ability of SOD to inhibit the reduction of WST-1 by superoxide. In brief, the reaction mixture contained the cytosolic fraction (100 g), phosphate buffer, xanthine, xanthine oxidase, EDTA and the water-soluble tetrazolium salt (WST-1). After incubation for 20 min. at 37ЊC, the optical absorption was measured at 450 nm. One unit was defined as the amount of enzyme that caused one-half of the maximum inhibition of WST reduction.
Mitochondria from left ventricles subjected to the ischemia protocol were isolated as previously described [21]. Two independent methods were used to evaluate the purity of the mitochondrial preparation. First, electron microscopic observations showed very little contamination from broken mitochondria or lysosomes. Second, western blotting indicated that the mitochondrial marker cytochrome c oxidase was enriched at least eight times in the mitochondria preparations, while the endoplasmic reticulum marker calreticulin was markedly reduced to insignificant levels in the purified mitochondria (data not shown). The enzyme activity assay of

Statistical analysis
Data are expressed as the mean Ϯ S.E. The time-dependent data were subjected to two-way ANOVA to determine whether there were any significant differences among the groups. In addition, a one sample t-test, chi-squared test or one-way ANOVA was conducted to evaluate the one-way layout data when appropriate. If a significant difference was found, Bonferroni's post hoc test was conducted to determine which groups differed significantly. P-values Ͻ0.05 were considered as statistically significant.

Taxol prevents ischemic ventricular arrhythmias
Taxol (0.1, 0.3 and 1 M), a microtubule stabilizer, effectively prevented ischemic ventricular arrhythmias, which could be identified from the reduced number of ventricular premature beats and the incidence and duration of ventricular tachycardia (P Ͻ 0.05) ( Table 1) function and did not have a protective effect, which could be considered as an overdose (data not shown).

Taxol preserves microtubule structure during ischemia
The microtubule disruption score was low (3.2 Ϯ 0.9%) in the control group, whereas it increased significantly to 28.8 Ϯ 6.7% in the ischemia group (P Ͻ 0.05). After Taxol treatment (0.1 M), the microtubule disruption score was reduced to 9.8 Ϯ 1.9%. However, the actin filaments were intact even in cells with microtubule disruption (Fig. 2).

Taxol decreases infarct size
Whether Taxol could increase cardiac repair in a myocardial infarction is a key factor for its potential clinical use in the treatment of ischemic ventricular arrhythmias. Therefore, the effects of Taxol on myocardial infarction were examined. Typical pictures of the size of the myocardial infarctions are shown in Figure 3A. Taxol (1 M) significantly reduced the infarct size in the isolated perfused rat heart subjected to 30 min. ischemia and 120 min. reperfusion compared with untreated ischemia/ reperfusion hearts (Fig. 3B).

2ϩ
] i and preserves the amplitude and decay time of Ca 2ϩ transients during the condition of simulated ischemia.
Microtubules are important regulators of Ca 2ϩ handling in the heart, and abnormal calcium homeostasis plays a causal role in the genesis of arrhythmias [5,23,24]. Therefore, the effects of Taxol on calcium homeostasis in the condition of simulated ischemia were studied. During the condition of simulated ischemia, [Ca 2ϩ ]i was increased by 62% (P Ͻ 0.05) (Fig. 4A) (Fig. 4A). Typical pictures of Ca 2ϩ transients are shown in Figure 4B. During the condition of simulated ischemia, the amplitude of transient decreased by 42%, whereas the decrease was only 13% with Taxol treatment (P Ͻ 0.05) (Fig. 4C). During the condition of simulated ischemia, the decay time of the Ca 2ϩ transients was 2.52 times that of the control, while Taxol increased the decay time by 0.59 times (P Ͻ 0.05) (Fig. 4D).

Taxol preserves SERCA2a activity during ischemia
SERCA2a mRNA and protein expression remained unchanged among the five groups studied (Fig. 5A-C) control group, SERCA2a activity was significantly decreased in the ischemia group. Taxol preserved the activity of SERCA2a during ischemia (Fig. 5D).

Taxol reduces ROS levels during ischemia
Microtubule disruption can be induced by oxidative stress, and Taxol is able to protect cultured adult mouse cardiac myocytes against oxidative stress induced by H2O2 [25,26]. Oxidative stress plays a pivotal role in ventricular arrhythmias [17,19,[27][28][29][30]. Therefore, the effects of Taxol on ROS generation in ischemia were studied. ROS levels were found to be increased during ischemia, and Taxol reduced this increase in the qualitative evaluation (Fig. 6A). Further study revealed that 0.1, 0.3 and 1 M Taxol reduced the level of ROS by 33%, 46% and 51%, respectively (P Ͻ 0.05) (Fig. 6 B).

Taxol increases the activity of mitochondrial electron transport chain complexes I and III during ischemia
The enzyme activities of mitochondrial electron transport chain complexes I and III were decreased during ischemia when compared with the control group (P Ͻ 0.05) ( Table 2). However, 0.3 and 1 M Taxol increased the activity of mitochondrial electron transport chain complex I, whereas 0.1, 0.3 and 1 M Taxol increased that of mitochondrial electron transport chain complex III (Table 2).

Discussion
The  [21,[31][32][33]. In this study, Taxol was found to reduce intracellular calcium accumulation and preserve calcium transients during the condition of simulated ischemia. SERCA2a is the main factor contributing to Ca 2ϩ transient decay in rat cardiac myocytes, and its function is critical in maintaining cardiac function [34]. SERCA2a overexpression may effectively prevent ventricular arrhythmias [1,31]. As predicted, the mRNA and protein levels of SERCA2a did not change after short ischemia. However, it was shown that SERCA2a activity was   Various ROS are generated during cardiac ischemia, and these deleterious species are highly reactive and prone to promoting oxidative changes in membrane lipids and ion pumps, which may further exacerbate the electrophysiological heterogeneity during ischemia and thus lead to the genesis of arrhythmias [17,28]. In the present study, Taxol was found to be able to reduce the levels of ROS during ischemia. The main cardiac sources of ROS are the mitochondrial electron transport chain complex I, the mitochondrial electron transport chain complex III ominant endogenous antioxidant is SOD [28]. During ischemia, antioxidant defences are overwhelmed by excess ROS, resulting in serious cellular injuries [28]. Our data showed that the enzyme activities of mitochondrial electron transport chain complexes I and III were decreased during ischemia, consistent with previous studies [27,35]. In our study, Taxol was found to increase the activities of mitochondrial electron transport chain complexes I and III. Microtubules are in close association with the mitochondria [23], which may be a basis for the effects of Taxol on the activities of complexes I and III. It should be mentioned that ROS generation in this study was measured after the addition of DCFH-DA to homogenates prepared from hearts subjected to ischemia. Due to the instability of ROS, it is possible that the above-mentioned ROS was generated during the in vitro assay. However, for the measurement of ROS, the left ventricles from the control group, ischemia group and Taxol groups were dealt with the same procedure. In the present study, 0.1, 0.3 and 1 M Taxol reduced the level of ROS by 33%, 46% and 51%, respectively. In view of the fact, ROS we measured were produced during preceding myocardial ischemia, but not during the in vitro assay.
In conclusion, the results of this study indicate that Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias likely through ameliorating abnormal calcium homeostasis and decreasing the level of ROS. This study provides evidence that Taxol may be a potential novel therapy for ischemic ventricular arrhythmias.
Of note, profound cardiac disturbances have already been reported in 5% of patients treated with Taxol. Symptomatic bradycardia has also been observed in 29% of ovarian cancer patients treated with Taxol [11]. Moreover, a previous study found that the probability of eliciting a stretch-induced arrhythmia increased in the hearts of rabbits treated with Taxol [12]. However, the present study found that the probability of eliciting ischemic ventricular arrhythmia decreased in rat hearts with Taxol. This difference may be attributed to the following reasons: (1) the Taxol doses used in this study are approximately one-tenth of those observed clinically in serum levels after continuous intravenous infusion, thus preventing the normal cardiac function from being affected [12]; (2) arrhythmias in the present study were elicited by ischemia, whereas those in the previous study were elicited by stretch [12]. The mechanisms of stretch-induced arrhythmia and ischemiainduced arrhythmia are somewhat different. In addition, the species difference also needs to be considered. In the present study, Taxol was found to prevent ischemic ventricular arrhythmias in rats, which is consistent with the fact that microtubule integrity is important for cardio-protection [9,10].
A potential weakness of the study was that the exact pathways by which Taxol preserves calcium handling in cardiomycytes and reduces oxidative stress remain elusive. Nevertheless, our analysis of the effects of Taxol on ameliorating abnormal calcium homeostasis and decreasing the level of ROS supports its predominant role in preventing ischemic ventricular arrhythmias. Moreover, the time at which Taxol should be given before anticipating ischemia and whether it can be used effectively during ischemia without preliminary treatment should be identified in the future. In addition, Taxol has imperfect specificity, as do almost all available drugs. Whether Taxol targets cardiac ion channels should also be determined in future studies.