Date Published: February 12, 2018
Publisher: Public Library of Science
Author(s): Stanisław Zajączkowski, Wiesław Ziółkowski, Piotr Badtke, Miłosz A. Zajączkowski, Damian J. Flis, Adam Figarski, Maria Smolińska-Bylańska, Tomasz H. Wierzba, Shama Ahmad.
It has long been suggested that reactive oxygen species (ROS) play a role in oxygen sensing via peripheral chemoreceptors, which would imply their involvement in chemoreflex activation and autonomic regulation of heart rate. We hypothesize that antioxidant affect neurogenic cardiovascular regulation through activation of chemoreflex which results in increased control of sympathetic mechanism regulating heart rhythm. Activity of xanthine oxidase (XO), which is among the major endogenous sources of ROS in the rat has been shown to increase during hypoxia promote oxidative stress. However, the mechanism of how XO inhibition affects neurogenic regulation of heart rhythm is still unclear.
The study aimed to evaluate effects of allopurinol-driven inhibition of XO on autonomic heart regulation in rats exposed to hypoxia followed by hyperoxia, using heart rate variability (HRV) analysis.
16 conscious male Wistar rats (350 g): control-untreated (N = 8) and pretreated with Allopurinol-XO inhibitor (5 mg/kg, followed by 50 mg/kg), administered intraperitoneally (N = 8), were exposed to controlled hypobaric hypoxia (1h) in order to activate chemoreflex. The treatment was followed by 1h hyperoxia (chemoreflex suppression). Time-series of 1024 RR-intervals were extracted from 4kHz ECG recording for heart rate variability (HRV) analysis in order to calculate the following time-domain parameters: mean RR interval (RRi), SDNN (standard deviation of all normal NN intervals), rMSSD (square root of the mean of the squares of differences between adjacent NN intervals), frequency-domain parameters (FFT method): TSP (total spectral power) as well as low and high frequency band powers (LF and HF). At the end of experiment we used rat plasma to evaluate enzymatic activity of XO and markers of oxidative stress: protein carbonyl group and 8-isoprostane concentrations. Enzymatic activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measures in erythrocyte lysates.
Allopurinol reduced oxidative stress which was the result of hypoxia/hyperoxia, as shown by decreased 8-isoprostane plasma concentration. XO inhibition did not markedly influence HRV parameters in standard normoxia. However, during hypoxia, as well as hyperoxia, allopurinol administration resulted in a significant increase of autonomic control upon the heart as shown by increased SDNN and TSP, with an increased vagal contribution (increased rMSSD and HF), whereas sympathovagal indexes (LF/HF, SDNN/rMSSD) remained unchanged.
Observed regulatory effects of XO inhibition did not confirm preliminary hypothesis which suggested that an antioxidant such as allopurinol might activate chemoreflex resulting in augmented sympathetic discharge to the heart. The HRV regulatory profile of XO inhibition observed during hypoxia as well as post-hypoxic hyperoxia corresponds to reported reduced risk of sudden cardiovascular events. Therefore our data provide a new argument for therapeutical use of allopurinol in hypoxic conditions.
Experimental and clinical data published to date have shown protective action of numerous antioxidants, which include antiatherogenic, antinflammatory and hypotensive effects. These molecules have also been shown to play a role in preventing endothelial dysfunction, vascular damage as well as protection from cardiac dysfunction caused by ischemia . One such enzyme, xanthine oxidase (XO; EC 126.96.36.199), has been shown to play a key role in purine metabolism and is among the major endogenous sources of reactive oxygen species (ROS). Moreover, it is implicated in inflammatory processes and ischemic injury. XO catalyzes oxidation of hypoxanthine to xanthine and xanthine to uric acid, while O2 acting as a cofactor is reduced to reactive superoxide radical (O2•−). Following spontaneous dismutation the latter is converted to hydrogen peroxide (H2O2) which is a major final ROS product of XO action, especially in hypoxic or inflammatory conditions. Inhibition of XO activity with allopurinol has been shown to reduce oxidative stress and prevent oxidative stress-driven cellular damage as well as ischemic complications . Allopurinol has been used to treat hyperuricemia and gout with relatively minor adverse effects. Beneficial effects of allopurinol, or its more stable metabolite oxypurinol, are evidenced in the case of vascular injury, inflammation , heart failure, ischemic heart disease [2,5], and also myocardial protection during cardiac or aortic surgery or post-ischemic reperfusion. Experimental and clinical data indicated reduction of the risk of ventricular arrhythmias related to prolonged allopurinol use [7,8]. In contrast to favorable effects of various antioxidants observed in experimental studies and in a wide range of clinical settings, a number of randomized multi-center clinical trials performed over the last two decades, disclosed that chronic use of common commercially available antioxidants such as beta-carotene, vitamin A, vitamin E, vitamin C or selenium has been associated with critical cardiovascular events, sudden death and an increase of all-cause mortality [9–11]. Intriguing point is that neither data from structural nor biochemical/biophysical studies have explained the nature of these adverse effects. ROS, and in particular O2•-, were shown to contribute in the arterial oxygen pressure sensing—thereby being involved in autonomic heart regulation. Systemic hypoxia apparently activates carotid bodies (CB), located in the carotid artery bifurcation, to trigger complex cardiorespiratory response, whereas hyperoxia inactivates the oxygen-sensing cells [12,13]. Although detailed mechanism of chemoreflex activation is still a matter of controversy, the evidence seems to indicate a pivotal role for inhibition of outward TASK K+ channel in CB, which results in an increased frequency of neural discharge from CB to the cardiorespiratory centers in the brainstem [14,15]. ROS such as O2•- or peroxides generated in the CB, do not only affect the redox state of the sensing cells but they can also oxidize TASK K+ channels, thus regulate their conductance. It is generally accepted that the chemoreflex activation depends on availability of oxygen and the redox state of the sensing cells. The literature suggests that increased local oxygen availability is proportional to enhanced ROS production and oxidative stress, whereas low O2 concentrations trigger the response to hypoxia as a result of compromised ROS generation. Suppression of endogenous oxygen radicals by antioxidants was hypothesized to induce the reflex cardiovascular response similar to that evoked by systemic hypoxia, with an increase of sympathetic drive of autonomic regulation of heart rhythm and ensuing increased risk of severe cardiac events [16–18]. Older experimental data which referred to the regulatory effects of antioxidants seemed to be inconsistent. Histidine, a powerful scavenger of singlet oxygen molecule (1O2), or trolox, a water soluble analogue of vitamin E, elicited an increase of sympathetic discharge to the rat heart [19,20], whereas ascorbic acid enhanced vagal control over the rat heart at doses up to 10 mg/kg, but at higher doses it increased sympathetic activity . On the other hand, prolonged supplementation with synthetic nitroxide antioxidant, tempol (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), increased autonomic control in hypoxia as well as hyperoxia in hypertensive rats (SHR, or nitric-oxide deficient) .
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Local Ethics Committee For Animal Experiments of the Medical University of Gdansk (consent no. 07/2011). Experiments were performed with all efforts made to minimize animal suffering. Sixteen male Wistar rats (350g) obtained from Medical University of Gdansk Breeding Laboratory (Gdansk, Poland) were used in this study. The animals were fed and watered ad libitum and kept under 12:12 hours light-dark cycle in standard atmospheric conditions. In the week preceding the proper procedure the rats were regularly habituated to the experimental environment. Then, the rats were anesthetized intraperitoneally with sodium pentobarbital (50mg/kg; Sigma-Aldrich Chemie GmbH, Munich, Germany) in accordance with institutional guidelines. Three silver ECG electrodes were implanted subcutaneously and exteriorized on occipital area. Following 48 hour recovery the experimental protocol was performed on conscious and unrestrained animals.
The major finding of this study, is that inhibition of XO significantly influenced neurogenic regulation of the heart rhythm during controlled hypobaric hypoxia and following hyperoxia. Allopurinol supplementation resulted in a significantly increased overall autonomic control including an increased vagal drive in the hypoxic conditions and also in the subsequent hyperoxia. Such profile of the regulatory response is most likely beneficial since dominant neurogenic control upon the intrinsic sinoatrial node pacemaker with prevalent vagal discharge is prerequisite for stable heart rhythm and is protective against ventricular arrhythmias [26,39,40].
The major issue of the experimental protocol was to provide experimental conditions for reliable assessment of undisturbed autonomic regulation with HRV analysis. In particular we tried to avoid excessive stress, injury or uncontrolled inflammation. For that reason the only invasive intervention was subcutaneous implantation of ECG electrodes of possibly minimal size. Such physiological variables of the regulatory relevance as arterial pressure or respiration has not been assessed due to high risk of unacceptable invasiveness of the surgical procedures. Blood samples useful for biochemical analyses were not taken during the experimental procedure. As a consequence our data are exclusively based on the ECG recording during the protocol and the biochemical assays from the final point of the experiment. Extrapolation of the obtained here data to humans needs caution. In the rat basal XO activity is much higher compared to humans and its devastating role as an oxidant is much higher . However, data from human studies showed that during ischemia or postischemic reperfusion generation of ROS by XO is rapidly increased so that the enzyme is an important trigger of oxidative stress.
Concluding, allopurinol reduced oxidative stress evoked by hypoxia and post-hypoxic hyperoxia in conscious unrestrained rats. In standard normoxic conditions XO inhibition does not seem to interfere with neurogenic heart regulation. Thus the concept that allopurinol acting as an oxidant perturbing oxygen sensing to activate chemoreflex has not been supported by our data. Moreover, in hypoxia, when chemoreflex is activated, XO inhibitor, allopurinol enhanced autonomic influence on the heart rhythm with increased vagal role. The observed regulatory effects provide an argument for using XO inhibitors in hypoxic conditions.