Promising effects of xanthine oxidase inhibition by allopurinol on autonomic heart regulation estimated by heart rate variability (HRV) analysis in rats exposed to hypoxia and hyperoxia

<div><p>Background</p><p>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.</p><p>Aim</p><p>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.</p><p>Material and methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>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.</p></div

HRV in the control and allopurinol group and allopurinol groups before allopurinol administration.

<p>HRV in the control and allopurinol group and allopurinol groups before allopurinol administration.</p

Promising effects of xanthine oxidase inhibition by allopurinol on autonomic heart regulation estimated by heart rate variability (HRV) analysis in rats exposed to hypoxia and hyperoxia - Fig 3

<p><b>Effect of XO inhibition by allopurinol on markers of oxidative stress in plasma: 8-isoprostanes (A) and protein carbonyl group (B).</b> Blue bar indicates control group, orange bar–rats treated with allopurinol. Data shown as Mean ± SEM according to Student t-test, * p<0.05, NS–not significant, allopurinol vs. control group.</p

Promising effects of xanthine oxidase inhibition by allopurinol on autonomic heart regulation estimated by heart rate variability (HRV) analysis in rats exposed to hypoxia and hyperoxia - Fig 2

<p><b>Effect of XO inhibition by allopurinol (50mg/kg) on activity of antioxidant enzymes in erythrocyte lysate: superoxide dismutase (A), catalase (B), glutathione peroxidase (C).</b> Blue bar indicates control group, orange bar–rats treated with allopurinol. Data shown as mean ± SEM * p<0.05, NS–non-significant, allopurinol vs. control group (N = 8; Fig A: Mann-Whitney test; Fig B and C: t-test).</p

Effects of hypoxia and hyperoxia on time-domain HRV parameters.

<p>Data shown as mean ± SEM. Only significant values are tagged. Red color indicates allopurinol group before allopurinol administration (AGB), green—allopurinol group after injection of 5mg/kg of allopurinol, blue—allopurinol group after injection of 50mg/kg of allopurinol. * p<0.05 hypoxia, hyperoxia or recovery vs. normoxia, ** p<0.01 hypoxia, hyperoxia or recovery vs. normoxia in each group; green <b>#</b> p<0.05 – 5mg/kg of allopurinol vs. AGB at the same condition, green <b>##</b> p<0.01 – 5mg/kg of allopurinol vs. AGB at the same condition; blue <b>#</b> p<0.05 – 50mg/kg of allopurinol vs. AGB at the same condition, blue <b>##</b> p<0.01 – 50mg/kg of allopurinol vs. AGB at the same condition. ♦ p<0.05 – 5mg/kg vs. 50mg/kg of allopurinol.</p

XO activity in plasma per 1 mg of hemoglobin (μU/mgHb).

<p>Blue bar indicates control group (N = 8), orange bar–rats pretreated with allopurinol (50 mg/kg ip.). Data shown as mean ± SEM *** p<0.001, allopurinol vs. control group according to Mann-Whitney test.</p