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There is growing evidence that stress contributes to cardiovascular disease and triggers the release of oxytocin. Moreover previous studies confirmed oxytocin mimics the protection associated with ischemic preconditioning. The present study was aimed to assess the possible cardioprotective effects of the centrally released oxytocin in response to stress and intracerebroventricular (i.c.v.) administration of exogenous oxytocin in ischemic-reperfused isolated rat heart.
Methods and subjects
Rats were divided in two main groups and all of them were subjected to i.c.v. infusion of vehicle or drugs: unstressed rats [control: vehicle, oxytocin (OT; 100 ng/5 μl), atosiban (ATO; 4.3 μg/5 μl) as oxytocin antagonist, ATO + OT] and stressed rats [St: stress, OT + St, ATO + St]. After anesthesia, hearts were isolated and subjected to 30 min regional ischemia and 60 min reperfusion (IR). Acute stress protocol included swimming for 10 min before anesthesia. Myocardial function, infarct size, coronary flow, ventricular arrhythmia, and biochemical parameters such as creatine kinase and lactate dehydrogenase were measured. Ischemia-induced ventricular arrhythmias were counted during the occlusion period.
The plasma levels of oxytocin and corticosterone were significantly elevated by stress. Unexpectedly hearts of stressed rats showed a marked depression of IR injury compared to control group. I.c.v. infusion of oxytocin mimicked the cardioprotective effects of stress, yet did not elevate plasma oxytocin level. The protective effects of both stress and i.c.v. oxytocin were blocked by i.c.v. oxytocin antagonist.
These findings suggest that i.c.v. infusion of exogenous oxytocin and centrally released endogenous oxytocin in response to stress could play a role in induction of a preconditioning effect in ischemic-reperfused rat heart via brain receptors.
Intensive cardiovascular research is set to identify a reliable cardioprotective intervention that can salvage ischemic myocardium.
Cardiac preconditioning represents the most potent and consistently reproducible method of rescuing heart tissue from undergoing irreversible ischemic damage. Several recent clinical studies documented the significant role of stress in evoking the severity of cardiovascular disease [
]. Anatomical studies of oxytocin pathways in the brain have revealed extensive innervation of the brain stem structures regulating the cardiovascular, behavioral, and neuroendocrine responses to stress by oxytocin fibers projecting from the paraventricular nucleus (PVN) [
Evidence for direct projections from oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus in the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and peptide produced by the same neurons.
]. Moreover exposure to various stressors such as swim stress (10 min, 19–21 °C) a combined emotional and physical stressor triggered the release of oxytocin within both supra optic nucleus (SON) and PVN, as studied in male and female rats [
The engagement of central oxytocin in the controlling neuroendocrine responses to stress, its putative contribution to the regulation of cardiovascular parameters, and its protective effect on ischemia/reperfusion-induced myocardial injury, raises the question whether central release of oxytocin in response to stress and central administration of exogenous oxytocin may also be involved in regulation of the cardiovascular system in ischemic-reperfused heart.
Therefore the present study was designed to evaluate the possible cardioprotective effects of oxytocin released into brain in response to stress and central administration of exogenous oxytocin on ischemic-reperfused isolated rat heart.
Materials and methods
Male Wistar rats (200–250 g) were obtained from Tehran University of Medical Sciences and were housed in an air-conditioned colony room on a light/dark (12/12 h) cycle (light on at 7 am) at 21–23 °C with free access to food and water. The rats were housed individually in stainless steel cages and anesthetized with sodium pentobarbital (60 mg/kg, 15 mg/0.5 ml, i.p.) and given heparin sodium (500 IU/0.5 ml, i.p.). All experiments were conducted in accordance with the institutional guidelines of Tehran University of Medical Sciences (Tehran, Iran) and the National Institutes of Health guidelines for the care and use of laboratory animals.
Implantation of the intracerebroventricular guide tube
Rats were deeply anesthetized with i.p. injection of a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) and then placed on a stereotaxic device. A 23-gauge stainless steel thin wall cannulae (9 mm long), was implanted into the lateral ventricle (intracerebroventricular; i.c.v.) using the following stereotaxic coordinates: 0.8 mm posterior to the bregma and 1.5 mm lateral from the midline, 3.2 mm below the surface of the skull. The guide tube was fixed to the skull using two stainless screws and dental acrylic cement and closed with a stainless steel stylet (30G). After surgery, the rats received antibiotic (penicillin 6.3.3., 30,000 U in 1 ml/rat i.m.) and were placed in their home cages. The animals were allowed 1 week of recovery after surgery.
At the beginning of each experimental session, the stylet was removed from the guide tube and the stainless steel tube (30G) that was 0.1 mm longer than the stylet was inserted into the guide tube and connected via the polyvinyl tubing to a micro syringe placed in an infusion pump (NE-1000, New Era Pump Systems Inc., Farmingdale, NY, USA). Each i.c.v. infusion was delivered at the rate of 5 μl/h. Rats received i.c.v. infusion of vehicle or drugs before anesthesia.
For stress induction, the rats were forced to swim for 10 min in deep water at 19–20 °C at 09:00 am. The forced swimming apparatus consisted of a Plexiglas cylinder that was 50 cm high and 30 cm in diameter. The cylinder was filled with tap water to a height of 35 cm. Rats were transferred to the cylinder from their home cages, forced to swim in the apparatus for 10 min and returned to their home cage [
After anesthesia hearts were rapidly excised and placed in an ice-cold buffer, and mounted on a constant pressure (80 mmHg) Langendorff-perfusion apparatus.
Hearts were perfused retrogradely with modified Krebs–Henseleit bicarbonate buffer containing (in mmol/l): NaHCO3 25; KCl 4.7; NaCl 118.5; MgSO4 1.2; KH2PO4 1.2; glucose 11; CaCl2 2.5 gassed with 95% O2/5% CO2 (pH 7.35–7.45 at 37 °C). A latex, fluid-filled, isovolumic balloon was inserted into the left ventricle through the left atrial appendage and inflated to give a diastolic pressure of 5–7 mmHg and connected to a pressure transducer (Harvard Apparatus, Holliston, MA, USA). To assess ventricular arrhythmia and heart rate monitoring, electrocardiographic recording was performed by fixation of thin stainless steel electrodes on ventricular apex and right atrium. A surgical needle (6-0 silk suture) was passed under the origin of the left anterior descending coronary artery, and the ends of the suture were passed through two plastic pipette tips to form a snare. Regional ischemia was induced by tightening the snare and reperfusion was performed by releasing the ends of the suture. The perfusion apparatus was water-jacketed to maintain constant perfusion temperatures of 37 °C. Hearts were allowed to beat spontaneously throughout the experiments. Hemodynamic parameters [left ventricular developed pressure (LVDP, the difference between left ventricular systolic and diastolic pressure) and heart rate (HR)] were monitored with a homemade program (Ossilo Graph Monitor, Biomed, Tehran, Iran). Left ventricular function was assessed by the rate pressure product (HR × LVDP). Ischemia-induced ventricular arrhythmias were counted during the occlusion period. Coronary effluent was collected at the end of reperfusion to measure enzymes including creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) as biochemical markers of myocyte necrosis.
Rats were randomly divided in two main stressed and non-stressed groups, and received i.c.v. infusion of vehicle or drugs before anesthesia and isolated hearts were subjected to 30 min ischemia and 60 min reperfusion.
Unstressed groups included: (1) control (n = 7), rats received vehicle; (2) OT (n = 7), oxytocin (OT) (100 ng/5 μl/h) was used; (3) ATO + OT (n = 5), atosiban (ATO) was administered (4.3 μg/5 μl/h) prior to infusion of oxytocin; (4) ATO (n = 6); atosiban was infused. Stressed groups exposed to swim stress for 10 min before anesthesia include: (1) St (stress) (n = 5), rats received vehicle 10 min prior to stress; (2) ATO + St (n = 5), atosiban was infused 10 min prior to stress; (3) OT + St (n = 6), oxytocin was infused 10 min prior to stress (Fig. 1).
Infarct size measurement
After completion of the reperfusion period, the left coronary artery was reoccluded, and Evans blue dye was infused via the aorta to differentiate the ischemic zone (area at risk; AAR) from the non-ischemic zone. Hearts were frozen overnight and then sliced into 2.0 mm (using stainless steel rat heart slicer matrix with 2.0 mm coronal section slice intervals) transverse sections from apex to base. Slices were then incubated with 1% triphenyl tetrazolium chloride (TTC in 0.1 M phosphate buffer, pH 7.4) for a period of 20 min at 37 °C. TTC reacts with viable tissue, producing a red formazan derivative, which is distinct from the white necrotic tissue once fixed in 10% formalin for 24 h. The areas of the left ventricle, AAR, and infarcted tissues were measured by method of planimetry from the scanned hearts by using Photoshop program. AAR was expressed as a percentage of left ventricular size for each heart and the infarct size was expressed as a percentage of AAR [
The coronary effluent was collected for cumulative CK and LDH release at 60 min of reperfusion. LDH and CK concentration in perfusate were determined by an autoanalyzer (Roche Hitachi Modular DP Systems, Mannheim, Germany) using specific kit (Sigma Chemical Co., St. Louis, MO, USA).
Assessment of ventricular arrhythmias
Ischemia-induced ventricular arrhythmias were determined in accordance with the Lambeth Conventions [
]. In this regard, three forms of ventricular arrhythmias were analyzed as below: ventricular ectopic beat (VEB), was identified as premature QRS complex; ventricular tachycardia (VT) was defined as four or more serial VEBs; and ventricular fibrillation (VF), was characterized as undetectable QRS complex. Multipart forms of VEBs such as bigeminy and salvos (couplet and triplet) were counted as separate episodes. The incidence, time of occurrence, and duration of arrhythmias were used to identify arrhythmia severity according to the following scoring system [
]: 0: 0–49 VEBs; 1: 50–499 VEBs; 2: >499 VEBs and/or 1 episode of spontaneously reverting VT or VF; 3: >1 episode of VT or VF or both with a total duration <60 s; 4: VT or VF or both 60–120 s total duration; 5: VT or VF or both >120 s duration; 6: fatal VF starting at >15 min after occlusion; 7: fatal VF starting between 4 and 14 min 59 s; 8: fatal VF starting between 1 and 3 min 59 s; 9: fatal VF starting <1 min after occlusion.
Blood samples for oxytocin and steroid analysis were taken from tails in control, OT, and St groups after anesthesia. Blood samples were centrifuged at 5000 rpm, 4 °C for 5 min in tubes containing EDTA (10% solution, 10 μl/100 μl blood), aprotinin (a protease inhibitor 10 μl/tube) and PMSF (5 μl/tube) and plasma aliquots were frozen at −70 °C until assay.
Oxytocin was analyzed in extracted plasma using an enzyme-linked immunoassay (ELISA; Phoenix Pharmaceuticals Inc., Burlingame, CA, USA) kit. The sensitivity of the assay was 0.11 pg/ml. The intraassay coefficients of variation were 6.8%.
Plasma corticosterone concentration as a stress marker was measured using an ELISA (DRG, Marburg, Germany) kit. The sensitivity of assay was 1.63 nmol/l. The intraassay coefficients of variation were 6.4%.
Atosiban, oxytocin acetate salt hydrate, TTC, and sodium pentobarbital were obtained from Sigma–Aldrich and heparin sodium was acquired from Caspian Tamin Pharmaceutical Co. (Tehran, Iran).
The hemodynamic parameters were analyzed using repeated measures of analysis of variance (ANOVA) with treatment and stress as grouping factors and time as a repeated factor followed by Tukey's post hoc test. Differences in infarct size, CK-MB, and LDH were evaluated by two-way ANOVA (stress and treatment as factors). When significant interaction was found, Tukey's post hoc test was used for comparison between pair groups. Statistical analysis of hormone levels was determined by one-way ANOVA and unpaired t-test.
Arrhythmia scores were analyzed with Kruskal–Wallis test followed by post hoc test Mann–Whitney, and the incidences of VT or VF were compared by Fisher exact test. All data were expressed as mean ± SEM. Statistical significance was defined as p < 0.05.
Cardiac function data observed at the baseline, end of ischemia, and reperfusion in all groups are reported in Table 1. Since HR and LVDP may recover to different degrees, rate pressure product (RPP) was calculated by multiplying heart rate by LVDP and data are presented as a reliable left ventricular function parameter for the isolated heart. In the baseline period all hearts had a similar RPP, LVDP, HR, and coronary flow (CF).
There were significant differences between RPP under baseline, end of ischemia, and end of reperfusion (repeated measures ANOVA, F(2,62) = 115.7, p < 0.001). The main effects of stress and treatment were not significant. Results showed a significant interaction between treatment and time on RPP (F(6,62) = 3.6, p < 0.001). After combining treatment and stress groups, Tukey's post hoc test revealed RPP in St hearts was significantly higher than control group at the end of reperfusion period (p < 0.05). Similarly oxytocin administration before stress significantly increased RPP (p < 0.05). I.c.v. administration of atosiban prior to stress caused a non-significant decrease in RPP in the ATO + St group compared to the St group. Central pretreatment with oxytocin in the OT group increased RPP in comparison with the control group (p < 0.05). Administration of atosiban prior to oxytocin restored RPP to that of the control group in the ATO + OT group (p < 0.01). No significant differences were shown between ATO and control groups.
Area at risk and infarct size
No significant treatment by stress interaction and no main effect of treatment and stress were revealed on AAR. Significant main effect was found for treatment on infarct size (two-way ANOVA, F(3,32) = 21.35, p < 0.001). The main effect of stress was not significant. The results showed a significant interaction between treatment and stress for infarct size (F(2,32) = 6.73, p < 0.05). For a combination of treatment and stress groups, all pairs of means were compared by Tukey's post hoc test adjusting for multiple comparisons. Stress significantly decreased infarct size in the St group compared to the control group (25.5 ± 2.6 IS/AAR % vs. 41.5 ± 2.8 IS/AAR %, p < 0.05). Oxytocin administration before stress significantly decreased the infarct size to 29.8 ± 4 IS/AAR % in the St + OT group from 41.5 ± 2 IS/AAR % in the control group (p < 0.05). Central administration of atosiban in the ATO + St group restored infarct size to those of the control group in comparison with the St group (48.3 ± 3.6 IS/AAR % vs. 25.5 ± 2.6 IS/AAR %, p < 0.001). Pretreatment with oxytocin prior to stress could not decrease infarct size compared to the St group. Central infusion of oxytocin in the OT group decreased significantly infarct size compared to the control group (22.5 ± 1.9 IS/AAR % vs. 41.5 ± 2.8 IS/AAR %, p < 0.001), whereas administration of atosiban prior to oxytocin caused a significant increase in infarct size in the ATO + OT group in comparison with the OT group (54.8 ± 5.8 IS/AAR % vs. 22.5 ± 2.6 IS/AAR %, p < 0.001). However, atosiban on its own had no significant effect on infarct size with respect to the control group (Fig. 2).
LDH and CK in coronary effluent
A significant main effect was found for treatment on CK-MB and LDH (two-way ANOVA; F(3,30) = 12.29, p < 0.001 and F(3,32) = 4.67, p < 0.01 respectively). The main effect of stress was not significant on CK-MB and LDH. The results showed a significant interaction between treatment and stress on CK-MB and LDH (F(2,30) = 11.38, p < 0.001 and F(2,32) = 9.34, p < 0.001, respectively). All pairs of means were compared by Tukey's post hoc test adjusting for multiple comparisons. Stress administration prior to ischemia/reperfusion significantly decreased CK and LDH levels in coronary effluent (64.8 ± 7.17 IU/l vs. 130 ± 17.2 IU/l and 47 ± 11.3 IU/l vs. 155.4 ± 22.9 IU/l, respectively, both p < 0.05). Similarly, significant differences were found in CK and LDH levels between St + OT and control groups (51 ± 9 IU/l vs. 130 ± 17 IU/l, p < 0.05 and 66.2 ± 26.3 IU/l vs. 155.4 ± 22 IU/l, p < 0.05, respectively). Central administration of atosiban prior to stress in the ATO + St group significantly increased CK and LDH levels compared to the St group (125 ± 15.3 IU/l vs. 64.8 ± 7.17 IU/l and 146.8 ± 11.8 IU/l vs. 47 ± 11.3 IU/l, respectively, both p < 0.05). CK and LDH levels in coronary effluent were markedly declined by central infusion of oxytocin compared to control at the end of reperfusion period (22.2 ± 6.6 IU/l vs. 130 ± 17.2 IU/l, p < 0.001 and 27.2 ± 3.6 IU/l vs. 155.4 ± 22.9 IU/l, p < 0.01 respectively). However i.c.v. administration of atosiban prior to oxytocin increased CK and LDH compared to the OT group (92.2 ± 10 IU/l vs. 22.25 ± 6.6 IU/l, p < 0.01 and 136.86 ± 29.6 IU/l vs. 27.2 ± 3.6 IU/l, p < 0.05, respectively, p < 0.05). Intraventricular infusion of oxytocin prior to stress in the OT + St group, had no significant effect on CK and LDH levels compared to the St group. Administration of atosiban alone prior to ischemia/reperfusion had no significant effect on CK and LDH in comparison with control (Fig. 3, Fig. 4).
Ventricular arrhythmias during ischemia
Severity of arrhythmias
Administration of stress prior to ischemia did not significantly decrease ischemia-induced ventricular arrhythmias severity with respect to control group (2.1 ± 0.55 arrhythmia score vs. 3.85 ± 0.45 arrhythmia score, p < 0.05). But administration of atosiban prior to stress in the ATO + St group intensified severity of arrhythmia compared to the St group (4.1 ± 0.65 arrhythmia score vs. 2.1 ± 0.55 arrhythmia score, p < 0.05). Arrhythmia severity was similar in hearts of the St and OT + St groups. Central administration of oxytocin alone had no significant effect on arrhythmia severity (Fig. 5).
Incidence of VT and VF
In the control group, the incidence of VT and VF occurred in 100% and 14.3% of hearts, respectively. No significant differences were observed in VF incidence among groups. Administration of stress prior to ischemia significantly decreased incidence of VT with respect to the control group. Central administration of atosiban prior to stress in the ATO + St group significantly increased VT incidence compared to the St group (p < 0.05). The incidence of VT markedly declined by central infusion of oxytocin compared to controls. However i.c.v. administration of atosiban prior to oxytocin increased VT incidence compared to the OT group (p < 0.05) (Fig. 6).
Plasma hormone level
The plasma concentrations of oxytocin and corticosterone were 7.38 ± 0.6 pg/ml and 26.2 ± 5.07 nmol/l, respectively in the control group. However, following induction of stress these concentrations significantly rose to mean concentrations of 17.9 ± 4.2 pg/ml and 41.7 ± 4.4 nmol/l, respectively. Administration of oxytocin in the OT group did not change the plasma oxytocin level in comparison to the control group (one way ANOVA, p < 0.05).
This study provides the first demonstration of the implication of central oxytocin released and its receptors in stress-induced cardioprotection. We observed that stress significantly decreased infarct size, incidence of VT, level of CK and LDH, and increased RPP in isolated rat hearts subjected to an ischemia–reperfusion sequence. Stress-induced myocardial tolerance to ischemia was abolished by i.c.v. administration of atosiban (as an oxytocin receptor antagonist) prior to stress exposure. To our knowledge this is the first report of the cardioprotective effect of i.c.v. infusion of oxytocin against IR injury in the heart which was inhibited by the administration of atosiban (i.c.v.).
It is well known that acute cardiovascular events (acute coronary syndrome and stroke) can be triggered by abrupt emotional or physical stressors [
In our study, an acute episode of stress experienced just before IR could provide myocardial protection against IR injury and decrease infarct size as the hardest end point of ischemic heart injury. The major effects of cardioprotection are reduction in infarct size (anti necrotic effect) [
]. Stress pretreatment improved contractile function, and reduced incidence of VT and biochemical parameters in the same direction as the infarct size in our experiment. Elevated levels of CK have been regarded as a specific biochemical marker of myocyte necrosis [
]. Our study showed that oxytocin plasma concentration was increased by swim stress, confirming the results of previous studies. Hence in the present experiment, central infusion of atosiban inhibited the protective effect of stress in the ATO + St group and abolished the effects of stress on infarct size, RPP, incidence of VT and release of CK and LDH. Since atosiban does not cross the blood–brain barrier [
], the inhibitory effects of atosiban may be mediated via brain oxytocin receptors. These findings seem to exclude the possibility that centrally released endogenous oxytocin in response to stress could protect and prevent worsening or development of IR injury by activation of central oxytocin receptors. Oxytocin is considered to be an endogenous stress-relieving compound [
]. Wsol et al. showed that oxytocin buffers the cardiovascular responses to stress and with central blockade of oxytocin receptors indicated that centrally released endogenous oxytocin significantly attenuates the intensity of the cardiovascular responses to acute stress in intact rats [
], whereas in others i.c.v. infusion of oxytocin for 5 days turned out to be hypotensive and reacted with a larger elevation of blood pressure and heart rate in response to unexpected and sudden noise [
Anatomical studies of oxytocin pathways in the brain have revealed extensive innervations of the brain stem structures regulating the cardiovascular, behavioral, and neuroendocrine responses to stress by oxytocin fibers projecting from the PVN [
]. However thus far, the role of central administration of oxytocin in IR injury has not been investigated.
In our study, central infusion of exogenous oxytocin increased recovery of LVDP and RPP at the end of reperfusion and decreased infarct size, the level of CK and LDH in comparison with the control group, without any changes in oxytocin plasma concentration. Central administration of an oxytocin receptor antagonist that does not cross the blood–brain barrier [
] eliminated the direct stimulatory influence of oxytocin on brain receptor. This implies that the cardioprotective effect of stress and i.c.v. infusion of oxytocin against IR injury may be transmitted in part by the actions of oxytocin in the brain, although oxytocin probably also has a direct cardioprotective effect. Therefore it may be deduced that oxytocin has a preconditioning effect via central action. Interestingly in the present experiment, administration of oxytocin prior to stress did not increase the protection of stress. There are two possible explanations for these findings: first, swimming could stimulate release of oxytocin in multiple ways and the release could be sufficient enough to saturate oxytocin receptors and second, since we have previously reported that oxytocin has a biphasic dose-dependent effect against IR injury [
], the combination of exogenous oxytocin and oxytocin released in response to stress may provide higher doses which show less activity.
In the present study, our model of stress could not cause a significant attenuation in severity of arrhythmias showing that the changes in infarct size do not correlate with the anti-arrhythmic effects of stress. In this regard some studies showed that preconditioning reduces infarct size, but accelerates time to ventricular fibrillation in ischemic heart [
]. Therefore, it seems that the extent of myocardial infarction may not directly affect arrhythmia.
Blockade of oxytocin receptor significantly offsets the effects of stress on ventricular arrhythmias. This observation implies beneficial effects of endogenous oxytocin on arrhythmia in stress condition. In this line some studies showed that adaptation to short-term non-damaging stress effect largely limits or prevents cardiac arrhythmias in acute ischemia and reperfusion [
] representing cardiac preconditioning. The present study showed that administration of stress prior to ischemia–reperfusion may have a preconditioning effect.
In conclusion these findings suggest that i.c.v. infusion of exogenous oxytocin and oxytocin released into the brain during stress could induce a preconditioning effect in ischemic-reperfused rat heart via brain receptors.
This study was supported by a grant from the Tehran University of Medical Science.
Role of neuropeptides in central control of cardiovascular responses to stress.
Evidence for direct projections from oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus in the medulla oblongata: immunohistochemical visualization of both the horseradish peroxidase transported and peptide produced by the same neurons.