The morbidity and mortality of myocardial infarction (MI) increase with increasing age 1, and the greatest risk factor for MI is age 2 partly because of an aging-related loss of the protective potency of cardiovascular strategies, for example preconditioning 3.
Reoxygenation ischemia–reperfusion (I/R) injury is a major complication of acute ischemic infarction, cardiopulmonary bypass, angioplasty, and heart transplantation 4. Previous studies have shown that aging hearts have reduced resistance to I/R injury in older rats compared with young rats 5, and after coronary angioplasty in humans, I/R injury was also marked in aged relative to younger patients 3. Moreover, I/R injury in aged rats was associated with lower reduced glutathione (GSH) to oxidized glutathione (GSSG) ratio and higher creatine kinase (CK) activity, together with lower leukocytic number and myeloperoxidase activity in perinecrotic areas in aged rats compared with young ones 6. Furthermore, in recent human studies, elderly patients had decreased cardiac functions and increased plasma apoptotic marker levels 7. During the period of ischemia, cellular pools of ATP decrease rapidly, triggering multiple apoptotic changes 8. The myocytic mitochondria located at the high-risk areas of the heart may show reversible dysfunction, manifested by a reduction in ATP production and increased release of reactive oxygen species 9.
There is accumulating evidence in recent years on the cardioprotective role of oxytocin including negative inotropy and chronotropy 10, parasympathetic neuromodulation 11, and release of atrial natriuretic peptide (ANP) 12 and nitric oxide (NO) 10. In addition, systemic oxytocin showed significant effects on blood pressure, vascular tone, and cardiovascular regulation 13. The protective role of oxytocin was shown by protecting isolated hearts from I/R injury in rats 14 and rabbits 15, where it decreased infarct size, ventricular arrhythmias, creatine kinase-MB levels, nitric oxide (NO) synthase activity, and matrix metalloprotein-1 (MMP-1) 16.
In this work, we aimed to test the effect of systemic oxytocin administration in alleviating I/R injury in aged rats.
Materials and methods
This randomized-controlled study was carried out in the Department of Physiology , Faculty of Medicine, Ain Shams University, Cairo, Egypt, from 1 March to 31 May 2012, and was approved by the Research Ethics Committee (REC) of Faculty of Medicine Ain Shams University (FMASU), Cairo, Egypt. The investigation conformed to the guide for the care and use of laboratory animals published by the United States National Institutes of Health. Forty-eight Wistar male rats were divided into four equal groups (n=12 rats each): young control, young oxytocin-treated (young OXT tr), old control, and old oxytocin-treated rats (old OXT tr). Young rats weighed 150–200 g and were 12–13 months old, whereas old rats weighed 270–350 g and were 23–24 months old. Oxytocin was injected intraperitoneally at a dose of 0.03 mg/kg/day for 7 days 17. Control rats received an equivalent amount of the vehicle by the same route and for the same duration.
On the day of the experiments, rats were weighed and injected intraperitoneally with heparin sodium, 1000 IU. One hour later, the rats were anesthetized with thiopental sodium (Sandoz GmbH, Kundl, Austria) at a dose of 40 mg/kg intraperitoneally. The hearts were removed using standard surgical techniques.
Perfusion of isolated hearts
The perfusion of isolated hearts was performed according to the method described before 18. The hearts were perfused in a Langendorff preparation, with retrograde perfusion under constant pressure 55 mmHg without recirculation. The perfusion medium used was the modified Krebs–Henseleit bicarbonate buffer of pH 7.4, equilibrated with O2 and CO2 95 : 5 at 37°C. Tension developed by the heart was measured using a lightweight 1–30 g range K-30 HSE isometric force transducer, which was connected through a strain gauge half-bridged Bioscience FC 117 coupler to a two-channel oscillograph (MD2 Bioscience, Washington, District of Columbia, USA). A 1-g weight was attached to the heart apex and left to hang freely, thus exerting resting tension of 1 g. The heart was left to stabilize for 10 min. A baseline recording was then obtained at a paper speed of 50 mm/s to determine the baseline heart beating rate (BR), developed peak tension (PT), time to peak tension (TPT), rate of tension development (dT/dt), and half relaxation time (½ RT). Myocardial flow rate (MFR) was determined by volumetric collection of the fluid passing out of the heart for 3 min, and expressed in ml/100 mg left ventricular weight/min. Ischemia was induced by stopping of the perfusion fluid for 30 min. Afterwards, the hearts were reperfused for an additional 30 min. At the end of 30 min of reperfusion, recordings were obtained at paper speed 50 mm/s for 1 min. In the meantime, MFR was measured at the same intervals by timed volumetric collection. Following heart perfusion, hearts were washed with isotonic saline, blotted dry by a filter paper, were further cleaned to remove fat and fibrous tissue, and then weighed. Results were expressed as the percentage change of the measured parameters relative to the baseline values to normalize individual differences between basal values among each group. The coronary effluent was collected and used for determination of CK and lactate dehydrogenase (LDH) according to the methodology described within kits supplied by Stanbio Laboratory Inc. (Boerne, Texas) by previous studies 19. Malondialdehyde (MDA) was estimated in cardiac homogenates using the double-heating method 20 and determination of plasma nitrates, as a stable end product of NO, was carried out using an end-point enzymatic one-step assay on the basis of the reduction of nitrate by nitrate reductase 21.
Statistical package for social sciences, version 16 for Windows (SPSS Inc., Chicago, Illinois, USA) was used for the statistical evaluation of the results. All data were expressed as mean±SEM. Statistical significance for data was determined using a one-way analysis of variance with a post-hoc test; significance was calculated using the least significant difference multiple-range test to find intergroup significance. A confidence level of 95% was considered statistically significant. Statistical significance of data of tissue nitrates as well as MDA was determined using a nonparametric Mann–Whitney U-test. P value less than 0.05 was considered significant.
The baseline values of cardiac parameters did not change significantly between young and old nontreated rats, except for a significantly lower MFR in old compared with young nontreated rats. Aged nontreated rats showed significant insult to the heart at 30 min reperfusion after 30 min of ischemia. The percentage change from baseline values of BR, dT/dt, and MFR was significantly higher in old compared with young nontreated controls, whereas the percentage change of ½ RT did not show statistical difference between the two groups. Administration of oxytocin to old rats resulted in a significant decrease in their basal heart rate compared with age-matched nontreated controls. Furthermore, old treated rats showed a significant improvement in cardiac activity after 30 min of ischemia, where the percentage change in BR and dT/dt was significantly less in old OXT tr rats compared with their age-matched nontreated controls. Despite improvement in the percentage change of ½ RT and MFR, they did not reach the level of significance between the two groups. In contrast, administration of oxytocin to young rats was not associated with statistically significant changes in cardiac parameters when compared with the age-matched nontreated group (Tables 1 and 2).
Biochemical results of the MDA assay in the heart homogenate indicated a higher MDA content in hearts of old compared with young nontreated rats (12.43±0.40 vs. 8.71±0.14, P<0.001). However, old OXT tr rats showed a significant decrease in the MDA content compared with age-matched nontreated controls (9.38±0.23 vs. 12.43±0.40, P<0.03) (Fig. 1). For plasma nitrate, old nontreated rats had significantly lower nitrate levels compared with young ones (184.3±5.37 vs. 225.89±2.06, P<0.001). On administration of oxytocin, nitrate levels were significantly increased in old OXT tr rats compared with nontreated age-matched controls (227.3±4.69 vs. 184.3±5.37, P<0.05). However, young OXT tr rats did not differ significantly from the age-matched nontreated group (Fig. 2). Postischemic levels of cardiac enzymes measured in the coronary effluent were significantly higher in old compared with young nontreated groups (P<0.05 for both LDL and CK). However, OXT tr old rats showed significantly lower levels of these enzymes when compared with age-matched nontreated controls (Figs 3 and 4).
The results of the present study show that the deterioration in cardiac function after I/R was more evident in old control rats as shown by the significant percentage decrease in dT/dt, BR, and MFR compared with young controls. The significant decrease in cardiac performance in aged rats in the present study was documented biochemically by a higher level of the cardiac enzymes, CK, and LDH, in the coronary effluent at 30 min reperfusion compared with their basal values, and compared with their matching levels in young control rats. The deleterious effect of age on I/R injury has been reported in previous studies 5. The worse recovery of old rats compared with young ones could be attributed to the increased oxidative stress caused by the higher levels of MDA in heart homogenates of old control rats. The formation of reactive oxygen species has been implicated as one of the pathomechanisms for tissue injury during I/R through induction of apoptosis 22. The release of reactive oxygen species causes increased permeability of the outer mitochondrial membrane, releasing the proapoptotic proteins from the mitochondria, with further activation of the mitochondria-dependent apoptosis pathway 23. An age-dependent impairment of mitochondrial function may comprise decreased electron transfer rates, increased H+ permeability of the inner membrane, and impaired H+-driven ATP synthesis 24.
A systemic (intraperitoneal) oxytocin injection in rats for 7 days exerted a powerful protective effect against I/R injury in old hearts, where the recovery improved significantly to values comparable with nontreated control young rats. There was a significant improvement in dT/dt, BR, and CRF and this improvement was confirmed biochemically by the significant decrease in cardiac enzymes in treated old rats compared with their age-matched controls. The beneficial effect of oxytocin preconditioning has been reported in several studies 16,25. Oxytocin injected in old rats was associated with a significant decrease in the MDA level in heart homogenates and a significant increase in plasma nitrate, an end product of nitric oxide, compared with their age-matched controls. The cardioprotective effect of oxytocin could exert a direct effect on oxytocin receptors (OXTR) in hearts of rats 26 and humans 27. The increased level of NO in old oxytocin-supplemented rats in the present study could reflect the protective effect of oxytocin in these rats, where NO might act to improve myocardial microvascular perfusion and limit cardiomyocyte apoptosis, thereby attenuating the myocardial dysfunction induced by myocardial I/R 7. Oxytocin also augments glucose uptake in cardiomyocytes by phosphoinositol 3 kinase (PI3K) 28, which is considered beneficial during myocardial injuries 29. Oxytocin can suppress inflammation, decrease neutrophils, macrophages, and T-lymphocytes with depression of the inflammatory cytokines tumor necrosis factor-α and IL-6 and promotion of IGF-β 30.
Several signaling pathways of OXTR in cardiac cells have been postulated in conjunction with specific functions in the heart. Oxytocin stimulates ANP release 12, which stimulates cGMP, which reduces the force and rate of contraction and increases vasodilation 31. A second signaling pathway for oxytocin receptors includes stimulation of phospholipase A2 (PLA2) 32, an agent that was related to protection against I/R in the heart through the production of eicosanoids 33. The protective effect of oxytocin could also be explained on the basis of its anti-inflammatory 27 and antioxidant activities 34. An emerging explanation for the protective role of oxytocin on the mitochondrial level is its ability to stimulate mitochondrial K+-ATP-sensitive channels, an effect that was abolished by K+-ATP-sensitive channel inhibitors 15. Indirect actions for oxytocin that may contribute toward minimizing I/R injury are the ability of OXT to stimulate muscarinic receptors 10, together with a decrease in sympathetic nervous system outflow to the heart and blood vessels evidenced by low plasma norepinephrine 35. Taken together, in addition to the higher sympathetic activity in old age 36, the aforementioned data could shed further light on our results in aging rats. Surprisingly, our results did not prove the protective role of oxytocin in young rats. This could be related to the lack of significant cardiac damage in young rats compared with old rats, which appears in low cardiac enzymes after I/R in young rats when compared with preischemic values.
In sum, the exaggerated I/R injury in old rats improved markedly by systemic administration of oxytocin, which might be because of its antioxidant effect, together with its enhanced release of nitric oxide. However, we could not detect such a protective effect of oxytocin in young rats probably because of the mild I/R injury in these rats.
Conflicts of interest
There are no conflicts of interest.
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