The role of gender and sex hormones in ischemic–reperfusion injury in isolated rat hearts☆
Introduction
Heart disease is the primary cause of death in developed countries in men and in women over the age of 60. While pre-menopausal women have a low incidence of cardiovascular disease compared to men, mortality in post-menopausal women rises to the same frequency as in men (Adams et al., 1995, Fraser et al., 2000, Gray et al., 2001, Wild and Bartholemew, 1988). This significant gender difference has mostly been attributed to the beneficial role of estrogens (Collins et al., 1993, Gray et al., 2001).
In cardiovascular tissues the protective effects of estrogen and testosterone are manifested through their receptors in coronary arteries, where estrogen directly relaxes arteries via endothelial nitric oxide (NO) mechanism (Dai et al., 2004, Santos et al., 2004, Woodman et al., 2004). It is probable that testosterone relaxes them via a different mechanism as it is in estrogens, also a non-genomic pathway, which does not involve the nuclear androgen receptors and is independent of the vascular endothelium. This response to testosterone is initiated at the specific binding sites in smooth muscle cell membranes. Testosterone directly inhibits voltage-gated calcium channels, with an additional inhibitory action upon store-operated calcium channels (Jones et al., 2004, Yildiz et al., 2005).
Estrogens in myocardium reduce the incidence of post-ischemic ventricular arrhythmias by reducing accumulation of intracellular Ca2+, protecting mitochondrial structures, inhibiting apoptosis, having antioxidant action and interacting with heat stress proteins, thus protecting the heart from injuries (Fraser et al., 2000, Kim et al., 1998, Knowlton and Sun, 2001, Zhai et al., 2000). Both the mitogen activated protein kinase and phosphoinositide-3-kinase are also believed to be involved in the regulation of NO synthesis by estrogen (Gray et al., 2001).
The action of androgens on cardiovascular systems has received relatively little attention and the authors disagree about possible detrimental or protective effects of testosterone in the heart (Pugh et al., 2000). Androgens have been associated with possible proatherogenic effects and an increased cardiovascular risk by adversely affecting the plasma lipid and lipoprotein profile, increasing the risk of thrombosis and cardiac hypertrophy (Adams et al., 1995). On the contrary, short-term administration of testosterone causes vasodilatation in a range of species including humans (Costarella et al., 1996, Crews and Khalil, 1999, Honda et al., 1999, Perusquia et al., 1996, Yue et al., 1995).
To compare the effects of androgens to estrogens on myocardial injuries of isolated rat hearts, we studied their effects on two models. Firstly, the rats were treated with supraphysiological/subtoxic levels of sex hormones each day for 10 days prior to the experiment (males with testosterone, females with estradiol) and then their isolated hearts were subjected to acute ischemia/reperfusion injury. Secondly, isolated rat hearts were perfused with both hormones directly; male hearts with testosterone and female hearts with estradiol and then exposed to 40 min of ischemia and 40 min of reperfusion. Markers for the estimation of ischemia/reperfusion injuries were the level of decrease in the incidence and duration of arrhythmias, the increase in coronary flows and the decrease in lactate dehydrogenase release rates.
Section snippets
Animals and perfusion technique
All experiments were conducted in accordance with guidelines set down by the Veterinary Administration of the Republic of Slovenia (permission No. 323-02-203/00). Male and female Wistar rats (270–330 g and 230–260 g in body weight respectively) were obtained from the Faculty of Medicine, Ljubljana. All animals were 9–12 weeks old, bred under constant housing conditions and fed with standard rat chow in the form of pellets (Krka d.o.o., Novo mesto, Slovenia). A regular circadian cycle was
The coronary flow
The effect of direct application or 10 days pretreatment with estradiol and testosterone on coronary flow was determined in experiments with 40 min ischemia and 40 min of reperfusion.
In hearts where animals were pretreated with both drugs subcutaneously 10 days prior to isolation a rapid increase in coronary flow after the start of reperfusion was induced, and the highest values were reached in the 77th minute of the experiment. The comparable increase in coronary flow was observed after direct
Discussion
The main finding of this study is that both estradiol and testosterone in supraphysiological (subtoxic) levels significantly decreased ischemia/reperfusion injuries of isolated rat hearts in accordance with Langendorff. Reperfusion injuries were reduced both by the direct application of, and 10 days pretreatment with sex hormones prior to the isolated heart experiment. However, protective effects against ischemia/reperfusion injuries were only observed in the male hearts of animals pretreated
Acknowledgements
This work was supported by grant P0-0508-0381 from the Slovenian Ministry of Education, Science and Sport. The authors are grateful to Dani Hamze Jokhadar for assistance in conducting the experiments and evaluation of results and to Prof. Metka V. Budihna for overall help in the research.
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Ischemic–reperfusion injury in isolated male rat hearts is reduced only after pretreatment of animals but not with direct administration of testosterone to the isolated heart.