Proteins released from liver after ischaemia induced an elevation of heart resistance against ischaemia-reperfusion injury: 2. Beneficial effect of liver ischaemia in situ

We have shown earlier that proteins released from the heart during preconditioning may protect non-preconditioned heart during sustained ischaemia, similarly as preconditioning itself. In other our experiments we have documented that also proteins released from isolated rat liver during reperfusion after global ischaemia performed a protective effect on isolated rat heart against ischaemia-reperfusion injury. In the current study we examined the effect of liver ischaemia in situ on resistance of rat heart to ischaemia and reperfusion injury. Wistar rats (male) were subjected to liver ischaemia maintained by occlusion of portal vein and hepatic artery for 20 min, followed with 30-min reperfusion after reopening of both vessels. Then the hearts were isolated and perfused according to Langendorf. Hearts, after initial stabilisation (15 min), were subjected to 20-min ischaemia and 30-min reperfusion. During reperfusion, the haemodynamic parameters of hearts were measured. The protein pattern of high soluble fraction (HS fraction) isolated from rat blood by precipitation with ammonium sulphate was detected by SDS-PAGE. Our results showed improved parameters of pressure and contractility in the group after liver ischaemia (ischaemic group), presented by decreased diastolic pressure and increased LVDP((S-D)) in comparison with levels of these parameters in the control group. We also observed improved heart contraction-relaxation cycles parameters (dP/dt)(max) and (dP/dt)(min) in ischaemic group as compared with the control group. On the other hand, there were no significant differences in heart rate and coronary flow between both experimental groups. SDS-PAGE showed changed protein pattern in HS fraction, particularly the levels of several low molecular weight proteins increased. We conclude that liver ischaemia induced a higher resistance of heart against ischaemia-reperfusion injury. We propose that release of some cardioprotective proteins present in HS fraction can also contribute to this cardioprotection.     

Gonadectomy prevents endothelial dysfunction in fructose-fed male rats, a factor contributing to the development of hypertension

Insulin resistance has been shown to be associated with increased blood pressure (BP). The sex hormones estrogen and testosterone have opposing effects in the development of increased BP. Since testosterone has been implicated in increased BP following insulin resistance, we have tried to dissect out the effects of insulin resistance on endothelium-dependent vasorelaxation in the presence and absence of testosterone. Both gonadectomized and sham-operated male Wistar rats fed with a high-fructose diet developed insulin resistance, but BP increased only in the sham-operated rats. Reintroduction of testosterone in vivo restored the increase in BP, thereby abolishing the protective effects of gonadectomy. Fructose feeding did not affect plasma testosterone levels. Insulin resistance induced endothelial dysfunction in the mesenteric arteries of sham-operated rats, which was prevented by gonadectomy, thus suggesting a key role for testosterone in the pathogenesis of secondary vascular complications. Subsequent to blocking the actions of endothelium-dependent hyperpolarizing factor (EDHF), relaxation to acetylcholine (ACh) was lower in sham-operated fructose-fed rats compared with other groups, suggesting the involvement of nitric oxide (NO) in vasorelaxation. Inhibition of NO synthesis nearly abolished the ACh-evoked relaxation in both fructose-fed groups, thus suggesting a testosterone-independent impairment of EDHF-mediated relaxation. The improvement in endothelial function following gonadectomy could be ascribed to a NO component, although plasma nitrite and nitrate levels were unchanged. In summary, testosterone is essential in vivo for the development of endothelial dysfunction and hypertension secondary to insulin resistance, suggesting a facilitatory role for testosterone in increasing BP in fructose-fed male rats.     

Is androgen-dependent aromatase activity sexually differentiated in the rat and dove preoptic area?

Aromatase activity is higher in the male than in the female anterior hypothalamic-preoptic area (POA) in both the avian and the rodent adult brain. This sex difference is abolished after castration of the male and restored by androgen treatment. Gonadectomy has no effect on POA aromatase in the female. The aim of this study was to find out whether sex dimorphism in adult POA aromatase is only due to a sex difference in circulating gonadal hormones or dependent upon sexual differentiation of the brain. Aromatase activity was measured in vitro in microdissected POA samples using a sensitive radiometric assay. We examined the effects of gonadectomy and testosterone treatment on enzyme activity in adult rats and doves of both sexes. We also studied the effects of neonatal gonadectomy and hormone substitution in male and female rats. The results suggest that levels of POA aromatase in the adult depend primarily on gonadal activity, but that mechanisms involved in the regulation of aromatase and enzyme induction may be sex-specific and could result from sexual differentiation of the brain in early life. Further work will be required to determine the developmental stage when this occurs and the exact mechanism(s) responsible for increased sensitivity of the adult male POA to the inductive effect of testosterone.     

Is androgen-dependent aromatase activity sexually differentiated in the rat and dove preoptic area?

Aromatase activity is higher in the male than in the female anterior hypothalamic-preoptic area (POA) in both the avian and the rodent adult brain. This sex difference is abolished after castration of the male and restored by androgen treatment. Gonadectomy has no effect on POA aromatase in the female. The aim of this study was to find out whether sex dimorphism in adult POA aromatase is only due to a sex difference in circulating gonadal hormones or dependent upon sexual differentiation of the brain. Aromatase activity was measured in vitro in microdissected POA samples using a sensitive radiometric assay. We examined the effects of gonadectomy and testosterone treatment on enzyme activity in adult rats and doves of both sexes. We also studied the effects of neonatal gonadectomy and hormone substitution in male and female rats. The results suggest that levels of POA aromatase in the adult depend primarily on gonadal activity, but that mechanisms involved in the regulation of aromatase activity and enzyme induction may be sex-specific and could result from sexual differentiation of the brain in early life. Further work will be required to determine the developmental stage when this occurs and the exact mechanism(s) responsible for increased sensitivity of the adult male POA to the inductive effect of testosterone.     

Chronic alcoholic cardiomyopathy. Protection of the isolated ischaemic working heart by ribose

The effects of ribose on the pre- and post-ischaemic functional performance of the isolated working heart from 24 month old chronically alcoholic animals was investigated. The improved perfusion model permitted the isolated heart to perform work analogous to that of the normal physiological load, in a system where systemic pressure and atrial pressure could be altered over a wide range and oxygen loss from the perfusion fluid was a minimum. There was a remarkable improvement in the performance of isolated hearts taken from alcoholic animals that were perfused with 1.7 mM ribose both before and after a 25.0 min period of global myocardial ischaemia (at 25 degrees C), however ribose treatment did not greatly affect the performance of hearts of isocaloric control aged rats. Chronic alcohol consumption significantly affected heart performance, causing a marked reduction in both cardiac and work output. After ischaemia the work of all hearts was notably decreased; there was no work output in untreated hearts of alcoholic animals, whereas in hearts of alcoholic animals treated with ribose work output was only decreased by 35%. The acute response to ribose by hearts of aged chronically alcoholic animals suggests a role for this compound as a positive inotropic agent and clearly indicates the beneficial potential of ribose for inclusion in cardioplegic solutions or for infusion in alcoholic subjects showing signs of heart failure or chronic heart disease.     

Sex hormone control of left ventricular structure/function: mechanistic insights using echocardiography, expression, and DNA methylation analyses in adult mice

Calcium flux into and out of the sarco(endo)plasmic reticulum is vitally important to cardiac function because the cycle of calcium entry and exit controls contraction and relaxation. Putative estrogen and androgen consensus binding sites near to a CpG island are present in the cardiac calsequestrin 2 (CSQ2) promoter. Cardiomyocytes express sex hormone receptors and respond to sex hormones. We hypothesized that sex hormones control CSQ2 expression in cardiomyocytes and so affect cardiac structure/function. Echocardiographic analysis of male and female C57bl6n mice identified thinner walled and lighter hearts in females and significant concentric remodeling after long-term gonadectomy. CSQ2 and sodium-calcium exchanger-1 (NCX1) expression was significantly increased in female compared with male hearts and decreased postovariectomy. NCX1, but not CSQ2, expression was increased postcastration. CSQ2 expression was reduced when H9c2 cells were cultured in hormone-deficient media; increased when estrogen receptor-α (ERα), estrogen receptor-β (ERβ), or androgen agonists were added; and increased in hearts from ERβ-deficient mice. CSQ2 expression was reduced in mice fed a diet low in the methyl donor folic acid and in cells treated with 5-azadeoxycytidine suggesting an involvement of DNA methylation. DNA methylation in CpG in the CSQ2 CpG island was significantly different in males and females and was additionally changed postgonadectomy. Expression of DNA methyltransferases 1, 3a, and 3b was unchanged. These studies strongly link sex hormone-directed changes in CSQ2 expression to DNA methylation with changed expression correlated with altered left ventricular structure and function.     

Comparison of cardiopulmonary responses of male and female rats to intermittent high altitude hypoxia

Intermittent high altitude hypoxia (8 hours a day, 5 days a week, stepwise up to the altitude of 7000 m, total number of exposures 24) induced in male and female rats, chronic pulmonary hypertension and right ventricular hypertrophy. No significant sex differences were found in both these parameters. A significant sex difference was demonstrated in the resistance of the cardiac muscle to acute anoxia in vitro: the myocardium of control female rats proved to be significantly more resistant to oxygen deficiency. Intermittent altitude hypoxia resulted in significantly enhanced resistance in both sexes, yet the sex difference was maintained. Sex differences were further observed in the growth response of experimental animals to the acclimatization process. Whereas the body weight of male rats exposed to intermittent altitude hypoxia was significantly lower, hypoxic females had body weights comparable to those of control animals.     

Influence of sex hormones on ethanol-induced gastric haemorrhagic erosions in rats.

The role of sex hormones in the pathogenesis of ethanol-induced gastric erosions was investigated following the recent observation that ethanol generates more severe gastric damage in male rats. Female and male Wistar rats aged 110 +/- 6 days were used. Intact female, ovariectomized female, intact male, orchidectomized male and cyproterone acetate-pretreated (this compound a testosterone antagonist) male rats were investigated. 1 ml of 75% ethanol was used to induce gastric lesions. The extent of the erosions was determined planimetrically 60 min after ethanol administration. The plasma testosterone and 17-beta-oestradiol levels were checked by radioimmunoassay (RIA) in gonadectomized rats. Ethanol generates more severe lesions in male rats. Orchidectomy and cyproterone acetate treatment each reduced the extent of ethanol-induced gastric erosions in male rats. Ovariectomy had no effect in this model. The plasma testosterone and 17-beta-oestradiol levels were significantly reduced after gonadectomy. It is concluded that endogenous testosterone plays an aggressive role in the pathogenesis of ethanol-induced gastric erosions in rats.     

Effects of adaptation to intermittent high altitude hypoxia on ischemic ventricular arrhythmias in rats

We compared the effects of adaptation to intermittent high altitude (IHA) hypoxia of various degree and duration on ischemia-induced ventricular arrhythmias in rats. The animals were exposed to either relatively moderate hypoxia of 5000 m (4 or 8 h/day, 2-3 or 5-6 weeks) or severe hypoxia of 7000 m (8 h/day, 5-6 weeks). Ventricular arrhythmias induced by coronary artery occlusion were assessed in isolated buffer-perfused hearts or open-chest animals. In the isolated hearts, both antiarrhythmic and proarrhythmic effects were demonstrated depending on the degree and duration of hypoxic exposure. Whereas the adaptation to 5000 m for 4 h/day decreased the total number of premature ventricular complexes (PVCs), extending the daily exposure to 8 h and/or increasing the altitude to 7000 m led to opposite effects. On the contrary, the open-chest rats adapted to IHA hypoxia exhibited an increased tolerance to arrhythmias that was even more pronounced at the higher altitude. The distribution of PVCs over the ischemic period was not altered by any protocol of adaptation. It may be concluded that adaptation to IHA hypoxia is associated with enhanced tolerance of the rat heart to ischemic arrhythmias unless its severity exceeds a certain upper limit. The opposite effects of moderate and severe hypoxia on the isolated hearts cannot be explained by differences in the occluded zone size, heart rate or degree of myocardial fibrosis. The proarrhythmic effect of severe hypoxia may be related to a moderate left ventricular hypertrophy (27 %), which was present in rats adapted to 7000 m but not in those adapted to 5000 m. This adverse effect can be overcome by an unknown protective mechanism(s) that is absent in the isolated hearts.     

Metabolic basis for contractile dysfunction following chronic partial bladder outlet obstruction in rabbits

Our study is designed to correlate nitrite concentration, an index of nitric oxide (NO) release with mast cell peroxidase (MPO), a marker of cardiac mast cell degranulation and cardioprotective effect of ischaemic preconditioning in isolated perfused rat heart subjected to 30 min of global ischaemia and 30 min of reperfusion. Ischaemic preconditioning, comprised of four episodes of 5 min global ischaemia and 5 min of reperfusion, markedly reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK) in coronary effluent and incidence of ventricular premature beats (VPBs) and ventricular tachycardia and fibrillation (VT/VF) during reperfusion phase. Ischaemia-reperfusion induced release of MPO was markedly reduced in ischaemic preconditioned hearts. Increased release of nitrite was noted during reperfusion phase after sustained ischaemia in preconditioned hearts as compared to control hearts. No alterations in the release of nitrite was observed immediately after ischaemic preconditioning. However, ischaemic preconditioning markedly increased the release of MPO prior to global ischaemia. It is proposed that cardioprotective and antiarrhythmic effect of ischaemic preconditioning may be ascribed to degranulation of cardiac mast cells. Depletion of cytotoxic mediators during ischaemic preconditioning and consequent decreased release of these mediators during sustained ischaemia-reperfusion may be associated with preservation of structures in isolated rat heart responsible for NO release.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Sex-related difference of kinin level in rat pituitary gland

The kinin level in the pituitary glands was compared in adult male and female rats. A sex-related difference in the bradykinin (BK)-like immunoreactivity was found in the posterior lobe. The posterior pituitaries of female rats contained a higher concentration of the immunoreactive kinin than those of males. Ovariectomy of female rats resulted in the disappearance of a sex difference in the posterior kinin level and about a 3-fold increase in the anterior one. Orchidectomy of adult male rats failed to alter the kinin levels in both lobes. Moreover, the constitution of pituitary kinins was determined using HPLC. The pituitary kinins consisted of BK, Lys-BK (L-BK) and Met-Lys-BK (ML-BK) in different proportions in both lobes of male and female rats. The gonadectomy altered the proportions of these kinins. These results suggest that the pituitary kinin system may be regulated by circulating gonadal steroid hormones.     

Association of renal damage and oxidative stress in a rat model of metabolic syndrome. Influence of gender

This study investigated the association between nephropathy and oxidative stress, by measurement of systolic blood pressure, lipid peroxidation, activities of catalase, manganese- and copper-zinc-superoxide dismutase and endothelial nitric oxide synthase expression and concentrations of nitrates/nitrites in kidneys from rats with Metabolic Syndrome. Weaning female or male rats had 30% sucrose to drink for 24 weeks (Metabolic Syndrome). Modulation by sex hormones was investigated by gonadectomy and hormone replacement. In Metabolic Syndrome, Castrated Metabolic Syndrome + Testosterone males and Ovariectomized Metabolic Syndrome females had increased blood pressure, proteinuria and lipid peroxidation. Nitrates/nitrites and activities of catalase, manganese and copper-zinc-superoxide dismutase decreased vs intact Control, Castrated Metabolic Syndrome males, intact Metabolic Syndrome and Ovariectomized Metabolic Syndrome + Estradiol females. The results suggest that sex hormones modulate the activity of superoxide-dismutase, catalase and endothelial nitric oxide-synthase. Ovariectomy decreased the protection against oxidative stress in females; the opposite occurred in castrated males.     

Prenatal diet determines susceptibility to cardiac ischaemia-reperfusion injury following treatment with diethylmaleic acid and N-acetylcysteine

Fetal undernutrition programmes increased risk of developing cardiovascular disease in adult life. We hypothesized that prenatal protein restriction would impair recovery in post-ischaemic cardiac function in adult offspring through antioxidant-mediated processes. Pregnant Wistar rats were fed control or maternal low protein diets (MLP) throughout gestation. The offspring of these rats were treated with either saline, N-acetylcysteine (NAC), diethylmaleate (DEM), or both NAC and DEM to manipulate glutathione status at 6 months of age. Hearts were rapidly excised and retro-perfused (Langendorff) to assess isolated cardiac function before (baseline), and during 30 min global ischaemia and 60 min reperfusion. Hearts from adult rats exposed to a MLP diet in utero suffered greater cardiac dysfunction than those from controls following 30 min ischaemia. Left ventricular developed pressure (LVDP) was significantly reduced upon early reperfusion (p<0.042) in MLP rats compared to controls. NAC pre-treatment had no effect on LVDP of hearts from control animal hearts but improved the revival of MLP hearts to the same level as controls. DEM treatment did not affect control hearts but significantly reduced recovery of LVDP of MLP hearts during early (p<0.008) and late reperfusion (0.035). Combined NAC and DEM treatment had no effect on LVDP between control and MLP fed offspring. Prenatal protein restriction throughout pregnancy increases the susceptibility of the adult rat heart to suffer a functional deficit following ischaemia-reperfusion injury. Pharmacologically improving antioxidant status prevented this injury. A nutritionally-imbalanced developmental environment may increase susceptibility to coronary heart disease through the programming of myocardial glutathione metabolism.     

Proteins released from liver after ischaemia induced an elevation of heart resistance against ischaemia-reperfusion injury: 1. Beneficial effect of protein fraction isolated from perfusate after ischaemia and reperfusion of liver

OBJECTIVES: Numerous mechanisms have been proposed to participate in adaptation of heart to ischaemia by ischaemic preconditioning. We have described previously a release of cardio-protective protein fraction during ischaemic preconditioning of dog heart. In the current study the effect of high soluble protein fraction (HS fraction) released from isolated perfused rat liver after ischaemia and reperfusion was examined on isolated perfused rat heart during ischaemia-reperfusion injury. METHODS: Livers were subjected to 30 or 60 min ischaemia followed with 120 min reperfusion. HS fraction was isolated using ammonium sulphate precipitation and dissolved in perfusion solution before Langendorf perfusion of isolated rat hearts. The protein pattern of HS fraction was detected with SDS-PAGE and western blot with ConA and anti ConA antibody. Hearts were then subjected to 20 min ischaemia followed by 20 min reperfusion. During reperfusion, the haemodynamic parameters of hearts were measured. Heart levels of adenine nucleotide were measured in HClO4 extracts using HPLC on C18 column. RESULTS: Liver ischaemia induced changes in protein pattern of HS fraction released from the liver during reperfusion period. Particularly, we registered an increase in amount of several low-molecular weight proteins and decreased amount of high-molecular weight proteins. Proteins in this fraction isolated from perfusate after liver ischaemia interact with ConA with lower intensity as proteins isolated from perfusate after control non-ischaemic condition. HS fraction isolated from perfusate after ischaemia and reperfusion of liver had beneficial effect on heart function during 20 min ischaemia and subsequent 20 min reperfusion, documented by: i) decrease of arrhythmia score from 2 to 1 in 5 min of reperfusion and from 2 to 0 in 10 min of reperfusion; ii) improved heart contractility monitored as stabilised [dP/dt]max and increased Q parameter; iii) increased coronary flow. Proteins isolated from liver perfused under control non-ischaemic condition did not induce similar effects. The stabilisation of heart haemodynamics, observed after administration of HS proteins isolated from perfusate after ischaemia and reperfusion was associated with slight increase in ATP and ADP levels as well as decrease in AMP level.     

Increased affinity to substrate in sarcolemmal ATPases from hearts acclimatized to high altitude hypoxia

It has been well documented that acclimatization to chronic high altitude hypoxia involves a complex of adaptation changes which are capable of protecting the myocardium in diverse situations such as in acute hypoxia, coronary occlusion-induced ischaemia or isoprenaline-induced calcium overload. Since many of the former changes concern membrane functions, namely those of the sarcolemma, the activities and kinetic properties of sarcolemmal Mg2+-, Ca2+- and (Na+ + K+)-ATPase were investigated in right heart ventricles of rats acclimatized to intermittent high altitude hypoxia simulated in a barochamber. In the course of the experiment, the ventricles were subjected to a special anoxic test in vitro. The high altitude induced increase in cardiac tolerance to anoxia was not accompanied by any preservation of the sarcolemmal ATPase activities. On the contrary, membrane preparations obtained from the right ventricles of hearts acclimatized to high altitude exhibited significantly lower ATPase activities in comparison to non-acclimatized controls. The significant diminution in Km values of ATPases established in acclimatized hearts points to an increase in the affinity of their active sites to ATP. The latter effect is in agreement with the lowered rate of both the decrease in ATPase activities and the reduction of contractility in acclimatized hearts during the anoxic test, as well as with the considerably improved postanoxic reparability of contractions as compared to the controls. It is being concluded that the sarcolemmal changes at the level of ATPases involved in ionic transport processes represent an integral part of the adaptation complex to chronic high altitude hypoxia.     

Gonadectomy reveals sex differences in circadian rhythms and suprachiasmatic nucleus androgen receptors in mice

In mammals, it is well established that circadian rhythms in physiology and behavior, including the rhythmic secretion of hormones, are regulated by a brain clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. While SCN regulation of gonadal hormone secretion has been amply studied, the mechanisms whereby steroid hormones affect circadian functions are less well known. This is surprising considering substantial evidence that sex hormones affect many aspects of circadian responses, and that there are significant sex differences in rhythmicity. Our previous finding that "core" and "shell" regions of the SCN differ in their expression of clock genes prompted us to examine the possibility that steroid receptors are localized to a specific compartment of the brain clock, with the discovery that the androgen receptor (AR) is concentrated in the SCN core in male mice. In the present study, we compare AR expression in female and male mice using Western blots and immunochemistry. Both of these methods indicate that ARs are more highly expressed in males than in females; gonadectomy eliminates and androgen treatment restores these sex differences. At the behavioral level, gonadectomy produces a dramatic loss of the evening activity onset bout in males, but has no such effect in females. Treatment with testosterone, or with the non-aromatizable androgen dihydrotestosterone, restores male locomotor activity and eliminates sex differences in the behavioral response. The results indicate that androgenic hormones regulate circadian responses, and suggest an SCN site of action.     

Changes in metabolite levels and morphology of teleost ventricular myocytes due to hypoxia, ischaemia, and metabolic inhibitors

Since fish hearts are resistant to the effects of hypoxia, comparison of the effects of hypoxia and ischaemia on fish and mammalian hearts may lead to better understanding of ischaemic injury in mammalian hearts. The ultrastructure and levels of ATP, creatine phosphate, and lactic acid were examined in hearts obtained from largemouth bass. Bass hearts were subjected to conditions of normoxia, ischaemia, hypoxia, and hypoxia in the presence of fluoride and cyanide. ATP levels remained stable during hypoxia and ischaemia, but fell during hypoxia in the presence of fluoride or fluoride plus cyanide. Changes in creatine phosphate and lactic acid indicated ATP was produced during hypoxia and ischaemia by glycolysis, by rephosphorylation from creatine phosphate, and by oxidative phosphorylation with oxygen obtained from myoglobin or the atmosphere. Ultrastructural changes were found similar to those reported in ischaemic mammalian heart, consisting of inter- and intracellular swelling, glycogen depletion, and mitochondrial alterations. Comparison of metabolic rates between fish and mammalian hearts suggests the lower rate in fish hearts may be the chief factor which permits stable ATP levels during hypoxia and ischaemia, and thus provides resistance to these conditions.