Analysis of Signaling Pathways of Necroptotic and Pyroptotic Cell Death in the Hearts of Rats With Type 2 Diabetes Mellitus.

Diabetes mellitus is known to produce various cell-damaging events and thereby underlie heart dysfunction and remodeling. However, very little is known about its inflammation-associated pathomechanisms due to necrosis-like cell death. For this purpose, we aimed to investigate signaling pathways of necroptosis and pyroptosis, known to produce plasma membrane rupture with the resultant promotion of inflammation. One-year old Zucker diabetic fatty (ZDF) rats did not exhibit significant heart dysfunction as revealed by echocardiographic measurement. On the other hand, there was a decrease in heart rate due to diabetes. Immunoblotting analysis showed that the left ventricles of ZDF rats overexpress neither the main necroptotic proteins including receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase-like pseudokinase (MLKL), nor the pyroptotic regulators including NLR family pyrin domain containing 3 protein (NLRP3), caspase-1, interleukin-1 beta (IL-1beta and the N-terminal gasdermin D (GSDMD-N). On the other hand, the increased activation of the RIP3 kinase due to phosphorylation was found in such hearts. In summary, we showed for the first time that the activation of cardiac RIP3 is upregulated due to disturbances in glucose metabolism which, however, did not proceed to necrosis-like cell death. These data can indicate that the activated RIP3 might also underlie other pleiotropic, non-necroptotic signaling pathways under basal conditions.

Effects of Iron Nanoparticles Administration on Ischemia/Reperfusion Injury in Isolated Hearts of Male Wistar Rats.

Iron is an essential mineral participating in numerous biological processes in the organism under physiological conditions. However, it may be also involved in the pathological mechanisms activated in various cardiovascular diseases including myocardial ischemia/reperfusion (I/R) injury, due to its involvement in reactive oxygen species (ROS) production. Furthermore, iron has been reported to participate in the mechanisms of iron-dependent cell death defined as "ferroptosis". On the other hand, iron may be also involved in the adaptive processes of ischemic preconditioning (IPC). This study aimed to elucidate whether small amounts of iron may modify the cardiac response to I/R in isolated perfused rat hearts and their protection by IPC. Pretreatment of the hearts with iron nanoparticles 15 min prior to sustained ischemia (iron preconditioning, Fe-PC) did not attenuate post-I/R contractile dysfunction. Recovery of left ventricular developed pressure (LVDP) was significantly improved only in the group with combined pretreatment with iron and IPC. Similarly, the rates of contraction and relaxation [+/-(dP/dt)max] were almost completely restored in the group preconditioned with a combination of iron and IPC but not with iron alone. In addition, the severity of reperfusion arrhythmias was reduced only in the iron+IPC group. No changes in protein levels of "survival" kinases of the RISK pathway (Reperfusion Injury Salvage Kinase) were found except for reduced caspase 3 levels in both preconditioned groups. The results indicate that a failure to precondition rat hearts with iron may be associated with the absent upregulation of RISK proteins and the pro-ferroptotic effect manifested by reduced glutathione peroxidase 4 (GPX4) levels. However, combination with IPC suppressed the negative effects of iron resulting in cardioprotection.

Properties of Na,K-ATPase in cerebellum of male and female rats: effects of acute and prolonged diabetes.

The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg-1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na+ and K+ gradients, depend on gender, age, and duration of diabetic impact.

Nrf2 as a key player of redox regulation in cardiovascular diseases.

The oxidative stress plays an important role in the development of cardiovascular diseases (CVD). In CVD progression an aberrant redox regulation was observed. In this regulation levels of reactive oxygen species (ROS) play an important role in cellular signaling, where Nrf2 is the key regulator of redox homeostasis. Keap1-Nrf2-ARE system regulates a great set of detoxificant and antioxidant enzymes in cells after ROS and electrophiles exposure. In this review we focus on radical-generating systems in cardiovascular system as well as on Nrf2 as a target against oxidative stress and a key player of redox regulation in cardiovascular diseases. We also summarize the current knowledge about the role of Nrf2 in pathophysiology of several CVD (hypertension, cardiac hypertrophy, cardiomyopathies) as well as in cardioprotection against myocardial ischemia/ reperfusion injury.

Cardioprotective effects of quercetin against ischemia-reperfusion injury are age-dependent.

Quercetin, a polyphenolic compound present in various types of food, has been shown to exert beneficial effects in different cardiac as well as non-cardiac ischemia/reperfusion (I/R) models in adult animals. However, there is no evidence about the effects of quercetin on I/R injury in non-mature animals, despite the fact that efficiency of some interventions against I/R is age-dependent. This study was aimed to investigate the effects of chronic quercetin treatment on I/R injury in juvenile and adult rat hearts. Juvenile (4-week-old) as well as adult (12-week-old) rats were treated with quercetin (20 mg/kg/day) for 4 weeks, hearts were excised and exposed to 25-min global ischemia followed by 40-min reperfusion. Functional parameters of hearts and occurrence of reperfusion arrhythmias were registered to assess the cardiac function. Our results have shown that quercetin improved post-ischemic recovery of LVDP, as well as recovery of markers of contraction and relaxation, +(dP/dt)max and -(dP/dt)max, respectively, in juvenile hearts, but not in adult hearts. Quercetin had no impact on incidence as well as duration of reperfusion arrhythmias in animals of both ages. We conclude that the age of rats plays an important role in heart response to quercetin treatment in the particular dose and duration of the treatment. Therefore, the age of the treated subjects should be taken into consideration when choosing the dose of quercetin and duration of its application in prevention and/or treatment of cardiovascular diseases.

Changes in activities of circulating MMP-2 and MMP-9 in patients suffering from heart failure in relation to gender, hypertension and treatment: a cross-sectional study.

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of heart failure (HF). Our aim was to determine the activities of circulating MMP-2 and MMP-9 in patients with HF in respect of gender, comorbidities and treatment (n=51). We did not reveal any differences in circulating pro-MMP-2 and pro-MMP-9 activities between the patients with HF and without it. However, there was a decrease in activity of pro-MMP-2 in treated hypertensive participants versus healthy ones. In contrast, we observed increased pro-MMP-2 activity in hypertensive participants with coexistent HF versus hypertensive participants without HF. In addition, a decrease in pro-MMP-2 activity was shown in women suffering from HF versus men suffering from HF. In conclusion, potential inhibitory effect of antihypertensive treatment on pro-MMP-2 activity was found. Coexistent HF with hypertension probably reduces the inhibitory effect of antihypertensive treatment on pro-MMP-2 activity. Our data also suggest the role of potential cardioprotective factors influencing the activity of pro-MMP-2 in women.

Remote preconditioning as a novel "conditioning" approach to repair the broken heart: potential mechanisms and clinical applications.

Remote ischemic preconditioning (RIPC) is a novel strategy of protection against ischemia-reperfusion (IR) injury in the heart (and/or other organs) by brief episodes of non-lethal IR in a distant organ/tissue. Importantly, RIPC can be induced noninvasively by limitation of blood flow in the extremity implying the applicability of this method in clinical situations. RIPC (and its delayed phase) is a form of relatively short-term adaptation to ischemia, similar to ischemic PC, and likely they both share triggering mechanisms, whereas mediators and end-effectors may differ. It is hypothesized that communication between the signals triggered in the remote organs and protection in the target organ may be mediated through substances released from the preconditioned organ and transported via the circulation (humoral pathways), by neural pathways and/or via systemic anti-inflammatory and antiapoptotic response to short ischemic bouts. Identification of molecules involved in RIPC cascades may have therapeutic and diagnostic implications in the management of myocardial ischemia. Elucidation of the mechanisms of endogenous cardioprotection triggered in the remote organ could lead to the development of diverse pharmacological RIPC mimetics. In the present article, the authors provide a short overview of RIPC-induced protection, proposed underlying mechanisms and factors modulating RIPC as a promising cardioprotective strategy.

Effect of yeast biomass with high content of carotenoids on erythrocyte deformability, NO production and Na,K-ATPase activity in healthy and LPS treated rats.

Measurements of red blood cell (RBC) deformability together with estimation of NO-synthase activity and Na,K-ATPase activity were used for characterization of RBC functionality in rats subjected to single dose of Escherichia coli lipopolysaccharides (LPS) at a dose of 1 mg/kg. We hypothesized that LPS might initiate a malfunction of RBC. We also investigated the potential effect of carotenoids (10 mg/kg/day) produced in red yeast biomass of Rhodotorula glutinis on RBC in LPS-challenged rats. LPS significantly reduced the deformability of RBC (by 14%) together with decrease of NO-synthase activity by 20%. Daily supplementation of carotenoids for 10 days attenuated the LPS-induced injury, as observed by 22% increase of RBC deformability and 23% increase of NO-synthase activity. The activity of Na,K-ATPase was also improved probably due to increased number of active enzyme molecules as indicated by 66% enhancement of Vmax value, hence maintaining the activity of erythrocyte Na,K-ATPase to the level even higher as compared with healthy control animals. It may be concluded that administration of yeast biomass with high content of carotenoids resulted in advanced function of erythrocytes as concerns their ability to squeeze through narrow capillaries of the circulation, better intrinsic production of NO and improvement of intracellular homeostasis of sodium.

Effect of duration of diabetes mellitus type 1 on properties of Na, K-ATPase in cerebral cortex.

Time sequence study was performed to characterize the effects of diabetes mellitus type 1 on properties of the Na, K-ATPase in cerebral cortex derived from normal and streptozotocin (STZ)-diabetic rats of both genders. The samples were excised at varying time intervals of diabetes induced by STZ (65 mg kg(-1)) for 8 days, and 8 and 16 weeks. Expression of α1-3 isoforms of Na, K-ATPase was not altered in statistically significant level during all stages of diabetes neither in female nor in male rats as revealed from Western blot analysis. Studies of kinetic properties of the enzyme resulted in variations in active number of Na, K-ATPase molecules as well as its qualitative properties. Sixteen-week-old control male rats showed better affinity to substrate as indicated by 13 % decrease of K m value. The effect persisted also in males subjected to 8 days lasting diabetes; however, in males subjected to 8 weeks lasting diabetes, the effect was lost. In 25-week-old rats, the Na, K-ATPase revealed again altered properties in males and females but the mechanism of the variation was different. In females, the number of active molecules of Na, K-ATPase was higher by 32 % in controls and by 17 % in rats with chronic diabetes when comparing to respective male groups as suggested by increased value of V max. So the properties of Na, K-ATPase in cerebral cortex, playing crucial role in maintaining intracellular homeostasis of Na(+) ions, depend on gender, age, and duration of diabetic insult.

Impaired PI3K/Akt signaling as a potential cause of failure to precondition rat hearts under conditions of simulated hyperglycemia.

The aim of the study was to evaluate the impact of simulated acute hyperglycemia (HG) on PI3K/Akt signaling in preconditioned and non-preconditioned isolated rat hearts perfused with Krebs-Henseleit solution containing normal (11 mmol/l) or elevated (22 mmol/l) glucose subjected to ischemia-reperfusion. Ischemic preconditioning (IP) was induced by two 5-min cycles of coronary occlusion followed by 5-min reperfusion. Protein levels of Akt, phosphorylated (activated) Akt (P-Akt), as well as contents of BAX protein were assayed (Western blotting) in cytosolic fraction of myocardial tissue samples taken prior to and after 30-min global ischemia and 40-min reperfusion. In "normoglycemic" conditions (NG), IP significantly increased P-Akt at the end of long-term ischemia, while reperfusion led to its decrease together with the decline of BAX levels as compared to non-preconditioned hearts. On the contrary, under HG conditions, P-Akt tended to decline in IP-hearts after long-term ischemia, and it was significantly higher after reperfusion than in non-preconditioned controls. No significant influence of IP on BAX levels at the end of I/R was observed under HG conditions. It seems that high glucose may influence IP-induced activation of Akt and its downstream targets, as well as maintain persistent Akt activity that may be detrimental for the heart under above conditions.

Severity of lethal ischemia/reperfusion injury in rat hearts subjected to ischemic preconditioning is increased under conditions of simulated hyperglycemia.

The aim of our study was to characterize resistance to ischemia/reperfusion (I/R) injury in Langendorff-perfused rat hearts and effectivity of ischemic preconditioning (PC) under condition of simulated acute hyperglycemia (SAHG) by perfusion of the hearts with Krebs-Henseleit (KH) solution with elevated glucose concentration (22 mmol/l). I/R injury was induced by 30-min coronary occlusion followed by 120-min reperfusion and PC by two cycles of 5-min occlusion/5-min reperfusion, prior to I/R. The severity of I/R injury was characterized by determination of the size of infarction (IS, expressed in % of area at risk size) and the amount of heart-type fatty acid binding protein (h-FABP, a marker of cell injury) released from the hearts to the effluent. Significantly smaller IS (8.8+/-1 %) and lower total amount of released h-FABP (1808+/-660 pmol) in PC group compared with IS 17.1+/-1.2 % (p<0.01) and amount of h-FABP (8803+/-2415 pmol, p<0.05) in the non-PC control hearts perfused with standard KH solution (glucose 11 mmol/l) confirmed protective effects of PC. In contrast, in SAHG groups, PC enhanced IS (21.4+/-2.2 vs. 14.3+/-1.3 %, p<0.05) and increased total amount of h-FABP (5541+/-229 vs. 3458+/-283 pmol, p<0.05) compared with respective non-PC controls. Results suggest that PC has negative effect on resistance of the hearts to I/R injury under conditions of elevated glucose in vitro.

Effect of quercetin on kinetic properties of renal Na,K-ATPase in normotensive and hypertensive rats.

The effect of quercetin, a plant-derived bioflavonoid with documented positive effect on the cardiovascular system, was examined after 4-week supplementation in the dose of 20 mg kg(-1) x day(-1) to young male normotensive control (C) and to spontaneously hypertensive rats (SHR) over the period of their 5(th)-8(th) week of age. The study was focused on the influence of quercetin on properties of the renal Na,K-ATPase, a key system in maintaining the homeostasis of sodium in the organism. Spontaneous hypertension by itself enhanced the activity of Na,K-ATPase probably as a consequence of a higher number of active enzyme molecules, as suggested by the 15% increase of V(max), along with improved affinity to ATP, as indicated by the 30% decrease in the value of Michaelis-Menten constant K(m) in untreated SHR vs. untreated normotensive rats. Quercetin induced a decrease of Na,K-ATPase activity in the presence of all ATP and Na(+) concentrations investigated. Evaluation of kinetic parameters resulted in a constant V(max) value. The ATP-binding properties of the enzyme were not influenced by quercetin, as suggested by statistically insignificant changes in the value of K(m) both in controls and in SHR. On the other hand, the affinity to sodium decreased, as suggested by an increase in the K(Na) value by 22% and 31% in normotensive and hypertensive groups, respectively. This impairment in the affinity of the Na(+)-binding site of Na,K-ATPase molecules was probably responsible for the deteriorated enzyme function in the kidneys of quercetin treated animals.

Simvastatin alleviates myocardial contractile dysfunction and lethal ischemic injury in rat heart independent of cholesterol-lowering effects.

Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are most frequently used drugs in the prevention of coronary artery disease due to their cholesterol-lowering activity. However, it is not exactly known whether these effects of statins or those independent of cholesterol decrease account for the protection against myocardial ischemia-reperfusion (I/R) injury. In this study, we investigated the effect of 5-day treatment with simvastatin (10 mg/kg) in Langendorff-perfused hearts of healthy control (C) and diabetic-hypercholesterolemic (D-H; streptozotocin + high fat-cholesterol diet, 5 days) rats subjected to 30-min global ischemia followed by 40-min reperfusion for the examination of postischemic contractile dysfunction and reperfusion-induced ventricular arrhythmias or to 30-min (left anterior descending) coronary artery occlusion and 2-h reperfusion for the infarct size determination (IS; tetrazolium staining). Postischemic recovery of left ventricular developed pressure (LVDP) in animals with D-H was improved by simvastatin therapy (62.7+/-18.2 % of preischemic values vs. 30.3+/-5.7 % in the untreated D-H; P<0.05), similar to the values in the simvastatin-treated C group, which were 2.5-fold higher than those in the untreated C group. No ventricular fibrillation occurred in the simvastatin-treated C and D-H animals during reperfusion. Likewise, simvastatin shortened the duration of ventricular tachycardia (10.2+/-8.1 s and 57.8+/-29.3 s in C and D-H vs. 143.6+/-28.6 s and 159.3+/-44.3 s in untreated C and D-H, respectively, both P<0.05). The decreased arrhythmogenesis in the simvastatin-treated groups correlated with the limitation of IS (in % of risk area) by 66 % and 62 % in C and D-H groups, respectively. However, simvastatin treatment decreased plasma cholesterol levels neither in the D-H animals nor in C. The results indicate that other effects of statins (independent of cholesterol lowering) are involved in the improvement of contractile recovery and attenuation of lethal I/R injury in both, healthy and diseased individuals.

The role of NO in ischemia/reperfusion injury in isolated rat heart.

UNLABELLED: Nitric oxide (NO) is an important regulator of myocardial function and vascular tone under physiological conditions. However, its role in the pathological situations, such as myocardial ischemia is not unequivocal, and both positive and negative effects have been demonstrated in different experimental settings including human pathology. The aim of the study was to investigate the role of NO in the rat hearts adapted and non-adapted to ischemia. Isolated Langendorff-perfused hearts were subjected to test ischemic (TI) challenge induced by 25 min global ischemia followed by 35 min reperfusion. Short-term adaptation to ischemia (ischemic preconditioning, IP) was evoked by 2 cycles of 5 min ischemia and 5 min reperfusion, before TI. Recovery of function at the end of reperfusion and reperfusion-induced arrhythmias served as the end-points of injury. Coronary flow (CF), left ventricular developed pressure (LVDP), and dP/dt(max) (index of contraction) were measured at the end of stabilization and throughout the remainder of the protocol until the end of reperfusion. The role of NO was investigated by subjecting the hearts to 15 min perfusion with NO synthase (NOS) inhibitor L-NAME (100 mmol/l), prior to sustained ischemia. At the end of reperfusion, LVDP in the controls recovered to 29.0 +/- 3.9 % of baseline value, whereas preconditioned hearts showed a significantly increased recovery (LVDP 66.4 +/- 5.7 %, p < 0.05). Recovery of both CF and dP/dt(max) after TI was also significantly higher in the adapted hearts (101.5 +/- 5.8 % and 83.64 +/- 3.92 % ) as compared with the controls (71.9 +/- 6.3 % and 35.7 +/- 4.87 %, respectively, p < 0.05). NOS inhibition improved contractile recovery in the non-adapted group (LVDP 53.8 +/- 3.1 %; dP/dt(max) 67.5 +/- 5.92 %) and increased CF to 82.4 +/- 5.2 %. In contrast, in the adapted group, it abolished the protective effect of IP (LVDP 31.8 +/- 3.1 %; CF 70.3 +/- 3.4 % and dP/dt(max) 43.25 +/- 2.19 %). Control group exhibited 100 % occurrence of ventricular tachycardia (VT), 57 % incidence of ventricular fibrillation (VF) - 21 % of them was sustained VF (SVF); application of L-NAME attenuated reperfusion arrhythmias (VT 70 %, VF 20 %, SVF 0 %). Adaptation by IP also reduced arrhythmias, however, L-NAME in the preconditioned hearts increased the incidence of arrhythmias (VT 100 %, VF 58 %, SVF 17 %). IN CONCLUSION: our results indicate that administration of L-NAME might be cardioprotective in the normal hearts exposed to ischemia/reperfusion (I/R) alone, suggesting that NO contributes to low ischemic tolerance in the non-adapted hearts. On the other hand, blockade of cardioprotective effect of IP by L-NAME points out to a dual role of NO in the heart: a negative role in the non-adapted myocardium subjected to I/R, and a positive one, due to its involvement in the mechanisms of protection triggered by short-term cardiac adaptation by preconditioning.

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.

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.