Hyperosmotic environment blunts effectivity of ischemic preconditioning against ischemia-reperfusion injury and improves ischemic tolerance in non-preconditioned isolated rat hearts.

Several studies have shown that diabetes mellitus modulates heart resistance to ischemia and abrogates effectivity of cardioprotective interventions, such as ischemic preconditioning (IP). The aim of this study was to evaluate whether the effect of hyperglycemic conditions on the severity of ischemia-reperfusion (I/R) injury in preconditioned and non-preconditioned hearts (controls, C) is related to changes in osmotic activity of glucose. Experiments were performed in isolated rat hearts perfused according to Langendorff exposed to 30-min coronary occlusion/120-min reperfusion. IP was induced by two cycles of 5-min coronary occlusion/5-min reperfusion, prior to the long-term I/R. Hyperosmotic (HO) state induced by an addition of mannitol (11 mmol/l) to a standard Krebs-Henseleit perfusion medium significantly decreased the size of infarction and also suppressed a release of heart fatty acid binding protein (h-FABP - biomarker of cell injury) from the non-IP hearts nearly to 50 %, in comparison with normoosmotic (NO) mannitol-free perfusion. However, IP in HO conditions significantly increased the size of infarction and tended to elevate the release of h-FABP to the effluent from the heart. The results indicate that HO environment plays a cardioprotective role in the ischemic myocardium. On the other hand, increased osmolarity, similar to that in the hyperglycemic conditions, may play a pivotal role in a failure of IP to induce cardioprotection in the diabetic myocardium.

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.

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.

Distinct effects of acute pretreatment with lipophilic and hydrophilic statins on myocardial stunning, arrhythmias and lethal injury in the rat heart subjected to ischemia/reperfusion.

Although both lipophilic and more hydrophilic statins share the same pathway of the inhibition of HMG-CoA reductase, their pleiotropic cardioprotective effects associated with the ability to cross cellular membranes, including membranes of heart cells, may differ. To test this hypothesis, isolated rat hearts were Langendorff-perfused either with simvastatin (S, 10 micromol/l) or pravastatin (P, 30 micromol/l), 15 min prior to ischemia. Control untreated hearts (C) were perfused with perfusion medium only. Postischemic contractile dysfunction, reperfusion-induced ventricular arrhythmias and infarct size were investigated after exposure of the hearts to 30-min global ischemia and 2-h reperfusion. Both lipophilic S and hydrophilic P reduced the severity of ventricular arrhythmias (arrhythmia score) from 4.3 +/- 0.2 in C to 3.0 +/- 0 and 2.7 +/- 0.2 in S and P, respectively, (both P < 0.05), decreased the duration of ventricular tachycardia and suppressed ventricular fibrillation. Likewise, the extent of lethal injury (infarct size) determined by tetrazolium staining and expressed in percentage of risk area, was significantly lower in both treated groups, moreover, the effect of P was more pronounced (27 +/- 2 % and 10 +/- 2 % in S and P groups, respectively, vs. 42 +/- 1 % in C; P < 0.05). In contrast, only S, but not P, was able to improve postischemic recovery of left ventricular developed pressure (LVDP; 48 +/- 12 % of preischemic values vs. 25 +/- 4 % in C and 21 +/ -7 % in P groups; P < 0.05). Our results suggest that differences in water solubility of statins indicating a different ability to cross cardiac membranes may underlie their distinct cardioprotective effects on myocardial stunning and lethal injury induced by ischemia/reperfusion.

Effect of Hemidesmus indicus on ischemia-reperfusion injury in the isolated rat heart.

The root extract of Hemidesmus indicus (Linn.) R. Br. (Asclepiadaceae) (HI) was studied for its cardioprotective effect in Langendorff-perfused rat hearts. HI was perfused for 15 min at a concentration of 0.09 g/L prior to 30 min global ischemia/120 min reperfusion (I/R). Recovery of functional parameters, reperfusion arrhythmias, and infarct size (TTC staining) served as the end-points. After 15 min of perfusion with HI, the left ventricular developed pressure (LVdevP) and HR (heart rate) were not altered significantly (p>0.05), as compared with the pre-drug values. During R, HI showed a significantly higher (p<0.05) recovery of LVdevP at nearly all time points. The recovery of maximal rate of pressure development (+dP/dtmax) and left ventricular end-diastolic pressure (LVEDP) at 40 min of R were significantly better than in non-treated controls. There was also a significant reduction in the total number of ventricular premature beats (VPB) and duration of ventricular tachycardia (VT). HI can protect ischemic myocardium against contractile dysfunction and reperfusion-induced arrhythmias and reduce the extent of irreversible tissue damage following I/R in rat hearts.

Reduced susceptibility to ischemia-induced arrhythmias in the preconditioned rat heart is independent of PI3-kinase/Akt.

We examined the involvement of phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (Akt) in cardioprotective effects of ischemic preconditioning (PC) with particular regards to its role in the protection against ischemia-induced arrhythmias in isolated perfused rat heart. PI3K/Akt inhibitor wortmannin (100 nM) was administered 15 min prior to 30-min regional (left anterior descending coronary artery occlusion) ischemia for the study of ischemic arrhythmias in the hearts perfused at constant coronary flow or prior to 30-min global ischemia followed by 2-h reperfusion for the infarct size (IS) determination (tetrazolium staining) in the hearts perfused at constant pressure. PC procedure (one cycle of ischemia/reperfusion, 5 min each) significantly reduced the total number of ventricular premature complexes (PVC) and severity of arrhythmias (arrhythmia score; AS) over the whole period of left anterior descending coronary artery occlusion in comparison with non-PC controls (PVC 166+/-40; AS 1.6+/-0.2 vs. 550+/-60 and 3.2+/-0.2; respectively; P<0.05). In a setting of global ischemia/reperfusion, PC decreased IS (in % of the left ventricle, LV) by 73 %. Pretreatment with wortmannin modified neither arrhythmogenesis nor IS in the non-PC hearts. Bracketing of PC with wortmannin did not abolish antiarrhythmic protection (PVC 92+/-25; AS 1.7+/-0.2; P<0.05 vs. non-PC hearts). On the other hand, wortmannin increased IS/LV in the PC hearts to 24+/-1.2 % as compared with 9 +/- 0.6 % in the untreated ones (P<0.05). In conclusion, PI3K/Akt inhibition did not affect reduced arrhythmogenesis during ischemia in the PC hearts indicating that in contrast to its positive role in the irreversible myocardial injury, PI3K/Akt activity is not required for protection induced by PC against ischemic arrhythmias in the rat heart.

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.

Protection against ischemia-induced ventricular arrhythmias and myocardial dysfunction conferred by preconditioning in the rat heart: involvement of mitochondrial K(ATP) channels and reactive oxygen species.

Ischemic preconditioning (I-PC) induced by brief episodes of ischemia and reperfusion (I/R) protects the heart against sustained I/R. Although activation of mitochondrial K(ATP) channels (mitoK(ATP)) interacting with reactive oxygen species (ROS) has been proposed as a key event in this process, their role in the antiarrhythmic effect is not clear. This study was designed: 1) to investigate the involvement of mito K(ATP) opening in the effect of I-PC (1 cycle of I/R, 5 min each) on ventricular arrhythmias during test ischemia (TI, 30-min LAD coronary artery occlusion) in Langendorff-perfused rat hearts and subsequent postischemic contractile dysfunction, and 2) to characterize potential mechanisms of protection conferred by I-PC and pharmacological PC induced by mito K(ATP) opener diazoxide (DZX), with particular regards to the modulation of ROS generation. Lipid peroxidation (an indicator of increased ROS production) was determined by measurement of myocardial concentration of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) in non-ischemic controls, non-preconditioned and preconditioned hearts exposed to TI, I-PC alone, as well as after pretreatment with DZX, mito K(ATP) blocker 5-hydroxydecanoate (5-HD) and antioxidant N-acetylcysteine (NAC). Total number of ventricular premature beats (VPB) that occurred in the control hearts (518+/-71) was significantly (P<0.05) reduced by I-PC (195+/-40), NAC (290+/-56) and DZX (168+/-22). I-PC and NAC suppressed an increase in CD and TBARS caused by ischemia indicating lower production of ROS. On the other hand, I-PC and DZX themselves moderately enhanced ROS generation, prior to TI. Bracketing of I-PC with 5-HD suppressed both, ROS production during PC and its cardioprotective effect. In conclusion, potential mechanisms of protection conferred by mito K(ATP) opening in the rat heart might involve a temporal increase in ROS production in the preconditioning phase triggering changes in the pro/antioxidant balance in the myocardium and attenuating ROS production during subsequent prolonged ischemia.

The effect of antioxidant treatment and NOS inhibition on the incidence of ischemia-induced arrhythmias in the diabetic rat heart.

Contrary to clinical trials, experimental studies revealed that diabetes mellitus (DM) may initiate, besides increased myocardial vulnerability to ischemia-reperfusion injury (I/R) and pro/antioxidant dysbalance, development of adaptation leading to an enhanced tolerance to I/R. The aims were to characterize 1) susceptibility to ischemia-induced ventricular arrhythmias in the diabetic rat heart 2) its response to antioxidant N-acetylcysteine (NAC) and a NOS inhibitor L-NAME, and 3) the effect of DM on endogenous antioxidant systems. Seven days after streptozotocin injection (65 mg/kg, i.p.), Langendorff-perfused control (C) and DM hearts were subjected to 30-min occlusion of the LAD coronary artery with or without prior 15-min treatment with L-NAME (100 microM) or NAC (4 mM). Total number of ventricular premature beats (VPB), as well the total duration of ventricular tachycardia (VT) were reduced in the DM group (from 533+/-58 and 37.9+/-10.2 s to 224.3+/-52.6 and 19+/-13.5 s; P<0.05). In contrast to the antiarrhythmic effects of L-NAME and NAC in controls group (VPB 290+/-56 and 74+/-36, respectively; P<0.01 vs. control hearts), application of both drugs in the diabetics did not modify arrhythmogenesis (L-NAME: VPB 345+/-136, VT 25+/-13 s; NAC: VPB 207+/-50, VT 12+/-3.9 s; P>0.05 vs non-treated diabetic hearts). Diabetic state was associated with significantly elevated levels of CoQ10 and CoQ9 (19.6+/-0.8 and 217.3+/-9.5 vs. 17.4+/- 0.5 and 185.0+/-5.0 nmol/g, respectively, in controls; P<0.05), as well as alpha-tocopherol (38.6+/-0.7 vs. 31.5+/-2.1 nmol/g in controls; P<0.01) in the myocardial tissue. It is concluded that early period of DM is associated with enhanced resistance to ischemia-induced arrhythmias. Diabetes mellitus might induce adaptive processes in the myocardium leading to lower susceptibility to antioxidant and L-NAME treatment.

Hypercholesterolemia abrogates an increased resistance of diabetic rat hearts to ischemia-reperfusion injury.

Both, diabetes mellitus (DM) and hypercholesterolemia (HCH) are known as risk factors of ischemic heart disease, however, the effects of experimental DM, as well as of HCH alone, on ischemia/reperfusion-induced myocardial injury are not unequivocal. We have previously demonstrated an enhanced resistance to ischemia-induced arrhythmias in rat hearts in the acute phase of DM. Our objectives were thus to extend our knowledge on how DM in combination with HCH, a model that is relevant to diabetic patients with altered lipid metabolism, may affect the size of myocardial infarction and susceptibility to arrhythmias. A combination of streptozotocin (STZ; 80 mg/kg, i.p.) and the fat-cholesterol diet (1% cholesterol, 1% coconut oil; FCHD) was used as a double-disease model mimicking DM and HCH simultaneosly occurring in humans. Following 5 days after STZ injection and FCHD leading to increased blood glucose and cholesterol levels, anesthetized open-chest diabetic, diabetic-hypercholesterolemic (DM-HCH) and age-matched control rats were subjected to 6-min ischemia (occlusion of LAD coronary artery) followed by 10 reperfusion to test susceptibility to ventricular arrhythmias in the in vivo experiments and to 30-min ischemia and subsequent 2-h reperfusion for the evaluation of the infarct size (IS) in the Langendorff-perfused hearts. The incidence of the most life-threatening ventricular arrhythmia, ventricular fibrillation, was significantly increased in the DM-HCH rats as compared with non-diabetic control animals (100% vs. 50%; p<0.05). Likewise, arrhythmia severity score (AS) was significantly higher in the DM-HCH rats than in the controls (4.9+/-0.2 vs. 3.5+/-0.5; p<0.05), but was not increased in the diabetic animals (AS 3.7+/-0.9; p>0.05 vs. controls). Diabetic hearts exhibited a reduced IS (15.1+/-3.0% of the area at risk vs. 37.6+/-2.8% in the control hearts; p<0.05), however, a combination of DM and HCH increased the size of myocardial infarction to that observed in the controls. In conclusion, HCH abrogates enhanced resistance to ischemia-reperfusion injury in the diabetic rat heart.

Ischemia-reperfusion injury--antiarrhythmic effect of melatonin associated with reduced recovering of contractility.

The effect of melatonin on reperfusion arrhythmias and postischemic contractile dysfunction was studied in the isolated rat heart. 25 min global ischemia was induced and followed by 30 min of reperfusion. Melatonin (10 micromol/l) was present in the perfusion solution during the whole experiment. Experiment revealed protective effect of melatonin on reperfusion-induced arrhythmias--arrhythmia score was significantly lower as well as the total time of arrhythmias duration was significantly shorter in melatonin group than in controls. On the other hand, post-ischemic recovering of contractility was significantly reduced in melatonin group.

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.

Effect of melatonin on the isolated heart in the standard perfusion conditions and in the conditions of calcium paradox.

UNLABELLED: The effect of melatonin (MLT) on the isolated rat heart was studied using the standard perfusion conditions (Langendorff preparation) and model of calcium paradox (Ca(2+)-paradox). Ca(2+)-paradox was induced by 1 minute perfusion with Ca(2+)-free Krebs-Henseleit (KH) solution and subsequent 20 minutes perfusion with a normal Ca(2+)-containing KH solution. In MLT group, MLT (10 micromol/l) was in the perfusion solution throughout the experiment. In controls, there was no MLT. VARIABLES: heart rate, coronary flow, systolic and diastolic pressure, +dP/dt max (index of contractility) and -dP/dt max (index of relaxation) were measured at the end of stabilization, i.e. after 30 minutes of standard perfusion and then in the 5th, 10th, 15th, 20th minute after perfusion with Ca(2+)-free KH solution. RESULTS: There was no difference between MLT group and controls in the standard perfusion conditions at the end of stabilization. After perfusion with Ca(2+)-free KH solution, systolic-diastolic difference (in the 10th, 15th, 20th minute), +dP/dt max (in the 5th, l0th, 15th, 20th minute) and -dP/dt max (in the 15th minute) were significantly decreased in MLT group in comparison to controls. CONCLUSION: Melatonin didn't influence rat isolated heart in standard perfusion conditions but it made the heart more susceptible to Ca(2+)-paradox.

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.

Preconditioning modulates susceptibility to ischemia-induced arrhythmias in the rat heart: the role of alpha-adrenergic stimulation and K(ATP) channels.

A new concept of cardioprotection based on the exploitation of endogenous mechanisms is known as ischemic preconditioning (IPC). It has been hypothesized that substances released during brief ischemic stress (e.g. catecholamines) stimulate the receptors and trigger multiple cell signaling cascades. Opening of ATP-sensitive K+ channels [K(ATP)] has been suggested as a possible final step in the mechanisms of protection. In this study, the role of adrenergic activation was tested in Langendorff-perfused rat hearts subjected to test ischemia (TI; 30 min occlusion of LAD coronary artery) by: 1) mimicking IPC (5 min ischemia, 10 min reperfusion) with short-term (5 min) administration of norepinephrine (NE, 1 microM), 15 min prior to TI; 2) blockade with beta- or alpha1-receptor antagonists, propranolol (10 microM) and prazosin (2 microM), respectively, applied 15 min prior to TI during IPC. The role of K(ATP) opening was examined by perfusion with a K(ATP) blocker glibenclamide (10 microM) during IPC. Both IPC and NE-induced PC effectively reduced the incidence of ventricular tachycardia (VT) to 33% and 37%, respectively, vs 100% in the non-PC controls, whereby ventricular fibrillation (VF) was totally abolished by IPC and markedly suppressed by PC with NE (0% and 10%, respectively, vs 70% in the non-PC hearts; P < 0.05). The severity of arrhythmias (arrhythmia score, AS) was also markedly attenuated by both interventions (IPC: AS 1.7 +/- 0.4; NE-PC: AS 1.8 +/- 0.3 vs AS 4.1 +/- 0.2 in the controls; P < 0.05). Protection was not suppressed by propranolol (VT 28%; VF 14%; AS 2.2 +/- 0.6), whereas prazosin reversed the protective effect of PC (VT 83%; VF 67%; AS 4.0 +/- 0.8). Antiarrhythmic protection afforded by NE-PC was abolished by pretreatment of rats with pertussis toxin (25 microg/kg, i.p.) given 48 h prior to the experiments. Glibenclamide did not suppress the IPC-induced protection. In conclusion, the sensitivity of the rat heart to ischemic arrhythmias can be modulated by IPC. Protection is mediated via stimulation of alpha1-adrenergic receptors coupled with Gi-proteins but glibenclamide-sensitive K(ATP) channels do not appear to be involved in the mechanisms of antiarrhythmic protection in this model.

Hypertension-related intermyocyte junction remodelling is associated with a higher incidence of low-K(+)-induced lethal arrhythmias in isolated rat heart.

The aim of this study was to characterise the arrhythmogenic mechanisms involved in hypokalaemia-induced sustained ventricular fibrillation (SVF), in hypertensive rats. The hearts from rats with hypertension induced by the nitric oxide synthase inhibitor L-NAME, and age-matched normotensive controls, were perfused in Langendorff mode with oxygenated Krebs-Henseleit solution followed by a K(+)-deficient solution. In additional experiments, free intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2 in conjunction with an epicardial optical probe. The epicardial electrocardiogram was continuously monitored during all experiments. The gap junction protein connexin-43 and the ultrastructure of the cardiomyocytes were examined, and selected enzyme activities were measured in situ. There was a higher incidence of low-K(+)-induced SVF in the hearts of hypertensive compared to normotensive rats (83 % vs. 33 %, P < 0.05). Perfusion with a low-K(+)-containing solution lead to elevation of diastolic [Ca(2+)](i) that was accompanied by premature beats, bigeminy, ventricular tachycardia and transient ventricular fibrillation. These events occurred earlier with increased incidence and duration in the hearts of hypertensive rats (arrhythmia scores: hypertensive, 4.9 +/- 0.7; normotensive, 3.1 +/- 0.1; P < 0.05), which exhibited apparent remodelling accompanied by a significant decrease in the density of connexin-43-positive gap junctions. Moreover, low-K(+)-related myocardial changes, including local impairment of intermyocyte junctions, ultrastructural alterations due to Ca(2+) overload and intercellular uncoupling, and decreased enzyme activities were more pronounced and more dispersed in hypertensive than normotensive rats. In conclusion, nitric oxide-deficient hypertension is associated with decreased myocardial coupling at gap junctions. The further localised deterioration of junctional coupling, due to low-K(+)-induced Ca(2+) disturbances, as well as spatial heterogeneity of myocardial alterations including interstitial fibrosis, probably provide the mechanisms for re-entry and sustaining ventricular fibrillation.

Modulation of cAMP level by tedisamil in guinea pig heart.

Tedisamil is antiarrhythmic class III drug with antifibrillating/defibrillating potency linked to enhancement of intermyocyte gap junctional electrical coupling most likely via its sympathomimetic cAMP-related mechanisms. This study was designed to examine the effect of tedisamil on cAMP level in guinea pig hearts in vivo and in vitro in Langendorff preparation. The drug was administered either as a bolus into vena jugularis in dosage 1.0 and 1.5 mg/kg or into the perfusion solution at a concentration of 1.5 x 10(-6) mol/l. In additional experiments, this period was followed by brief 10 min global ischemia, induced by clamping of the aorta or perfusion. After 10 min from the onset of tedisamil administration as well as after 10 min of ischemia the ventricular tissue was immediately frozen for cAMP immunoassay. Tedisamil caused in normal heart small but significant dose-dependent increase of myocardial cAMP (pmol/mg) level in vivo 1.8 and 2.5 vs. 1.4 as well as in vitro 1.1 vs. 0.8 (p < 0.05) conditions. Ischemia itself induced accumulation of cAMP in both, in vivo and in vitro experiments, 2.6 vs. 1.4 and 1.3 vs. 0.8, respectively. The preischemic elevation of cAMP by tedisamil was not potentiated by following ischemia, on the contrary, decline of the cyclic nucleotide was detected comparing to ischemia itself. In conclusion, tedisamil increased cAMP level in normal heart and prevented additional ischemia-related elevation of this nucleotide. The results indicate modulation of myocardial cAMP level by tedisamil, which may account for its protective effect on gap junctional electrical coupling.

Acute diabetes modulates response to ischemia in isolated rat heart.

Diabetic hearts are suggested to exhibit either increased or lower sensitivity to ischemia. Detrimental effects of prolonged ischemia can be attenuated by preconditioning, however, relatively little is known about its effects in the diseased myocardium. This study was designed to test the susceptibility to ischemia-induced arrhythmias and the effect of preconditioning in the diabetic heart. Rats were made diabetic with streptozotocin (45 mg/kg, i.v.). After 1 week, isolated Langendorff-perfused hearts were subjected to 30 min occlusion of LAD coronary artery without or with preceding preconditioning induced by one cycle of 5 min ischemia and 10 min reperfusion. Glycogen and lactate contents were estimated in the preconditioned and non-preconditioned hearts before and after ischemia. Diabetic hearts were more resistant to ischemia-induced arrhythmias: incidence of ventricular tachycardia (VT) decreased to 42% and only transient ventricular fibrillation (VF) occurred in 17% of the hearts as compared to the non-diabetic controls (VT 100% and VF 70% including sustained VF 36%; p < 0.05). Preconditioning effectively suppressed the incidence and severity of arrhythmias (VT 33%, VF 0%) in the normal hearts. However, this intervention did not confer any additional protection in the diabetic hearts. Despite higher glycogen content in the diabetic myocardium and greater glycogenolysis during ischemia, production of lactate in these hearts was significantly lower than in the controls. Preconditioning caused a substantial decrease in the accumulation of lactate in the normal hearts, whereby in the diabetic hearts, this intervention did not cause any further reduction in the level of lactate. In conclusion, diabetic rat hearts exhibit lower susceptibility to ischemic injury and show no additional response to preconditioning. Reduced production of glycolytic metabolites during ischemia can account for the enhanced resistance of diabetic hearts to ischemia as well as for the lack of further protection by preconditioning.