Effects of forskolin on contractile responses and protein phosphorylation in the isolated perfused rat heart.

Continuous perfusion of rat hearts with concentrations of forskolin between 0.1 and 12 microM resulted in transient increases in tension after 45 s, followed by a return to the control value after 5 min. In contrast, the content of cyclic AMP increased linearly with time over this period, reaching values up to 35 times control after 5 min. Increases in contractile force, intracellular cyclic AMP concentration and the proportion of phosphorylase in the a form were dependent on the concentration of forskolin when measured 45 s and 120 s after initiation of perfusion. In hearts perfused for 45 s with various concentrations of forskolin, the measured cyclic AMP-dependent protein kinase activity ratio and phosphorylase a content for a given measured intracellular cyclic AMP concentration were both much less than the corresponding values in hearts perfused for 30 s with various concentrations of isoprenaline. The phosphorylation of the contractile proteins troponin-I and C-protein also showed a concentration-dependent increase in hearts perfused with forskolin. There was a strong correlation between the cyclic AMP-dependent protein kinase activity ratios and the phosphorylation of the contractile proteins under all perfusion conditions. These results suggest that cyclic AMP is compartmented in perfused rat heart, and that much of the cyclic AMP produced in response to forskolin is unavailable to activate cyclic AMP-dependent protein kinase.     

Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart.

Isolated Langendorff-perfused rat hearts after 20 min of normoxic perfusion in the presence of 2.5 mM Ca++ and 11 mM glucose were subjected to 30 min of global normothermic ischemia followed by 30 min of normoxic reperfusion with the starting buffer. At the end of each perfusion condition, hearts were freeze-clamped and deproteinized by 0.6 M HClO4. Two-hundred microL of the neutralized tissue extracts were analyzed by a recently developed high-performance liquid chromatography (HPLC) method for the simultaneous determination of malondialdehyde (MDA), ascorbic acid, and adenine nucleotides. By means of this analytical technique, it was possible to demonstrate that MDA is undetectable in control hearts. In contrast, 30 min of ischemia induced a modest production of MDA (0.012 mumol/g dw), while a large amount of MDA (0.103 mumol/g dw) was observed in reperfused hearts. Values referring to ascorbic acid showed that the concentration of this antioxidant progressively decreased from 1.190 (control hearts) to 0.837 (ischemic hearts) and to 0.595 mumol/g dw (reperfused hearts). The overall conclusions of this study are that reperfusion induces an oxidative stress to the isolated myocardium, a decrease of ascorbate, and an increase of lipid peroxidation. Therefore, by means of a proper analytical method, MDA may represent a valid biochemical parameter to demonstrate the relationship between myocardial reperfusion and a detectable tissue damage.     

Effects of dietary polyunsaturated fatty acids and hepatic steatosis on the functioning of isolated working rat heart under normoxic conditions and during post-ischemic reperfusion

The purpose of this study was to modify the amount of 22:4 n-6, 22:5 n-6 and 20:5 n-3 in cardiac phospholipids and to evaluate the influence of these changes on the functioning of working rat hearts and mitochondrial energy metabolism under normoxic conditions and during postischemic reperfusion. The animals were fed one of these four diets: (i) 10% sunflower seed oil (SSO); (ii) 10% SSO + 1% cholesterol; (iii) 5% fish oil (FO, EPAX 3000TG, Pronova) + 5% SSO; (iv) 5% FO + 5% SSO + 1% cholesterol. Feeding n-3 PUFA decreased n-6 PUFA and increased n-3 PUFA in plasma lipids. In the phospholipids of cardiac mitochondria, this dietary modification also induced a decrease in the n-6/n-3 PUFA ratio. Cholesterol feeding induced marked hepatic steatosis (HS) characterized by the whitish appearance of the liver. It also brought about marked changes in the fatty acid composition of plasma and mitochondrial phospholipids. These changes, characterized by the impairment of 5- and 6-desaturases, were more obvious in the SSO-fed rats, probably because of the presence of the precursor of the n-6 family (linoleate) in the diet whereas the FO diet contained large amounts of eicosapentaenoic and docosahexaenoic acids. In the mitochondrial phospholipids of SSO-fed rats, the (22:4 n-6 + 22:5 n-6) to 18:2 n-6 ratio was decreased by HS, without modification of the proportion of 20:4 n-6. In the mitochondrial phospholipids of FO-fed rats, the amount of 20:5 n-3 tended to be higher (+56%). Cardiac functioning was modulated by the diets. Myocardial coronary flow was enhanced by HS in the SSO-fed rats, whereas it was decreased in the FO-fed animals. The rate constant k⁰¹² representing the activity of the adenylate kinase varied in the opposite direction, suggesting that decreased ADP concentrations could cause oxygen wasting through the opening of the permeability transition pore. The recovery of the pump function tended to be increased by n-3 PUFA feeding (+22%) and HS (+45%). However, the release of ascorbyl free radical during reperfusion was not significantly modified by the diets. Conversely, energy production was increased by ischemia/reperfusion in the SSO group, whereas it was not modified in the FO group. This supports greater ischemia/reperfusion-induced calcium accumulation in the SSO groups than in the FO groups. HS did not modify the mitochondrial energy metabolism during ischemia/reperfusion. Taken together, these data suggest that HS- and n-3 PUFA-induced decrease in 22:4 and 22:5 n-6 and increase in 20:5 n-3 favor the recovery of mechanical activity during post-ischemic reperfusion.     

Insulin improves postischemic recovery of function through PI3K in isolated working rat heart

Insulin improves contractile function after ischemia, but does not increase glucose uptake in the isolated working rat heart. We tested the hypothesis that the positive inotropic effect of insulin is independent of the signaling pathway responsible for insulin-stimulated glucose uptake. We inhibited this pathway at the level of phosphatidyl inositol 3-kinase (PI3K) with wortmannin. Hearts were perfused for 70 min at physiological workload with Krebs-Henseleit buffer containing [2-³H] glucose (5 mM, 0.05 Ci/ml) and oleate (0.4 mM, 1% BSA) in the presence (WM, n = 5) or absence (control, n = 7) of wortmannin (WM, 3 mol/L). After 20 min, hearts were subjected to 15 min of total global ischemia followed by 35 min of reperfusion. Insulin (1 mU/ml) was added at the beginning of reperfusion (WM + insulin n = 8, insulin n = 8). Cardiac power before ischemia was 8.1 ± 0.7 mW. Recovery of contractile function after ischemia was significantly increased in the presence of insulin (73.5 ± 8.9% vs. 38.5 ± 6.7%, p < 0.01). The addition of wortmannin completely abolished the effect of insulin on recovery (32.6 ± 6.4%). Glucose uptake was 1.84 ± 0.32 mol/min/g dry before ischemia and was slightly elevated during reperfusion (2.68 ± 0.35 mol/min/g dry, n.s.). Insulin did not affect postischemic glucose uptake. In the presence of wortmannin, glucose uptake was lowest during reperfusion (n.s.). The results suggest that PI3K is involved in the insulin-induced improvement in postischemic recovery of contractile function. This effect of insulin is independent of its effect on glucose uptake.     

Pharmacological evidence for catecholamine-independent contractile effects of X-537A on the isolated working rat heart preparation.

The ionophore X-537A increased heart rate and contractility of the isolated, working rat heart preparation. The increased heart rate appeared to be caused solely by release of catecholamines as the response was completely eliminated by reserpine pretreatment or addition of propranolol to the perfusate. The inotropic response, however, had an apparent catecholamine-independent component as neither propranolol, nor propranol in combination with phentolamine, completely eliminated the inotropic response to X-537A. On the other hand, reserpine pretreatment did abolish the inotropic effect of the ionophore but this action appeared to be a nonspecific one as the responses to norepinephrine and to CaCl2 were substantially diminished.     

Effects of the ionophore grisorixin on myocardial function and metabolism in isolated perfused working rat heart under normoxic and hypoxic conditions

The effects of grisorixin, a monocarboxylic ionophore, were studied on isolated working rat hearts perfused with a suspension of washed pig erythrocytes (10% hematocrit). Grisorixin (2.5 microM) induced a transient stimulation of heart work, maximal at 5 min, expressed by an increase in heart rate (+21%) and aortic flow (+17%) and by an increase in coronary flow, maximal at 10 min (+47%). Concomitantly, myocardial Vo2 was slightly enhanced and the myocardial creatine phosphate level dropped (2 min). The lactate production increased by 82% (5 min) then dropped to the control value (10 min) and increased again till the 45th min (+211%), indicating a cardiac metabolic drift towards anaerobic glycolysis due to partial inhibition of the oxidative metabolism. Owing to its properties as an ionophore, grisorixin also induced a strong and rapid increase of potassium concentration in the perfusate and a decrease of sodium. Grisorixin was tested on hearts submitted to 20 min of hypoxic conditions. The hypoxia was rather mild and induced only very slight modifications of the ultrastructure. In the control series, heart rate and aortic flow decreased regularly while coronary flow and lactate production increased. Upon reoxygenation, the heart performances were rapidly restored. Grisorixin was administered according to four different protocols. When injected at the onset of hypoxia or 5 min later, it was able to maintain the aortic flow during the first minutes and induce a higher coronary dilation. These beneficial effects were short-lasting and no deleterious effects were found on the ultrastructure of hearts subjected to grisorixin whether after hypoxia or after reoxygenation.     

Effect of exogenous N-stearoylethanolamine on fatty acid composition of individual phospholipids in the isolated rat heart under postischemic reperfusion

Changes of the individual phospholipid fatty acid composition under the normothermal short-time ischemia with following reperfusion were investigated. Modification of the phospholipid fatty acid (FA) composition under ischemia-reperfusion didn't bear total character and was more manifested in cardiolipin (CL) and phosphatidylethanolamine (PE). The decrease of short chain FA in these phospholipids (more than by 50%) was observed. The amount of unsaturated FA included in CL increased and whole the saturated ones decreased. This caused the rise of the unsaturation index. The selective type of the changes suggested that they had an adaptive character. The addition of the N-stearoilethanolamine (NSE) into the perfusion solution caused a normalization of saturated and unsaturated FA relative amount, as well as of omega-3 and omega-9 FA level in CL. The modification of the FA composition of phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI) was also found. The quantity of arachidonic acid in PC increased by 26% and the amount of stearinic acid enhanced in PS. The labeled N-([1-(14)C]-palmitoil)-ethanolamine was found in different lipid classes of the rat organs immediately 5 min following intraperitoneal injection. Approximately 1/3 of all incorporated label accumulated in the phospholipid fraction, and more than 50% of the labels were found in CL.     

Inhibition of PLC improves postischemic recovery in isolated rat heart

The Ca2+-dependent PLC converts phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. Because these products modulate Ca2+ movements in the myocardium, PLC may also contribute to a self-perpetuating cycle that exacerbates cardiomyocyte Ca2+-overload and subsequent cardiac dysfunction in ischemia-reperfusion (I/R). Although we have reported that I/R-induced changes in PLC isozymes might contribute to cardiac dysfunction, the present study was undertaken to examine the beneficial effects of the PLC inhibitor, U-73122, as well as determining the role of Ca2+ on the I/R-induced changes in PLC isozymes. Isolated rat hearts were subjected to global ischemia 30 min, followed by 5 or 30 min of reperfusion. Pretreatment of hearts with U-73122 (0.5 microM) significantly inhibited DAG and Ins(1,4,5)P3 production in I/R and was associated with enhanced recovery of cardiac function as indicated by measurement of left ventricular (LV) end-diastolic pressure (EDP), LV diastolic pressure (LVDP), maximum rate of pressure development (+dP/dtmax), and maximum rate of LV pressure decay (-dP/dtmax). Verapamil (0.1 microM) partially prevented the increase in sarcolemmal (SL) PLC-beta1 activity in ischemia and the decrease in its activity during the reperfusion phase as well as elicited a partial protection of the depression in SL PLC-delta1 and PLC-gamma1 activities during the ischemic phase and attenuated the increase during the reperfusion period. Although these changes were associated with an improved myocardial recovery after I/R, verapamil was less effective than U-73122. Perfusion with high Ca2+ resulted in the activation of the PLC isozymes studied and was associated with a markedly increased LVEDP and reduced LVDP, +dP/dtmax, and -dP/dtmax. These results suggest that inhibition of PLC improves myocardial recovery after I/R.     

Myocardial contractile function during postischemic low-flow reperfusion: critical thresholds of NADH and O2 delivery

The degree of myocardial oxygen delivery (Do2) that is necessary to reestablish functional contractile activity after short-term global ischemia in heart is not known. To determine the relationship between Do2 and recovery of contractile and metabolic functions, we used tissue NADH fluorometric changes to characterize adequacy of reperfusion flow. Isolated perfused rat hearts were subjected to global ischemia and were reperfused at variable flow rates that ranged from 1 to 100% of baseline flow. Myocardial function and tissue NADH changes were continuously measured. NADH fluorescence rapidly increased and plateaued during ischemia. A strong inverse logarithmic correlation between NADH fluorescence and reperfusion Do2 was demonstrated (r = -0.952). Left ventricular function (rate-pressure product) was inversely related to NADH fluorescence at reperfusion flows from 25 to 100% of baseline (r = -0.922) but not at lower reperfusion flow levels. An apparent reperfusion threshold of 25% of baseline Do2 was necessary to resume contractile function. At very low reperfusion flows (1% of baseline), another threshold flow was identified at which NADH levels increased beyond that observed during global ischemia (3.4 +/- 3.0%, means +/- SE, n = 9), which suggests further reduction of the cellular redox state. This NADH increase at 1% of baseline reperfusion flow was blocked by removing glucose from the perfusate. NADH fluorescence is a sensitive indicator of myocardial cellular oxygen utilization over a wide range of reperfusion Do2 values. Although oxygen is utilized at very low flow rates, as indicated by changes in NADH, a critical threshold of approximately 25% of baseline Do2 is necessary to restore contractile function after short-term global ischemia.     

Evidence of myofibrillar protein oxidation induced by postischemic reperfusion in isolated rat hearts

Although the contribution of reactive oxygen species to myocardial ischemia is well recognized, the possible intracellular targets, especially at the level of myofibrillar proteins (MP), are not yet fully characterized. To assess the maximal extent of oxidative degradation of proteins, isolated rat hearts were perfused with 1 mM H(2)O(2). Subsequently, the MP maximally oxidative damage was compared with the effects produced by 1) 30 min of no-flow ischemia (I) followed in other hearts by 3 min of reperfusion (I/R); and 2) I/R in the presence of a potent antioxidant N-(2-mercaptopropionyl)glycine (MPG). Samples from the H(2)O(2) group electrophoresed under nonreducing conditions and probed with actin, desmin, or tropomyosin monoclonal antibodies showed high-molecular mass complexes indicative of disulfide cross-bridges along with splitting and thickening of tropomyosin and actin bands, respectively. Only these latter changes could be detected in I/R samples and were prevented by MPG. Carbonyl groups generated by oxidative stress on MP were detected by Western blot analysis (oxyblot) under optimized conditions. The analyses showed one major band corresponding to oxidized actin, the density of which increased 1.2-, 2.8-, and 6.8-fold in I, I/R, and H(2)O(2) groups, respectively. The I/R-induced increase was significantly reduced by MPG. In conclusion, oxidative damage of MP occurs on reperfusion, although at a lower extent than in H(2)O(2) perfused hearts, whereas oxidative modifications could not be detected in ischemic hearts. Furthermore, the inhibition of MP oxidation by MPG might underlie the protective efficacy of antioxidants.     

Utilization of energy-providing substrates in the isolated working rat heart.

1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.     

Measurement and characterization of postischemic free radical generation in the isolated perfused heart

Electron paramagnetic resonance spectroscopy has been applied to measure radical generation in the postischemic heart; however, there is controversy regarding the methods used and the conclusion as to whether radicals are generated. In order to resolve this controversy, direct and spin trapping measurements of the time course and mechanisms of radical generation were performed in isolated perfused rabbit hearts. In reperfused tissue, 3 prominent radical signals are observed: A, isotropic g = 2.004 suggestive of a semiquinone; B, anisotropic g parallel = 2.033 and g perpendicular = 2.005 suggestive of ROO.; and C, a triplet g = 2.000 and aN = 24 G suggestive of a nitrogen centered radical. B and C, however, are highly labile and disappear at temperatures probably encountered in some previous studies. In normally perfused hearts, A is observed with only small amounts of B and C. During ischemia, B and C increase reaching a maximum after 45 min while A decreases. On reflow with oxygenated perfusate all 3 signals increase. With varying duration of ischemia and reflow, peak signal intensities occurred after 15 s of reflow following 30 min of ischemia. Reperfusion with superoxide dismutase, deferoxamine, or mannitol abolished the reperfusion increase of B. Measurements performed with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrated a similar time course of radical generation with prominent DMPO-OH and DMPO-R signals peaking between 10 and 20 s of reflow. Superoxide dismutase and deferoxamine also quenched these signals. Thus, .O2- derived .OH, R., and ROO. radicals are generated in postischemic myocardium. While the experimental techniques used can result in loss of intrinsic radicals and generation of extraneous radicals, with proper care and controls valid measurements of free radicals in biological tissues can be performed.     

Glutathione disulfide as an index of oxidative stress during postischemic reperfusion in isolated rat hearts

The objectives of this study were to determine 1) whether reactive oxygen species generated upon postischemic reperfusion lead to oxidative stress in rat hearts, and 2) whether an exogenous prooxidant present in the early phase of reperfusion causes additional injury. Isolated buffer-perfused rat hearts were subjected to 30 min of hypothermic no-flow ischemia followed by 30 min of reperfusion. Increased myocardial content of glutathione disulfide (GSSG) and increased active transport of GSSG were used as indices of oxidative stress. To impose a prooxidant load, cumene hydroperoxide (20 M) was administered during the first 10 min of reperfusion to a separate group of postischemic hearts. Reperfusion after 30 min of hypothermic ischemia resulted in a recovery of myocardial ATP from 28% at end-ischemia to 50–60%, a release of 5% of total myocardial LDH, and an almost complete recovery of both coronary flow rate and left ventricular developed pressure. After 5 and 30 min of reperfusion, neither myocardial content of GSSG nor active transport of GSSG were increased. These indices were increased, however, if cumene hydroperoxide was administered during early reperfusion. After stopping the administration of cumene hydroperoxide, myocardial GSSG content returned to control values and GSH content increased, indicating an unimpaired glutathione reductase reaction. Despite the induction of oxidative stress, reperfusion with cumene hydroperoxide did not cause additional metabolic, structural, or functional injury when compared to reperfusion without cumene hydroperoxide. We conclude that reactive oxygen species generated upon postischemic reperfusion did not lead to oxidative stress in isolated rat hearts. Moreover, even a superimposed prooxidant load during early reperfusion did not cause additional injury.     

Glibenclamide improves postischemic recovery of myocardial contractile function in trained and sedentary rats.

In this study, we sought to determine whether there was any evidence for the idea that cardiac ATP-sensitive K+ (K(ATP)) channels play a role in the training-induced increase in the resistance of the heart to ischemia-reperfusion (I/R) injury. To do so, the effects of training and an K(ATP) channel blocker, glibenclamide (Glib), on the recovery of left ventricular (LV) contractile function after 45 min of ischemia and 45 min of reperfusion were examined. Female Sprague-Dawley rats were sedentary (Sed; n = 18) or were trained (Tr; n = 17) for >20 wk by treadmill running, and the hearts from these animals used in a Langendorff-perfused isovolumic LV preparation to assess contractile function. A significant increase in the amount of 72-kDa class of heat shock protein was observed in hearts isolated from Tr rats. The I/R protocol elicited significant and substantial decrements in LV developed pressure (LVDP), minimum pressure (MP), rate of pressure development, and rate of pressure decline and elevations in myocardial Ca(2+) content in both Sed and Tr hearts. In addition, I/R elicited a significant increase in LV diastolic stiffness in Sed, but not Tr, hearts. When administered in the perfusate, Glib (1 microM) elicited a normalization of all indexes of LV contractile function and reductions in myocardial Ca(2+) content in both Sed and Tr hearts. Training increased the functional sensitivity of the heart to Glib because LVDP and MP values normalized more quickly with Glib treatment in the Tr than the Sed group. The increased sensitivity of Tr hearts to Glib is a novel finding that may implicate a role for cardiac K(ATP) channels in the training-induced protection of the heart from I/R injury.     

Ivabradine reduces heart rate while preserving metabolic fluxes and energy status of healthy normoxic working hearts

Heart rate reduction (HRR) is an important target in the management of patients with chronic stable angina. Most available drugs for HRR, such as β-blockers, have adverse effects, including on cardiac energy substrate metabolism, a well-recognized determinant of cardiac homeostasis. This study aimed at 1) testing whether HRR by ivabradine (IVA) alters substrate metabolism in the healthy normoxic working heart and 2) comparing the effect of IVA with that of the β-blocker metoprolol (METO). This was assessed using our well-established model of ex vivo mouse heart perfusion in the working mode, which enables concomitant evaluation of myocardial contractility and metabolic fluxes using (13)C-labeled substrates. Hearts were perfused in the absence (controls; n = 10) or presence of IVA (n = 10, 3 μM) with or without atrial pacing to abolish HRR in the IVA group. IVA significantly reduced HR (35 ± 5%) and increased stroke volume (39 ± 9%) while maintaining similar cardiac output, contractility, power, and efficiency. Effects of IVA on HR and stroke volume were reversed by atrial pacing. At the metabolic level, IVA did not impact on substrate selection to citrate formation, rates of glycolysis, or tissue levels of high-energy phosphates. In contrast, METO, at concentrations up to 40 μM, decreased markedly cardiac function (flow: 25 ± 6%; stroke volume: 30 ± 10%; contractility: 31 ± 9%) as well as glycolysis (2.9-fold) but marginally affected HR. Collectively, these results demonstrate that IVA selectively reduces HR while preserving energy substrate metabolism of normoxic healthy working mouse hearts perfused ex vivo, a model that mimics to some extent the denervated transplanted heart. Our results provide the impetus for testing selective HRR by IVA on cardiac substrate metabolism in pathological models.     

Transvalvular left ventricular pressure measurement in the isolated working rat heart.

A method of continuously measuring left ventricular (LV) pressure in an isolated buffer-perfused working rat heart is described. Transvalvular placement of a micromanometer through the aorta is the unique feature of this procedure. Advantages include catheter stability and lack of myocardial trauma. Changes in cardiac function were quantified by exposing hearts to either isoproterenol (10(-9) M) or halothane (1.5% vol/vol). To examine if any obstruction to LV outflow was caused by the micromanometer, cardiac performance was assessed during pullback from the ventricle to the aorta. Complications such as aortic insufficiency and ventricular arrhythmias were also studied. The results indicate that the transvalvular placement of a micromanometer can provide continuous, high-fidelity reproduction of LV pressure in this small-organ preparation. The presence of the micromanometer did not significantly alter cardiac performance, and proper catheter placement was achieved easily in a high percentage (> 90%) of cases.     

Acid hydrolase activity and cyclic nucleotide contents in the rat heart during myocardial ischemia and postischemic reperfusion

The acid phosphatase and cathepsin D activities and cAMP and cGMP levels in isolated perfused rat heart were investigated during various periods of ischaemic myocardial injury and postischaemic reperfusion. The effect of phosphodiesterase inhibitor--caffeine was also studied. Free acid hydrolases activities and cyclic nucleotide content were increased under 40 and 60 min ischemia and 20 min postischaemic reperfusion. Addition of 50 microM caffeine to perfusion solution after 30 min of ischaemia resulted in increase of cAMP level, cAMP/cGMP ratio, lysosomal bound activities of acid hydrolase and decrease of free acid hydrolase activities. The obtained results suggested that defect in cAMP synthesis might be present in lysosomal membranes labilization in cardiomyocytes injured during ischaemic conditions. Addition of such agents, as caffeine, which increased heart cAMP level, may be effective in lysosomal membranes stabilization under reversible heart ischaemia and reperfusion.     

Tricarboxylic acid cycle flux and enzyme activities in the isolated working rat heart.

Flux through the tricarboxylic acid cycle was calculated from oxygen consumption in hearts perfused near the physiological work load. Activities of citrate synthase, 2-oxoglutarate dehydrogenase and succinate dehydrogenase were measured in the same hearts. Only the activities of 2-oxoglutarate dehydrogenase correlated with calculated fluxes through the cycle.     

Disorders of the heart contractile function in chronic hemolytic anemia and their prevention

The coronary blood flow and heart contractile function were studied on rats with phenylhydrazine-induced chronic hemolytic anemia. The coronary blood flow in the animals' hearts was increased 2.5-fold, whereas the main parameters of contractile function were reduced but insignificantly. After the coronary blood flow dropped to the control level, the pressure and contraction rate fell by 40% and the relaxation rate diminished 2-fold. Thus, the enhanced coronary blood flow in the hearts of animals with hemolytic anemia appears to be a factor that compensates for the maintenance of myocardial contractility at the subnormal level. Administration of the antioxidant ionol, an inhibitor of lipid peroxidation, coupled with phenylhydrazine did not prevent the development of anemia but made the coronary blood flow descend in the hearts of anemic animal only by 80%. Since the iron-containing products of red cell dissolution activate lipid peroxidation during hemolytic anemia, this might play a role in the occurrence of heart muscle injuries. It is suggested that ionol prevents such injuries to a considerable extent, thereby preventing the development of compensatory enhancement of the coronary blood flow and heart contractile function disturbances during its normalization.