Prevention of myocardial reperfusion injury by increasing artificial trans-membrane sodium gradient in "calcium paradox" and post-ischemic reperfusion

An effect of the high sodium gradient during "calcium paradox" and postischemic reperfusion has been studied. A decrease of Na/Ca exchange by high sodium gradient (200 mM NaCl in the perfusion solution) resulted in the reduction of myoglobin release from the heart during "calcium paradox". High sodium concentration solution (200 mM) increased protective effect of ATP during "calcium paradox". Exogenous phosphocreatine (100 mumol/mol) increased myoglobin release from the heart. During perfusion of the heart by high sodium concentration, phosphocreatine efficiently decreased myoglobin release from the heart during "calcium paradox". Exogenous ATP (as Na-pump activator) and high Na+ concentration solution (180 mM) prevented the LDH release from the myocardium, decreased ATP hydrolysis, inhibited Ca influx, maintained total adenine nucleotides, phosphate potential, energy charge of the cardiomyocytes.     

Protective effect of adenosine against a calcium paradox in the isolated frog heart.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.     

The prevention of the development of myocardial contracture in the "calcium paradox" by action on Na-Ca metabolism]

The effect of artificial high sodium gradient on the rate of the myocardium contracture development during "calcium paradox" was studied in the experiments on the isolated heart of Langendorf-perfused rats. It is stated that artificial creation of a high sodium gradient decreases the rate of the myocardium contracture development. Exogenous nucleotides, activators of Na, K-ATPase, and their precursors intensified the protective action of the hypersodium medium. Phosphocreatine (100 mmol/l) had no protective effect during the "calcium paradox". However, under conditions of the high sodium gradient phosphocreatine efficiently prevented development of the contracture during the "calcium paradox". It is important to note that under analogous conditions creation of high osmosity of the solution adding 12 mmol/l of saccharose does not protect the heart from development of the myocardium contracture.     

Significance of extracellular concentration of sodium ions in the protective effect during the "calcium paradox"]

Increasing of extracellular sodium concentration up to 200 mM diminishes heart damage under "calcium paradox". Phosphocreatine (10(-4) M) potentiates the effect of high sodium perfusion media; in this case myoglobin release from the myocardium is minimal (5-9% of control). An the same time, ATP and phosphocreatine concentrations and oxidation to phosphorylation coupling in mitochondria remain at a sufficiently high level. Elevation of osmotic pressure by the effect of 120 mM sucrose enhances heart damage under "calcium paradox" both in the presence and absence of phosphocreatine. The protective effects of superhigh (200 mM) sodium concentrations and phosphocreatine are completely reversed by strophanthin or decreasing K+ concentration down to 0.5 mM.     

Protective effects of manganese, cobalt, nickel, and barium against a calcium paradox in the isolated frog heart

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.     

Dependence of myocardial damage on the anion composition and osmotic pressure in the extracellular fluid during "calcium paradox" in rats]

The data obtained reveal that elevation of extracellular osmolarity with sucrose during reintroduction of Ca-containing medium after 10 minutes of Ca2+ removal prevents loss of haemoglobin in a concentration-dependent mode. Reducing the extracellular osmolarity of the reperfusion medium by means of decreasing the concentration of sodium chloride and calcium chloride exacerbates the loss of haemoglobin from the cardiomyocytes. There is a close correlation between the water contents in tissues and the loss of haemoglobin during the "calcium paradox". The findings suggest dependence of the heart damage during the "calcium paradox" on anionic composition of extracellular space and activity of anionic transporters.     

Alterations in cardiac lysosomal hydrolases following induction of the calcium paradox

Although perfusion of the heart with calcium-free medium for a brief period followed by reperfusion with calcium-containing medium results in marked structural derangements (calcium paradox), the mechanisms for this cell damage are far from clear. Since activation of lysosomal enzymes has been associated with pathological damage, it was the purpose of this study to examine alterations in the activities of several lysosomal enzymes in rat hearts subjected to calcium paradox. No significant changes in the activities of beta-acetylglucosaminidase, beta-galactosidase, alpha-mannosidase, or acid phosphatase were seen in the homogenates of hearts exposed to the calcium paradox. However, there were dramatic alterations in the lysosomal enzyme activities in the sedimentable and nonsedimentable fractions during calcium paradox. The lysosomal enzyme activities were also detected in the perfusate collected during reperfusion with calcium-containing medium. These changes occurred during the reperfusion period since no alterations were apparent after calcium-free perfusion and were dependent upon the time of reperfusion with medium containing Ca2+ as well as the time of perfusion with Ca2+ -free medium before inducing Ca2+ paradox. These data indicate that alterations in lysosomal enzymes owing to reinstitution of calcium in Ca2+-deprived hearts may occur as a part of cardiac damage and general cellular disintegration.     

Extracellular calcium-dependent and -independent effects of adenosine in bovine coronary artery

Adenosine relaxes the coronary arteries of various species through A2 receptors. The aim of the present investigation was to evaluate the relaxing effects of adenosine in relation to the role of calcium in bovine coronary arteries by studying the vasodilatory effect of adenosine in normal and calcium-free medium and on calcium-45 efflux into calcium-free medium. Acetylcholine (ACh) and norepinephrine (NE) were used to induce tone in coronary artery rings. Adenosine, 5'-(N-ethylcarboxamido)adenosine (NECA), and N6-(L-phenylisopropyl)adenosine (L-PIA) produced concentration-dependent relaxations of the coronary artery rings. Both in normal and calcium-free medium, the order of potency for adenosine analogs (NECA greater than L-PIA greater than adenosine) was similar and 8-phenyltheophylline antagonized the relaxation responses to adenosine and its analogs. Removal of extracellular calcium shifted the concentration-response curves to the right in a parallel fashion, slowed the rate of relaxation, and in NE contracted rings reduced the maximum responses for adenosine and its analogs. In calcium-free medium, adenosine was without an effect on calcium-45 efflux in the presence of ACh. However, adenosine inhibited the stimulated calcium-45 efflux induced by NE. The data suggest that the vasodilatory action of adenosine in bovine coronary smooth muscle has both extracellular calcium-dependent and -independent components.     

Calcium exposure required for full expression of injury in the calcium paradox

Isolated hearts repleted with calcium after a short period of perfusion with a calcium-free medium undergo the injury of the calcium paradox and release cellular protein. In the present experiments, 15 min perfusion with a calcium containing HEPES solution subsequent to 10 min calcium-free perfusion resulted in the loss of 42.7 +/- 3.9 mg of protein/g heart. If hearts were repleted with calcium for 30 s only, then returned to calcium-free perfusion, resultant protein loss was the same. When calcium repletion was further decreased to 20 s, 23.9 +/- 1.3 mg/g of protein was lost. This critical period coincided with the onset of contracture but was prior to major ion movements. It is concluded that the transition to irreversible injury occurs within 30 s of calcium repletion and that subsequent removal of extra-cellular calcium does not alter the course of events leading to cell death.     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in the energy metabolism of the isolated perfused frog heart during calcium depletion and subsequent repletion

Summary The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.Abbreviations ADP Adenosine diphosphate - AMP Adenosine monophosphate - ATP Adenosine triphosphate - EDTA Ethylene-diamino-tetraacetic acid - NAD ⁺ Nicotinamide-adeninedinucleotide - NADH Nicotinamide-adenine-dinucleotide (reduced form) - TRA Triethanolamine     

Dependence of myocardium injury on extracellular K+ concentration during calcium paradox

It is well-known that the first stage of the calcium paradox involves decreasing of Na+ gradient. The decreased sodium gradient is a cause of activation of the Na(+)-Ca+ exchange and formation of cardiac injury during the calcium repletion. Potassium ions are natural extracellular activators of Na(+)-pump. It has been shown that heart perfusion by Ca(2+)-free medium evoked extrusion from cells of hydrophilic amino acids whose transport-depends on sodium gradient. The heart reperdusion with Ca(2+)-containing agent leads to myofibrillar contracture and extensive myoglobin release. The simultaneous events are: elevation in tissue water contents, decreasing of intracellular concentration of adeninnucleotides, uncoupling of oxidation and phosphorylation in mitochondria. The decreasing of K+ level to 0.5 mM exacerbates myocardial damage during the calcium paradox, despite absence of myocardial contracture. The elevation of K+ (to 10 mM or 20 mM) attenuated the calcium paradox development in the heart. The elevated K+ concentration protected isolated heart from extensive myoglobin release, development of myocardial contracture. The high K+ concentrations alleviate mitochondrial damage and elevate contents of adeninnucleotide in the tissue. The positive effect of the elevated K+ concentration can be completely blocked by strophanthine, the selective Na+, K(+)-pumb blocker.     

Mitochondrial and cytoplasmic aspartate aminotransferase enzyme release in the calcium paradox

The release of mitochondrial and cytoplasmic aspartate aminotransferase (AST) enzymes from the myocardium was studied in the isolated rabbit heart under conditions of the calcium paradox. Four different periods of calcium-free perfusion for 10, 15, 20, and 25 min were selected to produce different degrees of the calcium paradox and the associated myocardial damage which was indicated by impairment in the left ventricular contractile function. Calcium-free perfusion periods of less than 20 min were associated with partial recovery of ventricular function, while periods of 20 min or greater were associated with little or no recovery of contraction after reperfusion with calcium. Mitochondrial (ASTm) and cytoplasmic (ASTc) aspartate aminotransferase were released from the heart beginning within 1 min of reintroduction of Ca2+. The cumulative amount of ASTm release was about one-tenth the amount of ASTc release. The cumulative amount of ASTm and ASTc released were significantly (p less than 0.05) related to the duration of calcium-free perfusion. The time to 90% of maximum AST release was slightly longer for ASTm compared with ASTc (6.8 +/- 0.6 vs. 5.7 +/- 0.5 min, 0.10 greater than p greater than 0.05). ASTc but not ASTm correlated significantly (p less than 0.05) with total protein release from the myocardium, while ASTm was not as consistently related to protein loss. The cumulative amount of ASTm and ASTc were inversely related to the extent of recovery of left ventricular contractile function. Disparities did occur as the longest duration of the calcium-free period, which did not produce any further damage to left ventricular function, was nonetheless associated with more enzyme release from the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the ion composition of the calcium-free medium and cardiomyocyte damage during "calcium paradox" in rats]

The findings reveal that the degree of myocardial damage in the "calcium paradox" does not depend on the contracture strength, that the contracture attenuation due to a decreased concentration in the Ca-free medium is not equal to the cardiocytes protection as compared with other means. Mg2+ and the studied means of myocardial protection seem to play a major role in assessment of the "calcium paradox" development and explain the difference in results of the hyposodium medium effects in the "calcium paradox".     

Possible mechanisms involved in the development of the calcium paradox

Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in excessive influx of calcium into the cells and irreversible cell damage (calcium paradox). This paper describes the possible routes of calcium entry that occurs during the phase of calcium repletion, and the possible mechanisms involved in the development of the calcium paradox damage. The routes of calcium entry include the glycocalyx, the slow channels, the Na+-Ca2+ exchange mechanism, passive diffusion, and abnormal sites of calcium entry. In addition to an increased influx of calcium, a loss in the ability of the sarcolemma to remove calcium from the cells may contribute to the net gain of tissue calcium. The calcium paradox damage itself, which follows the massive influx of calcium into the myocardial cells, may be a result of calcium-triggered energy dependent reactions and a concomitant acidification of the cytoplasm. Mechanical factors may also be involved in the development of the calcium paradox.     

Effect of calcium depletion and calcium paradox on myocardial energy metabolism

Both phases of the calcium paradox were associated with major alterations in myocardial energy metabolism. During calcium-free perfusion contractility of the heart ceased, resulting in a dramatic decrease in anaerobic and aerobic metabolism but no change in tissue high energy phosphate levels. Tissue content of most citric acid cycle intermediates were elevated, while there was a net decrease in the content of transaminase-linked amino acids. Reperfusion of the calcium-depleted heart with calcium-containing buffer failed to restore either the contractile or the metabolic state of the heart. Within seconds following calcium repletion, tissue high energy phosphate content plummeted. This occurred even though glucose utilization increased significantly and aerobic metabolism remained at levels observed in the calcium-depleted heart. Analogous to changes seen in acidosis and ischemia, alpha-ketoglutarate and citrate levels decreased abruptly. After a short delay, the levels of several key amino acids also dropped. The results support the hypothesis that the impairment of mitochondrial function contributes to the depletion of high energy phosphate stores during the calcium paradox.     

The role of calcium in the initiation of superoxide release from alveolar macrophages

The role of calcium in the release of superoxide anion (O2-) was examined in alveolar macrophages after stimulation with the soluble stimuli: concanavalin A (Con A), N-formyl methionyl phenylalanine (FMP), and the calcium ionophore. A23187. The release of O2- by Con A was unaffected over a wide range of extracellular calcium concentrations (20 microM to 3 mM), whereas increasing the extracellular calcium above 2 mM inhibited FMP-stimulated O2- release. In contrast, A23187 did not stimulate O2- release in calcium-free medium (less than or equal to 30 microM). The addition of EGTA (50 microM) to calcium-free medium had no effect on Con A stimulation of O2- release or FMP-stimulated O2- release. These results suggest that, for the three soluble stimuli, there are different roles for Ca+2 in the activation and transmission of stimulatory signals across the cell membrane. Con A- or FMP-stimulated calcium efflux from calcium-loaded cells in either calcium-free medium or 0.5 mM calcium-containing medium. In calcium-free medium, FMP transiently retarded 45Ca+2 uptake, while in 0.5 mM calcium-containing medium, FMP transiently stimulated 45Ca+2 uptake. For either Con A or FMP, calcium efflux preceded O2- release by 30-45 sec. Quinine, an agent that blocks membrane hyperpolarization in macrophages, completely blocked O2- release by concanavalin A or FMP and inhibited 45CA+2 efflux by 50% or more for both agents. These results support the hypothesis that redistribution of cellular Ca+2 is one of the initial steps leading to the release of O2-.     

Enhanced early after-myocardial infarction concentration of TNF-alpha subsequently increased circulating and myocardial adrenomedullin in spontaneously hypertensive rats

Both inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the cardiac protective peptide adrenomedullin (AM) are increased in cardiac tissues and plasma in patients with myocardial infarction (MI) and chronic heart failure. Recently they have been increasingly recognized as important factors in the pathophysiology of MI and resultant congestive heart failure. Compared with sham-operated spontaneously hypertensive rats (SHR), we investigated myocardial immunoreactivity of TNF-alpha and AM and also their mutual relations in vivo in SHR+MI. Residual myocardial depression after MI was studied also in isolated perfused hearts. In chronic experiments, 24 and 48 h after permanent ligation of the descending anterior branch of the left coronary artery, we examined hemodynamics, plasma and myocardial peptide levels. Left ventricular function was assessed in isolated perfused hearts subjected to "global ischemia and reperfusion" and after induction of "calcium paradox". Circulating and myocardial TNF-alpha concentrations increased early after MI in SHR. Studies with global ischemia and calcium paradox in isolated heart showed early myocardial depression and calcium-dependent gradual increase of left-ventricular end-diastolic pressure. In the SHR+MI myocardial AM concentrations were increased 9- and 49-fold after respective 24 h and culminated 48 h following MI. Circulating and myocardial AM was increased in SHR+MI in association with TNFalpha-induced myocardial depression. The both studied cardiac parameters displayed the beneficial effect of the enhanced myocardial AM concentration.     

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of prolactin stimulated pig oocytes

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of pig oocytes activated by prolactin was investigated, using the fluorescent dye chlortetracycline. In the presence of extracellular calcium, the inhibitor of protein kinase C Ro 31-8220 increased calcium exit from intracellular stores in pig oocytes after prolactin treatment. In calcium-free medium, Ro 31-8220 exerted effect on calcium release from intracellular stores. In calcium-free medium, prolactin did not stimulate calcium release from intracellular stores of oocytes in the presence of thimerosal, while in the presence of protein kinase C inhibitor, prolactin increased Ca2+ content from intracellular stores in such oocytes. These data suggest a direct involvement of protein kinase C in the processes of regulation of Ca2+ exit from intracellular stores of pig oocytes stimulated by prolactin.     

Dependence of myocardial contracture on energy resources during the calcium paradox

Perfusion of the rat isolated hearts with calcium-free and calcium containing solution revealed a complex and deep myocardial damage called the calcium paradox. The reperfusion of the rat heart with calcium rich media resulted in myoglobin loss from the heart, significant decreasing of ATP and phosphocreatine level, complete uncoupling of respiration and phosphorylation in mitochondria, occurrence of myocardial contracture. Decreasing of sodium level to 30 mM--80 mM in calcium free media exacerbates the heart damage due to the calcium paradox with absence of contracture. Addition of phosphocreatine (1 mM, 5 mM, 10 mM) evoked some restoration of ATP contents in the tissue with appearance of significant contracture. Phosphocreatine exacerbated the loss of myoglobin from the heart subjected to the calcium paradox. A discrepancy between myocardial contracture and degree of cellular damage has been observed during the calcium paradox.