Effects of ouabain on calcium paradox in rat hearts

The effects of ouabain (10(-7) to 10(-5) M) on the interrelationship between cell-cell contacts, resting tension, and creatine phosphokinase (CK) leakage owing to myocardial cell injury during Ca2+ paradox were studied in isolated perfused rat heart preparations. After perfusing for 15 min with Ca2+ -containing medium, hearts were perfused for 5 min with Ca2+ -free medium followed by a reperfusion with Ca2+ -containing medium for 5 min. This resulted in a transient increase in resting tension and a substantial release of CK into the perfusate during the calcium reperfusion period. These changes were accompanied by extensive structural damage in the myocardial cell, including formation of contraction bands, swelling of the mitochondria, and cell-cell separation. Inclusion of 10(-5) M ouabain for 5 min in the Ca2+ -containing perfusion medium prior to the start of Ca2+ -free perfusion resulted in a higher and sustained resting tension that was accompanied by a reduced loss of CK from the heart during Ca2+ reperfusion. In a histological examination of these ouabain exposed hearts, most of the structural changes owing to calcium paradox were apparent, but the cell-cell contacts were maintained. The results are consistent with the hypothesis that the loss of cell-cell contacts in the intercalated disc during the occurrence of Ca2+ paradox may be the cause of the delayed decline in the resting tension and is only partially responsible for the loss of CK. These differences in myocardial changes during Ca2+ paradox with or without ouabain may be due to the retention of calcium at certain crucial sites under the influence of ouabain.     

Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury

Adaptation to intermittent high altitude (IHA) hypoxia can protect the heart against ischemia-reperfusion injury. In view of the fact that both Ca2+ paradox and ischemia-reperfusion injury are associated with the intracellular Ca2+ overload, we tested the hypothesis that IHA hypoxia may protect hearts against Ca2+ paradox-induced lethal injury if its cardioprotection bases on preventing the development of intracellular Ca2+ overload. Langendorff-perfused hearts from normoxic and IHA hypoxic rats were subjected to Ca2+ paradox (5 min of Ca2+ depletion followed by 30 min of Ca2+ repletion) and the functional, biochemical and pathological changes were investigated. The Ca2+ paradox incapacitated the contractility of the normoxic hearts, whereas the IHA hypoxic hearts significantly preserved contractile activity. Furthermore, the normoxic hearts subjected to Ca2+ paradox exhibited a marked reduction in coronary flow, increase in lactate dehydrogenase release, and severe myocyte damage. In contrast, these changes were significantly prevented in IHA hypoxic hearts. We, then, tested and confirmed our hypothesis that the protective mechanisms are mediated by mitochondria ATP-sensitive potassium channels (mitoKATP) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), as the protective effect of IHA hypoxia was abolished by 5-hydroxydecanoate, a selective mitoKATP blocker, and significantly attenuated by KN-93, a CaMKII inhibitor. In conclusion, our studies offer for the first time that IHA hypoxia confers cardioprotection against the lethal injury of Ca2+ paradox and give biochemical evidence for the protective mechanism of IHA hypoxia. We propose that researches in this area may lead a preventive regimen against myocardial injury associated with Ca2+ overload.     

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.     

Oxygen derived radicals related injury in the heart during calcium paradox

The effects of oxygen-derived radical scavengers (ODRS) on the heart was investigated during the calcium paradox. Perfusion with Ca2+-free medium caused cell separation at the intercalated discs and changes in the endothelial cells. Upon Ca2+ reintroduction, a massive cell damage occurred. The cytosolic enzyme, creatine phosphokinase (CPK), was released in large amounts (p less than 0.001). The tissue adenosine triphosphate (ATP) was reduced to 3.7 mumol/g dry weight from the control value of 21.6 mumol/g dry weight and tissue Ca2+ content was increased threefold. The treatment with superoxide dismutase (SOD) and catalase (CAT) increased percentage of normal cells (62.2%) compared to nontreated Ca2+ paradox group (0.2%) and caused negligible leakage of CPK. Tissue ATP was preserved (p less than 0.03), and Ca2+ content was also reduced in the hearts treated with SOD and CAT (p less than 0.03). The cell membranes and vascular endothelium were well preserved in the hearts treated with SOD and CAT. Boiled SOD and CAT administered were totally ineffective. It is suggested that oxygen-active species may have a role in the Ca2+ paradox injury.     

Regulation of calcium transport in drug-induced taurine-depleted hearts

Drug-induced taurine depletion of rat heart led to the accumulation of free CoA, free carnitine and long-chain acylcarnitine, but a small decrease in long-chain fatty acyl-CoA. Although elevations in total tissue long-chain acylcarnitine levels have been linked to defective membrane function and the association of long-chain acylcarnitines with extramitochondrial membranes, these effects were absent in isolated sarcoplasmic reticulum prepared from taurine-depleted hearts. In contrast to the sarcoplasmic reticulum data, taurine depletion was associated with a significant decrease in ATP-dependent calcium uptake by isolated sarcolemmal vesicles. The major effect of taurine depletion on the sarcolemma was a 2-fold decrease in both the Vmax of calcium transport and the activity of the Ca2+ -stimulated ATPase. Sarcolemmal vesicles prepared from taurine-depleted hearts also exhibited a decreased capacity to transport calcium in exchange for sodium, although the initial rate of the process was unaffected by taurine depletion. Since incubation of sarcolemma from taurine-depleted hearts with taurine could not overcome the effects of taurine depletion, it was concluded that the effects of taurine were not caused by a direct interaction of it with the calcium pump. Possible mechanisms of taurine action are discussed.     

Role of calcium ions in the evolution of the anticonvulsant effect of taurine

The decreased microsomal Ca++Mg++ATPase activity and lowered level of Ca++ binding by the brain cortex microsomes in seizure prone rats as compared with normal animals have been revealed. Taurine increases these parameters in experiments in vitro. Injection of taurine into the penicillin-provoked epileptogenic focus prevents the seizure reaction in rabbits. This effect is not observed after injection of taurine together with EGTA. The data obtained demonstrate the important role of calcium ions in the anticonvulsant action of taurine.     

Ethanol protects the heart against the calcium paradox injury

Rat hearts were depleted of Ca2+ (less than 10(-9) M) for 10 min, followed by 15 min of Ca2+-repletion. The calcium paradox injury occurs during Ca2+-repletion, after a period of calcium depletion. The calcium paradox injury was assessed by percent recovery (hemodynamics, [Ca2+]i, and energy levels) during Ca2+-repletion. A decrease in Na+ concentration during Ca2(+)-depletion did not allow for recovery during Ca2(+)-repletion, however 2.5% and 5% ethanol during Ca2(+)-depletion allowed for an approximate 50% recovery during Ca2(+)-repletion. A combination of ethanol (2.5% or 5%) with a low extracellular Na+ concentration (88 mM) allowed for complete recovery. Ethanol prevented a depletion of diastolic [Ca2+]i during Ca2(+)-depletion, and allowed for a return of normal diastolic [Ca2+]i during Ca2(+)-repletion. Ethanol modulates the activity of the Na+/Ca2+ exchanger and protects against the Ca2(+)-paradox injury.     

Myocardial taurine,development and vulnerability to ischemia

Summary. Depleting intracellular taurine in heart cells improves their resistance to ischemia and reperfusion injury. The aim of this work was to see whether physiologically low levels of endogenous taurine also reflect a reduced vulnerability of the myocardium to cardiac insults. The myocardial concentration of taurine was measured during different stages of development and compared with vulnerability to ischemia and reperfusion injury in the rat and in pediatric patients undergoing cardiac surgery.Rat hearts with relatively lower levels of taurine were significantly more resistant to an ischemic inult and there was a strong negative correlation between taurine content and recovery. Childrens hearts had significantly lower taurine levels compared to infants hearts which was consistent with their known increased resistance to an ischemic cardioplegic insult (Imura et al., 2001). This work shows that the changes in the concentration of myocardial taurine during development correlate with vulnerability to ischemia where low myocardial taurine is associated with improved recovery upon reperfusion.     

Production of hydroxyl radicals and their disassociation from myocardial cell injury during calcium paradox.

The production of hydroxyl radicals during calcium paradox injury was investigated by measuring the production of 2,5-dihydroxybenzoic acid (2,5-DHBA) from salicylate. Four groups of rats were analyzed. In the first group, isolated hearts were perfused with calcium-free medium for 10 minutes followed by perfusion with medium containing Ca++ for 10 minutes. In the other groups, 0.25 microM N,N'-diphenyl-1,3-phenylenediamine (DPPD), 80 microM cytochrome c, or 450 U/ml catalase was added. Coronary effluent was analyzed for the presence of 2,5-DHBA, and tissue sections were examined using light microscopy. In the first group, 2,5-DHBA production began during the calcium-free period, peaked tenfold 60-90 sec. into the Ca repletion period, and declined thereafter. The increase in 2,5-DHBA was accompanied by severe cell damage. Cytochrome c reduced 2,5-DHBA production, and catalase almost completely inhibited 2,5-DHBA production, while DPPD had no effect on 2,5-DHBA production. None of the three additives provided any complete morphological protection. The data provide evidence for the production of hydroxyl radicals during calcium-paradox injury, that their production is dependent upon the presence of hydrogen peroxide, and that cell damage in the calcium paradox is not primarily mediated by the extracellular hydroxyl radicals.     

Effects of taurine on the electrical and mechanical activities of embryonic chick heart

The effect of taurine (2-aminoethanesulphonic acid) on myocardial slow action potentials (APs) and accompanying contractions was examined in isolated perfused chick hearts and reaggregated cultured cells. Isoproterenol (ISO), histamine (HIS), or tetraethylammonium (TEA) induced slow APs and contractions in hearts whose fast Na+ channels had been inactivated by elevated K+. Taurine (10 mM) not only failed to induce slow APs, but actually decreased ISO (10(-8) M), HIS (10(-4) M), or TEA (10 mM) induced slow APs and contractions transiently (about 30s-2 min after the addition of taurine). The properties of the slow APs recovered to control levels by 7-13 min after the addition of the taurine; at this time, there was an increase in developed tension of the contraction accompanying the slow APs. These results suggest that the positive inotropic action of taurine is not mediated through an increase in the slow inward Ca2+ current. However, the transient depression of Ca2+-dependent slow APs by taurine probably explains the transient negative inotropic effect of taurine.     

Release of fatty acid-binding protein from isolated rat heart subjected to ischemia and reperfusion or to the calcium paradox

The release of cardiac fatty acid-binding protein (cFABP) and of fatty acids from isolated rat hearts was measured during both reperfusion following 60 min of ischemia and the calcium paradox (readmission of Ca2+ after a period of Ca2+-free perfusion). Total cFABP release was much more pronounced after Ca2+ readmission (over 50% of tissue content) than during post-ischemic reperfusion (on average, 3% of tissue content), but in both cases, it closely paralleled the release of lactate dehydrogenase. Only minor amounts of long-chain fatty acids, if any, were released from the heart. These observations are challenging the idea that cFABP plays a fatty acid-buffering role under the pathophysiological conditions studied.     

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.     

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.     

Assessment of membrane protection by 31P-NMR effects of lidocaine on calcium-paradox in myocardium

In studying calcium paradox, perfused rat hearts were used to investigate the myocardial protective effects of lidocaine. Intracellular contents of phosphates were measured using the 31P-NMR method. In hearts reexposed to calcium, following 3 minute calcium-free perfusion, a rapid contracture occurred, followed by rapid and complete disappearance of intracellular phosphates with no resumption of cardiac function. In hearts where lidocaine was administered from the onset of the calcium-free perfusion until 2 minutes following the onset of reexposure to calcium, both intracellular phosphates and cardiac contractility were maintained. Therefore, it can be said that cell membranes were protected by lidocaine.     

Dose-Dependent, K+-Stimulated Efflux of Endogenous Taurine from Primary Astrocyte Cultures Is Ca2+-Dependent

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.     

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.     

Contracture and cell damage in calcium paradox is not caused by lipid peroxidation

The role of oxygen radicals and lipid peroxidation in calcium-paradox injury in isolated perfused rat hearts was studied by examining the effects of mannitol and (or) allopurinol on this phenomenon. Myocardial changes due to calcium paradox were characterized by contractile failure, a rise in resting tension, and cell damage. These changes were also accompanied by increased lipid peroxidation, as indicated by an increase in malondialdehyde content. Mannitol (an effective quencher of hydroxyl radicals) treatment resulted in a dose-dependent decrease in lipid peroxidation but did not affect other changes due to calcium paradox. Allopurinol (an inhibitor of xanthine oxidase) neither affected lipid peroxidation nor modified any of the structure-function changes due to calcium paradox. These data demonstrate the occurrence of lipid peroxidation which, however, may not be involved in the observed structure-function changes due to calcium paradox. It is also suggested that in this experimental model, xanthine oxidase may not be the inducer of oxygen radicals or of lipid peroxidation.     

Anaerobic rat heart: mitochondrial role in calcium uptake and contractility.

In order to evaluate the manner by which fumarate enhances contractility in the anaerobic heart, we examined Ca++ movements in isolated heart mitochondria and in the isolated perfused heart. Our experiments showed that in isolated antimycin A plus cyanide treated mitochondria: (a) Ca++ uptake was promoted by electron transport generated by fumarate-dependent oxidation of NADH, (b) Ca++ stimulated fumarate-dependent oxidation of NADH, (c) the ratio of Ca++ uptake:NADH oxidized was 1.7 and (d) the Ca++ sequestered is transiently highly mobile and is rapidly released upon collapse of the membrane potential. In anaerobic hearts perfused with glucose plus fumarate, malate and glutamate Ca++ levels were the same as those in oxygenated hearts while in anaerobic organs perfused with or without glucose Ca++ content was appreciably lower. Succinate production in anaerobic heart perfusions was related to: (a) an increased retention of Ca++ by the heart, (b) a diminution in peak aortic pressure generated by cardiac contractions and (c) an increase in heart rate. The information obtained indicates that mitochondria have a capability for Ca++ movement which be used physiologically, particularly in fumarate perfused anaerobic hearts, to assist the mechanism for contraction and relaxation of the heart.     

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.     

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.