Contracture and the calcium paradox in the rat heart

The role of contracture in the manifestation of calcium paradox-induced damage was examined using 2,3-butanedione monoxime (BDM) to inhibit myofibrillar activity. Calcium and sodium gain, loss of intracellular components, and changes in structure were monitored. If 30 mM BDM was added at the time of calcium repletion after 10 minutes of calcium-free perfusion, some protection was afforded, particularly at the early stages of calcium repletion. However, much greater protection was obtained if BDM was present during the final 2 minutes of calcium-free perfusion and throughout repletion. Sodium gain and loss of intracellular components were markedly attenuated, as was the incidence of severely contracted cells. Calcium gain, although significantly reduced during the period of repletion, was not abolished. After 10 minutes of repletion, a calcium content of 11.44 +/- 1.57 mumol/g dry wt was observed. This suggests that other noncontracture related routes of calcium entry are involved. If BDM is removed after 5 minutes of calcium repletion and perfusion is continued with BDM-free perfusate, there is a rapid gain of sodium, further gain of calcium, loss of intracellular components and the cells contract severely, tearing away from neighboring cells. It is evident, therefore, that returning calcium to hearts after a period of calcium-free perfusion under conditions that significantly reduce the typical calcium paradox-associated damage does not necessarily repair the underlying defect. These results support the hypothesis that contracture-induced sarcolemmal disruption may be responsible for the terminal manifestation of the calcium paradox.     

MAPK activation and apoptotic alterations in hearts subjected to calcium paradox are attenuated by taurine

OBJECTIVE: The Ca2+ -paradox is an important phenomenon to study cell injury induced by Ca2+ -overload in myocardium. Although intracellular Ca2+ -overload acts as a trigger and modulator of cell death due to apoptosis under various pathophysiological conditions, the presence of apoptosis in hearts subjected to Ca2+ -paradox has not been demonstrated. Since taurine attenuates the changes in cardiac function due to Ca2+ -paradox, this study investigated the occurrence and mechanisms of apoptosis in Ca2+ -paradoxic hearts treated in the absence and presence of taurine. METHODS: Ca2+ -paradox was induced by perfusing the isolated rat heart with Ca2+ -free medium for 5 min followed by reperfusion with Ca2+ -containing medium for 30 min. Apoptosis related signal transduction mechanisms were determined in Ca2+ -paradoxic hearts perfused with or without 10 mM taurine. RESULTS: Marked alterations in cardiac function and the presence of apoptosis were seen in Ca2+ -paradoxic hearts reperfused for 30 min. Unlike the total protein contents in hearts subjected to Ca2+ -paradox, the contents of phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK)1, ERK2 and c-jun amino-terminal kinase were increased by 125 +/- 8.6%, 296 +/- 14.3%, 213 +/- 8.5% and 133 +/- 4.2%, respectively vs. control. Caspase-3 and phosphorylated Bcl-2 contents were also increased by 193 +/- 10.2% and 134 +/- 5.0% vs. control whereas phosphorylated Bad and the ratio of Bcl-2/Bad were depressed by 32 +/- 10.8% and 0.23 +/- 0.5% vs. control in Ca2+ -paradoxic hearts. The apoptosis as seen in Ca2+ -paradoxic hearts reperfused for 30 min was not evident in hearts at 10 min Ca2+ -repletion but was similar to hearts subjected to 60 min Ca2+ -repletion. These changes in the apoptotic pathway in cardiomyocytes subjected to Ca2+ -paradox were prevented by taurine. Furthermore, taurine attenuated the KCl- or ATP-induced increase in intracellular concentration of Ca2+ in cardiomyocytes. CONCLUSIONS: This study suggests that cardiac dysfunction due to Ca2+ -paradox may be associated with apoptosis. In addition, the beneficial effects of taurine on cardiac function may be related to the attenuation of changes in MAPK and apoptotic signal transduction mechanisms in Ca2+ -paradoxic hearts.     

Transmural distribution of calcium accumulation and glucose uptake in the calcium paradox in the rat left ventricle

The calcium paradox was induced in the isolated perfused rat heart and the transmural distributions of glucose uptake and calcium accumulations were determined using 2-deoxy-D-[3H]glucose and 45Ca2+ as tracers. The regional distribution of coronary flow was measured using radiolabelled microspheres. A significant transmural gradient was observed in left ventricular glucose uptake both in the control and the calcium paradox hearts: subendocardial and subepicardial glucose uptake rates were 5.70 +/- 0.65 mumol/min/g protein (mean +/- S.E.M.) and 4.29 +/- 0.35 mumol/min/g protein (P less than 0.05) in the control hearts, and 8.64 +/- 0.79 mumol/min/g protein and 4.09 +/- 0.71 mumol/min/g protein (P less than 0.01) in the paradox hearts. This increase in subendocardial glucose uptake in the calcium paradox was also significant (P less than 0.05), while the average left ventricular calcium accumulation was about 10-fold in the paradox hearts compared to the controls mumol x 10(-1)/min/g protein in the subendocardium and 14.02 +/- 0.93 mumol x 10(-1)/min/g protein in the subepicardium (P less than 0.001). The subendocardium received initially 30% more perfusion than subepicardium (P less than 0.01), but the subendocardial flow during the calcium depletion and replenishment periods was only 40% of that in the subepicardium (P less than 0.001) and the average myocardial flow was reduced by c. 60% (P less than 0.001). The data show clear transmural differences in the alterations associated with the calcium paradox. This suggests that the calcium paradox induced myocardial injury is unevenly distributed across the left ventricular wall, this uneven distribution possibly being due to regional differences in the damage of mitochondrial energy production.     

Myocardial cAMP and calcium levels in the calcium paradox

Summary In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP.This work was supported by grants from the Norwegian Research Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanites.     

Slow calcium channel blockers and the calcium paradox: comparative studies in the rat with seven drugs

In studies of the calcium paradox, the isolated perfused rat heart was used to characterize the relationship between myocardial protein leakage and the concentration of calcium antagonist (verapamil and D600) or calcium in the perfusion fluid during a cycle of calcium depletion and repletion. The results indicated a dose-dependency such that protein leakage could be progressively reduced by decreasing the concentration of calcium during calcium repletion and/or by increasing the concentrations of drug. Detailed dose-response studies with seven calcium antagonists (verapamil, D600, nifedipine, terodiline, diltiazem, fendiline and prenylamine) and a calcium concentration of 1.0 mmol/l during a 20 min period of calcium repletion following a 10 min period of calcium depletion revealed complex dose-response characteristics for each drug. In the dose range studied (0 to 40 mumol/l) all drugs with the exception of prenylamine were able to reduce protein leakage by up to 25 to 30% Optimal concentrations for verapamil, nifedipine, D600, diltiazem and terodiline were all between 2.0 and 4.0 mumol/l. With the exception of D600, which provided a constant reduction of protein leakage at all concentrations above this optimum, all drugs exhibited bell-shaped dose-response curves with a loss of efficacy at higher concentrations. Fendiline also had a bell-shaped dose-response curve with 23% as a maximal reduction of leakage; however, the optimal concentration for this drug was 21.0 mumol/l. Additional studies with verapamil revealed that the drug acted during the calcium repletion phase and did not influence events during calcium depletion. Simultaneous use of two drugs, verapamil and nifedipine, at their optimal concentrations failed to improve the reduction in protein leakage over and above that observed with one drug alone.     

Effects of the free radical scavenger DMTU and mannitol on the oxygen paradox in perfused rat hearts

The oxygen paradox refers to the abrupt release of cytoplasmic enzymes and severe cellular disruption that occurs following reoxygenation of anoxic perfused hearts. In this study, the ability of a series of oxygen-derived free radical inhibitors and scavenging agents to protect isolated perfused rat hearts from the oxygen-induced enzyme release following 30 or 60 mins of anoxic perfusion (oxygen paradox) and cumene hydroperoxide-induced injury was evaluated. Malondialdehyde (MDA) release, an indicator of lipid peroxidation, and creatine kinase (CK) release, an indicator of cellular injury, were monitored. We evaluated five agents previously reported to scavenge or inhibit the formation of oxygen free radicals. The putative hydroxyl radical scavengers dimethylthiourea (DMTU) and mannitol; catalase, an agent protective against peroxide injury; allopurinol, an inhibitor of xanthine oxidase; and albumin, a non-specific protein control, were evaluated. Coronary flow rates and myocardial temperature were continuously monitored to ensure uniform perfusion conditions. The MDA assay was carefully monitored by constructing standard curves on each experimental day. Addition of 20 microM cumene hydroperoxide to oxygenated perfused hearts caused peroxidative cell injury as evidenced by significant MDA and CK release in the coronary effluent. DMTU and catalase provided near complete protection from cumene hydroperoxide-induced cell injury but did not reduce CK release from hearts subjected to either the mild (30-min) or the severe (60-min) oxygen paradox (reoxygenation-induced injury). Allopurinol caused a significant reduction in MDA release but not CK release from oxygen paradox-injured hearts. Allopurinol and albumin had no significant effect on MDA release from cumene-hydroperoxide-injured hearts. Catalase (300 U/ml) caused a mild but not statistically significant reduction in MDA release from cumene hydroperoxide injury but did not provide protection from the oxygen paradox at either injury level. Mannitol (120 mM), in contrast to DMTU, was ineffective in reducing cumene-induced injury but showed a significant protective effect against oxygen paradox-induced damage. It is concluded that the ability of mannitol to reduce reoxygenation-induced CK release in the oxygen paradox may be due to its osmotic activity and consequent ability to prevent cellular swelling rather than its activity as an oxygen-free radical scavenger.     

Changes in calcium handling in atrophic heterotopically isotransplanted rat hearts

Atrophy of the rat heart induced by hemodynamic unloading after heterotopic transplantation is associated with impaired relaxation while systolic function remains normal when compared to the heart of the recipient animal. To identify possible underlying mechanisms for the above, we studied some aspects of membrane calcium handling using postextrasystolic potentiation of contractions in the isolated right ventricular papillary muscle and in the left ventricle of the Langendorff-perfused heart. We also compared the alterations of the unloaded heart with those of overloaded hypertrophic hearts of rats with suprarenal aortic constriction. In the atrophic heart the degree of potentiation after one extrasystole, considered to be proportional to the trans-sarcolemmal influx of Ca²⁺ during an action potential, was increased by 125% when compared with recipient hearts. The rate of decay of potentiation which reflects the fraction of activator Ca²⁺ recirculating in the cells via the sarcoplasmic reticulum, negatively correlated with the degree of potentiation, although its mean value was not significantly altered. In hypertrophic hearts the decay of potentiation was faster when compared with the hearts of sham-operated animals, indicating a decreased recirculating fraction of Ca²⁺. The data suggest that the relative importance of trans-sarcolemmal Ca²⁺ fluxes is increased both in cardiac atrophy and hypertrophy, but their quantitative role in the control of cardiac contraction might differ.     

Calcium entry in the calcium paradox

In this review the mechanisms that are involved in the aetiology of the Ca²⁺ paradox are discussed, with particular reference to the route and consequences of the massive influx of Ca²⁺ that occurs during the phase of Ca²⁺ repletion. The precise route of Ca²⁺ entry is unknown and it may be abnormal. The voltage activated slow channels do not appear to be the major route of entry, although this route may be important for Ca²⁺ entry during the early phase (the first 1 or 2 min) of Ca²⁺ repletion. The entry of large amount of Ca²⁺ appears to trigger a sequence of events that results in cell destruction, and this may involve acidification of the cytosol due to the breakdown of ATP. To a lesser extent cytosolic acidification may be due to the ejection of H⁺ from mitochondria in exchange for Ca²⁺. Abnormal mechanical stresses may also contribute to cell disruption.     

Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes

The androgenic steroid hormone testosterone induced an early (less than 30-60 seconds) stimulation of endocytosis, hexose transport, and amino acid transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, 2-deoxyglucose, and alpha-aminoisobutyrate, respectively, in rat ventricle cubes and acutely isolated ventricular myocytes. This stimulation was time- and concentration-dependent and was maximal at 10(-9) to 10(-8) M testosterone, consistent with androgen-receptor mediation. EGTA (2.5 mM), La3+ (1 mM), and verapamil (100 microM) ablated the hormonal response. The calcium ionophore A23187 (10 microM) induced an acute stimulation of endocytosis, amino acid transport, and hexose transport which was not further increased by testosterone (10(-8) M), suggesting a common effector pathway. Testosterone (10(-8) M) also evoked a rapid (less than 30 seconds) stimulation of 45Ca influx and efflux. Testosterone (10(-8) M) induced a rapid (less than 5 seconds) transient increase in ornithine decarboxylase (ODC) activity peaking (twofold to threefold) at 60 seconds, and an early (15 seconds) transient accumulation of polyamines peaking at 60 seconds in isolated myocytes. The specific, irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO, 5-10 mM) blocked the testosterone-evoked increase in ODC activity and polyamine levels and the stimulation of Ca2+ fluxes, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5-1 mM), the ODC product, reversed DFMO inhibition and restored the increase in polyamines, 45Ca fluxes, and Ca2+-dependent membrane transport processes. These results demonstrate that rapid, transient ODC-regulated polyamine synthesis is essential for androgenic stimulation of Ca2+ fluxes and membrane transport processes in ventricular myocytes. These findings support a model for signal transduction in which newly synthesized polyamines serve as intracellular messengers to regulate transmembrane Ca2+ movements, Ca2+-dependent membrane transport functions, and other Ca2+- and polyamine-sensitive processes in cardiac myocytes.     

Fasting limits the increase in intracellular calcium during ischemia in isolated rat hearts

Introduction Fasting has been shown to limit ischemic injury and improve functional activity after global ischemia. Because calcium overload is considered a mechanism of ischemic injury, we hypothesized that fasting would limit the accumulation of intracellular calcium [Ca]_i during ischemia, potentially due to reduced accumulation of intracellular sodium [Na]_i. Methods To address this hypothesis, hearts isolated from rats fed either a normal diet or fasted for 24 hours underwent 20 min of global ischemia at 37°. In addition to functional parameters, [Na]_i and [Ca]_i were measured using ²³Na and ¹⁹F spectroscopy using thulium-DOTP⁻⁵ and 5F-BAPTA, respectively. In vitro measurement of sarcoplasmic reticulum calcium uptake and release, as well as activity of the sarcolemmal Na-Ca exchanger, was performed in hearts from fed and fasted animals under baseline and ischemic conditions. Results Hearts from fasted animals showed greater recovery of developed pressure (37 ± 9 vs. 11 ± 6 cm H₂O, p < 0.05) and less contracture (end-diastolic pressure 25 ± 2 vs. 47 ± 2 cm H₂O, p < 0.05) by the end of the reperfusion period. [Na]_i was similar in the 2 groups during the first half of the ischemic period, albeit with a higher concentration of [Na]_i in hearts from fed compared to fasted animals at reperfusion. Fasting markedly limited calcium accumulation during ischemia, with end-ischemic calcium being 419 ± 46 nM in the hearts from fasted animals and 858 ± 140 nM in the hearts from fed animals (p < 0.01). There was no significant effect of fasting on calcium uptake or release by the SR, nor on sarcolemmal Na-Ca exchange activity. Conclusions Fasting for 24 hours improves functional recovery and markedly limits [Ca]_i accumulation during ischemia and early reperfusion. The mechanism for this phenomenon remains to be elucidated. Received: 7 December 2000, Returned for revision: 22 December 2000, Revision received: 5 February 2001, Accepted: 8 February 2001     

Cobalt, manganese and the calcium paradox

The reperfusion of hearts with Ca2+-containing buffers after relatively short periods of Ca2+-free perfusion results in tissue damage, Ca2+ overload, loss of mechanical function and displacement of intracellular components into the extracellular phase. Using isolated, spontaneously beating Langendorff perfused rat hearts we have investigated whether Mn2+ and Co2+ exert a dose dependent inhibitory effect on this gain in Ca2+ and loss of intracellular constituents (assayed as myoglobin). We have also determined if the timing of the addition of Co2+ or Mn2+ is critical, and whether their protective effect is Ca2+-sensitive. When added only at Ca2+ repletion neither Co2+ nor Mn2+ provided protection. When present during the entire period of Ca2+ depletion and repletion, Mn2+ (005 to 4.0 mM) and Co2+ (0.1 to 4.0 mM) exerted a dose-dependent protective effect, indicted by a reduced and delayed onset of myoglobin release, a reduced gain in Ca2+, and an improved recovery of mechanical function. This protection was proportionally diminished if the cations were added late during the 10 min period of Ca2+ depletion and further reduced if after their late addition, Mn2+-free or Co2+-free Ca2+ repletion buffer was used. Mn2+ was more effective than Co2+, and decreasing the Ca2+ content of the perfusion buffer from 2.5 to 1.3 mmol.1-1 shifted the dose-response curve for this protection to the left. These results are discussed in terms of the possible mechanisms involved in the dose and time dependent protective effect of Mn2+ and Co2+.     

A protective effect of verapamil on the calcium paradox in the isolated perfused rat heart

Experiments were undertaken to examine whether and how verapamil, one of the well known slow-channel calcium-antagonists, protected cardiac muscles against the calcium paradox in isolated perfused rat hearts.Following a 5-min perfusion with calcium-free buffer at pressure of 80 cm H₂O (high flow) and 40 cm H₂O (low flow), severe and mild calcium paradoxical injuries were produced, respectively, by reperfusion with buffer containing 2.5 mm of calcium for 10 min at 80 cm H₂O. When verapamil (1 mg/l) was added to the perfusion medium during both the calcium depletion and repletion periods, a marked protective effect on the mild calcium paradox was observed, evidenced by a higher recovery in the contractility, an about 50% reduction in development of the contracture, and almost complete prevention in release of creatine kinase, an inhibition of tissue calcium accumulation, and much larger tissue stores of high energy phosphates; whereas no protective effect was observed on the severe calcium paradox. In the mild calcium paradox, administration of verapamil up to 2 min after the readmission of calcium was sufficient for protection against the calcium paradox.It was suggested that verapamil could substantially protect heart muscles from injuries associated with the calcium paradox probably due to an inhibition of sudden calcium influx into the cardiac cells in early calcium repletion period.     

Sarcolemmal disruption during the calcium paradox

Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in irreversible cell damage (calcium paradox). The ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox was studied by using fast freezing devices. This method excluded ultrastructural changes induced by chemical fixation and cryoprotection. In addition, thin-section and conventional freeze-fracture electron microscopy were used. During reperfusion with calcium-containing solution disruption of the sarcolemma was observed, which was attended with formation of unilamellar and multilamellar vesicles and aggregation of intramembrane particles. These ultrastructural changes are explained in terms of calcium- and proton-induced lateral phase separation and fusion processes in the lipid bilayer of the sarcolemma.     

Energy dependence of the calcium paradox

Reperfusion of isolated rat hearts with Ca²⁺-containing medium, after a short Ca²⁺-free perfusion period, results in irreversible cell damage (calcium paradox). In this study this type of cell damage was studied in the anoxic rat heart, in the presence and absence of glucose. Creatine kinase release and ultrastructural changes were used to define cell damage.During anoxic perfusion in the presence of glucose, myocardial ATP was maintained at a fairly high concentration. Successive perfusion with Ca²⁺-free and Ca²⁺-containing medium resulted in the calcium paradox.During anoxic perfusion in the absence of glucose, myocardial ATP was reduced to almost zero. Under these conditions the calcium paradox did not occur unless the hearts were reoxygenated. Potassium cyanide (KCN) completely inhibited this oxygen-induced calcium paradox.It is concluded that the presence of either ATP or electron transport is a prerequisite for the occurrence of this type of Ca²⁺-induced cell damage. Mitochondria most likely play a key role in the occurrence of the calcium paradox because of their ability to accumulate huge amounts of Ca²⁺ under these conditions.