Calcium-free Perfusion Research Articles

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.

Polyamines and the calcium paradox in rat hearts

Polyamine levels were measured by means of high-performance liquid chromatography in Langendorff-perfused rat hearts subjected to the calcium paradox protocol. The concentrations of putrescine, spermidine and spermine did not change significantly during calcium-free perfusion but decreased when calcium was readmitted. This decrease was due to membrane disruption and release of the polyamines into the coronary effluent. The sum of released and remaining spermidine exceeded the concentration of spermidine in control hearts, but, for spermine, this sum was lower than the control level. The addition of 0.5 m m EGTA to the calcium-free solution raised the myocardial concentrations of putrescine and spermidine and enhanced the net increase of spermidine on calcium repletion. DL-α-Difluoromethylornithine (DFMO) inhibited these increases and lowered the putrescine level during all perfusion stages. External polyamines had a negative inotropic effect and inhibited the loss of myoglobin on calcium repletion (order of effectiveness: spermine > spermidine > putrescine). Inhibition of contractions by the combined action of verapamil and ryanodine or by potassium depolarization did not prevent myoglobin loss. External polyamines had no effect on high K/low Na contractures, which were mediated mainly by NaCa exchange. Calcium-free perfusion in the presence of 0.5 to 1 m m EGTA improved the membrane protection by polyamines or by diamines and analogues, like ornithine, 1,3-diaminopropane, or DFMO, which, in the absence of EGTA, gave no clear protection. It is concluded that calcium depletion and repletion influences myocardiaal polyamine concentrations by (1) membrane disruption and release of polyamines into the coronary effluent, and (2) probably by a stimulation of ornithine decarboxylase activity. The changes in polyamine concentrations do not seem to have any causal role in calcium overload and cell death. Exogenous polyamines protect against membrane damage.

Intracellular sodium during ischemia and calcium-free perfusion: A 23Na NMR study

Accumulation of sodium-ions (Na +) in myocardial cells during both ischemia and calcium (Ca 2+)-free perfusion has been suggested to play an important role in the damage occurring during subsequent reperfusion and calcium repletion, respectively. We have used 23Na NMR spectroscopy in combination with shift reagents to determine intracellular Na +-concentration ([Na +] i) in isolated rat hearts during either control perfusion followed by ischemia and reperfusion, or during control perfusion, Ca 2+-free perfusion and subsequent ischemia. [Na +] i during control perfuson was found to be 10.5 ± 0.6 mmol/l. During 30 min of ischemia [Na +] i rose substantially to 25.0 ± 3.2 mmol/l. During 15 min of reperfusion [Na +] i initially decreased, but leveled off after approximately 3 min and was 17.9 ± 3.7 mmol/l at the end of the reperfusion period. Most surprisingly, however, no significant increase of [Na +] i was observed during 30 min of Ca 2+-free perfusion, although severe calcium paradox damage was shown to occur under the used conditions, when calcium was readmitted to the heart. The absence of a rise of [Na +] i during Ca 2+-free perfusion was substantiated when during subsequent ischemia a similar rise of [Na +] i was observed as during ischemia without previous Ca 2+-depletion. We conclude that an increased [Na +] i during Ca 2+-depletion is not a prerequisite for the calcium paradox to occur, but that increased [Na +] i during ischemia may influence the subsequent reperfusion damage through Na +-Ca 2+ exchange.

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

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, alpha-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.

Role of calcium channels and protein kinase C for release of norepinephrine and neuropeptide Y

The role of calcium for the release of norepinephrine (NE, determined by high-pressure liquid chromatography) and neuropeptide Y (NPY, determined by radioimmunoassay) was investigated in guinea pig perfused hearts with intact sympathetic innervation. In the presence of extracellular calcium (1.85 mM), electrical stimulation of the left stellate ganglion (12 Hz, 1 min) induced a closely related release of NE and NPY with the molar ratio of approximately 400-600 (NE) to 1 (NPY). The stimulation-evoked overflow of both transmitters was dependent from the extracellular calcium concentration and was almost completely suppressed by calcium-free perfusion. The corelease of both transmitters was not affected by the L-type calcium channel blocker felodipine (1-10 microM). However, the overflow of NE and NPY was markedly attenuated by the unselective calcium antagonist flunarizine (1-10 microM) and completely prevented by the neuronal (N-type) calcium channel blockers omega-conotoxin (1-100 nM) and cadmium chloride (10-100 microM), indicating a key role for N-type calcium channels in the exocytotic release of transmitters from cardiac sympathetic nerve fibers. Possibly due to unspecific actions, such as interference with sodium channels or uptake1-blocking properties, the phenylalkylamines verapamil (0.01-10 microM) and gallopamil (1-10 microM) reduced NPY overflow with only a minor effect on NE overflow. The stimulation-induced transmitter release was increased up to twofold by activation of protein kinase C (phorbol 12-myristate 13-acetate, 3 nM-3 microM) and completely suppressed by inhibition of protein kinase C (polymyxin B, 100 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes

Microdialysis of dopamine was performed in the striatum with four different microdialysis cannulas: a trans-cerebral probe, a U-shaped probe, a I-shaped probe and a commercially available I-shaped probe (Carnegie). The commercial cannula was studied with as well as without a guide cannula. The effect of infusion of tetrodotoxin (TTX) or calcium free-Ringer solution on the dialysate content of dopamine released was determined 2 h after implantation (day 1) as well as 24 h after implantation (day 2). Two hours after implantation, all cannulas displayed a certain amount of TTX-independent overflow of dopamine. The best results were obtained with the I-shaped cannula: already 2 h after implantation of the probe, about 85% of the release of dopamine was TTX-dependent. In case of the trans-cerebral cannula and the Carnegie cannula (implanted without guide in anesthetized rats), 70-75% of the output of dopamine was TTX-dependent. When the Carnegie cannula was implanted with a guide cannula, only 45% of the dopamine output was TTX-dependent. The responses to calcium-free perfusion were much more variable. Dopamine output in I-shaped and the U-shaped probes was virtually insensitive to calcium-free perfusion in acute experiments. Twenty-four hours after implantation of the probes, the calcium- and TTX-dependency was much more pronounced. All types of cannulas studied now sampled dopamine that was completely TTX-dependent. Calcium-free perfusion caused a reproducible disappearance of dopamine from the dialysates to 30% of controls, in all cannulas studied. When the removable Carnegie cannula was re-implanted 24 h after its first implantation, 91% of the dopamine overflow was TTX-sensitive. These results confirm earlier conclusions that microdialysis experiments should be carried out at least 24 h after implantation of the probe.

Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic, ionic, contractile and electrophysiological studies

The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37 degrees C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22 degrees C and 27 degrees C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37 degrees C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24 +/- 2.49 IU.g dry wt-1, and the total amount of protein lost was 70.37 +/- 0.73 mg.g dry wt-1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37 degrees C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.

Effects of repeated low calcium perfusion on the rat heart: A grandual induction of calcium related damage

The calcium paradox occurs immediately upon perfusion with calcium-containing medium after a period of calcium-free perfusion. The sequence of events occur so rapidly that it is difficult to distinguish causal factors from resultant. The present study describes a model in which calcium induced damage is produced more gradually. Isolated perfused rat hearts were subjected to brief periods of hypocalcium perfusion (< 50 μ m) alternated with normal calcium perfusion (1.25 m m) over 60 min. Changes in high energy phosphate content were monitored using 31P-NMR. With repeated sequential 2-min periods of alternate hypo- and normocalcium perfusion, there was a gradual reduction in ATP and phosphocreatine with a concomitant loss of function and decline in coronary flow. There was no change in inorganic phosphate content and a small degree of acidosis. Increasing the hypocalcium concentration from 5 to 40 μ m resulted in a more gradual depletion in energy stores. Trifluoroperazine (a calmodulin inhibitor) had no effect on the energetic changes. Electron microscopic studies reveal that this model of damage induced by repeated low and normal calcium perfusion has some features in common with the calcium paradox. The extent of damage induced is greater when lower calcium concentrations (5 μ m) are used.

Tissue protection by verapamil in the calcium paradox

The effect of verapamil (+/- 2 mumol/l verapamil) on calcium paradox injuries, as well as on hearts subjected to calcium-free perfusion alone, was studied in isolated perfused rat hearts. Three different protocols for calcium depletion (5 min) were followed: 5 or 45 ml of nominally calcium-free solution (1 or 9 ml/min), or 45 ml of nominally calcium-free solution to which 20 mumol/l CaCl2 was added. Ultrastructural analyses showed that verapamil protects against cellular injuries upon readmission of calcium to hearts subjected to partial calcium depletion (5 ml calcium-free solution, or 45 ml to which 20 mumol/l CaCl2 was added) by reducing the number of affected cells. No protection was found after more extensive calcium washout. However, in all groups examined, we found an inverse relationship between the number of injured cells and ATP content. Verapamil protected against contracture development and reduced the increase in tissue calcium content observed after readmission of calcium to hearts perfused with 5 ml calcium free solution, whereas no significant effect of verapamil on tissue calcium content was found in hearts perfused with 45 ml nominally calcium-free solution. In hearts studied after calcium-free perfusion no effect of verapamil treatment on the separation of the intercalated disc was found. The protective effect of verapamil could not be explained by differences in calcium or cAMP levels after calcium-free perfusion.

Extracellular sodium and chloride depletion enhances nonexocytotic noradrenaline release induced by energy deficiency in rat heart.

The effect of either extracellular sodium or extracellular chloride reduction on the release of endogenous noradrenaline and its deaminated metabolite dihydroxyphenylglycol (DOPEG) has been studied in the isolated perfused rat heart under conditions of ischaemia and cyanide intoxication. The overflow of noradrenaline and DOPEG was determined by high pressure liquid chromatography. The efflux of DOPEG, the predominant neuronal noradrenaline adrenaline metabolite, served as indicator of the free axoplasmic plasmic amine concentration. A calcium-free perfusion buffer was used to avoid exocytotic noradrenaline release. Sodium and chloride in the perfusion buffer were replaced by lithium and isethionate, respectively. (1) Reduction of extracellular sodium or chloride increased noradrenaline overflow in ischaemia. The release was suppressed by the uptake1 blocker cocaine indicating carrier-mediated outward transport of noradrenaline. (2) In cyanide intoxication sodium or chloride reduction accelerated the onset of DOPEG efflux reflecting increased axoplasmic noradrenaline concentrations. This was accompanied by increased noradrenaline release. The ratio of noradrenaline/DOPEG overflow was increased by reduced sodium or chloride, indicating facilitation of carrier-mediated noradrenaline net outward transport. (3) In the presence of unaltered energy metabolism overflow of both, noradrenaline and DOPEG, was not enhanced by sodium or chloride reduction. The results demonstrate that reduction of extracellular sodium or chloride has two effects on noradrenaline release from the sympathetic neuron with reduced energy supply. First, reduced sodium or chloride induces increased axoplasmic noradrenaline concentrations by interference with vesicular storage function. Second, both interventions enhance carrier-mediated noradrenaline release.

Assessment of membrane protection by 3 1P-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 3 1P-NMR method. In hearts reexposed to calcium, following 3 minute calcium-free perfusion, a rapid contracture occured, 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.

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)

Intracellular sodium during ischemia and calcium-free perfusion: A 23Na NMR study on isolated rat hearts : C.J.A. van Echteld, J.H. Kirkels, P. van der Meer, T.J.C. Ruigrok, F.L. Meijler. Interuniversity Cardiology Institute of The Netherlands and Department of Cardiology, University Hospital, Utrecht, The Netherlands

Intracellular sodium during ischemia and calcium-free perfusion: A 23Na NMR study on isolated rat hearts : C.J.A. van Echteld, J.H. Kirkels, P. van der Meer, T.J.C. Ruigrok, F.L. Meijler. Interuniversity Cardiology Institute of The Netherlands and Department of Cardiology, University Hospital, Utrecht, The Netherlands

Phospholipid reorganization and bilayer destabilization during myocardial ischemia and reperfusion: A hypothesis

The ultrastructure of myocardial tissue was studied during ischemia and reperfusion, and during reperfusion with a calcium-containing solution after a short period of calcium-free perfusion (calcium paradox). After ischemia an aggregation of the sarcolemmal intramembranous particles was observed. Subsequent reperfusion resulted in further aggregation of the intramembranous particles and disruption of the sarcolemma, which was attended with the formation and extrusion of multilamellar, lipidic structures. Similar ultrastructural changes of the sarcolemma were observed during the calcium paradox. Our hypothesis is that these changes are a result of lateral phase separation of the membrane phospholipids and destabilization of the lipid bilayer. This reorganization of phospholipids may be induced by a decrease of the intracellular pH during ischemia, and an increase of the intracellular calcium content during reperfusion after ischemia and during calcium repletion after calcium-free perfusion. These ultrastructural changes of the sarcolemma are, in our view, a consequence of the physicochemical behaviour of the sarcolemmal phospholipids.

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.

Polyamines mediate uncontrolled calcium entry and cell damage in rat heart in the calcium paradox.

Brief perfusion of heart with calcium-free medium renders myocardial cells calcium-sensitive so that readmission of calcium results in uncontrolled Ca2+ entry and acute massive cell injury (calcium paradox). We investigated the hypothesis that polyamines may be involved in the mediation of abnormal Ca2+ influx and cell damage in the calcium paradox. The isolated perfused rat heart was used for these studies. Calcium-free perfusion promptly (less than 5 min) decreased the levels of polyamines and the activity of their rate-regulating synthetic enzyme, ornithine decarboxylase (ODC), and calcium reperfusion abruptly (less than 15-180 s) increased these components. alpha-Difluoromethylornithine (DFMO), a specific suicide inhibitor of ODC, suppressed the calcium reperfusion-induced increase in polyamines and the concomitant increase in myocardial cellular 45Ca influx, loss of contractility, release of cytosolic enzymes, myoglobin, and protein, and structural lesions. Putrescine, the product of ODC activity, nullified DFMO inhibition and restored the calcium reperfusion-induced increment in polyamines and the full expression of the calcium paradox. Putrescine itself enhanced the reperfusion-evoked release of myoglobin and protein in the absence of DFMO. Hypothermia blocked the changes in heart ODC and polyamines induced by calcium-free perfusion and calcium reperfusion and prevented the calcium paradox. These results indicate that rapid Ca2+-directed changes in ODC activity and polyamine levels are essential for triggering excessive transsarcolemmal transport of Ca2+ and explosive myocardial cell injury in the calcium paradox.

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.

Tissue protection by allopurinol in the myocardial calcium paradox.

The aim of the present study was to evaluate the protective properties of the xanthine oxidase inhibitor allopurinol in the myocardial calcium paradox. Two injury levels, minimal and total calcium paradox, caused by different volumes (5 ml and 45 ml) of calcium-free perfusion (5 min) prior to calcium repletion (15 min) were examined +/- allopurinol (0.15 mmol/l) in the normothermic isolated rat heart model. Allopurinol supplementation (5 min prior to, during and 5 min following Ca2+-free perfusion) had no effect upon tissue injury in the total calcium paradox, but afforded considerable protection as assessed by enzymatic, physiologic, and metabolic parameters in the minimal calcium paradox. When allopurinol was omitted during calcium repletion, tissue protection was less apparent. The presence of verapamil (2 mumol/l) in addition to allopurinol (5 min prior to, during, and 5 min following calcium depletion) afforded only a marginal further protection in the minimal calcium paradox. It is concluded from the present study that tissue protection by allopurinol in the calcium paradox is limited to minimal or less severe calcium paradox models and that the protective action of allopurinol may indicate an inhibition of the xanthine oxidase reaction and the generation of free oxygen radicals.

Calcium dependence of muscarinic receptor-mediated catecholamine secretion from the perfused rat adrenal medulla.

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.

Calcium repletion-induced arrhythmias after short periods of calcium-free perfusion in the isolated rat heart.

The occurrence of arrhythmias was studied in the "calcium-paradox" model in the isolated rat heart. Clear relationships were found between the duration of calcium-free perfusion and (a) the occurrence of calcium-free-induced electrophysiological changes, (b) the incidence and duration of subsequently induced calcium-repletion arrhythmias and (c) mechanical recovery at the end of the repletion period. The first signs of electrophysiological changes (i.e. decreased heart rate, T-wave amplitude and increased PQ-interval) and irreversible loss of myocardial recovery occurred during or after 60-90 s of calcium-free perfusion. The occurrence of calcium-repletion induced ventricular tachycardia parallelled this onset of irreversible cardiac injury. These results suggest that the process of calcium washout and subsequent sudden calcium overloading may play a role as a trigger in the pathogenesis of ventricular arrhythmias.