thapsigargin and Myocardial-Ischemia

Angiotensin-converting enzyme inhibitors (ACE-I) improve clinical outcome in patients with myocardial infarction (MI) and chronic heart failure. We investigated potential anti-arrhythmic (AA) benefits in a mouse model of ischemic HF. We hypothesized that normalization of diastolic calcium (Ca(2+)) by ACE-I may prevent Ca(2+)-dependent reduction of inward rectifying K(+) current (IK1) and occurrence of arrhythmias after MI. Mice were randomly assigned to three groups: Sham, MI, and MI-D (6 weeks of treatment with ACE-I delapril started 24h after MI). Electrophysiological analyses showed that delapril attenuates MI-induced prolongations of electrocardiogram parameters (QRS complex, QT, QTc intervals) and conduction time from His bundle to ventricular activation. Delapril improved the sympatho-vagal balance (LF/HF) and reduced atrio-ventricular blocks and ventricular arrhythmia. Investigations in cardiomyocytes showed that delapril prevented the decrease of IK1 measured by patch-clamp technique. IK1 reduction was related to intracellular Ca(2+) overload. This reduction was not observed when intracellular free-Ca(2+) was maintained low. Conversely, increasing intracellular free-Ca(2+) in Sham following application of SERCA2a inhibitor thapsigargin reduced IK1. Thapsigargin had no effect in MI animals and abolished the benefits of delapril on IK1 in MI-D mice. Delapril prevented both the prolongation of action potential late repolarization and the depolarization of resting membrane potential, two phenomena known to trigger abnormal electrical activities, promoted by MI. In conclusion, early chronic therapy with delapril after MI prevented Ca(2+)-dependent reduction of IK1. This mechanism may significantly contribute to the antiarrhythmic benefits of ACE-I in patients at risk for sudden cardiac death.

Topics: Action Potentials; Angiotensin-Converting Enzyme Inhibitors; Animals; Calcium Signaling; Cells, Cultured; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Indans; Male; Mice; Myocardial Contraction; Myocardial Ischemia; Myocytes, Cardiac; Potassium; Potassium Channels, Inwardly Rectifying; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Ventricular Fibrillation

The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.

Topics: Animals; Calcium; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; HeLa Cells; Humans; Male; Membrane Glycoproteins; Mice; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nerve Growth Factors; Protein Binding; Protein Sorting Signals; Protein Structure, Tertiary; Proteins; Rats; Sarcoplasmic Reticulum; Thapsigargin; Tunicamycin

The type 2 chloride intracellular channel, CLIC-2, is a member of the glutathione S-transferase structural family and a suppressor of cardiac ryanodine receptor (RyR2) Ca2+ channels located in the membrane of the sarcoplasmic reticulum (SR). Modulators of RyR2 activity can alter cardiac contraction. Since both CLIC-2 and RyR2 are modified by redox reactions, we speculated that the action of CLIC-2 on RyR2 may depend on redox potential. We used a GSH:GSSG buffer system to produce mild changes in redox potential to influence redox sensors in RyR2 and CLIC-2. RyR2 activity was modified only when both luminal and cytoplasmic solutions contained the GSH:GSSG buffer and the effects were reversed by removing the buffer from one of the solutions. Channel activity increased with an oxidizing redox potential and decreased when the potential was more reducing. Addition of cytoplasmic CLIC-2 inhibited RyR2 with oxidizing redox potentials, but activated RyR2 under reducing conditions. The results suggested that both RyR2 and CLIC-2 contain redox sensors. Since cardiac ischemia involves a destructive Ca2+ overload that is partly due to oxidation-induced increase in RyR2 activity, we speculate that the properties of CLIC-2 place it in an ideal position to limit ischemia-induced cellular damage in cardiac muscle.

Topics: Animals; Buffers; Caffeine; Calcium Signaling; Chloride Channels; Glutathione; Glutathione Disulfide; Lipid Bilayers; Membrane Potentials; Myocardial Ischemia; Myocytes, Cardiac; Oxidation-Reduction; Rabbits; Ruthenium Red; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sheep; Thapsigargin

There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability.. The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion.. In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion.. These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.

Topics: Analysis of Variance; Animals; Calcium; Cell Death; Cell Size; Cells, Cultured; Dose-Response Relationship, Drug; Male; Models, Animal; Myocardial Ischemia; Myocardial Reperfusion; Myocardium; Perfusion; Random Allocation; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Swine; Thapsigargin; Thiourea; Time Factors

Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC.. Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC.. In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i).. The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Cell Size; Cells, Cultured; Coronary Vessels; Cytosol; Endoplasmic Reticulum; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Hydrogen-Ion Concentration; Lactic Acid; Male; Manganese; Myocardial Ischemia; Rats; Rats, Wistar; Ryanodine; Sodium-Hydrogen Exchangers; Sulfones; Thapsigargin

Acute ischemia is associated with rapidly decreasing contractility and Ca2+-transients. Diastolic intracellular Ca2+, however, only mildly increases until development of contracture. The purpose of this study was to investigate whether changes of cellular calcium handling during the early phase of ischemia are associated with active sarcolemmal calcium transport.. Changes of extracellular concentration of calcium ([Ca2+]o) and tetramethylammonium ([TMA+]o), to estimate extracellular space, were simultaneously measured with ion-specific electrodes in the globally ischemic rat heart. The magnitude and direction of sarcolemmal calcium transport were calculated from [Ca2]o corrected for changed extracellular water content. Energy dependence of sarcolemmal calcium transport was investigated by application of iodoaceticacid (IAA) to inhibit anaerobic glycolysis, and the involvement of the sarcoplasmic reticulum (SR) was studied by application of thapsigargin. The effect of anoxia and thapsigargin on cytosolic and SR calcium was studied in isolated myocytes with the fluorescent indicator indo-1.. [Ca2+]o increased and extracellular space gradually decreased in the ischemic intact heart. During the first 7 min, the increase of [Ca2+]o was associated with net outward transport of calcium. Subsequently, net re-uptake occurred. IAA completely abolished outward transport and influx was accelerated and enhanced. Application of thapsigargin attenuated outward transport. In electrically-stimulated myocytes, anoxia caused little change of diastolic calcium and depletion of SR. Thapsigargin reduced both calcium transient amplitude and SR calcium without affecting diastolic calcium. During three successive short episodes of ischemia/reperfusion (preconditioning), outward transport of calcium progressively decreased.. During the early phase of global ischemia, energy dependent transport of calcium to the extracellular space occurs. At least part of this calcium originates from SR. During the later stage of ischemia, re-uptake of calcium occurs, which is associated with development of contracture.

Topics: Animals; Biological Transport; Calcium; Energy Metabolism; Extracellular Matrix; In Vitro Techniques; Iodoacetic Acid; Male; Myocardial Ischemia; Myocardium; Quaternary Ammonium Compounds; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Thapsigargin

We tested for the first time the hypothesis that lessened uptake of Ca2+ into sarcoplasmic reticulum by inhibitors of the Ca(2+)-ATPase pump can decrease the severity of reperfusion stunning (postischemic mechanical dysfunction). We used two novel inhibitors of the Ca(2+)-ATPase pump: (a) cyclopiazonic acid (CPA, 10(-6)-10(-8)M), and (b) thapsigargin (10(-6) or 2.5 x 10(-8)M). The isolated working rat heart was subjected to 20-min global ischemia before 20-min reperfusion. The inhibitor was added either before onset of ischemia or at time of reperfusion. Reperfusion mechanical function (aortic output, AO) was measured and compared with the preischemia values. Pretreatment with CPA improved recovery of AO after 20-min reperfusion from 78.2 +/- 3.0 (n = 12) to 93.3 +/- 1.6% (n = 7) (p < 0.002) while CPA added during reperfusion only, improved AO recovery from 78.2 +/- 3.0 (n = 12) to 90.2 +/- 2.7% (n = 6) (p < 0.05). Pretreatment with thapsigargin (2.5 x 10(-8) M) improved reperfusion AO recovery from 63.5 +/- 1.1 (n = 6) to 96.8 +/- 4.2% (n = 6) (p < 0.002), but when thapsigargin was added only during reperfusion AO recovery did not change. We conclude that inhibition of the Ca2+ uptake pump represents a new principle of control of cell calcium fluxes and that CPA is more effective than thapsigargin. The proposed mechanism of protection against stunning may include inhibition of oscillations of intracellular calcium, and/or depletion of calcium in sarcoplasmic reticulum (SR).

Topics: Animals; Anti-Arrhythmia Agents; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Heart Rate; Indoles; Male; Myocardial Ischemia; Myocardial Stunning; Rats; Sarcoplasmic Reticulum; Terpenes; Thapsigargin

The hypothesis tested was that sequestration of calcium by the sarcoplasmic reticulum and internal calcium oscillations may play a role in the genesis of ischemic and reperfusion ventricular arrhythmias.. Previous data suggest that inhibition of the release of intracellular calcium from the sarcoplasmic reticulum by ryanodine may prevent ventricular fibrillation.. The isolated Langendorff perfused rat heart was treated with two specific inhibitors of the calcium ATPase pump of the sarcoplasmic reticulum (thapsigargin [10(-6) mol/liter] or cyclopiazonic acid [10(-7) mol/liter]) for 5 min before left anterior descending coronary artery ligation was performed. One group of hearts was subject to 30 min of coronary artery ligation, and ischemic arrhythmias were monitored. In a second group, the incidence of reperfusion arrhythmias was measured after 10, 15, 20, 25 and 30 min of coronary artery ligation.. Thapsigargin treatment during ischemia and reperfusion decreased the incidence of reperfusion ventricular fibrillation after 10 min of coronary artery ligation from 67% (n = 6) to 0% (n = 6) (p < 0.05), after 15 min from 81% (n = 16) to 25% (n = 20) (p < 0.002) and after 20 min of ischemia from 90% (n = 10) to 46% (n = 13) (p < 0.05). Thapsigargin treatment also decreased the incidence of ischemic ventricular fibrillation from 83% (n = 12) to 0% (n = 12) (p < 0.002). Cyclopiazonic acid treatment during ischemia and reperfusion likewise decreased the incidence of ischemic and reperfusion arrhythmias.. The highly specific inhibitors of the calcium uptake pump of the sarcoplasmic reticulum--thapsigargin and cyclopiazonic acid--have antifibrillatory properties in the isolated perfused rat heart. They appear to act by restriction of calcium oscillations between the sarcoplasmic reticulum and the cytosol.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium-Transporting ATPases; Coronary Vessels; Drug Evaluation, Preclinical; Heart; In Vitro Techniques; Indoles; Ligation; Male; Myocardial Ischemia; Myocardium; Rats; Rats, Inbred Strains; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Time Factors