thapsigargin and Arrhythmias--Cardiac

Measurement and quantification of cardiomyocyte or cardiac contractions as important (patho) physiologic parameters require highly specialized and expensive setups of fully integrated hard- and software that may be very difficult to use and may also depend on highly sophisticated methods of further data evaluation. With MYOCYTER (MC) we present a complete and highly customizable open-source macro for ImageJ, enabling fast, reliable user-friendly large scale analysis extracting an extensive amount of parameters from (even multiple) video recorded contracting cells or whole hearts, gained from a very competitive experimental setup. The extracted parameters enable extensive further (statistical) analysis to identify and quantify the effects of pathologic changes or drugs. Using videos following known mathematical functions, we were able to demonstrate the accuracy of MYOCYTER's data extraction, also successfully applied the software to both cellular and animal models, introducing innovations like dynamic thresholding, automatic multi-cell recognition, "masked" evaluation and change of applied parameters even after evaluation.

Topics: Animals; Arrhythmias, Cardiac; Daphnia; Ethanol; Image Processing, Computer-Assisted; Male; Mice, Inbred C57BL; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Reproducibility of Results; Software; Thapsigargin

Atrial fibrillation (AF) is the most common arrhythmia in humans, yet; treatment has remained sub-optimal due to poor understanding of the underlying mechanisms. Cardiac alternans precede AF episodes, suggesting an important arrhythmia substrate. Recently, we demonstrated ventricular SERCA2a overexpression suppresses cardiac alternans and arrhythmias. Therefore, we hypothesized that atrial SERCA2a overexpression will decrease cardiac alternans and arrhythmias.. Adult rat isolated atrial myocytes where divided into three treatment groups 1) Control, 2) SERCA2a overexpression (Ad.SERCA2a) and 3) SERCA2a inhibition (Thapsigargin, 1μm). Intracellular Ca2+ was measured using Indo-1AM and Ca2+ alternans (Ca-ALT) was induced with a standard ramp pacing protocol.. As predicted, SR Ca2+ reuptake was enhanced with SERCA2a overexpression (p< 0.05) and reduced with SERCA2a inhibition (p<0.05). Surprisingly, there was no difference in susceptibility to Ca-ALT with either SERCA2a overexpression or inhibition when compared to controls (p = 0.73). In contrast, SERCA2a overexpression resulted in increased premature SR Ca2+ (SCR) release compared to control myocytes (28% and 0%, p < 0.05) and concomitant increase in SR Ca2+ load (p<0.05). Based on these observations we tested in-vivo atrial arrhythmia inducibility in control and Ad.SERCA2a animals using an esophageal atrial burst pacing protocol. There were no inducible atrial arrhythmias in Ad.GFP (n = 4) animals though 20% of Ad.SERCA2a (n = 5) animals had inducible atrial arrhythmias (p = 0.20).. Our findings suggest that unlike the ventricle, SERCA2a is not a key regulator of cardiac alternans in the atrium. Importantly, SERCA2a overexpression in atrial myocytes can increase SCR, which may be arrhythmogenic.

Topics: Animals; Arrhythmias, Cardiac; Atrial Fibrillation; Brugada Syndrome; Calcium; Cardiac Conduction System Disease; Gene Expression Regulation; Glucans; Heart Atria; Heart Conduction System; Humans; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Topics: Acidosis; Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dantrolene; Disease Models, Animal; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Peptides; Phosphorylation; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Time Factors; Ventricular Function, Left; Ventricular Pressure

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.

Topics: Action Potentials; Adrenergic beta-Agonists; Animals; Arrhythmias, Cardiac; Calcium Channel Blockers; Calcium Signaling; Carbachol; Cardiac Pacing, Artificial; Cholinergic Agonists; Enzyme Inhibitors; Gestational Age; Heart; Heart Ventricles; Isoproterenol; Mice; Ryanodine; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Verapamil

Aconitine is an effective ingredient in Aconite tuber, an important traditional Chinese medicine. Aconitine is also known to be a highly toxic diterpenoid alkaloid with arrhythmogenic effects. In the present study, we have characterized the properties of arrhythmic cytotoxicity and explored the possible mechanisms of aconitine-induced cardiomyocytes. Results show that aconitine induces significant abnormity in the spontaneous beating rate, amplitude of spontaneous oscillations and the relative intracellular Ca(2+) concentration. Also, mRNA transcription levels and protein expressions of SR Ca(2+) release channel RyR(2) and sarcolemmal NCX were elevated in aconitine-induced cardiomyocytes. However, co-treatment with ruthenium red (RR), a RyR channel inhibitor, could reverse the aconitine-induced abnormity in intracellular Ca(2+) signals. These results demonstrate that disruption of intracellular Ca(2+) homeostasis in the cardiac excitation-contraction coupling (EC coupling) is a crucial mechanism of arrhythmic cytotoxicity in aconitine-induced cardiomyocytes. Moreover, certain inhibitors appear to play an important role in the detoxification of aconitine-induced Ca(2+)-dependent arrhythmias.

Topics: Aconitine; Animals; Arrhythmias, Cardiac; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cells, Cultured; Heart Rate; Homeostasis; Myocardial Contraction; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Ruthenium Red; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Thapsigargin

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca(2+) release from intracellular stores. These Ca(2+)-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of approximately 370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to Ca(2+) sparks), LCCs also showed some different characteristics. With simultaneous Ca(2+) imaging and current recording, the localized fluorescence increase due to Ca(2+) entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.

Topics: Animals; Arrhythmias, Cardiac; Caffeine; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cell Membrane; Cresols; Enzyme Inhibitors; Heart Atria; Heart Ventricles; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Mice; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Tetracaine; Thapsigargin

Early afterdepolarizations are seen during the repolarization phases of the action potential and delayed afterdepolarizations appear later, usually following complete repolarization of the cell membrane potential. Both forms of afterdepolarization are linked to the occurrence of aftercontractions, seem to play a role in the generation of ventricular arrhythmias and are believed to be the result of abnormalities of intracellular Ca handling. Suggestions for the mechanisms responsible vary from both types of afterpotential being mediated by Ca release from the sarcoplasmic reticulum, to early afterdepolarization formation being due to reactivation of the L-type sarcolemmal Ca channels during the action potential. We tried to assess the functional importance of the sarcoplasmic reticulum or Ca influx in the development of afterpotentials and abnormal contractile activity in guinea-pig cardiac myocytes. Ca influx was increased using isoproterenol, Bay K8644 or increasing extracellular [Ca]. Sarcoplasmic reticulum Ca content was measured using rapid cooling contractures or caffeine-induced Na/Ca exchange current and the sarcoplasmic reticulum was inhibited using caffeine or thapsigargin. Aftercontractions associated with either early or delayed afterdepolarizations could be induced by increasing Ca influx. The increased Ca influx produced increases in sarcoplasmic reticulum Ca content and aftercontractions were associated with a larger SR Ca content. However, the sarcoplasmic reticulum was no more loaded with Ca when aftercontractions occurred than when aftercontractions did not occur. Preventing Ca sequestration by the sarcoplasmic reticulum inhibited the formation of aftercontractions. The results suggest that alterations to both Ca influx and sarcoplasmic reticulum Ca content are required to produce aftercontractions.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Action Potentials; Adrenergic beta-Agonists; Animals; Arrhythmias, Cardiac; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channels, L-Type; Cells, Cultured; Enzyme Inhibitors; Guinea Pigs; Heart; Ion Transport; Isoproterenol; Membrane Potentials; Myocardial Contraction; Myocardium; Receptors, Adrenergic, beta; Sarcoplasmic Reticulum; Thapsigargin

Various studies on humans and experimental mammals showed that d-sotalol and tedisamil (class III antiarrhythmic drugs with positive inotropic effect) facilitate spontaneous ventricular defibrillation. Following our previous results, we summarized that spontaneous ventricular defibrillation requires high level of intercellular coupling and synchronization, both of which depends on intracellular free Ca2+ concentration. We hypothesized that any antiarrhythmic compound that facilitates spontaneous defibrillation, including d-sotalol and tedisamil, should prevent intracellular free Ca2+ overload most likely by elevating cAMP level and enhancing cAMP-related Ca2+ uptake of the sarcoplasmic reticulum (SR). The aim of the present study was to examine the role of the SR uptake function in their effect against Ca2+ overload.. The effect of d-sotalol, tedisamil and dbcAMP on increased intracellular Ca2+ level were examined in cultured rat cardiomyocytes during blockade of SR Ca2+ uptake by administration of thapsigargin (TG), a selective inhibitor of Ca2+-ATPase.. Administration of 3 x 10(-6) mol/l TG, prior to d-sotalol, tedisamil and dbcAMP, significantly increased intracellular free Ca2+ concentration and prevented the effect of d-sotalol, tedisamil or dbcAMP to decrease intracellular Ca2+ level to its beseline, while 10(-6) mol/l TG prevented it only partially. Administration of either d-sotalol or tedisamil (at concentration of 10(-5) mol/l) before the administration of 10(-6) mol/l TG prevent the TG induced elevation of [Ca2+]i.. These results support our hypothesis that d-sotalol and tedisamil prevent Ca2+ overload by the cAMP dependent SR Ca2+ uptake.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Bridged Bicyclo Compounds, Heterocyclic; Bucladesine; Calcium; Cells, Cultured; Cyclopropanes; Heart; Humans; Ion Transport; Myocardium; Rats; Sarcoplasmic Reticulum; Sotalol; 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