ryanodine and Tachycardia

The possibility to generate cardiomyocytes (CMs) from disease-specific induced pluripotent stem cells (iPSCs) is a powerful tool for the investigation of various cardiac diseases in vitro. The pathological course of various cardiac conditions, causatively heterogeneous, often converges into disturbed cellular Ca(2+) cycling. The gigantic Ca(2+) channel of the intracellular Ca(2+) store of CMs, the ryanodine receptor type 2 (RyR2), controls Ca(2+) release and therefore plays a crucial role in Ca(2+) cycling of CMs. In the present protocol we describe ways to measure and analyze global as well as local cellular Ca(2+) release events in CMs derived from a patient carrying a CPVT-causing RyR2 mutation.

Topics: Aniline Compounds; Animals; Calcium; Calcium Signaling; Cell Culture Techniques; Cell Differentiation; Cellular Reprogramming; Fibroblasts; Gene Expression; Humans; Induced Pluripotent Stem Cells; Isoproterenol; Mice; Molecular Imaging; Mutation; Myocytes, Cardiac; Primary Cell Culture; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tachycardia; Xanthenes

The mechanisms of sinoatrial node (SAN) dysfunction in patients with chronically elevated sympathetic tone and reduced pacemaker current (I(f); such as heart failure) are poorly understood. We simultaneously mapped membrane potential and intracellular Ca(2+) in the Langendorff-perfused canine right atrium (RA). Blockade of either I(f) (ZD-7288) or sarcoplasmic reticulum Ca(2+) release (ryanodine) alone decreased heart rate by 8% (n = 3) and 16% (n = 3), respectively. Combined treatment of ZD-7288 and ryanodine consistently resulted in prolonged (> or =3 s) sinus pauses (PSPs) (n = 4). However, the middle SAN remained as the leading pacemaking site after these treatments. Prolonged exposure with isoproterenol (0.01 micromol/l) followed by ZD-7288 completely suppressed SAN but triggered recurrent ectopic atrial tachycardia. Cessation of tachycardia was followed by PSPs in five of eight RAs. Isoproterenol initially increased heart rate by 75% from baseline with late diastolic intracellular Ca(2+) elevation (LDCAE) from the superior SAN. However, after a prolonged isoproterenol infusion, LDCAE disappeared in the superior SAN, the leading pacemaker shifted to the inferior SAN, and the rate reduced to 52% above baseline. Caffeine (2 ml, 20 mmol/l) injection after a prolonged isoproterenol infusion produced LDCAE in the SAN and accelerated the SAN rate, ruling out sarcoplasmic reticulum Ca(2+) depletion as a cause of Ca(2+) clock malfunction. We conclude that in an isolated canine RA preparation, chronically elevated sympathetic tone results in abnormal pacemaking hierarchy in the RA, including suppression of the superior SAN and enhanced pacemaking from ectopic sites. Combined malfunction of both membrane and Ca(2+) clocks underlies the mechanisms of PSPs.

Topics: Analysis of Variance; Animals; Bradycardia; Calcium; Cardiotonic Agents; Dogs; Heart Atria; Isoproterenol; Membrane Potentials; Pyrimidines; Ryanodine; Tachycardia

Few experimental models produce spontaneous tachycardia in normal left atria to allow the study of the cellular mechanisms underlying this contributor to atrial fibrillation. We reported 2-aminoethoxydiphenyl borate (2-APB) that provokes sporadic spontaneous mechanical activity and calcium leak in isolated rat left atria. Since sarcoplasmic reticulum calcium leak in the presence of high calcium load may trigger tachyarrhythmias, we tested how conditions that increase calcium load affect 2-APB-induced ectopic activity. Exposing superfused rat left atria to (i) 30 nM isoproterenol, (ii) 3 microM forskolin, (iii) 300 nM (-)BayK 8644 ((4S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluormethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), (iv) 300 nM FPL-64176 (2,5-Dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester) or (v) 120 microM ouabain increases their force of contraction, evidence of calcium loading, but does not produce ectopic activity. Spontaneous mechanical activity occurs in left atria superfused with 20 microM 2-APB at 47+/-6 contractions/min in the absence of pacing. Any of these five agents increase rates of 2-APB-induced spontaneous mechanical activity to >200 contractions/min in the absence of pacing. Washing tachycardic left atria with superfusate lacking 2-APB restores normal function, demonstrating the reversibility of these effects. Decreasing superfusate sodium reverses this tachycardia and two hyperpolarization-activated current (I(f)) inhibitors blunt this ectopic activity. Thus conditions that increase atrial calcium load increase the frequency of spontaneous mechanical activity. Decreasing extracellular sodium and I(f) inhibitors suppress this spontaneous tachycardia suggesting forward-mode sodium-calcium exchange and I(f)-like activities underlie this activity. This model may help define cell pathways that trigger atrial tachyarrhythmias.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Benzazepines; Boron Compounds; Calcium; Calcium Channel Agonists; Cardiotonic Agents; Cyclic Nucleotide-Gated Cation Channels; Heart Atria; In Vitro Techniques; Male; Ouabain; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; RNA, Messenger; Ryanodine; Tachycardia

We have previously demonstrated that brief episodes of tachycardia prior to a prolonged occlusion of a coronary artery, followed by reperfusion, substantially reduce the infarct size. Adenosine receptors and mitochondrial ATP-dependent K(+) channels mediate this effect. Since preconditioning can be induced or reverted by maneuvers that increase or decrease [Ca(2+)](i), respectively, and tachycardia increases [Ca(2+)](i), we studied the participation of sarcoplasmic reticulum and Ca(2+) in the preconditioning effect of tachycardia. We measured the effect of ischemia and tachycardia on Ca(2+) uptake and release by sarcoplasmic reticulum vesicles isolated from left ventricular canine myocardium. Myocardial ischemia increased Ca(2+)-release rate constants and decreased both the initial rates of Ca(2+) uptake and [(3)H]-ryanodine binding by sarcoplasmic reticulum. In addition, ischemia induced a decrease in the pentameric form of phospholamban and in the content of ryanodine-receptor Ca(2+)-release channel protein. All these effects were reverted in hearts preconditioned with tachycardia. Furthermore, tachycardia by itself increased [(3)H]-ryanodine binding, Ca(2+)-release rate constants and the protein levels of ryanodine-receptor Ca(2+)-release channels and the ATP-dependent Ca(2+) pump. These results suggest that tachycardia preserves the integrity of the sarcoplasmic reticulum preventing the excess of release and the decrease of uptake of Ca(2+) produced by ischemia, thereby avoiding cytosolic Ca(2+) overload. This sarcoplasmic reticulum protection could partly explain the preconditioning effect of tachycardia.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Dogs; Heart Ventricles; Ischemic Preconditioning, Myocardial; Kinetics; Myocardial Ischemia; Myocardium; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Tachycardia

We have previously shown that amiloride suppresses the induction of sustained ventricular tachycardia both in dogs late following myocardial infarction and in patients. In those studies the only electrophysiologic correlate of amiloride's antiarrhythmic activity observed was prolongation of ventricular effective refractory period at the zone bordering the infarct. The purpose of this study was to assess the pharmacologic effects of amiloride associated with antiarrhythmic efficacy. However, amiloride has multiple pharmacologic effects, including inhibition of the slow inward calcium current (ICa), inhibition of the sodium-calcium and sodium-hydronium ion exchangers, acidification of intracellular pH resulting in partial inhibition of the inwardly rectifying potassium current (IK1), and increase in serum potassium and magnesium. The approach used in this study was to use selective pharmacologic probes to produce the known components of amiloride's pharmacologic effects. The selective agents consisted of verapamil (partial blockade of ICa), 3',4'-dichlorobenzamil (partial inhibition of the Na-Ca exchanger), 5-(N-ethyl-N-isopropyl) amiloride (partial inhibition of the Na-H exchanger), the combination of these congeners, KCl infusions to increase serum potassium, MgSO4 infusions to increase serum magnesium, the combination of KCl and MgSO4 infusions, barium (partial block of IK1), ryanodine (partial blockade of sarcoplasmic reticulum calcium release), and placebo. In this study only barium produced antiarrhythmic and electrophysiologic effects similar to those of amiloride. However, amiloride prolongs border zone refractoriness selectively, whereas barium prolongs refractoriness diffusely throughout the myocardium. Blockade of ICa by verapamil, increases in serum magnesium and potassium alone or in combination, and partial blockade of sarcoplasmic reticulum by ryanodine were not antiarrhythmic in this model. Monotherapies that produced partial blockade of the Na-Ca and Na-H exchangers separately did not produce antiarrhythmic activity. However, the combination of these amiloride congeners reproduced the antiarrhythmic activity of amiloride but did not prolong border zone refractoriness. From these studies we conclude that the antiarrhythmic activity of amiloride relates to (a) selective blockade of IK1 in the border zone and/or (b) combined inhibition of sodium-calcium and sodium-hydronium ion exchangers.

Topics: Amiloride; Animals; Anti-Arrhythmia Agents; Barium; Dogs; Dose-Response Relationship, Drug; Drug Interactions; Electrophysiology; Magnesium; Potassium; Ryanodine; Tachycardia; Ventricular Function; Verapamil