cytochalasin-d and Arrhythmias--Cardiac

AF (atrial fibrillation) is the most common sustained arrhythmia, and the PVs (pulmonary veins) play a critical role in triggering AF. Stretch causes structural remodelling, including cytoskeleton rearrangement, which may play a role in the genesis of AF. Lat-B (latrunculin B), an inhibitor of actin polymerization, is involved in Ca(2+) regulation. However, it is unclear whether Lat-B directly modulates the electrophysiological characteristics and Ca(2+) homoeostasis of the PVs. Conventional microelectrodes, whole-cell patch-clamp, and the fluo-3 fluorimetric ratio technique were used to record ionic currents and intracellular Ca(2+) within isolated rabbit PV preparations, or within isolated single PV cardiomyocytes, before and after administration of Lat-B (100 nM). Langendorff-perfused rabbit hearts were exposed to acute and continuous atrial stretch, and we studied PV electrical activity. Lat-B (100 nM) decreased the spontaneous electrical activity by 16±4% in PV preparations. Lat-B (100 nM) decreased the late Na(+) current, L-type Ca(2+) current, Na(+)/Ca(2+) exchanger current, and stretch-activated BKCa current, but did not affect the Na(+) current in PV cardiomyocytes. Lat-B reduced the transient outward K(+) current and ultra-rapid delayed rectifier K(+) current, but increased the delayed rectifier K(+) current in isolated PV cardiomyocytes. In addition, Lat-B (100 nM) decreased intracellular Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content in PV cardiomyocytes. Moreover, Lat-B attenuated stretch-induced increased spontaneous electrical activity and trigger activity. The effects of Lat-B on the PV spontaneous electrical activity were attenuated in the presence of Y-27632 [10 μM, a ROCK (Rho-associated kinase) inhibitor] and cytochalasin D (10 μM, an actin polymerization inhibitor). In conclusion, Lat-B regulates PV electrophysiological characteristics and attenuates stretch-induced arrhythmogenesis.

Topics: Amides; Animals; Arrhythmias, Cardiac; Atrial Fibrillation; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Cytochalasin D; Electrophysiological Phenomena; Heart Atria; In Vitro Techniques; Male; Myocytes, Cardiac; Perfusion; Pulmonary Veins; Pyridines; Rabbits; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thiazolidines

Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether α-smooth muscle actin (α-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes.. We investigated whether pharmacological ablation of α-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast-cardiomyocyte cross-talk.. Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of α-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes.. The results suggest that α-SMA containing stress fibers importantly contribute to myofibroblast arrhythmogeneicity. After ablation of this cytoskeletal component, cells lose their arrhythmic effects on cardiomyocytes, even if heterocellular electrotonic coupling is sustained. The findings identify α-SMA containing stress fibers as a potential future target of antiarrhythmic therapy in hearts undergoing structural remodeling.

Topics: Actins; Action Potentials; Animals; Animals, Newborn; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Bridged Bicyclo Compounds, Heterocyclic; Cell Communication; Cell Shape; Cells, Cultured; Coculture Techniques; Cytochalasin D; Depsipeptides; Dose-Response Relationship, Drug; Gap Junctions; Myocytes, Cardiac; Myofibroblasts; Phenotype; Rats; Rats, Wistar; Stress Fibers; Thiazolidines; Time Factors

Cardiomyocytes may experience significant cell swelling during ischemia and reperfusion. Such changes in cardiomyocyte volume have been shown to affect the electrical properties of the heart, possibly leading to cardiac arrhythmia. In the present study the regulatory volume decrease (RVD) response of neonatal rat cardiomyocytes was studied in intact single cells attached to coverslips, i.e. with an intact cytoskeleton. The potential contribution of KCNQ (Kv7) channels to the RVD response and the possible involvement of the F-actin cytoskeleton were investigated. The rate of RVD was significantly inhibited in the presence of the KCNQ channel blocker XE-991 (10 and 100 microM). Electrophysiological experiments confirmed the presence of an XE-991 sensitive current and Western blotting analysis revealed that KCNQ1 channel protein was present in the neonatal rat cardiomyocytes. Hypoosmotic cell swelling changes the structure of the F-actin cytoskeleton, leading to a more rounded cell shape, less pronounced F-actin stress fibers and patches of actin. In the presence of cytochalasin D (1 microM), a potent inhibitor of actin polymerization, the RVD response was strongly reduced, confirming a possible role for an intact F-actin cytoskeleton in linking cell swelling to activation of ion transport in neonatal rat cardiomyocytes.

Topics: Actins; Animals; Animals, Newborn; Anthracenes; Arrhythmias, Cardiac; Cell Adhesion; Cell Size; Cells, Cultured; Cytochalasin D; Electric Conductivity; Ion Transport; KCNQ Potassium Channels; Membrane Potentials; Myocardium; Myocytes, Cardiac; Rats; Rats, Wistar; Reperfusion Injury; Stress Fibers

Investigation of the mechanisms of arrhythmia genesis and maintenance has benefited from the use of optical mapping techniques that employ excitation-contraction uncouplers. We investigated the effects of the excitation-contraction uncouplers 2,3-butanedione monoxime (BDM) and cytochalasin D (Cyto D) on the induction and maintenance of arrhythmia by electric shocks. Electrical activity was optically mapped from anterior epicardium of rabbit hearts (n = 9) during shocks (-100 V, 8 ms) applied from a ventricular lead at various phases of action potential duration (APD). Restitution curves were obtained using S1-S2 protocol and measurement of APD values at 70% of repolarization. Compared with Cyto D, BDM significantly shortened APD at 90% of repolarization, although no significant difference in dispersion of repolarization was observed. Wavelength was also shortened with BDM. In general, shock-induced arrhythmias with BDM and Cyto D were ventricular tachycardic in nature. With respect to shock-induced sustained arrhythmias, the vulnerable window was wider and the incidence was higher with BDM than with Cyto D. There was also a difference in the morphology of ventricular tachycardia (VT) between the two agents. The arrhythmias with BDM usually resembled monomorphic VT, especially those that lasted >30 s. In contrast, arrhythmias with Cyto D more resembled polymorphic VT. However, the average number of phase singularities increased under Cyto D vs. BDM, whereas no significant difference in the dominant frequency of shock-induced sustained arrhythmia was observed. BDM reduced the slope of the restitution curve compared with Cyto D, but duration of arrhythmia under BDM was significantly increased compared with Cyto D. In conclusion, BDM increased arrhythmia genesis and maintenance relative to Cyto D.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Cytochalasin D; Diacetyl; Electroshock; In Vitro Techniques; Rabbits; Reaction Time

Chemical uncouplers diacetyl monoxime (DAM) and cytochalasin D (cyto-D) are used to abolish cardiac contractions in optical studies, yet alter intracellular Ca(2+) concentration ([Ca(2+)](i)) handling and vulnerability to arrhythmias in a species-dependent manner. The effects of uncouplers were investigated in perfused mouse hearts labeled with rhod-2/AM or 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) to map [Ca(2+)](i) transients (emission wavelength = 585 +/- 20 nm) and action potentials (APs) (emission wavelength > 610 nm; excitation wavelength = 530 +/- 20 nm). Confocal images showed that rhod-2 is primarily in the cytosol. DAM (15 mM) and cyto-D (5 microM) increased AP durations (APD(75) = 20.0 +/- 3 to 46.6 +/- 5 ms and 39.9 +/- 8 ms, respectively, n = 4) and refractory periods (45.14 +/- 12.1 to 82.5 +/- 3.5 ms and 78 +/- 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min and DAM by 10% gradually in 1 h (n = 5 each). Uncouplers did not alter the direction and gradient of repolarization, which progressed from apex to base in 15 +/- 3 ms. Peak systolic [Ca(2+)](i) increased with cyto-D from 743 +/- 47 (n = 8) to 944 +/- 17 nM (n = 3, P = 0.01) but decreased with DAM to 398 +/- 44 nM (n = 3, P < 0.01). Diastolic [Ca(2+)](i) was higher with cyto-D (544 +/- 80 nM, n = 3) and lower with DAM (224 +/- 31, n = 3) compared with controls (257 +/- 30 nM, n = 3). DAM prolonged [Ca(2+)](i) transients at 75% recovery (54.3 +/- 5 to 83.6 +/- 1.9 ms), whereas cyto-D had no effect (58.6 +/- 1.2 ms; n = 3). Burst pacing routinely elicited long-lasting ventricular tachycardia but not fibrillation. Uncouplers flattened the slope of AP restitution kinetic curves and blocked ventricular tachycardia induced by burst pacing.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Cytochalasin D; Diacetyl; Electrophysiology; Fluorescent Dyes; Heart; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Kinetics; Mice; Mice, Inbred Strains; Myocardial Contraction; Nucleic Acid Synthesis Inhibitors; Organ Preservation Solutions; Perfusion

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Cytochalasin D; Heart Conduction System; Heart Ventricles; Myocardial Contraction; Rabbits

We sought to determine whether potassium (K(+)) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes.. Because KBs attenuate K(+) outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca(2+)) transient ([Ca(2+)](i)) in cardiomyocytes. Elevation of intracellular Ca(2+) levels can trigger various signaling events. Influx of Ca(2+) through L-type Ca(2+) channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown.. Primary cultured rat cardiomyocytes were stimulated with four different KBs-4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine-and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca(2+))(i) was monitored by the fluorescent calcium indicator Fluo-4.. E-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca(2+))(i) by 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not.. These findings indicate that KBs induce ERK activation, which starts with Ca(2+) entry through the L-type Ca(2+) channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.

Topics: 4-Aminopyridine; Action Potentials; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium Channels, L-Type; Cells, Cultured; Cytochalasin D; Drug Evaluation, Preclinical; ErbB Receptors; Focal Adhesion Kinase 2; Mitogen-Activated Protein Kinases; Myocardium; Phosphorylation; Piperidines; Potassium Channel Blockers; Protein-Tyrosine Kinases; Pyridines; Quinazolines; Quinidine; Rats; Rats, Wistar; Tetraethylammonium; Time Factors; Tyrphostins