Arrhythmogenic Responses Research Articles

A Multiwell Cardiac μGMEA Platform for Action Potential Recordings from Human iPSC-Derived Cardiomyocyte Constructs.

SummaryMultielectrode array (MEA) technology has been extensively used for field potential recordings from excitable cells. However, its application for action potential (AP) measurements has not been harnessed. Here, we report a novel platform for high-resolution intracellular AP recordings from induced pluripotent stem cell-cardiomyocyte constructs derived from human cardiac fibroblasts. To gain intracellular access, micro-gold MEAs were used to electroporate multiple constructs simultaneously. High-throughput AP measurements were obtained from 41 multicellular constructs. Repeated electroporations of the same cells did not affect the signal stability. Our model has the capability to distinguish subtle differences in AP morphology to characterize the network profile. Furthermore, we confirm the reliability of the system by recapitulating known drug-induced physiological and arrhythmogenic responses. Overall, the model provides a unique cardio-electronic interface for non-invasive measurements of AP dynamics for drug screening and disease modeling. This technology opens the door for identifying novel cardio-factors to enhance electrophysiological maturation.

Anatomical dilatation of the superior vena cava associated with an arrhythmogenic response induced by SVC scan pacing after atrial fibrillation ablation

BackgroundThe relationship between pulmonary vein (PV) arrhythmogenicity and its anatomy has been reported. However, that of the superior vena cava (SVC) has not been well discussed. Arrhythmogenic response induced by pacing stimulation at SVC might help with identifying SVC arrhythmogenicity. The purpose of this study was to investigate the relationship between the anatomical dilatation of SVC and the arrhythmogenic response induced by pacing at SVC. MethodsForty-three patients who underwent atrial fibrillation (AF) ablation were enrolled in this study. After PV isolation, scan pacing (up to triple extra stimulation following intrinsic sinus beats) was performed at SVC. The arrhythmogenic response was defined as following: (1) repetitive atrial responses, (2) non-sustained, and (3) sustained AF/ atrial tachycardia. To assess the dilatation of SVC, we measured the cross-sectional area of the SVC (SVC-area) using multi-planar reconstruction CT imaging. ResultsArrhythmogenic responses were documented in 24 patients (Group 1). No arrhythmogenic responses were documented in the remaining 19 patients (Group 2). The SVC-area was significantly larger in Group 1 than Group 2 (3.1±0.9 vs. 2.2±0.8cm2, P=0.004). A multivariate analysis revealed only SVC-area was associated with arrhythmogenic responses (odds ratio=2.87, CI 1.05–7.82, P=0.04). Furthermore, AF recurrence rate was significantly higher in patients with SVC-area>2.56cm2 than those with SVC-area <2.56cm2 (9 [42.9%] of 21 vs. 3 [13.6%] of 22, P=0.026). ConclusionDilatation of SVC was associated with an arrhythmogenic response, and the AF recurrence rate was significantly higher in patients with large SVC-area. Adjunctive catheter intervention for the SVC might be indicated in patients with a dilated SVC and an arrhythmogenic response.

Flecainide-induced prolongation of ventricular repolarization contributes to the proarrhythmic profile of action

Flecainide is class Ic Na + channel blocker that is used to restore sinus rhythm and to prevent the arrhythmia recurrence in patients with atrial fibrillation. Antiarrhythmic effect of flecainide is attribut- ed to prolonged atrial effective refractory period and depressed car- diac action potential upstroke. Nevertheless, flecainide has been reported to produce arrhythmogenic responses in ventricular myo- cardium. In this regard, the Cardiac Arrhythmia Suppression Trial (CAST) has shown that flecainide therapy is associated with marked- ly increased risk of sudden arrhythmic death in patients with healed myocardial infarction, an effect possibly accounted for by the drug- induced anisotropic conduction slowing in the peri-infarct zone. Based on these findings, indications for flecainide therapy are cur- rently limited to the atrial fibrillation control in patients with no structural heart disease. Although flecainide-induced conduction abnormalities are unlikely to occur in the absence of myocardial ischemia or fibrosis, there is in- creasing evidence to suggest that flecainide may promote arrhythmo- genic responses in patients with structurally normal hearts. Flecainide administration has been reported to induce torsade de pointes, a poten- tially life-threatening polymorphic ventricular tachycardia, in patients with no coronary artery disease or severe heart failure (1,2). This kind of arrhythmia is thought to be attributed to the prolonged QT interval and increased spatial dispersion of ventricular repolarization. However, the contribution of these factors to flecainide-induced proarrhythmia is often overlooked, because flecainide (along with other class Ic Na + channel blockers) is generally regarded asanagentproducingonly little or no effect on ventricular repolarization.

A dynamic action potential model analysis of shock-induced aftereffects in ventricular muscle by reversible breakdown of cell membrane.

To elucidate the subcellular mechanism underlying the aftereffects of high-intensity dc shocks, a small pore, which mimics reversible breakdown of the cell membrane (electroporation), was incorporated into the phase-2 Luo-Rudy (L-R) model of ventricular action potentials. The pore size was set to occupy 0.15%-4.25% of the total cell membrane during the 10-ms shock. The pore was assumed to decrease after the shock exponentially with a time constant of 100-1,400 ms to simulate resealing process. In normal myocytes, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by prolonged depolarization and oscillation of membrane potential like early afterdepolarization (EAD). Time- and voltage-dependent changes in the delayed rectifier K+ currents (IKr, IKs) in combination with those of L-type Ca2+ current (ICa,(L)) and ion flux through the pore (I(pore)) are responsible for the potential changes. Spontaneous excitation from the oscillation depends on activation of ICa,(L). In myocytes overloaded with Na+ and Ca2+ secondary to 90% inhibition of Na+-K+ pump, the pore formation results in a delay of repolarization of the shocked action potential, which is followed by slower cyclic depolarization in response to spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). This delayed afterdepolarization-type oscillation is abolished by complete block of Ca2+ release from the SR. These findings suggest that high-intensity electric field application will cause arrhythmogenic responses through a transient rupture of sarcolemma with different subcellular events in ventricular cells under normal and pathological conditions.

Inositol 1,4,5-trisphosphate and reperfusion arrhythmias.

1. The present review focuses on the role of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate (IP3) in initiating arrhythmias during early reperfusion following a period of myocardial ischaemia. 2. Evidence for an arrhythmogenic action of IP3 was provided by studies showing a correlation between the extent of the increase in IP3 and the incidence of arrhythmias in early reperfusion. In addition, phospholipase C inhibitors selective for thrombin receptor stimulation were anti-arrhythmic only when arrhythmias were thrombin initiated. 3. Mechanisms by which IP3 could initiate arrhythmias are discussed, with particular emphasis on the role of slow and unscheduled Ca2+ release. 4. The reperfusion-induced IP3 and arrhythmogenic responses can be initiated through either alpha1-adrenoceptors or thrombin receptors, but endothelin receptor stimulation was ineffective. Further studies have provided evidence that the noradrenaline-mediated response was mediated by alpha1A-receptors, while the alpha1B-adrenoceptor subtype appeared to be protective. 5. Reperfusion-induced IP3 responses could be inhibited by procedures known to reduce the incidence of arrhythmias under these conditions, including preconditioning, inhibiting Na+/H+ exchange or by dietary supplementation with n-3 polyunsaturated fatty acids. 6. Inositol 1,4,5-trisphosphate generation in cardiomyocytes can be facilitated by raising intracellular Ca2+ and it seems likely that the rise in Ca2+ in ischaemia and reperfusion is responsible for the generation of IP3, which will, in turn, further exacerbate Ca2+ overload.

Functional InsP3 receptors that may modulate excitation-contraction coupling in the heart.

The roles of the Ca2+-mobilising messenger inositol 1,4,5-trisphosphate (InsP3) in heart are unclear, although many hormones activate InsP3 production in cardiomyocytes and some of their inotropic, chronotropic and arrhythmogenic effects may be due to Ca2+ release mediated by InsP3 receptors (InsP3Rs) [1-3]. In the present study, we examined the expression and subcellular localisation of InsP3R isoforms, and investigated their potential role in modulating excitation-contraction coupling (EC coupling). Western, PCR and InsP3-binding analysis indicated that both atrial and ventricular myocytes expressed mainly type II InsP3Rs, with approximately sixfold higher levels of InsP3Rs in atrial cells. Co-immunostaining of atrial myocytes with antibodies against type II ryanodine receptors (RyRs) and type II InsP3Rs revealed that the latter were arranged in the subsarcolemmal space where they largely co-localised with the junctional RyRs. Stimulation of quiescent or electrically paced atrial myocytes with a membrane-permeant InsP3 ester, which enters cells and directly activates InsP3Rs, caused the appearance of spontaneous Ca2+-release events. In addition, in paced cells, the InsP3 ester evoked an increase in the amplitudes of action potential-evoked Ca2+ transients. These data indicate that atrial cardiomyocytes express functional InsP3Rs, and that these channels could modulate EC coupling.

A model analysis of aftereffects of high-intensity DC stimulation on action potential of ventricular muscle.

The mechanism for aftereffects of high-intensity dc stimulation on ventricular muscle was studied by using Beeler-Reuter's action potential model. A leak conductance (Gpore, maximal value from 40 to 80 microS for 1 cm2 of membrane), which mimics reversible dielectric breakdown of the cell membrane by the shock, was incorporated into the model. To simulate resealing process, Gpore was assumed to decrease after the shock exponentially at a time constant (tau pore) of 5-50 s. The simulation results are qualitatively consistent with our experimental observations in guinea pig papillary muscle [1]; they include prolonged depolarization, diastolic depolarization or oscillation of membrane potential leading to a single or multiple spontaneous excitation. The phase-independence and shock intensity-dependence can also be reproduced. Analysis of current components has revealed that: 1) a large inward leak current (Ileak) is responsible for the prolonged depolarization; 2) time-dependent decay of outward current (IX1) in combination with Ileak and slow inward current (I(s)) results in diastolic depolarization or oscillation of membrane potential; 3) spontaneous excitation depends on an activation of I(s). These findings support our hypothesis that strong shocks (> 15 V/cm) will produce abnormal arrhythmogenic responses in ventricular muscle through a transient rupture of sarcolemmal membrane.

Ins(1,4,5)P3 and arrhythmogenic responses during myocardial reperfusion: evidence for receptor specificity.

Reperfusion of ischemic rat hearts initiates the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and arrhythmias, provided that either norepinephrine or thrombin is present. In the current study, effects on endothelin-1 (ET-1) responses were investigated. Reperfusion of catecholamine-depleted, [3H]inositol-labeled hearts in the presence of ET-1 caused an increase in [3H]inositol phosphates (7,073 +/- 1,004 to 17,300 +/- 206 counts.min-1.g tissue-1, means +/- SE, n = 4, P < 0.01), which was quantitatively greater than the release observed under normoxic conditions, but there was no increase in [3H]Ins(1,4,5)P3. Gentamicin (150 microM) inhibited inositol phosphate responses in the presence of either norepinephrine or thrombin but did not inhibit the response to ET-1, providing additional evidence that the inositol phosphate response to ET-1 does not involve formation of Ins(1,4,5)P3, even under reperfusion conditions. In contrast to norepinephrine and thrombin, ET-1 did not initiate reperfusion arrhythmias (4.4% ventricular fibrillation compared with 0% ventricular fibrillation in catecholamine-depleted controls). The data provide strong evidence that the effect of ischemia-reperfusion on inositol phosphate responses is specific for particular receptor types and eliminates G proteins, phospholipase C enzymes, and substrate availability as the primary factors responsible for Ins(1,4,5)P3 generation under reperfusion conditions.

Dietary fish oil prevents reperfusion Ins(1,4,5)P3 release in rat heart: possible antiarrhythmic mechanism.

Dietary enrichment with fish oil-derived n-3 polyunsaturated fatty acids has been shown to suppress the arrhythmias that occur during postischemic reperfusion. We have recently implicated a rapid release of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] during postischemic reperfusion in the generation of these arrhythmias. The effects of dietary supplementation with fish oil on both cardiac Ins(1,4,5)P3 and arrhythmogenic responses to reperfusion were therefore investigated in perfused rat hearts. Comparisons were made with control and n-6 polyunsaturated or saturated fat-supplemented diets. In control hearts, reperfusion increased Ins(1,4,5)P3 levels [from 9 +/- 2 at 20 min ischemia to 26 +/- 3 counts per minute (cpm)/mg protein with 2 min of reperfusion] and produced a high incidence of ventricular tachycardia (92% VT) and ventricular fibrillation (85% VF). Dietary fish oil supplementation, which increased composition of n-3 fatty acids in myocardial membrane phospholipids, prevented the reperfusion-induced rise in Ins(1,4,5)P3 (11 +/- 1 at 20 min ischemia and 12 +/- 2 cpm/mg protein after 2-min reperfusion) and significantly suppressed reperfusion arrhythmias (38% VT, 13% VF; P < 0.01 vs. control group). Thus the inhibition of reperfusion-induced rises in Ins(1,4,5)P3 by n-3 polyunsaturated fatty acids after dietary fish oil supplementation provides a possible mechanism for the inhibitory effect of n-3 fatty acids on reperfusion-induced arrhythmias.

GABAergic and Glutaminergic Modulation of Centrally Evoked Arrhythmias in Rats

A standard electrical stimulus applied to the posterior hypothalamus evoked cardiac arrhythmogenic responses in the spontaneously hypertensive rat. Isolated premature ventricular beats or doublets and nonsustained ventricular tachycardic salvos were observed. This effect was associated with a large rise in blood pressure (79 +/- 3 mm Hg). The same stimulus in normotensive Wistar-Kyoto rats produced no significant cardiac arrhythmias, and the rise in blood pressure was smaller (36 +/- 2 mm Hg). We investigated the influence of baclofen, a GABAB receptor agonist, and two N-methyl-D-aspartate receptor antagonists on the arrhythmogenic response to hypothalamic stimulation. Intravenous baclofen (3 mg/kg) had no effect in the normotensive Wistar-Kyoto rats, but in the spontaneously hypertensive rats it enhanced the adjusted mean value of the number of extrasystoles from 0.5 +/- 0.5 to 18 +/- 1 (P < .001). This value was also increased (from 3 +/- 1 to 17 +/- 1, P < .001) by an intracisternal injection of baclofen (1 micrograms/kg). This facilitatory effect of baclofen was prevented by treatment with atenolol (0.5 mg/kg). Two glutamate receptor antagonists, ketamine (7.5 mg/kg IV) and kynurenic acid (200 micrograms/kg intracerebroventricularly), prevented both the arrhythmogenic response to the hypothalamic stimulation and its facilitation by baclofen. The study confirms that hypothalamic stimulation facilitates the development of arrhythmias through a sympathetic drive and that these arrhythmias are easier to induce in spontaneously hypertensive rats than in normotensive Wistar-Kyoto rats. Both the central GABAergic and the glutamatergic systems are implicated in the development of these ventricular arrhythmias, since baclofen could disinhibit the glutamatergic central pathway. These results could account for the ability of the spontaneously hypertensive rats to develop ventricular arrhythmias of central origin.

Aftereffects of high-intensity DC stimulation on the electromechanical performance of ventricular muscle.

To clarify the mechanisms underlying cardiac dysfunction after electrical defibrillation, we investigated the effects of direct current field stimulation (10 ms, 1-80 V/cm) on isolated guinea pig papillary muscles. Shocks (S2) > 15 V/cm lowered the plateau height of the S2-induced action potential and inhibited its terminal repolarization. Subsequent responses to basic stimuli (S1, 1.0 Hz) for 1-3 min were characterized by a decrease in the maximum diastolic potential, a shortening of action potential duration, and an increase of the developed tension. With S2 > 30 V/cm, a marked delay in repolarization of the S2-induced action potential was followed by oscillation of membrane potential, resulting in repetitive spontaneous activity and often refractoriness to S1 stimulation. The aftereffects were independent of the phase of S2 application. Most of the aftereffects were preserved in the presence of nifedipine (1 microM) or ryanodine (1 microM). Only sodium channel blockade by tetrodotoxin (10 microM) modified the aftereffects by depressing the generation of spontaneous activity. These findings suggest that strong shocks (> 15 V/cm) will produce abnormal arrhythmogenic responses probably through a transient rupture of sarcolemmal membrane (electroporation) leading to a disturbance of the ionic equilibrium of the myocyte.

Role of dietary magnesium deficiency in the pressor and arrhythmogenic response to epinephrine in the intact dog

The effect of dietary magnesium deficiency on the pressor and arrhythmogenic responses to epinephrine was investigated in 19 dogs maintained either on a normal diet (11 dogs) or a diet deficient in magnesium (8 dogs). Magnesium-deficient dogs had significantly lower serum magnesium levels than the control dogs on a normal diet. Magnesium-deficient dogs showed an increased pressor sensitivity to epinephrine as determined by the dose of epinephrine required to cause a maximal pressor response (3.4 μg/kg/min compared to 13.4 μg/kg/min, p < 0.05). Magnesium-deficient dogs also had a significantly lower threshold dose for ventricular premature beats (0.8 μg/kg/min compared to 2.7 μg/kg/min, p < 0.05). Acute administration of magnesium sulfate restored pressor sensitivity and ventricular premature beat threshold to normal levels in the magnesium-deficient dogs. Threshold dose for ventricular tachycardia beat was similar in both normal and magnesium-deficient dogs, and threshold was raised significantly in both groups by acute administration of magnesium.

Effect of Halothane on Arrhythmogenic Responses Induced by Sympathomimetic Agents in Single Rat Heart Cells

The combination of catecholamines and halothane has long been recognized as arrhythmogenic. The purpose of this study was to evaluate whether the mechanism of this interaction originates at the single cell level. The incidence of spontaneous contractile waves occurring between stimulated beats (interbeat waves), early aftercontractions, and late aftercontractions was measured in rat myocytes exposed to sympathomimetics with and without halothane. Each of these endpoints in single cells has the potential to produce arrhythmias in multicellular preparations. Interbeat waves and late aftercontractions were observed with isoproterenol (1 X 10(-7) M) and norepinephrine (1-3 X 10(-7) M). The incidence of these phenomena was significantly reduced in the presence of 0.30 mM halothane. Early aftercontractions occurred in the presence of isoproterenol (1 X 10(-7) M), norepinephrine (1-3 X 10(-7) M), and phenylephrine (5-10 X 10(-6) M). There was a statistically significant decrease in the incidence of early aftercontractions in the presence of 0.30 mM halothane. These results indicate that the mechanism behind the clinically observed increased arrhythmogenicity of catecholamines with halothane does not arise at the level of single ventricular cells because halothane inhibited sympathomimetic-induced arrhythmogenic activity in this model. The probable mechanisms rather include altered impulse propagation, which might lead to phenomena such as reentry.

A direct role of endogenous prostaglandins in reperfusion-induced cardiac arrhythmias.

The possible role of prostaglandins (PG) in arrhythmias associated with ischemia and reperfusion was studied in isolated, superfused canine Purkinje tissues. Ischemic conditions caused partial depolarization and decrease of excitability. Neither inhibition of PG synthesis (ibuprofen, 30 micrograms/mL) nor addition of exogenous PGF2 alpha (1 ng/mL) modified responses to "ischemia." Reperfusion with normal Tyrode's solution stimulated PG production (measured as 6-keto-PGF1 alpha) and induced a series of electrophysiological events. Under control conditions, Purkinje fibres rapidly repolarized. Subsequently, these tissues began to depolarize and oscillatory afterpotentials appeared. Purkinje tissues depolarized further and became temporarily inexcitable. Return of activity was associated with depolarization-induced automaticity. Ibuprofen prevented reperfusion-stimulated PG release. Ibuprofen also increased the magnitude of early repolarization and greatly attenuated subsequent depolarization. Depolarization-induced automaticity was not observed under these conditions; however, oscillatory afterpotentials were not abolished by ibuprofen. Addition of PGF2 alpha to "ischemic" and reperfusion solutions in the presence of ibuprofen restored the arrhythmogenic responses. We conclude that release of endogenous prostaglandins contributes to electrophysiological changes elicited by reperfusion in canine Purkinje fibres.

Potentiation of toxicity and positive inotropic effect of ouabain by furosemide in guinea pig heart.

The present study was undertaken to elucidate the potentiation by furosemide of toxicity and positive inotropic effect of ouabain in guinea pigs. Arrhythmogenic responses to ouabain as well as the lowering of its lethal dose were potentiated by pretreatment with furosemide in guinea pigs. The potentiation of ouabain toxicity after furosemide administration was inhibited by pretreatment with potassium sparing diuretic, prorenoate. Furosemide-induced potentiations of contractile force and arrhythmogenic effect of ouabain were also observed in isolated guinea pig papillary muscle. However after pretreatment with furosemide, ouabain produced arrhythmias without any significant changes in either left ventricular or subcellular fractions binding of 3H-ouabain in guinea pigs. These findings suggest that furosemide-induced potentiation of ouabain toxicity is at least in part associated with the decreased intracellular potassium content of guinea pig heart. It was also demonstrated that the positive inotropic action induced by subtoxic dose of ouabain was potentiated by furosemide in guinea pig papillary muscle preparation.

Cardiac arrhythmias of sympathetic origin in the dog.

The effects of ventrolateral and ventromedial cardiac nerve (left sympathetics) stimulation on cardiac force, on rate, and on arrhythmogenic responses were characterized and quantitated. The stimulation of left sympathetic nerves produced augmentation in cardiac contraction in 45% of the experiments, an augmentation of both a cardiac rate and force in 47%, and in cardioacceleration alone in 8%. Two characteristic patterns of arrhythmogenic responses were elicited from stimulations of 100 sympathetic nerves. The two types of neurally induced arrhythmias were atrioventricular junctional or ventricular in origin. The onset and duration of the arrhythmias were quantitated. Both types of neurally induced arrhythmias were prevented either by blocking the beta receptors with propranolol or by preventing the neural release of norepinephrine with bretylium tosylate. The neurally induced arrhythmias were probably the result of enhanced automaticity in the atrioventricular junction area and in the ventricles produced by stimulating the sympathetic nerve fibers. This report thus implicates the ventromedial cardiac nerve in the genesis of cardiac arrhythmias.