Gary L. Aistrup

Neuronal nicotinic acetylcholine receptors: a new target site of ethanol.

Whereas a variety of neuroreceptors and ion channels have been demonstrated to be affected by ethanol including GABAA receptors, NMDA receptors, non-NMDA glutamate receptors, 5-HT3 receptors and voltage-gated calcium channels, neuronal nicotinic acetylcholine receptors (nnAChRs) have recently emerged as a new target site of ethanol. The nnAChRs are different from the muscle type nicotinic AChRs with respect to their molecular architecture and pharmacology. This article briefly reviews the structure, distribution and function of nnAChRs for which a considerable amount of information has been rapidly accumulated during the past 5-10 years. The potent and unique action of ethanol on nnAChRs has been unveiled only during the past few years. Most recent developments along this line of ethanol action are discussed in this paper.

Pacing-induced Heterogeneities in Intracellular Ca2+ Signaling, Cardiac Alternans, and Ventricular Arrhythmias in Intact Rat Heart

Optical mapping studies have suggested that intracellular Ca2+ and T-wave alternans are linked through underlying alternations in Ca2+ cycling-inducing oscillations in action potential duration through Ca2+-sensitive conductances. However, these studies cannot measure single-cell behavior; therefore, the Ca2+ cycling heterogeneities within microscopic ventricular regions are unknown. The goal of this study was to measure cellular activity in intact myocardium during rapid pacing and arrhythmias. We used single-photon laser-scanning confocal microscopy to measure Ca2+ signaling in individual myocytes of intact rat myocardium during rapid pacing and during pacing-induced ventricular arrhythmias. At low rates, all myocytes demonstrate Ca2+ alternans that is synchronized but whose magnitude varies depending on recovery kinetics of Ca2+ cycling for each individual myocyte. As rate increases, some cells reverse alternans phase, giving a dyssynchronous activation pattern, even in adjoining myocytes. Increased pacing rate also induces subcellular alternans where Ca2+ alternates out of phase with different regions within the same cell. These forms of heterogeneous Ca2+ signaling also occurred during pacing-induced ventricular tachycardia. Our results demonstrate highly nonuniform Ca2+ signaling among and within individual myocytes in intact heart during rapid pacing and arrhythmias. Thus, certain pathophysiological conditions that alter Ca2+ cycling kinetics, such as heart failure, might promote ventricular arrhythmias by exaggerating these cellular heterogeneities in Ca2+ signaling.

Autonomic Remodeling in the Left Atrium and Pulmonary Veins in Heart Failure – Creation of a Dynamic Substrate for Atrial Fibrillation

Background—Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results—Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, &bgr;-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and &bgr;-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in &bgr;1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. &bgr;-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions—In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.Background— Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. Methods and Results— Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular–paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, β-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and β-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in β1-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. β-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. Conclusions— In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.

Potent modulation of neuronal nicotinic acetylcholine receptor-channel by ethanol.

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 microM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.

Variability in timing of spontaneous calcium release in the intact rat heart is determined by the time course of sarcoplasmic reticulum calcium load.

Background: Abnormalities in intracellular calcium (Ca) cycling during Ca overload can cause triggered activity because spontaneous calcium release (SCR) activates sufficient Ca-sensitive inward currents to induce delayed afterdepolarizations (DADs). However, little is known about the mechanisms relating SCR and triggered activity on the tissue scale. Methods and Results: Laser scanning confocal microscopy was used to measure the spatiotemporal properties of SCR within large myocyte populations in intact rat heart. Computer simulations were used to predict how these properties of SCR determine DAD magnitude. We measured the average and standard deviation of the latency distribution of SCR within a large population of myocytes in intact tissue. We found that as external [Ca] is increased, and with faster pacing rates, the average and SD of the latency distribution decreases substantially. This result demonstrates that the timing of SCR occurs with less variability as the sarcoplasmic reticulum (SR) Ca load is increased, causing more sites to release Ca within each cell. We then applied a mathematical model of subcellular Ca cycling to show that a decrease in SCR variability leads to a higher DAD amplitude and is dictated by the rate of SR Ca refilling following an action potential. Conclusions: Our results demonstrate that the variability of the timing of SCR in a population of cells in tissue decreases with SR load and is dictated by the time course of the SR Ca content.

Mechanisms of alcohol-nicotine interactions: alcoholics versus smokers.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Bo Söderpalm. The presentations were (1) Nicotinic mechanisms and ethanol reinforcement: Behavioral and neurochemical studies, by Bo Söderpalm, M. Ericson, P. Olausson, and J. A. Engel; (2) Chronic nicotine and ethanol: Differential regulation in gene expression of nicotinic acetylcholine receptor subunits, by X. Zhang and A. Nordberg; (3) Nicotine-ethanol interactions at neuronal nicotinic acetylcholine receptors, by Toshio Narahashi, William Marszalec, and Gary L. Aistrup; (4) Relapse prevention in alcoholics by cigarette smoking? Treatment outcome in an observational study with acamprosate, by L.G. Schmidt, U. Kalouti, M. Smolka, and M. Soyka; and (5) Effect of nicotine on voluntary ethanol intake and development of alcohol dependence in male rats, by L. Hedlund and G. Wahlström.

Ultrastructural and cellular basis for the development of abnormal myocardial mechanics during the transition from hypertension to heart failure

Although the development of abnormal myocardial mechanics represents a key step during the transition from hypertension to overt heart failure (HF), the underlying ultrastructural and cellular basis of abnormal myocardial mechanics remains unclear. We therefore investigated how changes in transverse (T)-tubule organization and the resulting altered intracellular Ca(2+) cycling in large cell populations underlie the development of abnormal myocardial mechanics in a model of chronic hypertension. Hearts from spontaneously hypertensive rats (SHRs; n = 72) were studied at different ages and stages of hypertensive heart disease and early HF and were compared with age-matched control (Wistar-Kyoto) rats (n = 34). Echocardiography, including tissue Doppler and speckle-tracking analysis, was performed just before euthanization, after which T-tubule organization and Ca(2+) transients were studied using confocal microscopy. In SHRs, abnormalities in myocardial mechanics occurred early in response to hypertension, before the development of overt systolic dysfunction and HF. Reduced longitudinal, circumferential, and radial strain as well as reduced tissue Doppler early diastolic tissue velocities occurred in concert with T-tubule disorganization and impaired Ca(2+) cycling, all of which preceded the development of cardiac fibrosis. The time to peak of intracellular Ca(2+) transients was slowed due to T-tubule disruption, providing a link between declining cell ultrastructure and abnormal myocardial mechanics. In conclusion, subclinical abnormalities in myocardial mechanics occur early in response to hypertension and coincide with the development of T-tubule disorganization and impaired intracellular Ca(2+) cycling. These changes occur before the development of significant cardiac fibrosis and precede the development of overt cardiac dysfunction and HF.

Multiple Defects in Intracellular Calcium Cycling in Whole Failing Rat Heart

Background—A number of defects in excitation-contraction coupling have been identified in failing mammalian hearts. The goal of this study was to measure the defects in intracellular Ca2+ cycling in left ventricular epicardial myocytes of the whole heart in an animal model of congestive heart failure (CHF). Methods and Results—Intracellular Ca2+ transients were measured using confocal microscopy in whole rat hearts from age-matched Wistar-Kyoto control rats and spontaneously hypertensive rats at ≈23 months of age. Basal Ca2+ transients in myocytes in spontaneously hypertensive rats were smaller in amplitude and longer in duration than Wistar-Kyoto control rats. There was also greater variability in transient characteristics associated with duration between myocytes of CHF than Wistar-Kyoto controls. Approximately 21% of CHF myocytes demonstrated spontaneous Ca2+ waves compared with very little of this activity in Wistar-Kyoto control rats. A separate population of spontaneously hypertensive rat myocytes showed Ca2+ waves that were triggered during pacing and were absent at rest (triggered waves). Rapid pacing protocols caused Ca2+ alternans to develop at slower heart rates in CHF. Conclusions—Epicardial cells demonstrate both serious defects and greater cell-to-cell variability in Ca2+ cycling in CHF. The defects in Ca2+ cycling include both spontaneous and triggered waves of Ca2+ release, which promote triggered activity. The slowing of Ca2+ repriming in the sarcoplasmic reticulum is probably responsible for the increased vulnerability to Ca2+ alternans in CHF. Our results suggest that defective Ca2+ cycling could contribute both to reduced cardiac output in CHF and to the establishment of repolarization gradients, thus creating the substrate for reentrant arrhythmias.

Mechanisms Underlying the Formation and Dynamics of Subcellular Calcium Alternans in the Intact Rat Heart

Optical mapping of intact cardiac tissue reveals that, in some cases, intracellular calcium (Ca) release can alternate from one beat to the next in a large-small-large sequence, also referred to as Ca transient (CaT) alternans. CaT alternans can also become spatially phase-mismatched within a single cell, when one part of the cell alternates in a large-small-large sequence, whereas a different part alternates in a small-large-small sequence, a phenomenon known as subcellular discordant alternans. The mechanisms for the formation and spatiotemporal evolution of these phase-mismatched patterns are not known. We used confocal Ca imaging to measure CaT alternans at the sarcomeric level within individual myocytes in the intact rat heart. After a sudden change in cycle length (CL), 2 distinct spatial patterns of CaT alternans emerge. CaTs can form spatially phase-mismatched alternans patterns after the first few beats following the change in CL. The phase mismatch persists for many beats, after which it gradually becomes phase matched via the movement of nodes, which are junctures between phase-mismatched cell regions. In other examples, phase-matched alternans gradually become phase-mismatched, via the formation and movement of nodes. In these examples, we observed large beat-to-beat variations in the cell activation times, despite constant CL pacing. Using computer simulations, we explored the underlying mechanisms for these dynamical phenomena. Our results show how heterogeneity at the sarcomeric level, in conjunction with the dynamics of Ca cycling and membrane voltage, can lead to complex spatiotemporal phenomena within myocytes of the intact heart.

Ranolazine Antagonizes the Effects of Increased Late Sodium Current on Intracellular Calcium Cycling in Rat Isolated Intact Heart

Pathological conditions, including ischemia and heart failure, are associated with altered sodium channel function and increased late sodium current (INa,L), leading to prolonged action potential duration, increased intracellular sodium and calcium concentrations, and arrhythmias. We used anemone toxin (ATX)-II to study the effects of increasing INa,L on intracellular calcium cycling in rat isolated hearts. Cardiac contraction was abolished using paralytic agents. Ranolazine (RAN) was used to inhibit late INa. Hearts were loaded with fluo-4-acetoxymethyl ester, and myocyte intracellular calcium transients (CaTs) were measured using laser scanning confocal microscopy. ATX (1 nM) prolonged CaT duration at 50% recovery in hearts paced at a basal rate of 2 Hz and increased the sensitivity of the heart to the development of calcium alternans caused by fast pacing. ATX increased the time required for recovery of CaT amplitude following a previous beat, and ATX induced spontaneous calcium release waves during rapid pacing of the heart. ATX prolonged the duration of repolarization from the initiation of the activation to terminal repolarization in the pseudo-electrocardiogram. All actions of ATX were both reversed and prevented by subsequent or prior exposure, respectively, of hearts to RAN (10 μM). Most importantly, the increased vulnerability of the heart to the development of calcium alternans during rapid pacing was reversed or prevented by 10 μM RAN. These results suggest that enhancement of INa,L alters calcium cycling. Reduction by RAN of INa,L-induced dysregulation of calcium cycling could contribute to the antiarrhythmic actions of this agent in both reentrant and triggered arrhythmias.