Stefan Dhein

Effects of chronic atrial fibrillation on gap junction distribution in human and rat atria.

OBJECTIVES To elucidate the structural basis for the electrophysiologic remodeling induced by chronic atrial fibrillation (AF), we investigated connexin40 and connexin43 (Cx40 and Cx43) expression and distribution in atria of patients with and without chronic AF and in an animal model of AF with additional electrophysiologic investigation of anisotropy (ratio of longitudinal and transverse velocities). BACKGROUND Atrial fibrillation is a common arrhythmia that has a tendency to become persistent. Since gap junctions provide the syncytial properties of the atrium, changes in expression and distribution of intercellular connections may accompany the chronification of AF. METHODS Atrial tissues isolated from 12 patients in normal sinus rhythm at the time of cardiac surgery and from 12 patients with chronic AF were processed for immunohistology and immunoblotting for the detection of the gap junction proteins. The functional study of the cardiac tissue anisotropy was performed in rat atria in which AF was induced by 24 h of rapid pacing (10 Hz). RESULTS Immunoblotting revealed that AF did not induce any significant change in Cx43 content in human atria. In contrast, a 2.7-fold increase in expression of Cx40 was observed in AF. Immunohistologic analysis indicated that AF resulted in an increase in the immunostaining of both connexins at the lateral membrane of human atrial cells. A similar spatial redistribution of the Cx43 signal was seen in isolated rat atria with experimentally-induced AF. In addition, AF in rat atria resulted in decreased anisotropy with slightly enhanced transverse conduction velocity. CONCLUSIONS This experimental study showed that AF is accompanied by spatial remodeling of gap junctions that might induce changes in the biophysical properties of the tissue.

Chronic effects of endothelin 1 and angiotensin II on gap junctions and intercellular communication in cardiac cells.

Endothelin‐1 (ET‐1) and angiotensin‐II (AT‐II) participate in the pathophysiology of cardiovascular diseases. Regulation of gap junctional intercellular communication may influence heart function and its response to cardiac injury. In this study, we examined the effects of ET‐1 and AT‐II on connexin43 (Cx43) and connexin40 (Cx40) in cultured neonatal rat ventricular cardiomyocytes (NRCs) and the role of mitogen‐activated protein kinase signaling in the ET‐1‐and AT‐II‐induced responses. NRCs were incubated for 24 h with either ET‐1 or AT‐II (each at concentrations ranging from 10 to 1000 nM), and Cx43 expression and phosphorylation increased with increasing concentrations of both. ET‐1 effects were significantly blocked by ETA (BQ123), but not by ETB (BQ788), receptor antagonists. AT‐II‐induced Cx43 induction could be completely inhibited by the AT1 receptor antagonist losartan. In contrast to Cx43, Cx40 expression did not change in either ET‐1‐ or in AT‐II‐treated NRCs. Thus, these two connexins were differentially regulated. ET‐1 and AT‐II increased the gap junctional conductance between the cardiomyocytes in culture as measured using a dual‐cell voltage clamp. Mitogen‐activated protein kinase inhibition revealed that ERK1/2 was critical for up‐regulation of Cx43 in response to ET‐1, whereas both ERK and p38 signal pathways were involved in the regulation of Cx43 by AT‐II. Thus, stimulation of the ERK and p38 signal pathways via ETA and AT1 receptors may partcipate in the regulation of cardiac gap junctions under (patho)physiological conditions.

Blunted Cardiac Responses to Receptor Activation in Subjects With Thr164Ile β2-Adrenoceptors

Background —Recent evidence indicates that certain genotypes of &bgr;2-adrenoceptors (AR) may indicate an increased risk of cardiovascular disease or an increased rate of disease progression. Of particular importance, the Thr164Ile polymorphism, which is found in ≈4% of humans, shows decreased receptor signaling, blunted cardiac response when expressed in transgenic mice, and is associated with a decreased survival rate in patients with congestive heart failure. Methods and Results —In this study, we compared functional activity, ie, chronotropic (heart rate increases) and inotropic (duration of the electromechanical systole) responses to intravenously administered terbutaline, in 6 subjects (4 women and 2 men) who were heterozygous for Thr164Ile with the responses in 12 volunteers (6 women and 6 men) who were homozygous for wild-type (WT) &bgr;2-AR (ie, Arg16, Gln27, and Thr164). The &bgr;2AR polymorphism significantly affected the dose-response curves for terbutaline-induced inotropic and chronotropic responses: compared with WT individuals, subjects with the Thr164Ile receptor had substantial blunting in maximal increases in heart rate (WT, 29.7±3.9 beats/min; Ile164, 20.7±1.9 beats/min;P =0.016) and a shortening of the duration of electromechanical systole (WT, 51.9±4.5 ms; Ile164, 37.9±4.6 ms;P =0.02). Conclusions —These data show that humans with the Ile164 genotype show blunted cardiac &bgr;2-AR responsiveness, which may help explain the decreased survival of patients with this genotype in the setting of congestive heart failure.

Comparative study on the proarrhythmic effects of some antiarrhythmic agents.

BackgroundA main side effect of antiarrhythmic drug therapy is the tendency of these drugs to promote arrhythmia within the therapeutic concentration range, i.e., the proarrhythmic activity of these drugs. However, a model for in vitro assessment, quantification, and comparison of proarrhythmic drug activities was still lacking, and only sparse data were available. Methods and ResultsTo analyze the arrhythmogenic risk of common antiarrhythmic drugs in a quantitative and comparative manner, isolated perfused rabbit hearts were treated with increasing concentrations of antiarrhythmic drugs corresponding to low, medium, and high therapeutic concentrations. For analysis of the epicardial activation process, an epicardial mapping (256 unipolar leads) was performed. For each electrode, the activation time was determined. From these data, the origins of epicardial activation (“breakthrough points” [BTPJ) were determined. At each electrode, an activation vector (VEC) was calculated giving direction and velocity of the local excitation wave. The beat similarity of various heartbeats (under treatment) compared with control was evaluated by determination of the percentage of identical BTPs (deviation .1 mm) and of similarVECs (deviation c5°)* BTP and VEC were reduced by all antiarrhythmic agents tested (propafenone=flecainide>quinidine>ajmaline> procainamide>disopyramide>mexiletine=lidocaine>sotalol), indicating a more or less pronounced disturbance of the epicardial activation process. Treatment with propafenone, quinidine, and disopyramide and to a lesser extent sotalol prolonged the activation-recovery interval (ARI). ARI dispersion was greatly enhanced by flecainide and was reduced by sotalol. In addition, it could be shown that propranolol is able to reduce the proarrhythmic action of flecainide. This effect seemed to be due to a reduction of the flecainide-induced increase in ARI dispersion. ConclusionFrom the results of our study, we propose the following rank order of the arrhythmogenic risk: flecainide> propafenone > quinidine > ajmaline> disopyramide> procainamide> mexiletine, lidocaine>sotalol. Moreover, we conclude that propranolol given additionally may be helpful in reducing the proarrhythmic risk of flecainide.

Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation

Antiarrhythmic peptides enhance gap junction current in pairs of cardiomyocytes and coupling in cardiac tissue. To elucidate the underlying mechanisms, we investigated the effects of the antiarrhythmic peptide AAP10 (GAG‐4Hyp‐PY‐CONH2) on pairs of adult guinea pig ventricular cardiomyocytes and pairs of HeLa cells transfected with rat cardiac connexin 43 (Cx43). By using a double‐cell voltage‐clamp technique in pairs of cardiomyocytes, we found that under control conditions the gap junction conductance (gj) steadily decreased with time (by ?0.292 ± 0.130 nS/min). Use of 50 nmol/L AAP10 reversed this rundown and increased gj (by +0.290 ± 0.231 nS/min, P<0.05). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM) and by the protein kinase C (PKC) subtype‐specific inhibitor CGP54345 (PKCα). In HeLa‐Cx43 cells, AAP10 exerted the same electrophysiological effect. In these cells, AAP10 activated PKC (determined by using ELISA) in CGP54345‐sensitive manner and significantly enhanced incorporation of 32P into Cx43 with dependence on PKC. If G‐protein coupling was inhibited with 1 mM GDP‐βS, we found the effects of AAP10 on 32P incorporation were also completely abolished. Next, we performed a radioligand binding study with 14C‐AAP10 as radioligand and AAPnat as competitor. We found saturable binding of 14CAAP10 to cardiac membrane preparations, which could be displaced with AAPnat. The Kd of AAP10 was 0.88 nmol/L. We conclude that 1) AAP10 increases gj both in adult cardiomyocytes and in transfected HeLa‐Cx43 cells, 2) AAP10 exerts its effect via enhanced PKC‐dependent phosphorylation of Cx43, 3) AAP10 activates PKCα, and 4) a membrane receptor exists for antiarrhythmic peptides in cardiomyocytes.

A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns induced by regional ischemia. A mapping study.

Common antiarrhythmic agents affect ionic membrane channels and thereby alter cellular electrical activity. Since this accounts for the proarrhythmic effects as well we tried to find new substances with different profiles of actions. A new antiarrhythmic peptide, H2N-Gly-Ala-Gly-4Hyp-Pro Tyr-CONH2 (AAP 10), was synthetized using the Fmoc-strategy. This peptide was analyzed for its electrophysiological profile of action in normal isolated rabbit hearts perfused according to the Langendorff technique either under control conditions or after induction of a regional ischemia. For this purpose 256 channel epicardial mapping was employed allowing the determination of the timepoints of activation at each electrode thus identifying the origins of epicardial activation (socalled breakthrough-points, BTP). Epicardial spread of activation was then described mathematically by activation vectors which gave direction and velocity of the epicardial activation wave at each electrode. Single heart beats were analyzed under control conditions and under treatment with AAP 10 or under regional ischemia with or without AAP 10-pretreatment (10−8 mol/l). We calculated the percentage of similar vectors (VEC) with unaltered direction (deviation <-5°) and the percentage of identical breakthroughpoints (deviation ≤ 1 mm) compared to control conditions. In addition, apparent epicardial velocities, total activation time of a given region and activation-recovery interval (ARI) as well as dispersion of ARI (i.e. standard deviation of ARI) and distribution of ARI were analyzed. Under control conditions treatment with AAP 10 (10−10 to 3*10−7 mol/l) led to a significant decrease in ARI-dispersion without alteration of any of the other parameters under investigation. Left ventricular regional ischemia resulted in a marked alteration of the activation patterns (a significant decrease in vectorfield-and breakthroughpoint-similarity) which could be significantly inhibited by pretreatment with AAP 10. In addition, we found that AAP 10 depressed the increase in ARI-dispersion during the first minutes of ischemia and accelerated normalization of ARI-dispersion during reperfusion. In additional experiments, it could be shown that AAP 10 did not alter action potential duration, maximum dU/dt, amplitude or resting membrane potential of isolated guinea pig muscles using a common intracellular action potential recording technique.From these results it is concluded that (a) AAP 10 inhibits ischemia induced alterations of the activation pattern (b) that it decreases ARI-dispersion (c) that this effect seems not to be due to an action on ionic channels (d) that the effect of AAP 10 may be due to an improvement of cellular coupling and finally (e) that AAP 10 may be an interesting new approach to the problem of prophylaxis of ischemia-associated ventricular arrhythmias.

ACE-inhibitor treatment attenuates atrial structural remodeling in patients with lone chronic atrial fibrillation.

AbstractObjectiveChronic atrial fibrillation (AF) is characterized by a remodeling process which involves the development of fibrosis. Since angiotensin II has been suspected to be involved in this process, the aim of our study was to investigate a possible influence of an ACE–I therapy in patients with chronic AF regarding the occurrence of left atrial structural remodeling.MethodsAtrial tissue samples were obtained from patients with lone chronic AF or sinus rhythm (SR). Collagen I, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) protein expression were measured by quantitative Western Blotting techniques and calculated as mean ± SEM. Histological tissue samples were used for calculating microvessel density (microvessel/mm2 ± SEM).ResultsIn AF, the collagen amount was higher (1.78 ± 0.21; p = 0.01) vs. SR (0.37 ± 0.07) accompanied by declining microcapillary density (AF: 145 ± 13 vs. SR: 202 ± 9; p = 0.01). Additionally, a negative correlation (p = 0.01) between collagen content and microcapillary density was observed. To investigate the influence of an ACE–I therapy on this remodeling process, patient groups were divided into AF and SR both with or without ACE–I. Interestingly, there was a significantly lower expression of collagen I in AF with ACE–I (1.04 ± 0.26) vs. AF without ACE–I treatment (2.07 ± 0.24, p = 0.02). The microcapillaries were not diminished in AF with ACE–I (180 ± 15) vs. SR with ACE–I (196 ± 9), but there was a significant rarification in AF without ACE–I (123 ± 18; p = 0.03). The expression of VEGF and bFGF did not reveal any significant differences.ConclusionIn patients undergoing ACE–I treatment: atrial structural remodeling was attenuated and the loss of atrial microcapillaries was prevented.

Effects of the gap junction uncoupler palmitoleic acid on the activation and repolarization wavefronts in isolated rabbit hearts

The heart normally acts as an electrical syncytium coupled via gap junctional channels. Since closure of these channels has been considered arrhythmogenic, we wanted to elucidate, how activation and repolarization wavefronts are altered during progressive pharmacological gap junctional uncoupling. We used the well known gap junction uncoupler palmitoleic acid (PA). The specificity of PA was tested in rabbit papillary muscles, which exhibited slowed conduction without affecting action potential morphology. We submitted isolated rabbit hearts (Langendorff‐technique) to increasing concentrations of palmitoleic acid (0.2, 1, 2, 5, 10, 20 μM), while 256 channel epicardial potential mapping was carried out. In presence of PA activation recovery intervals (ARI) at the 256 electrodes became highly inhomogeneous with a dramatic increase in the dispersion of activation recovery intervals (from 6 to 35 ms, P>0.01; EC50=7 μM), while the mean ARI‐duration at 256 sites remained stable. PA led to marked alterations of the activation pattern, expressed as percentage of unchanged activation vectors (reduction from 32 to 10%, P<0.01, EC50=3.3 μM), to prolongation of atrioventricular conduction time (from 58 to 107 ms, P<0.01; EC50=8 μM) of total activation time (from 7 to 14 ms, P<0.05, EC50=11 μM) and of QRS‐complex‐duration. In additional experiments the ventricle was paced via a bipolar electrode during the mapping procedure. From the isochrones longitudinal and transversal velocities were assessed showing that PA reduced transversal conduction velocity more distinctly than longitudinal. With regard to maximum effects and EC50 values we conclude that gap junction uncoupling by PA mainly affects atrioventricular conduction, ARI‐dispersion and ventricular activation pattern. As important arrhythmogenic effects of uncoupling enhancement of dispersion with concomitant disturbation of the normal activation pattern and slowing of conduction might be considered.

Actions of the antiarrhythmic peptide AAP10 on intercellular coupling

Abstract Disturbances in gap junction distribution and a decrease in the connexin43 content of the heart were shown to occur after myocardial infarction and in ischemic heart disease, respectively. These changes are now thought to play an important role in the genesis of arrhythmias associated with these diseases. It is thought that agents that can increase cellular coupling might be beneficial in these situations. Recently, we presented data showing that the synthetic peptide AAP10 acts antiarrhythmically in a model of regional ischemia. The data suggested that AAP10 might act via an increase in cellular coupling. The goal of this study was to establish whether AAP10 can interact with cardiac gap junctions. Measurements of the stimulus-response-interval (SRI) in guinea pig papillary muscle showed that high concentrations of AAP10 (1 µM) can decrease the SRI by about 10% under normoxic conditions. At lower concentrations (10 nM) AAP10 had no effect on SRI under normoxic conditions but prevented the increase in the SRI induced by perfusion with hypoxic, glucose-free Tyrode’s solution. Double-cell voltage-clamp experiments confirmed that AAP10 can interact with cardiac gap junctions. 10 nM AAP10 could either diminish or reverse the run-down of gap junction conductance normally observed in pairs of guinea pig ventricular myocytes. During control gap junction conductance decreased with a rate of -2.5 ± 2.0 nS/min. After application of 10 nM AAP10 gap junction conductance increased with a rate of +1.0 ± 0.7 nS/min (p < 0.01). After washout of AAP10 gap junction conductance decreased again with a rate not significantly different from control. Our results show that AAP10 does interact with gap junctions. Because no other effects of AAP10 on other electrophysiological parameters could be found, this action on gap junctions might be the basis of AAP10’s antiarrhythmic effect seen in previous studies

Increase in gap junction conductance by an antiarrhythmic peptide

Impaired cellular coupling is thought to be a very important factor for the genesis of cardiac arrhythmia. Cellular coupling is mediated by gap junctions. However, there are no therapeutic agents or experimental substances yet that increase cellular coupling. In addition, it has been shown that most antiarrhythmic drugs available now possess serious adverse effects. Thus, there is an urgent need for new antiarrhythmic agents. Previous studies using epicardial mapping in isolated rabbit hearts provided indirect evidence supporting the hypothesis that a newly synthesised antiarrhythmic peptide (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH2 = AAP10) might act via an increase in cellular, i.e., gap junctional coupling. The aim of the present study was to test this hypothesis. Measurement of the stimulus-response interval in papillary muscle showed a decrease of about 10% after application of 1 microM AAP10. These results are compatible with the hypothesis of AAP10 acting on gap junctions. In order to prove this hypothesis, gap junction conductance was measured directly by performing double-cell voltage-clamp experiments in isolated pairs of guinea-pig myocytes. During a 10 min control period gap junction conductance slowly decreased with a rate of -2.5 +/- 2.0 nS/min. After application of 10 nM AAP10 this behaviour reversed and gap junction conductance now increased with +1.0 +/- 0.7 nS/min. Upon washout of AAP10 gap junction conductance again decreased with a rate similar to that under control conditions. Another important finding was that we could not detect any other actions of AAP10 on cardiac myocytes. All parameters of the transmembrane action potential remained unchanged and, similarly, no changes in the IV relationship of single cardiac myocytes treated with 10 nM AAP10 could be observed. We conclude that AAP10 increases gap junction conductance, i.e., cellular coupling in the heart. This finding might be the first step towards the development of a new class of antiarrhythmic agents.