Comparison of electrophysiological models for human ventricular cells and tissues

In this paper we briefly review currently published models for human ventricular cells and tissues. We discuss the Priebe–Beuckelmann (PB) model and the reduced version of this model constructed by Bernus et al. (redPB), the Ten Tusscher–Noble–Noble–Panfilov (TNNP) model and the Iyer–Mazhari–Winslow (IMW) model. We compare several characteristics of these models such as: sources of experimental data the models are based on, action potential morphology, action potential duration (APD) and conduction velocity (CV) restitution and computational efficiency. Finally, we discuss the application of a subset of these models—the redPB and the TNNP model—to study simulated spiral wave dynamics in 2D tissue sheets and in the human ventricles. We discuss the suitability of the different models for particular research questions and their limitations.     

Caveolae, ion channels and cardiac arrhythmias

Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids which are present in multiple cell types including cardiomyocytes. Along with the essential scaffolding protein caveolin-3, a number of different ion channels and transporters have been localized to caveolae in cardiac myocytes including L-type Ca²⁺ channels (Ca_v1.2), Na⁺ channels (Na_v1.5), pacemaker channels (HCN4), Na⁺/Ca²⁺ exchanger (NCX1) and others. Closely associated with these channels are specific macromolecular signaling complexes that provide highly localized regulation of the channels. Mutations in the caveolin-3 gene (CAV3) have been linked with the congenital long QT syndrome (LQT9), and mutations in caveolar-localized ion channels may contribute to other inherited arrhythmias. Changes in the caveolar microdomain in acquired heart disease may also lead to dysregulation and dysfunction of ion channels, altering the risk of arrhythmias in conditions such as heart failure. This review highlights the existing evidence identifying and characterizing ion channels localized to caveolae in cardiomyocytes and their role in arrhythmogenesis.     

Stereoselective effects of (R)- and (S)-carvedilol in humans

Carvedilol is currently used as the racemic mixture, (R,S)-carvedilol, consisting of equal amounts of (R)-carvedilol, an alpha-blocker, and (S)-carvedilol, an alpha- and beta-blocker, which have never been tested in their optically pure forms in human subjects. We performed a randomized, double-blind, placebo-controlled, crossover study in 12 healthy male volunteers. Subjects received single oral doses of 25 mg (R,S)-carvedilol, 12.5 mg (R)-carvedilol, 12.5 mg (S)-carvedilol, and placebo at 8 AM as well as at 8 PM. Exercise was performed at 11 AM, and heart rate and blood pressure were measured at rest and after 10 min of exercise. Urine was collected between 10 AM and 6 PM, as well as between 10 PM and 6 AM, and the amounts of urinary 6-hydroxy-melatonin sulfate (aMT6s) were determined by RIA. Compared to placebo, (R)-carvedilol increased heart rate during exercise (+4%, P < 0.05) and recovery (+10%, P < 0.05); (S)-carvedilol decreased heart rate during exercise (-14%, P < 0.05) and recovery (-6%, P < 0.05), and systolic blood pressure during exercise (-12%, P < 0.05); (R,S)-carvedilol decreased heart rate during exercise (-11%, P < 0.05), and systolic blood pressure at rest (-7%, P < 0.05) and during exercise (-10%, P < 0.05). None of the agents had any significant effect on the release of aMT6s. Our results indicate that only (S)-carvedilol causes beta-blockade, whereas (R)-carvedilol appears to increase sympathetic tone, presumably as a physiological reaction to the decrease of blood pressure caused by alpha-blockade. None of the drugs had any influence on melatonin release. The weak clinical net effect of beta-blockade of (R,S)-carvedilol at rest might be one reason why this drug causes fewer side effects than other beta-blockers, such as a reduction of nocturnal melatonin release.     

Comparative study of cardiac electrophysiological effects of atrial natriuretic peptide

The effects of atrial natriuretic peptide (ANP) on action potential characteristics were studied in various (human, rabbit, guinea-pig) atrial and guinea-pig right ventricular papillary muscles. ANP (1–100 nM) did not modify the resting membrane potential nor the maximum rate of depolarization phase (V_max). Up to 10 nM, ANP dose-dependently decreased the action potential amplitude both in guinea-pig atrial and ventricular muscles, but it did not affect this parameter in the other atrial preparations. ANP caused a dose-dependent, marked decrease of action potential duration (APD) in practically every cardiac preparation studied (exception of guinea-pig left atrium). The strongest effect on APD can be observed in human atrial and guinea-pig ventricular fibers. The K⁺ channel blocker 4-aminopyridine (1 mM) and the ATP-dependent K⁺ channel inhibitor glibenclamide (10Nl) prevented the effect of ANP on APD in both ventricular atrial preparations. ANP prevented the appearance of isoprenaline (0.5 M) induced slow AP in K⁺ depolarized myocardium. The present data suggest that ANP may inhibit the slow inward Ca²⁺ channel activity and facilitate the K⁺ channel activity.     

Modulatory role of verapamil treatment on the cardiac electrophysiological effects of cisapride

The role of transport proteins in the distribution of drugs in various tissues has obvious implications for drug effects. Recent reports indicate that such transporters are present not only in the liver, intestine, or blood-brain barrier but also in the heart. The objective of our study was to determine whether treatment of animals with verapamil, a well-known L-type calcium channel blocker with modulatory properties of membrane transporters, would alter distribution and cardiac electrophysiological effects of an I(Kr) blocker. Male guinea pigs (n = 72) were treated with either saline or verapamil at various doses (1.5 to 15 mg/kg) and for various durations (1 to 7 d). Animals were sacrificed 24 h after the last dose of verapamil (or saline), and their hearts were isolated and retroperfused with cisapride, a gastrokinetic drug with I(Kr) blockade properties. In hearts obtained from animals treated with vehicle, 50 nmol/L cisapride prolonged MAPD90 by 15 +/- 5 ms vs. 36 +/- 8 ms in hearts from animals treated with verapamil 15 mg.kg(-1).d(-1) for 5 d (p < 0.01). Treatment effects were dose- and time-dependent. Cardiac myocytes isolated from animals treated with vehicle or verapamil were incubated for 3 h with 100 ng/mL cisapride. Intracellular concentrations of cisapride in cardiac myocytes from animals treated with verapamil were 1.6-fold higher than those measured in myocytes from animals treated with vehicle (p < 0.01). The increase in intracellular concentrations of cisapride and potentiation of cisapride electrophysiological effects suggest that chronic treatment with drugs such as verapamil may modulate drug effects on the QT interval because of an increased access to intracellular binding sites on I(Kr) channels.     

An electromechanical model of cardiac tissue: constitutive issues and electrophysiological effects

We present an electromechanical model of myocardium tissue coupling a modified FitzHugh-Nagumo type system, describing the electrical activity of the excitable media, with finite elasticity, endowed with the capability of describing muscle contractions. The high degree of deformability of the medium makes it mandatory to set the diffusion process in a moving domain, thereby producing a direct influence of the deformation on the electrical activity. Various mechano-electric effects concerning the propagation of cylindrical waves, the rotating spiral waves, and the spiral breakups are discussed.     

An ultrastructural and electrophysiological study of the development of neuro-muscular junctions between previously dissociated foetal rat cells in vitro

Summary The development of neuro-muscular junctions between previously dissociated foetal rat spinal cord and somatic muscle has been investigated. The first indications of junction formation, both ultrastructurally and electrophysiologically, were observed after circa 18 days in vitro. The junctions contained numerous vesicles, but no secondary folds were developed even after 6 weeks in culture, and synaptic densities were not well marked. Functional endplates were found, and action potentials, endplate potentials and miniature endplate potentials recorded.The authors wish to thank Mr. D. Fraser, B. Sc., for valued technical help, and Mr. S. Waterman for photographic printing.     

Clonidine effects on protein and carbohydrate electrophysiological responses of labellar and tarsal sensilla in Phormia regina

The electrophysiological response of labellar and tarsal chemosensilla in the blowfly Phormia regina was studied in response to a complex stimulus naturally encountered by flies such as sheep faeces, and to beef liver, a proteinaceous feeding source. Responses were investigated both before or after injection of clonidine, an octopamine agonist previously shown to enhance sucrose ingestion, while decreasing that of proteins. As assessed by single sensillum recordings, the four different chemosensory - "salt", "sugar", "deterrent" and "water" - cells were all activated by both stimuli, regardless of sex and sensillum type, the "sugar" one being in all cases the most sensitive to beef liver before clonidine injection. Clonidine treatment affected neither labellar nor tarsal sensitivity to sucrose. Conversely, clonidine-injected flies showed a significant increase in the activity of the "deterrent" cell to beef liver, thus accounting for a decrease in protein ingestion. This study for the first time provides evidence of a key role of a clonidine-sensitive peripheral taste sensitivity in down-regulation of protein ingestion in blowflies. Correlation between peripheral sensitivity and behavioural output is discussed.     

How the choice of method influence on the results in electrophysiological studies of insect olfaction

In identifying the volatiles that insects use to locate suitable host plants, electrophysiological recordings of olfactory responses to plant volatiles may give important information. However, divergent results may be obtained with different recording techniques. To illustrate these differences, the results of a previous investigation using single cell recordings linked to a gas chromatograph (SCR-GC) are compared with the results obtained with an electroantennogram linked to a gas chromatograph (EAG-GC). Testing insects of the same species (Hylobius abietis) for the same test sample, 30 potent volatile compounds were identified by SCR-GC and 18 by EAG-GC. Of the 34 different compounds, 14 were identified by both techniques. Furthermore, when the same compound elicited detectable responses by both techniques, the response strength was usually not the same relative to the strongest response recorded by each technique. This shows that both EAG-GC and SCR-GC are important techniques in the identification of potent plant volatiles for insects. However, by using SCR-GC more information was obtained, information that can be crucial for understanding the insect-plant relationship.     

The effects of endogenous diuretic and antidiuretic peptides and their second messengers in the Malpighian tubules of Tenebrio molitor: an electrophysiological study

The Malpighian tubules of Tenebrio molitor provide a model system for interpreting the actions of endogenous diuretic and antidiuretic peptides. The effects of diuretic (Tenmo-DH(37)) and antidiuretic (Tenmo-ADFa) peptides and their respective second messengers (cyclic AMP and cyclic GMP) on basolateral (V(bl)) and transepithelial (V(te)) potentials of Tenebrio Malpighian tubules were determined using conventional microelectrodes. In the presence of 6 mmol l(-1) Ba(2+), Tenmo-DH(37) (100 nmol l(-1)) reversibly hyperpolarized V(bl) and depolarized V(te). A similar response was seen with the addition of 1 mmol l(-1) cyclic AMP; however, the apical membrane potential (V(ap)) then showed a hyperpolarization, whereas a depolarization of V(ap) was observed with Tenmo-DH(37). Bafilomycin A(1) (5 micromol l(-1)) inhibited fluid secretion of stimulated tubules and reversed the hyperpolarization of V(bl) in response to Tenmo-DH(37). In response to 100 nmol l(-1) Tenmo-ADFa or 1 mmol l(-1) cyclic GMP, V(bl) and V(te) depolarized, although cyclic GMP affected membrane potentials somewhat differently by causing an initial hyperpolarization of V(bl) and V(te). In high [K(+)]-low [Na(+)] Ringer, 1 mmol l(-1) amiloride decreased fluid secretion rates, and depolarized both V(bl) and V(te). Amiloride significantly decreased luminal pH in paired experiments, indicating the presence of a K(+)/nH(+) exchanger in tubule cells of Tenebrio. The results suggest that the endogenous factors and their second messengers stimulate/inhibit fluid secretion by acting on the apical V-ATPase, basolateral K(+) transport, and possibly Cl(-) transport.     

The actions of opiates in the rat substantia nigra: An electrophysiological analysis

Single unit recording and micropressure ejection techniques were used to investigate the actions of opiates on dopaminergic and non-dopaminergic neurons in the rat substantia nigra. Systemic administration of morphine, 1 to 4 mg/kg, led to a naloxone-reversible increase in firing rate of all zona compacta dopaminergic (ZC) neurons examined (n=10). In a specifically defined subpopulation of non-dopaminergic nigral zona reticulata (ZR) neurons, systemically administered morphine led to a naloxone reversible decrease in activity (n=9). D-Ala²-d-leu⁵ (DADL)-enkephalin, when applied directly onto ZC neurons by micropressure ejection techniques, had no effect on their firing rate. In contrast, micropressure ejection of DADL enkephalin onto ZR neurons produced a decrease in firing rate which was blocked by systemically administered naloxone. Morphine sulfate applied by pressure ejection onto both ZC and ZR neurons produced mixed results which were not always blocked by naloxone. These results suggest that one of the mechanisms by which opiates increase dopaminergic neurotransmission is through disinhibition of dopaminergic neurons in the substantia nigra.     

The effects of felodipine and bepridil on calcium-stimulated calmodulin binding and calcium pumping ATPase of cardiac sarcolemma before and after removal of endogenous calmodulin

Summary The Ca²⁺ channel blockers felodipine and bepridil are known to affect selectively functions of calmodulin. We studied their effects on calmodulin binding and ATPase activities of calmodulin-containing and calmodulin-depleted rabbit heart sarcolemma. Both drugs as well as the specific anti-calmodulin drug calmidazolium at a concentration of 50 µM, inhibited the Ca²⁺-stimulated calmodulin binding to calmodulin-depleted sarcolemma. Within the concentration range of 3 to 100 µM all three drugs also progressively inhibited Ca²⁺ pumping ATPase in calmodulin containing sarcolemma, although the enzyme was assayed at saturating Ca²⁺ (100 µM). The inhibitory potency of calmidazolium and bepridil, but not that of felodipine, increased when the membrane protein concentration in the ATPase assay was lowered. At low membrane protein concentration 30 µM calmidazolium completely blocked calmodulin-dependent Ca²⁺ pumping ATPase, whereas the inhibition caused by 30 µM felodipine or bepridil remained partially. A similar inhibition pattern of the drugs was found in the calmodulin binding experiments. Within a concentration range of 3 to 30 µM, all three drugs had negligible effects on the basal Ca²⁺ pumping ATPase which was measured in calmodulin-depleted sarcolemma. In conclusion, the characteristics of the anti-calmodulin action of felodipine on the rabbit heart sarcolemmal Ca²⁺ pumping ATPase are not different from those of bepridil. Both drugs may inhibit the enzyme by interference with the Ca²⁺-stimulated binding of calmodulin.Abbreviations Ca²⁺ pumping ATPase Ca²⁺ stimulated Mg²⁺-dependent ATP hydrolyzing activity - Na⁺ pumping ATPase Na⁺-stimulated K⁺- and Mg²⁺-dependent ATP hydrolyzing activity - Tris-maleate tris (hydroxymethyl) aminomethane hydrogen maleate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino) ethane sulfonic acid and Egta, ethylene glycol bis (p-amino ethylether)-N,N,N,N tetraacetic acid     

Nicotinic and muscarinic responses of embryonic chick ventricular myocardium to acetylcholine: chronotropic and electrophysiological effects

Sensitivity of 7-day-old chick embryo ventricular heart fragments to acetylcholine was investigated. Low doses mainly produced a positive chronotropic effect, whereas high doses of acetylcholine provoked a decrease in the heart beat rhythm. The positive chronotropic effect of acetylcholine was related to the presence of nicotinic receptors that were evidenced within ventricular myocardium by autoradiography. Membrane potential recording showed that acetylcholine hyperpolarizes the diastolic membrane potential when the drug had a negative chronotropic effect. This effect of acetylcholine on the membrane potential was not observed when the drug had a positive chronotropic effect. In many cases, the diastolic membrane potential exhibited spontaneous small depolarizing potentials. Their amplitude was low and their frequency was irregular. These potentials were suppressed by treatment with alpha-bungarotoxin, suggesting that they are triggered by nicotinic receptor activation.     

Synthesis of some new C2 substituted dihydropyrimidines and their electrophysiological evaluation as L-/T-type calcium channel blockers

Drugs targeting different calcium channel subtypes have strong therapeutic potential for future drug development for cardiovascular disorders, neuropsychiatric diseases and cancer. This study aims to design and synthesize a new series of C2 substituted dihydropyrimidines to mimic the structure features of third generation long acting dihydropyridine calcium channel blockers and dihydropyrimidines analogues. The target compounds have been evaluated as blockers for Ca_V1.2 and Ca_V3.2 utilizing the whole-cell patch clamp technique. Among the tested compounds, compound 7a showed moderate calcium channel blockade activity against Ca_V3.2. Moreover, the predicted physicochemical properties and pharmacokinetic profiles of the target compounds recommend that they can be considered as drug-like candidates. The results highlight some significant information for the future design of lead compounds as calcium channel blockers.     

Clozapine interacts with the glycine site of the NMDA receptor: electrophysiological studies of dopamine neurons in the rat ventral tegmental area

Clozapine has a remarkable efficacy in treatment-resistant schizophrenia and is one of the most effective antipsychotic drugs used today. The clinical effects of clozapine are suggested to be related to a unique interaction with a variety of receptor systems, including the glutamatergic receptors. Kynurenic acid (KYNA) is an endogenous blocker of alpha7 nicotinic receptors and a glutamate-receptor antagonist, preferentially blocking N-methyl-D-aspartate (NMDA) receptors. In the present in vivo electrophysiological study, changes in endogenous concentration of brain KYNA were utilized to analyze an interaction between clozapine and the glycine site of NMDA receptors. In control rats intravenously administered clozapine (0.078-10 mg/kg) increased the firing rate and the burst firing activity of dopamine (DA) neurons in the ventral tegmental area (VTA). Pretreatment with indomethacin (50 mg/kg, i.p., 1-3.5 h), a cyclooxygenase (COX)-inhibitor with a preferential selectivity for COX-1, which produced a significant elevation in brain KYNA levels, reversed the excitatory action of clozapine into an inhibitory response. In contrast, pretreatment with the COX-2 selective inhibitor parecoxib (25 mg/kg, i.v., 1-1.5 h) decreased brain KYNA formation and furthermore, clearly potentiated the excitatory effect of clozapine. Our results show that endogenous levels of brain KYNA are of importance for the response of clozapine on VTA DA neurons. On the basis of the present data we propose that clozapine is able to interact with glutamatergic mechanisms, via actions at the NMDA/glycine receptor.     

Lack of effect of receptor blockers on the formation of long-lasting associations between sympathetic nerves and cardiac muscle cells in vitro

Summary Sympathetic nerves in vitro form long-lasting, intimate, functional relationships with cardiac muscle cells, but not with fibroblasts. In the presence of an adrenergic -blocker and a cholinergic muscarinic blocker, long-lasting relationships still take place. It was concluded that neurotransmitter receptors are not involved in the mechanism of recognition of cardiac muscle cells by sympathetic nerves.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Smad signaling in the neural crest regulates cardiac outflow tract remodeling through cell autonomous and non-cell autonomous effects

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.