inositol-1-4-5-trisphosphate and Arrhythmias--Cardiac

Synthetic compounds open up new avenues to interrogate and manipulate intracellular Ca2+ signalling pathways. They may ultimately lead to drug-like analogues to intervene in disease. Recent advances in chemical biology tools available to probe Ca2+ signalling are described, with a particular focus on those synthetic analogues from our group that have enhanced biological understanding or represent a step towards more drug-like molecules. Adenophostin (AdA) is the most potent known agonist at the inositol 1,4,5-trisphosphate receptor (IP3R) and synthetic analogues provide a binding model for receptor activation and channel opening. 2-O-Modified inositol 1,4,5-trisphosphate (IP3) derivatives that are partial agonists at the IP3R reveal key conformational changes of the receptor upon ligand binding. Biphenyl polyphosphates illustrate that simple non-inositol surrogates can be engineered to give prototype IP3R agonists or antagonists and act as templates for protein co-crystallization. Cyclic adenosine 5'-diphosphoribose (cADPR) can be selectively modified using total synthesis, generating chemically and biologically stable tools to investigate Ca2+ release via the ryanodine receptor (RyR) and to interfere with cADPR synthesis and degradation. The first neutral analogues with a synthetic pyrophosphate bioisostere surprisingly retain the ability to release Ca2+, suggesting a new route to membrane-permeant tools. Adenosine 5'-diphosphoribose (ADPR) activates the Ca2+-, Na+- and K+-permeable transient receptor potential melastatin 2 (TRPM2) cation channel. Synthetic ADPR analogues provide the first structure-activity relationship (SAR) for this emerging messenger and the first functional antagonists. An analogue based on the nicotinic acid motif of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonizes NAADP-mediated Ca2+ release in vitro and is effective in vivo against induced heart arrhythmia and autoimmune disease, illustrating the therapeutic potential of targeted small molecules.

Topics: Adenosine; Adenosine Diphosphate Ribose; Arrhythmias, Cardiac; Calcium; Calcium Channel Blockers; Calcium Signaling; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; NADP; Ryanodine Receptor Calcium Release Channel; Small Molecule Libraries; Structure-Activity Relationship

Calcium channels are critical to normal cardiac function. They are involved in the generation and conduction of the action potential and in contraction. Three surface membrane channels have been identified. The L-type Ca channel is most abundant and is responsible for Ca entry into the cell that triggers contraction. T-type Ca channels are most prevalent in the conduction system and are probably involved in automaticity. A newly described TTX-sensitive calcium current may be important in "boosting" or enhancing conduction and contraction. The main intracellular Ca channel resides in the sarcoplasmic reticulum and is responsible for the release of the Ca that activates contraction. Oscillatory behavior of this channel influences the sarcolemmal membrane, causing delayed aftercontractions and arrhythmias such as those seen in digoxin toxicity. The on-going molecular characterization of these channels will enhance our knowledge of their normal function and dysfunction in disease states, leading to the development of new therapeutic agents to treat arrhythmias and contractile dysfunction.

Topics: Adrenergic beta-Agonists; Animals; Arrhythmias, Cardiac; Calcium; Calcium Channels, L-Type; Calcium Channels, T-Type; Calcium Signaling; Humans; Inositol 1,4,5-Trisphosphate; Magnesium; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Structure-Activity Relationship; Terminology as Topic; Tetrodotoxin

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.

Topics: Animals; Arrhythmias, Cardiac; Calcium Signaling; Humans; Inositol 1,4,5-Trisphosphate; Myocardial Reperfusion Injury

Topics: Apoptosis; Arrhythmias, Cardiac; Calcium; Heart Failure; Humans; Inositol 1,4,5-Trisphosphate; Models, Cardiovascular; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Signal Transduction

Augmented inositol 1,4,5-trisphosphate (IP

Topics: Animals; Arrhythmias, Cardiac; Calcium; Calcium Signaling; Cells, Cultured; Excitation Contraction Coupling; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Myocytes, Cardiac; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum

Topics: Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium; Excitation Contraction Coupling; Humans; Inositol 1,4,5-Trisphosphate; Sarcoplasmic Reticulum

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that regulates intracellular Ca(2+) release through IP(3) receptors located in the sarco(endo)plasmic reticulum of cardiac myocytes. Many prohypertrophic G protein-coupled receptor (GPCR) signaling events lead to IP(3) liberation, although its importance in transducing the hypertrophic response has not been established in vivo.. Here, we generated conditional, heart-specific transgenic mice with both gain- and loss-of-function for IP(3) receptor signaling to examine its hypertrophic growth effects following pathological and physiological stimulation.. Overexpression of the mouse type-2 IP(3) receptor (IP(3)R2) in the heart generated mild baseline cardiac hypertrophy at 3 months of age. Isolated myocytes from overexpressing lines showed increased Ca(2+) transients and arrhythmias in response to endothelin-1 stimulation. Although low levels of IP(3)R2 overexpression failed to augment/synergize cardiac hypertrophy following 2 weeks of pressure-overload stimulation, such levels did enhance hypertrophy following 2 weeks of isoproterenol infusion, in response to Galphaq overexpression, and/or in response to exercise stimulation. To inhibit IP(3) signaling in vivo, we generated transgenic mice expressing an IP(3) chelating protein (IP(3)-sponge). IP(3)-sponge transgenic mice abrogated cardiac hypertrophy in response to isoproterenol and angiotensin II infusion but not pressure-overload stimulation. Mechanistically, IP(3)R2-enhanced cardiac hypertrophy following isoproterenol infusion was significantly reduced in the calcineurin-Abeta-null background.. These results indicate that IP(3)-mediated Ca(2+) release plays a central role in regulating cardiac hypertrophy downstream of GPCR signaling, in part, through a calcineurin-dependent mechanism.

Topics: Age Factors; Angiotensin II; Animals; Arrhythmias, Cardiac; Calcineurin; Calcium Signaling; Cardiomegaly; Disease Models, Animal; Endothelin-1; GTP-Binding Protein alpha Subunits, Gq-G11; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Isoproterenol; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; Phenotype; Physical Exertion

We have recently reported that left atrial injections of the thromboxane A(2) (TXA(2)) mimetic, (5Z)-7-[(1R,4S,5S,6R)-6-[(1E,3S)-3-hydroxy-1-octenyl]-2 -oxabicyclo[2.2.1]hept-5-yl]-5-heptenoic acid (U46619), induced ventricular arrhythmias in the anesthetized rabbit. Data from this study led us to hypothesize that TXA(2) may be inducing direct actions on the myocardium to induce these arrhythmias. The aim of this study was to further elucidate the mechanism responsible for these arrhythmias. We report that TXA(2)R is expressed at both the gene and protein levels in atrial and ventricular samples of adult rabbits. In addition, TXA(2)R mRNA was identified in single, isolated ventricular cardiac myocytes. Furthermore, treatment of isolated cardiac myocytes with U46619 increased intracellular calcium in a dose-dependent manner and these increases were blocked by the specific TXA(2)R antagonist, 7-(3-((2-((phenylamino)carbonyl)hydrazino)methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ29548). Pretreatment of myocytes with an inhibitor of inositol trisphosphate (IP(3)) formation, gentamicin, or with an inhibitor of IP(3) receptors, 2-aminoethoxydiphenylborate (2-APB), blocked the increase in intracellular calcium. In vivo pretreatment of anesthetized rabbits with either gentamicin or 2-APB subsequently inhibited the formation of ventricular arrhythmias elicited by U46619. These data support the hypothesis that TXA(2) can induce arrhythmias via a direct action on cardiac myocytes. Furthermore, these arrhythmogenic actions were blocked by inhibitors of the IP(3) pathway. In summary, this study provides novel evidence for direct TXA(2)-induced cardiac arrhythmias and provides a rationale for IP(3) as a potential target for the treatment of TXA(2)-mediated arrhythmias.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arrhythmias, Cardiac; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Fatty Acids, Unsaturated; Heart Atria; Heart Ventricles; Hydrazines; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Myocytes, Cardiac; Rabbits; Receptors, Thromboxane A2, Prostaglandin H2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thromboxane A2; Vasoconstrictor Agents

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Calcium Channels, L-Type; Calcium Signaling; Cardiomegaly; Cell Differentiation; Cell Line; Heart Rate; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Myocardial Contraction; Myocytes, Cardiac; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Time Factors; Type C Phospholipases

Although ventricular cardiomyocytes express inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors, it is unclear how these Ca2+ channels contribute to the effects of Gq-coupled agonists. Endothelin-1 augmented the amplitude of pacing-evoked Ca2+ signals (positive inotropy), and caused an increasing frequency of spontaneous diastolic Ca2+-release transients. Both effects of endothelin-1 were blocked by an antagonist of phospholipase C, suggesting that Ins(1,4,5)P3 and/or diacylglycerol production was necessary. The endothelin-1-mediated spontaneous Ca2+ transients were abolished by application of 2-aminoethoxydiphenyl borate (2-APB), an antagonist of Ins(1,4,5)P3 receptors. Incubation of electrically-paced ventricular myocytes with a membrane-permeant Ins(1,4,5)P3 ester provoked the occurrence of spontaneous diastolic Ca2+ transients with the same characteristics and sensitivity to 2-APB as the events stimulated by endothelin-1. In addition to evoking spontaneous Ca2+ transients, stimulation of ventricular myocytes with the Ins(1,4,5)P3 ester caused a positive inotropic effect. The effects of endothelin-1 were compared with two other stimuli, isoproterenol and digoxin, which are known to induce inotropy and spontaneous Ca2+ transients by overloading intracellular Ca2+ stores. The events evoked by isoproterenol and digoxin were dissimilar from those triggered by endothelin-1 in several ways. We propose that Ins(1,4,5)P3 receptors support the development of both inotropy and spontaneous pro-arrhythmic Ca2+ signals in ventricular myocytes stimulated with a Gq-coupled agonist.

Topics: Animals; Arrhythmias, Cardiac; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Digoxin; Endothelin-1; Heart Ventricles; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Isoproterenol; Myocytes, Cardiac; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Type C Phospholipases

Inositol-1,4,5-trisphosphate (IP(3))-dependent Ca(2+) release represents the major Ca(2+) mobilizing pathway responsible for diverse functions in non-excitable cells. In the heart, however, its role is largely unknown or controversial. In intact cat atrial myocytes, endothelin (ET-1) increased basal [Ca(2+)](i) levels, enhanced action potential-evoked [Ca(2+)](i) transients, caused [Ca(2+)](i) transients with alternating amplitudes (Ca(2+) alternans), and facilitated spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) in the form of Ca(2+) sparks and arrhythmogenic Ca(2+) waves. These effects were prevented by the IP(3) receptor (IP(3)R) blocker aminoethoxydiphenyl borate (2-APB), suggesting the involvement of IP(3)-dependent SR Ca(2+) release. In saponin-permeabilized myocytes IP(3) and the more potent IP(3)R agonist adenophostin increased basal [Ca(2+)](i) and the frequency of spontaneous Ca(2+) sparks. In the presence of tetracaine to eliminate Ca(2+) release from ryanodine receptor (RyR) SR Ca(2+) release channels, IP(3) and adenophostin triggered unique elementary, non-propagating IP(3)R-dependent Ca(2+) release events with amplitudes and kinetics that were distinctly different from classical RyR-dependent Ca(2+) sparks. The effects of IP(3) and adenophostin were prevented by heparin and 2-APB. The data suggest that IP(3)-dependent Ca(2+) release increases [Ca(2+)](i) in the vicinity of RyRs and thus facilitates Ca(2+)-induced Ca(2+) release during excitation-contraction coupling. It is concluded that in the adult mammalian atrium IP(3)-dependent Ca(2+) release enhances atrial Ca(2+) signalling and exerts a positive inotropic effect. In addition, by facilitating Ca(2+) release, IP(3) may also be an important component in the development of Ca(2+)-mediated atrial arrhythmias.

Topics: Animals; Arrhythmias, Cardiac; Atrial Function; Calcium; Calcium Channels; Calcium Signaling; Cats; Endothelin-1; Female; Heart Atria; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Male; Myocardium; Myocytes, Cardiac; Osmolar Concentration; Receptors, Cytoplasmic and Nuclear; Signal Transduction

Activation of the Fas receptor in various cell types, including myocytes, triggers apoptotic as well as nonapoptotic effects. Recent studies suggest that Fas activation in the heart participates in the development of major pathologies such as myocarditis and ischemic/reperfusion insults, which are manifested by arrhythmias and mechanical dysfunction. To decipher the contribution of the Fas/FasL pathway to myocardial pathologies, we have investigated the functional consequences of Fas activation in normoxic and hypoxic ventricular myocytes. Our major findings were as follows. (1) Although Fas is constitutively expressed in ventricular myocytes, normoxic myocytes are resistant to Fas-mediated apoptosis. In contrast, hypoxia predisposes myocytes to apoptosis induced by Fas activation. The underlying mechanism is a shift in the balance between proapoptotic proteins (including Fas) and antiapoptotic proteins toward the former. (2) In normoxic myocytes, Fas activation causes a wide range of functional disturbances, which include reduction in resting potential and action potential amplitude, prolonged action potential duration, development of delayed and early after-depolarizations, occasionally culminating into arrhythmias, diastolic [Ca(2+)](i) level increase, decreased I(to) and increased I(Ca,L). (3) The above-mentioned effects in normoxic myocytes (but not Fas-mediated apoptosis in hypoxic myocytes) depend on the phospholipase C --> 1,4,5-IP(3) --> SR [Ca(2+)](i) release cascade. (4) Inhibition of tyrosine kinases with genistein blocks both the apoptotic and nonapoptotic consequences of Fas activation in ventricular myocytes. Based on these studies we propose that tyrosine phosphorylation in ventricular myocytes can serve as a novel potential target for attenuating Fas-mediated dysfunction in normoxic and hypoxic myocardium.

Topics: Animals; Arrhythmias, Cardiac; Cardiac Output, Low; fas Receptor; Inositol 1,4,5-Trisphosphate; Phosphorylation; Protein-Tyrosine Kinases

Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca(2+) signalling to the ability of various stimuli to induce diastolic, premature extra Ca(2+) transients (ECTs). For this, we recorded global and spatially resolved Ca(2+) signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca(2+) response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the beta-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca(2+) responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca(2+) transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The beta-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP(3)) receptors with a membrane-permeable InsP(3) ester (InsP(3) BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP(3) receptors using 2 microM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca(2+) signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP(3) BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP(3)Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca(2+) sparks by subsarcolemmal InsP(3)Rs. Summation of such sparks efficiently generates delayed after depolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca(2+) signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP(3) signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Calcium Channels; Calcium Signaling; Cardiotonic Agents; Digoxin; Electric Stimulation; Electrophysiology; Endothelin-1; Heart Atria; Immunohistochemistry; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Membrane Potentials; Microscopy, Fluorescence; Patch-Clamp Techniques; Rats; Receptors, Cytoplasmic and Nuclear; Subcellular Fractions

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P(3)] and the development of arrhythmias, following stimulation of alpha(1)-adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na(+)/H(+) exchanger, which might reflect a central role for increased Ca(2+)following reverse mode activation of the Na(+)/Ca(2+) exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P(3) measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [(3)H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P(3) (1266+/-401 to 3387+/-256 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01) and the development of arrhythmias. Inhibition of NCX either by reperfusion at low Ca(2+) (1133+/-173 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) or by adding 10 microm KB-R7943, an inhibitor of reverse mode Na(+)/Ca(2+) exchange, prevented the Ins(1,4,5)P(3) response (1151+/-243 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) and the development of ventricular fibrillation. Lower concentrations of KB-R7943 were less effective. Reverse mode activation of NCX is therefore required for the enhanced Ins(1,4,5)P(3) response in early reperfusion, and inhibitors of this transporter may be useful in the prevention of arrhythmias under such conditions.

Topics: Animals; Arrhythmias, Cardiac; Calcium; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Heart; Inositol 1,4,5-Trisphosphate; Lithium Chloride; Male; Models, Biological; Myocardium; Norepinephrine; Perfusion; Rats; Rats, Sprague-Dawley; Reperfusion; Sodium-Calcium Exchanger; Thiourea

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Disease Models, Animal; Humans; Inositol 1,4,5-Trisphosphate; Myocardial Reperfusion; Rats; Sodium-Calcium Exchanger; Thiourea

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P(2)) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P(3)) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na(+)/H(+)exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1, 4,5)P(3)content was measured by mass analysis or by anion-exchange HPLC following [(3)H]inositol labeling. Reperfusion caused generation of [(3)H]Ins(1,4,5)P(3)(1732+/-398 to 3103+/-214, cpm/g tissue, mean+/-S.E.M., n=5, P<0.01) and the development of arrhythmias. Inhibition of Na(+)/H(+)exchange, by reperfusing at pH 6.3 or by pretreating with HOE-694 (10 n M-3 microM) or HOE-642 (3 microM) prevented the [(3)H]Ins(1,4,5)P(3)generation, without causing any suppression of norepinephrine release. Increases in Ins(1,4,5)P(3)mass were similarly reduced by inhibition of Na(+)/H(+)exchange. Thus, activation of Na(+)/H(+)exchange is required for the enhanced Ins(1,4,5)P(3)response observed under reperfusion conditions, and prevention of Ins(1,4,5)P(3)generation may be an important contributor to the anti-arrhythmic actions of inhibitors of Na(+)/H(+)exchange.

Topics: Animals; Arrhythmias, Cardiac; Chromatography, High Pressure Liquid; Guanidines; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Male; Models, Biological; Myocardial Reperfusion; Norepinephrine; Perfusion; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium-Hydrogen Exchangers; Sulfones

Reperfusion of ischemic rat hearts in the presence of thrombin or norepinephrine but not endothelin-1 causes the generation of inositol 1,4,5-trisphosphate (Ins 1,4,5P3) and arrhythmias. The present study investigates the effect of endothelin-1 on these responses.. Ins 1,4,5P3 generation was quantified by use of [3H] labeling and high-performance liquid chromatography as well as by mass analysis. Twenty minutes of global ischemia followed by 2 minutes of reperfusion increased [3H]Ins 1,4,5P3 from 2828+/-265 to 5033+/-650 cpm/g tissue in the presence of thrombin 2.5 IU/mL and to 4561+/-286 cpm/g tissue in response to release of norepinephrine (n=4, P<0.01) in both cases. Reperfusion in the presence of endothelin-1 alone caused no change in Ins 1,4,5P3 (2762+/-240 cpm/g tissue), but when added together with thrombin or norepinephrine, endothelin-1 reduced the Ins 1,4,5P3 responses to 2313+/-197 and 1764+/-168 cpm/g tissue, respectively (n=4, P<0.01 in both cases). Similar inhibitory interactions between endothelin-1 10 nmol/L and thrombin 2.5 IU/mL were observed under normoxic conditions in nonperfused ventricle, eliminating the possibility that excessive vasoconstriction was responsible. In parallel studies, endothelin-1 suppressed the development of reperfusion arrhythmias initiated by either thrombin (ventricular fibrillation, 75% to 39%, n=16 to 18) or norepinephrine (83% to 8%, n=12 to 22) (P<0.01 in both cases).. Inhibition of Ins 1,4,5P3 generation during myocardial reperfusion by endothelin-1 represents a novel antiarrhythmic mechanism.

Topics: Animals; Arrhythmias, Cardiac; Drug Interactions; Endothelin-1; Hemostatics; Inositol 1,4,5-Trisphosphate; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Norepinephrine; Oxygen; Phospholipids; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha; Sympathomimetics; Thrombin

To determine whether the phospholipase C (PLC)/inositol 1,4,5 trisphosphate (IP3)/Ca2+ pathway mediates cardiac arrhythmias induced by kappa-opioid receptor stimulation, the effects of U50,488H, a selective kappa-opioid receptor agonist, on cardiac rhythm in a isolated perfused rat heart, intracellular calcium ([Ca2+]i) in a single ventricular myocyte and IP3 production in myocytes were studied in the presence and absence of PLC inhibitors. U50,488H, the effects of which had been shown to be abolished by a selective kappa-receptor antagonist, nor-binaltorphimine, induced arrhythmias dose-dependently and increased both [Ca2+]i and IP3-production in the heart. More importantly, the effects of U50,488H were blocked by PLC inhibitors, neomycin and streptomycin. To further confirm the selectivity of action of the PLC inhibitor, the effects of another PLC inhibitor U73122 and its inactive structural analog, U73343, on cardiac rhythm in the isolated perfused rat heart were compared. The former did, while the latter did not, block the arrhythmogenic effect of U50,488H. We also determined whether the effects of kappa-receptor stimulation involves a pertussis toxin (PTX)-sensitive G-protein. We found that pretreatment with PTX at 4 microg/l for 10 min, a treatment shown to affect PTX sensitive G-protein-mediated functions, attenuated significantly the U50,488H-induced arrhythmias. The present study provides evidence that kappa-receptor stimulation-induced cardiac arrhythmias involves, at least partly, the PLC/IP3/Ca2+ pathway as well as a PTX sensitive G-protein.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Arrhythmias, Cardiac; Calcium; Enzyme Inhibitors; Estrenes; Heart Rate; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Naltrexone; Narcotic Antagonists; Neomycin; Pertussis Toxin; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Signal Transduction; Streptomycin; Type C Phospholipases; Virulence Factors, Bordetella

Cardiac reperfusion initiates release of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and arrhythmogenesis via norepinephrine stimulation of alpha1-adrenergic receptors. The present study examines arrhythmogenic effects of thrombin-stimulated Ins(1,4,5)P3 release under these conditions.. [3H]Ins(1,4,5)P3 release was measured in [3H]inositol-labeled rat hearts by high-performance liquid chromatography. Arrhythmia studies were performed in buffer-perfused rat hearts. Two-minute reperfusion after 20 minutes of global ischemia increased [3H]Ins(1,4,5)P3 from 1123 +/- 77 to 2238 +/- 44 cpm/mg tissue. No increase was observed in catecholamine-depleted hearts (755 +/- 89 cpm/mg). The addition of thrombin (5 IU/mL) or thrombin receptor agonist peptide (TRAP(1-6), 50 micromol/L) restored the reperfusion Ins(1,4,5)P3 response (thrombin, 1518 +/- 68 cpm/mg and TRAP(1-6), 1755 +/- 128 cpm/mg). Ins(1,4,5)P3 release initiated by norepinephrine or thrombin was inhibited by gentamicin (150 micromol/L; 986 +/- 52 and 868 +/- 125 cpm/mg, respectively). The thrombin response was inhibited by the phospholipase C inhibitor U-73122 (5 micromol/L; 394 +/- 59 cpm/mg) but not by its inactive isomer U-73343. The norepinephrine response was not inhibited by U-73122 (2126 +/- 74 cpm/mg). Ventricular tachycardia and ventricular fibrillation were observed in intact hearts but not in hearts from catecholamine-depleted rats (ventricular fibrillation duration, 110 +/- 19 versus 0 +/- 0 seconds). The addition of thrombin or TRAP(1-6) increased arrhythmias in catecholamine-depleted hearts (112 +/- 32 and 89 +/- 28 seconds, respectively). Gentamicin and U-73122 but not U-73343 prevented thrombin-induced arrhythmias. Gentamicin inhibited norepinephrine-initiated arrhythmias, but U-73122 was ineffective.. This study demonstrates that the development of reperfusion arrhythmias under these conditions depends on the release of Ins(1,4,5)P3.

Topics: Animals; Arrhythmias, Cardiac; Inositol 1,4,5-Trisphosphate; Male; Myocardial Reperfusion Injury; Norepinephrine; Rats; Rats, Sprague-Dawley; Thrombin

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.

Topics: Adenosine Triphosphate; Animals; Arrhythmias, Cardiac; Dietary Fats, Unsaturated; Fish Oils; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lactic Acid; Male; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Norepinephrine; Phosphatidylinositols; Phospholipids; Rats; Rats, Sprague-Dawley

Reperfusion of globally ischemic rat hearts in vitro causes release of inositol(1,4,5) trisphosphate (Ins(1,4,5)P3) which is associated with the development of reperfusion arrhythmias. Both of these responses require the presence of a receptor agonist, either norepinephrine or thrombin, and both responses are inhibited by the aminoglycoside, gentamicin and the polyamine, spermine. In the current study, the role of Ins(1,4,5)P3 in the development of arrhythmias under ischemic conditions was addressed. Arrhythmias [ventricular premature beats, ventricular tachycardia and ventricular fibrillation (VF)] occurring over 25 min subsequent to coronary artery ligation were shown to be independent of endogenous norepinephrine or adrenergic receptor stimulation but were effectively inhibited by gentamicin (0.15-1.5 mM, 95% VF in controls compared with 0% VF, at 1.5 mM, P < 0.01) and spermine (5 mM, 40% VF, P < 0.01). Depletion of Ca2+ stores, including Ins(1,4,5)P3-sensitive Ca2+ stores, with thapsigargin (300 nM) reduced the incidence of ischemic arrhythmias (40% VF, P < 0.01). [3H]-Inositol-labeled right atria incubated under conditions of simulated ischemia retained the ability to respond to norepinephrine by releasing inositol phosphates. Under ischemic conditions, gentamicin (1.5 mM) caused a reduction in [3H]Ins(1,4,5)P3 without any effect on the other inositol phosphates. Similar effects of gentamicin were observed under ischemic conditions in the absence of norepinephrine (95 +/- 8 cpm/mg, mean +/- S.E.M., n = 4, v 29 +/- 4, P < 0.0] for 1.5 mM gentamicin). Agonist independent release of [3H]Ins(1,4,5)P3 under ischemic conditions required extracellular Ca2+ suggesting the operation of a Ca(2+)-activated phospholipase C. In agreement with this, release of [3H]Ins(1,4,5)P3 could be initiated by Ca2+ overload under normoxic conditions and this was inhibited by gentamicin. These findings show that Ca2+ overload can enhance release of Ins(1,4,5)P3 under ischemic conditions and provide evidence that this release is involved in the genesis of arrhythmias under these conditions.

Topics: Animals; Arrhythmias, Cardiac; Calcimycin; Calcium; Gentamicins; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ionophores; Male; Myocardial Ischemia; Norepinephrine; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic

Preconditioning the heart by brief episodes of ischemia and reperfusion can be cardioprotective to injury from subsequent sustained ischemia and reperfusion. Such protective effects include the reduction of reperfusion-induced arrhythmias. We have previously reported a rapid rise in the second messenger inositol(1,4,5) trisphosphate with post-ischemic reperfusion, which has been implicated in the generation of reperfusion-induced arrhythmia. Given the possible importance of inositol(1,4,5)trisphosphate, studies were performed in isolated perfused rat hearts to determine if preconditioning could reduce the reperfusion-induced rise in inositol(1,4,5) trisphosphate. [3H]Inositol labeled hearts underwent 20 min global ischemia with or without 2 min reperfusion. This was preceded by preconditioning of two or three cycles of 2 or 5 min ischemia separated by 5 or 10 min reperfusion, or by time matched control perfusions. Two min reperfusion following 20 min ischemia in time matched controls caused an increase of [3H]inositol(1,4,5)trisphosphate from 20 +/- 3 cpm/mg protein to 52 +/- 5 cpm/mg protein (mean +/- S.E.M. n = 4, P < 0.01). Preconditioning of three cycles of 5 min ischemia and 5 min reperfusion inhibited the 2 min reperfusion-induced rise in [3H]inositol(1,4,5)trisphosphate (26 +/- 4 cpm/mg protein. P < 0.01 relative to non-preconditioned hearts), however protocols that involved either fewer ischemic cycles or shorter ischemic periods were ineffective. Preconditioning did not affect myocardial ATP levels or norepinephrine release with sustained ischemia and reperfusion. Inhibition of the reperfusion-induced rise in inositol(1,4,5) trisphosphate may provide an explanation for the inhibitory effects of preconditioning on reperfusion-induced arrhythmias.

Topics: Adenosine Triphosphate; Animals; Arrhythmias, Cardiac; Heart Ventricles; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lactates; Male; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Norepinephrine; Perfusion; Rats; Rats, Sprague-Dawley; Time Factors