Isolated Heart Muscle Preparations Research Articles

Green Tea Catechins Increase the Force of Contraction in Isolated Guinea Pig Atrial Muscle Preparations by Increasing the Amplitude of Intracellular Ca<sup>2+</sup> Concentration

It has been reported that green tea catechins enhance the force of contraction of isolated heart muscle preparations. However, it remains controversial whether or not the increase in force of contraction is related to an increase in the intracellular Ca(2+) concentration ([Ca(2+)]i). In this study, the relationship was investigated using a left atrial muscle preparation isolated from guinea pig heart. In the left atrial muscle preparations without fura-2/AM loading, neither EGC (epigallocatechin) nor EC (epicatechin) influenced the force of contraction, but EGCG (epigallocatechin gallate) and ECG (epicatechin gallate) increased the force of contraction in a dose-dependent manner. The ED(50) value of EGCG was significantly higher than that of ECG. In the atrial muscle preparations loaded with fura-2/AM, EGCG and ECG increased the amplitude of [Ca(2+)]i(peak [Ca(2+)]i minus diastolic [Ca(2+)]i) which is associated with the increase in force of contraction. Simple regression analysis between the degree of increase in the force of contraction and the increase in the amplitude of [Ca(2+)]i revealed a positive correlation in EGCG, ECG and CaCl(2). In addition, the slopes of the regression lines of EGCG and ECG were comparable with those of CaCl(2). It was suggested that atrial muscle preparations had a higher affinity for ECG than EGCG, and that the increase in the force of contraction by EGCG and ECG was closely related to the increase in the amplitude of [Ca(2+)]i.

Synthesis and Biological Activity of Thienodiltiazem in Isolated Heart Muscle Preparations of Guinea Pigs

The new compound thienodiltiazem was synthesized and investigated regarding structure-activity relations and calcium antagonistic properties. Isometric contraction force was measured in guinea-pig papillary muscles and chronotropic activity was studied in right atria of guinea pigs. Compared to the parent drug diltiazem (CAS 42399-41-7) the thieno derivative had a more potent negative chronotropic effect on spontaneously beating right atria and a more potent inotropic effect on papillary muscles. The negative inotropic action was reversed by increasing the extracellular calcium concentration.

On the cumulation of digitoxin and digitoxigenin in isolated heart muscle preparations

On the cumulation of digitoxin and digitoxigenin in isolated heart muscle preparations

Ouabain attenuates cardiotoxicity induced by other cardiac steroids

All cardiac steroids have a similar structure, bind to and inhibit the ubiquitous transmembrane protein Na(+), K(+)-ATPase and increase the force of contraction of heart muscle. However, there are diverse biological responses to different cardiac steroids both at the cellular and at the molecular level. Moreover, we have recently shown that ouabain inhibits digoxin- and bufalin-induced changes in membrane traffic. The present study was designed to test the hypothesis that ouabain also has an inhibitory effect on cardiotoxicity induced by other cardiac steroids. The hypothesis was tested in isolated heart muscle preparations and in an in vivo model of cardiotoxicity in guinea pigs. Ouabain at a low dose attenuated the toxicity induced by bufalin and digoxin in heart muscle preparations. In addition, ouabain at the low dose (91 ng.kg(-1).h(-1)), but not at a higher dose (182 ng.kg(-1).h(-1)), delayed the development of digoxin-induced (500 microg.kg(-1).h(-1)) cardiotoxicity in anaesthetized guinea pigs, as manifested by delayed arrhythmia and terminal ventricular fibrillation, as well as a reduced heart rate. In addition, as observed with ouabain, the phosphoinositide 3-kinase inhibitor wortmannin (100 microg.kg(-1).h(-1)) delayed the digoxin-induced arrhythmia in anaesthetized guinea pigs. The present study demonstrates the inhibitory effect, probably through signal transduction pathways, of ouabain on digoxin- and bufalin-induced cardiotoxicity in guinea pigs. Further understanding of this phenomenon could be beneficial for increasing the therapeutic window for cardiac steroids in the treatment of chronic heart failure.

ChemInform Abstract: Synthesis and Biological Activity of Thienodiltiazem in Isolated Heart Muscle Preparations of Guinea Pigs.

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FK506 does not affect cardiac contractility and adrenergic response in vitro

FK506 (tacrolimus) is a new immunosuppressant being used in cardiac allograft transplantation. While cyclosporine A has been shown to exert an acute negative inotropic effect on isolated heart muscle preparations, little is known of the inotropic influence of FK506. The Ca 2+ release channel of human skeletal muscle and cardiac muscle is associated with FK506 binding proteins (FKBP), FKBP12 and FKBP12.6, respectively. FKBPs can be dissociated by treatment with FK506. As a consequence of FK506 exposure, isolated skeletal muscle and cardiac muscle ryanodine receptors show altered gating characteristics. Therefore, we analyzed the direct inotropic effect of FK506 exposure to isolated, intact heart muscle preparations from the human and rabbits. Experiments were performed on isolated, electrically stimulated right atrial auricular muscle strips obtained from human myocardium during elective open heart surgery and on intact right ventricular trabeculae from rabbit hearts. The human preparations were exposed to concentrations of 8×10 −9, 8×10 −8 and 8×10 −6 M FK506 followed by a cumulative dose–response curve with isoprenaline as a non-selective β-adrenoceptor agonist. Our data suggest that FK506 does not exert any positive or negative inotropic effect in either human or rabbit myocardium.

Doxorubicin: an antagonist of muscarinic receptors in guinea pig heart

While studying the mechanisms of doxorubicin-induced cardiotoxicity, we observed that doxorubicin inhibited the negative inotropic effect of acetylcholine in isolated heart muscle preparations. We therefore examined the effects of doxorubicin on muscarinic acetylcholine receptors. In left atrial muscle preparations isolated from guinea pig heart and stimulated at 2 Hz at 30°C, doxorubicin caused a parallel right-ward shift of the dose-response curves for the negative inotropic effects of acetylcholine. The inhibitory action was reversed by an additional incubation in the absence of doxorubicin. Doxorubicin reversed the carbachol-induced inhibition of developed tension: a high concentration of doxorubicin brought the force back to its original strength. Doxorubicin inhibited specific [ 3H]quinuclidinyl benzilate (QNB) binding to membrane preparations obtained from ventricular muscle of guinea pig hearts. The pA 2 value for doxorubicin obtained in the inotropic study corresponded to the IC 50 value for doxorubicin observed in the [ 3H]QNB binding assay. These results indicate that doxorubicin acts as a weak competitive antagonist on muscarinic acetylcholine receptors.

Phosphodiesterase inhibition and positive inotropy in failing human myocardium.

Positive inotropic effects of phosphodiesterase inhibitors like 3-isobutyl-1-methylxanthine (IBMX), pimobendan, adibendan, milrinone, saterinone, and enoximone are greatly diminished in isolated heart muscle preparations from human failing myocardium as compared to nonfailing myocardium. This is accompanied by a reduced increase in cAMP content in intact isometrically contracting human trabeculae. With anion exchange chromatography four peaks of phosphodiesterase activities (PDE I-IV) could be separated from both nonfailing and failing human myocardium. Substrate specificity, Km, and Vmax were similar in nonfailing and failing myocardium. Furthermore, the PDE inhibitors investigated exhibited similar IC50-values in both tissues, indicating that the sensitivity of the enzymes from nonfailing and failing tissue was unchanged. Thus, changes in PDE are probably not responsible for the reduced positive inotropic and cAMP-increasing effects of PDE inhibitors in human failing heart muscle preparations. Instead, an increase in signal transducing inhibitory G-proteins may keep the adenylyl cyclase at reduced activity, resulting in an attenuated formation of cAMP, even in the presence of PDE inhibitors.

Factors released from endocardium of the ferret and pig modulate myocardial contraction.

1. In isolated heart muscle preparations, selective removal of the endocardium results in a characteristic and unusual negative inotropic effect. Possible mechanisms for this effect were investigated in this study. 2. In endocardium-intact preparations of ferret papillary muscle, 8-bromo-cyclic GMP, sodium nitroprusside, atrial natriuretic peptide (ANP) and substance P each induced changes in contractile behaviour similar to selective endocardial removal, and each significantly elevated myocardial cyclic GMP levels. Substance P failed to elevate myocardial cyclic GMP levels following removal of endocardium or in the presence of haemoglobin, suggesting that it may act by releasing endothelium-derived relaxing factor (EDRF) from endocardium. However, there was no change in myocardial cyclic GMP levels following endocardium removal alone. 3. In cascade bioassay experiments, it was confirmed that porcine cultured endocardial cells released an unstable humoral agent whose effects on an endothelium-denuded pig coronary artery were indistinguishable from EDRF. 4. The negative inotropic effects of endocardium removal were reversed in bioassay experiments where an endocardium-denuded papillary muscle was exposed to the effluent from a column of porcine cultured endocardial cells on microcarrier beads. This demonstrates for the first time the release of a 'contraction prolonging factor' from endocardium, the tonic release of which would explain the negative inotropic effect of endocardium removal. 5. It is concluded that elevation of ferret papillary muscle cyclic GMP (as for example with EDRF) produces changes in contractile performance similar to those induced by endocardium removal. We also demonstrate that superfused porcine cultured endocardial cells release a humoral agent (provisionally named 'endocardin') which causes reversal of the changes in mechanical properties seen after endocardial removal.

Endocardial Endothelium Modulates Inotropic Responses of Subjacent Myocardium

In contrast to vascular endothelium, the functional role of endocardial endothelium has been largely underestimated. It was now been demonstrated that selective removal of the endocardial endothelium from isolated heart muscle preparations resulted in reduction of twitch duration and peak twitch tension, but did not significantly alter maximal unloaded velocity of shortening (V max ). Alternatively, the presence of an intact endocardial endothelium increased peak twitch tension mainly by prolonging twitch duration.

Endocardial Endothelium Modulates Inotropic Responses of Subjacent Myocardium

Summary: In contrast to vascular endothelium, the functional role of endocardial endothelium has been largely underestimated. It has now been demonstrated that selective removal of the endocardial endothelium from isolated heart muscle preparations resulted in reduction of twitch duration and peak twitch tension, but did not significantly alter maximal unloaded velocity of shortening (Vmax). Alternatively, the presence of an intact endocardial endothelium increased peak twitch tension mainly by prolonging twitch duration. In the present study, we have investigated the effect of several hormones normally present in the blood on myocardial performance of isolated cat papillary muscle in the presence and absence of a functional endocardial endothelium. The results can be subdivided into three arbitrary groups: (a) The effect was dependent upon the presence of an intact endocardium. (b) The effect was modulated or could be reversed by the endocardium. (c) The effect revealed the existence of a complex role of the endocardium.

Mercury compounds: lipophilicity and toxic effects on isolated myocardial tissue

Lipophilicity is suggested to modulate the diffusion and the cytotoxic effects of mercury compounds. To investigate this, the positive inotropic effect of four Hg compounds (HgCl2, CH3HgCl, chlormerodrin, bromomercurihydroxypropane) was studied in catecholamine-depleted isolated heart muscle preparations. The rate of development of the positive effect was inversely correlated to the concentration in the case of HgCl2 and chlormerodrin, i.e. the product of concentration (c) and time to half-maximal effect (t50) remained constant. This was in accordance with the assumption of a permeation-controlled rate of action, as was shown earlier for p-chloromercuriphenyl-sulfonic acid. In addition, the c X t50 values of the individual mercurials decreased hyperbolically with the increase in lipophilicity as measured by the octanol/water partition. The results support the view that the toxicity of mercurials increases with their lipid solubility. In conjunction with the previously reported negative inotropic effect of Hg compounds, a model is proposed allocating thiol groups responsible for the negative inotropic action to lipid compartments within the cell membrane, while SH groups conveying the increase in contraction force are thought to be situated at the internal surface of the sarcolemma.

Mechanism of ECG changes and arrhythmogenic properties of low osmolality contrast media during coronary arteriography in dog

The aim of the study was to investigate the mechanisms behind ECG changes and ventricular arrhythmias during coronary arteriography. Transmembrane action potentials were recorded from isolated heart muscle preparations superfused with contrast media. Conductivity of plasma diluted with contrast media was determined in vitro. Epicardial ECG recordings were made during coronary arteriography. Epicardial ECG recordings were made in eight mongrel dogs of either sex, weight 14-22 kg. Atrial appendages were excised from the same dogs and used for heart muscle preparation studies. Iohexol and ioxaglate affected the action potentials similarly: resting potential, amplitude, rate of depolarisation, and action potential duration increased; effective refractory period decreased. Both contrast media reduced plasma conductivity. During coronary arteriography both media increased R wave amplitude, depressed ST segment, and prolonged QT time on epicardial ECG. ST segment deviation on ECG reflects hyperpolarization and increased amplitude of action potential rather than depolarisation consistent with ischaemia. Increased rate of depolarisation and amplitude of action potential together with reduced conductivity after contrast media may explain increased amplitude of QRS voltage on ECG. Observed regional changes in depolarisation, repolarisation, and refractoriness may be important in the genesis of ventricular arrhythmias.

On the mechanism of positive inotropic effects of alpha-adrenoceptor agonists

The positive inotropic effect of the alpha 1-adrenoceptor agonist phenylephrine is accompanied by an increase in the presumed second messengers inositol 1,4,5-trisphosphate (1,4,5-IP3) and inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4). Both 1,4,5-IP3 and 1,3,4,5-IP4 sensitize myocardial contractile proteins in chemically skinned fibers. In addition to the Ca++ releasing effect of 1,4,5-IP3 from the sarcoplasmic reticulum the Ca++-sensitizing effect of the inositol phosphates may play a role in alpha 1-adrenergic positive inotropism. In isolated heart muscle preparations from patients with endstage heart failure (due to dilated cardiomyopathy) beta-adrenergic as well as alpha 1-adrenergic effects are reduced compared to preparations from healthy hearts. The reduced beta-adrenergic effects can in part be explained by an increased content of signal transducing G1-proteins. It is tempting to investigate whether other G proteins are also altered in severe congestive heart failure.

Adrenoceptor-Mediated Changes of Excitation and Contraction in Isolated Heart Muscle Preparations

The inotropic effects of sympathetic stimulation on the heart are mainly ascribed to the activation of beta-adrenoceptors. However, several findings suggest that alpha-adrenoceptors also may help mediate the inotropic response to catecholamines under certain conditions. The onset of the positive inotropic effect mediated by beta-adrenoceptors occurs within seconds and is associated with a faster rate of relaxation. Both beta 1- and beta 2-adrenoceptors are stimulatorily coupled to the enzyme adenylate cyclase, thereby leading to the generation of cyclic AMP. Cyclic AMP increases the slow inward calcium current and enhances the uptake of calcium into the sarcoplasmic reticulum. GTP-binding protein Gs is important for the transmembrane signal transduction. Muscarine and adenosine receptors are inhibitorily coupled to adenylate cyclase, thereby decreasing the inotropic response to catecholamines. Another GTP-binding protein, Gi, is involved in this pathway. The stimulation of the myocardium through the activation of alpha-adrenoceptors seems to differ both qualitatively and quantitatively. Myocardial alpha-adrenoceptors of most species are, if existent, predominantly of the alpha 1-subtype; the functional role of these receptors obviously considerably varies among species.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipophilicity and pharmacodynamics of cardiotonic steroids in guinea-pig isolated heart muscle preparations.

1. The inhibitory potency of cardiotonic steroids on myocardial Na+-K+-ATPase increases with increasing lipophilicity. Employing the inotropic action on guinea-pig isolated left atria, the relationship between lipophilicity, chemical structure and the pharmacodynamic properties of cardiac glycosides was further examined. Nineteen digitoxigenin- and digoxigenin-derivatives, whose lipophilic nature and inhibitory effects on the myocardial Na+-K+-ATPase have been previously investigated, were tested. 2. All steroids exhibited positive inotropic effects which varied with the lipophilicity of these drugs. The dependence of the relationship between these two parameters on structural transformations of the steroids showed qualitatively very close parallelism to that between lipophilicity and their inhibitory effects on myocardial Na+-K+-ATPase. 3. The positive inotropic effects and the inhibitory effects on myocardial Na+-K+-ATPase also correlated very well, exhibiting similar patterns in their respective correlations with the individual lipophilicity parameters. 4. It is inferred that (a) the positive inotropic effects of the cardiac glycosides vary with their lipophilicity, exhibiting trends similar to those shown for their inhibitory effects on myocardial Na+-K+-ATPase, (b) this interdependence is secondary to the influence of structural changes, particularly on the steroid nucleus, and (c) both the lipophilic nature and pharmacodynamic behaviour of the cardiac glycosides almost exclusively depend on the steroid nucleus itself.

The effect of stimulation frequency and calcium concentration on maintenance of developed tension in isolated heart muscle preparations

The ability of isolated cardiac muscle preparations to maintain force of contraction was assessed at different extracellular Ca 2+ concentrations and at various stimulation frequencies. Preparations were incubated in Krebs-Henseleit buffer at 32°C and electrically stimulated at frequencies between 0.25 and 3.0 Hz. Extracellular Ca 2+ was maintained at given concentrations between 0.8 and 2.4 mM, a range that included the plasma-ionized Ca 2+ levels determined for rat and guinea pig. Developed tension of atrial or papillary muscle preparations of guinea pig heart was more stable at higher stimulation frequencies. Rat atria and ventricular strips were more stable at 0.25 than at 0.5 or 1.0 Hz, and the stability increased at 2.0 and 3.0 Hz. Guinea pig atrial muscle was maintained for a longer time at higher media Ca 2+ concentrations; however, no such effect was observed with rat atrial tissue. Thus, the decrementing of developed tension in isolated cardiac muscle preparations is dependent on stimulation frequency and Ca 2+ concentrations and can be retarded by selection of appropriate incubation conditions.

Class III Antiarrhythmic Action Linked with Positive Inotropy

Most antiarrhythmic drugs are more or less negatively inotropic. Positively inotropic properties, however, have been demonstrated for some class III antiarrhythmic drugs. To test the hypothesis that class III antiarrhythmic effect and positive inotropy may be linked, we used the sea-anemone polypeptide ATX II, which in isolated heart muscle preparations has been shown to specifically inhibit the inactivation of the sodium channel and thereby increase action potential duration and inotropy. We used 12 pentobarbital-anesthetized dogs. Atrial arrhythmias were induced by high-rate stimulation of the right atrium in 5 dogs. Cardiac electrophysiological effects were studied by His-bundle electrography, programmed electrical stimulation, and monophasic action potential (MAP) recordings in 7 autonomically blocked dogs. ATX II (1.0-5.0 micrograms/kg i.v.) converted the arrhythmias, and in the autonomically blocked dogs markedly increased atrial and ventricular refractoriness and ventricular MAP duration without influencing atrial or ventricular conduction velocities, heart rate, or AV-nodal refractoriness. ATX II induced a marked increase in left ventricular dP/dt max. The study indicates that ATX II has class III antiarrhythmic effect, and that the electrophysiological and positive inotropic effects of ATX II have a common mechanism.

Development of myocardial dysfunction in endotoxin shock

Isolated heart muscle preparations were used to investigate the onset and development of myocardial inotropic dysfunction during endotoxin shock in guinea pigs. Left atrial muscles were removed from separate groups of animals at increasing time intervals after administration of either 4 mg/kg purified Escherichia coli endotoxin (shock groups) or an equivalent volume of isotonic saline (control groups). Peak developed contractile tension (CT) and maximal rate of tension development (+dT/dtmax) were significantly depressed in shock tissues as early as 2 h postendotoxin (P less than 0.01), with the magnitude of the contractile deficit progressively increasing during 4, 6, and 12 h postendotoxin. Contractility remained significantly depressed (P less than 0.001) at 16 and 24 h postendotoxin but progressively recovered toward control levels during 16, 24, 48, and 72 h postendotoxin. Shock-induced myocardial dysfunction was characterized by altered contractile responsiveness to low-Ca2+ medium (0.5 mM), gentamicin (4 mM), and hypoxia; altered inotropic reactivity to these interventions followed similar temporal development as the postendotoxin changes in basal contractile parameters. Left ventricular papillary muscles obtained at 16 h postendotoxin corroborated the shock-induced contractile depression observed in atria. These studies provide evidence for early and progressive intrinsic myocardial dysfunction in endotoxin shock and demonstrate that this dysfunction can be unmasked through the study of in vitro atrial and ventricular heart muscle preparations isolated from in vivo shocked animals.

Erythrosin B inhibits high affinity ouabain binding in guinea-pig heart Na+-K+-ATPase without influence on cardiac glycoside induced contractility.

Binding of [3H]-ouabain to guinea-pig heart membranes enriched in Na+-K+-ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 X 10(-7) mol 1(-1) (Bmax = 16.8 pmol ouabain mg-1 protein) whereas low affinity ouabain binding occurred at a KD much greater than 10(-6) mol 1(-1). To discover whether the two ouabain binding sites are functional in guinea-pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea-pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+ (Na+)-dependent-, as well as Na+-K+-activated ATPase (ID50 = 9 X 10(-6) mol 1(-1). Contractility of guinea-pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 X 10(-5) mol 1(-1). Only very high concentrations (4 X 10(-4) mol 1(-1) resulted in a slightly negative inotropic effect (about 20%). Erythrosin B dose-dependently inhibited [3H]-ouabain binding to the Na+-K+-ATPase (KD = - 3.6 X 10(-6) mol 1(-1). In a concentration of 1 X 10(-5) mol 1(-1) the dye abolish high affinity [3H]-ouabain binding without affecting the low affinity binding sites. In contrast, in guinea-pig isolated atria, no functional antagonism between erythrosin B (5 X 10(-5) mol 1(-1) and ouabain was observed. 5 As there is a coincidence between the high affinity binding (KD = 2.2 x 10-7moll ') and the concentration for half maximum inotropic effects of ouabain (EDIo = 1.6 x 10-7 mol I), the lack of effect oferythrosin B on ouabain-induced inotropy may be caused by an inaccessibility of the dye to the (internal) ATP-site of the Na+-K+-ATPase.