Birgitt Stein

Muscarinic and beta-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase.

>Nitric oxide (NO) is an ubiquitous signaling molecule produced from L-arginine by NO synthase (NOS). In the vasculature, NO mediates parasympathetic endothelium-dependent vasodilation. NO may also mediate the parasympathetic control of myocardial function. This is supported by the observations that NOS3, the endothelial constitutive NOS, is expressed in normal cardiac myocytes from rodents and human, and NOS and/or guanylyl cyclase inhibitors antagonize the effect of muscarinic agonists on heart rate, atrio–ventricular conduction, contractility and L-type calcium current. Here we examine the autonomic regulation of the heart in genetically engineered mice deficient in NOS3 (NOS3-KO)(ref. 8). We show that the chronotropic and inotropic responses to both β-adrenergic and muscarinic agonists were unaltered in isolated cardiac tissue preparations from NOS3-KO mice, although these mice have a defective parasympathetic regulation of vascular tone. Similarly, β-adrenergic stimulation and muscarinic inhibition of the calcium current did not differ in cardiac myocytes from NOS3-KO mice and those from wild-type mice. RT–PCR did not demonstrate upregulation of other NOS isoforms. Similarly, Gi/Go proteins and muscarinic receptor density were unaltered. These data refute the idea that NOS3 is obligatory for the normal autonomic control of cardiac muscle function.

Basal and isoprenaline-stimulated cAMP content in failing versus nonfailing human cardiac preparations

We measured force of contraction and cAMP content in human isolated electrically driven right ventricular trabeculae carneae with and without the addition of isoprenaline (0.2 microM). Basal cAMP content was approximately 200% higher in preparations from nonfailing hearts than from hearts with end-stage myocardial failure. Isoprenaline was less effective in increasing force of contraction in failing (by approximately 100%) than in nonfailing cardiac preparations (by approximately 500%). With isoprenaline, cAMP content was approximately 50% lower in failing than in nonfailing preparations. We conclude that the reduced increase in force of contraction of failing human cardiac preparations with isoprenaline added may be causally related to an inadequately increased cAMP content.

Reduced α1- and β2-adrenoceptor-mediated positive inotropic effects in human end-stage heart failure

1 α1‐Adrenoceptor (phenylephrine in the presence of propranolol) and β2‐adrenoceptor (fenoterol)‐mediated positive inotropic effects were investigated in human ventricular preparations isolated from five nonfailing (prospective organ donors) and from eight explanted failing hearts with end‐stage idiopathic dilative cardiomyopathy (NYHA IV). 2 For comparison, the nonselective β‐adrenoceptor agonist isoprenaline, the phosphodiesterase (PDE) inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX), the cardiac glycoside dihydroouabain, and calcium were studied. 3 Furthermore, the influence of IBMX on adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) PDE activity as well as total β‐adrenoceptor density, β1‐ and β2‐adrenoceptor subtype distribution, and α1‐adrenoceptor density were compared in nonfailing and failing human heart preparations. The radioligands (−)‐[125I]‐iodocyanopindolol for β‐adrenoceptor binding and [3H]‐prazosin for α1‐adrenoceptor binding were used. 4 The inotropic responses to calcium and dihydroouabain in failing human hearts were unchanged, whereas the maximal α1‐ and β2‐adrenoceptor‐mediated positive inotropic effects were greatly reduced. The inotropic effects of the other cyclic AMP increasing compounds, i.e. isoprenaline and IBMX, were also reduced to about 60% of the effects observed in nonfailing controls. The potency of these compounds was decreased by factors 4–10. 5 The basal PDE activity and the PDE inhibition by IBMX were similar in nonfailing and failing preparations. 6 The total β‐adrenoceptor density in nonfailing hearts was about 70 fmol mg−1 protein. In failing hearts the total number of β‐adrenoceptors was markedly reduced by about 60%. The β1/β2‐adrenoceptor ratio was shifted from about 80/20% in nonfailing to approximately 60/40% in failing hearts which was due to a selective reduction of β1‐adrenoceptors. The β2‐adrenoceptor population remaining unchanged. α1‐Adrenoceptor density was increased from about 4 fmol mg−1 protein in nonfailing to 10 fmol mg−1 protein in failing hearts. 7 Changes in PDE activity and adrenoceptor downregulation cannot completely explain the reduced positive inotropic effects of α1‐ and β2‐adrenoceptor agonists in failing human hearts. This supports the hypothesis that impairment of other processes such as the coupling between receptor and effector system, i.e. the respective G‐proteins, are equally important in end‐stage heart failure.

Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations.

SummaryThe present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the aβ-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydroouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors.To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I–III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I–III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored.These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure. Furthermore, the concomitant application of a β-adrenoceptor agonist and a phosphodiesterase inhibitor might be beneficial in terminal heart failure refractory to conventional therapeutic regimens.

Receptor mechanisms involved in the 5‐HT‐induced inotropic action in the rat isolated atrium

1 The effects of 5‐hydroxytryptamine (5‐HT) in rat cardiac preparations were studied. 5‐HT up to 10 μM failed to affect contractility in papillary muscles. However, in electrically driven (1 Hz) left atria 5‐HT exerted a positive inotropic effect that started at 1 μM and attained its maximum at 10 μM (312±50% of predrug value, n=8). 2 5‐HT 10 μM stimulated the content of inositol‐1,4,5‐trisphosphate but not of cyclic AMP in rat left atria. 3 Plasma and serum levels of 5‐HT amounted to about 0.3 μM and 15 μM, respectively. 4 The selective 5‐HT4 receptor antagonists GR 125487 (10 nM and 1 μM) and SB 203186 (1 μM) did not attenuate the positive inotropic effect of 5‐HT in rat left atria. In contrast, the 5‐HT2 receptor antagonist ketanserin (5 nM, 50 nM, 1 μM) resulted in a concentration‐dependent diminution of the positive inotropic effect of 5‐HT in rat left atria. 5 Reverse transcriptase polymerase chain reaction with specific primers detected mRNA of the 5‐HT2A receptor in rat atria and ventricles, while expression of the 5‐HT4 receptor was confined to atria. 6 It is suggested that the positive inotropic effect of 5‐HT in electrically driven rat left atria is mediated by ketanserin‐sensitive 5‐HT2A receptors and not through 5‐HT4 receptors.

Diminution of contractile response by κ-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein

Opioids directly decrease the contractile response of isolated ventricular cardiomyocytes to electrical stimulation. To investigate whether these effects are mediated via GTP-binding Gi/o proteins we examined the influence of pertussis toxin on the effects of the κ-opioid receptor agonist trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U-50,488) methanesulphonate and on the as yet undescribed effects of the opioid peptide dynorphin A (1–8) on contraction. In isolated, electrically driven, rat ventricular cardiomyocytes both agents concentration dependently reduced cell shortening within 15 min, decreasing the contractile response by 79±4% (n=5) and 62±2% (n=6) of control values at maximal effective concentrations of 10 µM (U-50,488) and 1 µM [dynorphin A (1–8)], respectively. Pertussis toxin pre-treatment (200 ng/ml; 4.5–5 h) completely abolished the effects of U-50,488 and dynorphin A (1–8) on the contractile response, indicating that these effects are mediated via Gi/o proteins. In addition, the non-selective opioid receptor antagonist (–)-naloxone and the κ-opioid receptor antagonist nor-binaltorphimine antagonized the effects of U-50,488 and dynorphin A (1–8) on the contractile response. Furthermore, the µ- and δ-opioid receptor agonist (D-Ala2, D-Leu5)-enkephalin (DADLE) had no effects on contraction. These results indicate that the decrease in cell shortening is due to stimulation of κ-opioid receptors. The direct effect of κ-opioid receptor agonists on the contractile response thus represents an additional mechanism for decreasing cardiac contractility, besides the M-cholinoceptor- or adenosine receptor-mediated pathway. It is conceivable that increased release of endogenous dynorphins from the heart during hypoxia may protect the heart in a similar manner to adenosine.

Effects of isomazole on force of contraction and phosphodiesterase isoenzymes I-IV in nonfailing and failing human hearts.

The phosphodiesterase (PDE) inhibitor isomazole increased the force of contraction to 278.3 ± 89.1% (n = 7) of the predrug value in ventricular trabeculae carneae isolated from nonfailing human hearts. This effect can be attributed mainly to a PDE III or a combined PDE III/IV inhibition since at the concentration of the maximal positive inotropic effect of isomazole, PDE III and PDE IV were completely inhibited. In explanted failing human hearts (end-stage myocardial failure, NYHA IV), isomazole increased the force of contraction only marginally to 110.1 ± 10.7% of the predrug value. The lack of a distinct positive inotropic efficacy of isomazole in failing human hearts could not be explained by an impairment of PDE inhibition since the properties of the PDE I-IV isoenzymes separated by DEAE-Sepharose chromatography and the inhibitory effects of isomazole did not differ in both preparations. The positive inotropic effect of the β-adrenoceptor agonist isoprenaline was also reduced in failing hearts. However, in the presence of isomazole, the diminished positive inotropic effect of isoprenaline was restored to values obtained with isoprenaline alone in nonfailing hearts. Thus, the decreased effect of inotropic drugs like isoprenaline or isomazole in preparations from failing human heart might be explained mainly by a diminished cAMP formation due to a defect in receptor-adenylate cyclase coupling.

Novel halogenated dihydropyridine derivatives with high vascular selectivity

SummaryCalcium channel antagonists of the dihydropyridine type exhibit preferential vasodilator properties. To study whether this vascular selectivity is due to distinct steric modifications or may be influenced by the physicochemical nature of these drugs, contractility in guinea pig heart isolated papillary muscles, vasodilator properties in isolated rabbit femoral arteries and the lipophilicity of some novel halogenated dihydropyridines have been examined. All newly synthesized derivatives exhibited dose-dependent negative inotropic and vasodilator effects. The negative inotropic potency of all the halogenated derivatives was weaker than that of the parent compound nitrendipine. In contrast, compared to nitrendipine the vasodilator potency of the ester substituted derivatives was slightly increased, while halogen substitution in position 2 and 6 of the dihydropyridine nucleus decreased the vasodilator potency. As a result of the different influence on cardiac and vascular smooth muscle an improved vascular selectivity of the drugs was attained. The ester-substituted dihydropyridine derivatives showed a 9 times (3-bromoethyl-nitrendipine) or 11 times (3-chloroethyl-nitrendipine) higher vascular selectivity with respect to nitrendipine. Correlation of the lipophilicity with the physiological properties showed an increase in biological activity with decreasing lipophilicity. Within the ester-halogenated dihydropyridine derivatives an inverse trend was observed (increasing vasodilation with increasing lipophilicity), indicating a different influence of lipophilicity with the ester-substituted compounds on the different tissues examined. The improved vascular selectivity of the novel halogenated dihydropyridines may be at least in part a consequence of the different lipophilicity of the drugs. In addition, differences in the binding affinities of the dihydropyridines subordinate to distinct voltage dependent conformation states of the calcium channel may contribute.

Dissociation of the effects of forskolin and dibutyryl cAMP on force of contraction and phospholamban phosphorylation in human heart failure.

Forskolin and dibutyryl cyclic adenosine monophosphate (cAMP) stimulate force of contraction independent of beta-adrenoceptor stimulation. We studied their effects on force of contraction and phosphorylation of regulatory proteins in isolated electrically driven trabeculae carneae from failing human ventricles. The phosphorylation state of the regulatory protein phospholamban was studied because its phosphorylation usually faithfully follows contractility. For comparison, the phosphorylation state of the inhibitory subunit of troponin was studied. The phosphorylation state was inferred from in vitro phosphorylation of homogenates with cAMP-dependent protein kinase in the presence of radioactive gamma[32P]ATP Proteins were separated by electrophoresis, and radioactivity in the proteins of interest was quantified. The maximal positive inotropic effects occurred at 30 microM forskolin and were attenuated in comparison with the maximal effects to dibutyryl cAMP (1 mM). Both forskolin and dibutyryl cAMP enhanced phospholamban phosphorylation. However, phospholamban phosphorylation in intact trabeculae treated with 30 microM forskolin and 1 mM dibutyryl cAMP was comparable. It is suggested that phospholamban phosphorylation can be dissociated from inotropy at least in isolated trabeculae from failing human hearts.

Regulation of Adenosine Receptor Subtypes and Cardiac Dysfunction in Human Heart Failure

A defective adenosine receptor-mediated autoregulation of the heart has been hypothesized to cause cardiomyopathy. Regulation of gene expression of the four adenosine receptor subtypes A1, A2A, A2B, A3 and the A1-mediated antiadrenergic signal transduction pathway has been studied in nonfailing and failing human hearts. Gene expression of myocardial A1-adenosine receptors and A1-mediated inhibitory effects on cAMP levels or negative inotropic effects were not altered in failing compared to nonfailing human hearts, indicating that an impairment in the A1-mediated pathway may not be involved in the pathogenesis of heart failure. Furthermore, A3-adenosine receptor gene expression was unchanged in failing compared to nonfailing hearts, while A2B mRNA was not detectable. However, A2A-adenosine receptor gene expression was increased by about 60% in patients with dilated cardiomyopathy. Thus, the A2A-upregulation may indicate a pathophysiological role for adenosine and adds therefore to the increasingly complex picture of molecular alterations in heart failure.