PDE3 inhibition by C-type natriuretic peptide-induced cGMP enhances cAMP-mediated signaling in both non-failing and failing hearts.

We have previously shown that the natriuretic peptide receptor B (NPR-B) agonist C-type natriuretic peptide (CNP) enhances cyclic adenosine 3´,5´-monophosphate (cAMP)-mediated signaling in failing hearts, through cyclic guanosine 3´,5´-monophosphate (cGMP)-mediated phosphodiesterase (PDE) 3 inhibition. As several signaling pathways are importantly changed in failing hearts, it could not be taken for granted that this crosstalk would be the same in non-failing hearts. Thus, we wanted to clarify to which extent this effect of CNP occurred also in non-failing hearts. Inotropic and lusitropic responses were measured in muscle strips and cGMP levels, localized cAMP levels, cAMP-PDE activity and mRNA levels were analyzed in isolated cardiomyocytes from left ventricles of non-failing and failing rat hearts. CNP increased cGMP and enhanced ß1- and ß2-adrenoceptor-mediated inotropic and ß1-adrenoceptor-mediated lusitropic responses, in non-failing and failing hearts. The NPR-A agonist brain natriuretic peptide (BNP) increased cGMP, but did not affect inotropic or lusitropic responses, indicating different compartmentation of cGMP from the two natriuretic peptide receptors. cAMP-PDE activity of PDE3 was concentration-dependently inhibited by cGMP with the same potency and to the same extent in non-failing and failing cardiomyocytes. CNP enhanced ß1-adrenoceptor-induced cAMP increase in living cardiomyocytes in the absence, but not in the presence of a PDE3 inhibitor indicating involvement of PDE3. In summary, CNP sensitizes cAMP-mediated signaling in non-failing as in failing hearts, via NPR-B-mediated increase of cGMP that inhibits the cAMP-PDE activity of PDE3.

Prostaglandin E1 facilitates inotropic effects of 5-HT4 serotonin receptors and ß-adrenoceptors in failing human heart.

Prostaglandins have displayed both beneficial and detrimental effects in clinical studies in patients with severe heart failure. Prostaglandins are known to increase cardiac output, but the mechanism is not clarified. Here, we tested the hypothesis that prostaglandins can increase contractility in human heart by amplifying cAMP-dependent inotropic responses. Contractility was measured ex vivo in isolated left ventricular strips and phosphodiesterase (PDE) and adenylyl cyclase (AC) activity was measured in homogenates or membranes from failing human left ventricles. PGE(1) (1 µM) alone did not modify contractility, but given prior, amplified maximal serotonin (5-HT)-evoked (10 µM) contractile responses mediated by 5-HT(4) receptors several fold (24 ± 7 % with PGE(1) vs. 3 ± 2 % above basal with 5-HT alone). The 5-HT(4)-mediated inotropic response was amplified by the PDE3 inhibitor cilostamide and further amplified in combination with PGE(1) (26 ± 6 vs. 56 ± 12 % above basal). PGE(1) reduced the time to reach 90 % of both the maximal 5-HT- and isoproterenol-evoked inotropic response compared to 5-HT or isoproterenol alone. PGE(1) did not modify PDE activity in the homogenate, either alone or when given simultaneously with PDE3 and/or PDE4 inhibitors. Neither 5-HT- nor isoproterenol-stimulated AC activity was significantly amplified by PGE(1). Sensitivity of ventricular strips to Ca(2+) was not enhanced in the presence of PGE(1). Our results show that PGE(1) can enhance cAMP-mediated responses in failing human left ventricle, through a mechanism independent of PDE inhibition, amplification of AC activity or increasing sensitivity to calcium. This effect of PGE(1) possibly contributes to the increase of cardiac output, independent of decreased afterload, observed after prostaglandin administration in humans.

Agents increasing cyclic GMP amplify 5-HT4-elicited positive inotropic response in failing rat cardiac ventricle.

Activation of 5-HT(4) receptors in failing ventricles elicits a cAMP-dependent positive inotropic response which is mainly limited by the cGMP-inhibitable phosphodiesterase (PDE) 3. However, PDE4 plays an additional role which is demasked by PDE3 inhibition. The objective of this study was to evaluate the effect of cGMP generated by particulate and soluble guanylyl cyclase (GC) on the 5-HT(4)-mediated inotropic response. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats, exhibiting heart failure 6 weeks after surgery. Contractility was measured in left ventricular preparations. Cyclic GMP was measured by EIA. In ventricular preparations, ANP or BNP displayed no impact on 5-HT(4)-mediated inotropic response. However, CNP increased the 5-HT(4)-mediated inotropic response as well as the ß(1)-adrenoceptor (ß(1)-AR)-mediated response to a similar extent as PDE3 inhibition by cilostamide. Pretreatment with cilostamide eliminated the effect of CNP. Inhibition of nitric oxide (NO) synthase and soluble GC by L-NAME and ODQ, respectively, attenuated the 5-HT(4)-mediated inotropic response, whereas the NO donor Sin-1 increased this response. The effects were absent during PDE3 inhibition, suggesting cGMP-dependent inhibition of PDE3. However, in contrast to the effects on the 5-HT(4) response, Sin-1 inhibited whereas L-NAME and ODQ enhanced the ß(1)-AR-mediated inotropic response. cGMP generated both by particulate (NPR-B) and soluble GC increases the 5-HT(4)-mediated inotropic response in failing hearts, probably through inhibition of PDE3. ß(1)-AR and 5-HT(4) receptor signalling are subject to opposite regulatory control by cGMP generated by soluble GC in failing hearts. Thus, cGMP from different sources is functionally compartmented, giving differential regulation of different G(s)-coupled receptors.

Differential regulation of ß2 -adrenoceptor-mediated inotropic and lusitropic response by PDE3 and PDE4 in failing and non-failing rat cardiac ventricle.

BACKGROUND AND PURPOSE: ß-Adrenoceptors play a major role in regulating myocardial function through cAMP-dependent pathways. Different phosphodiesterases (PDEs) regulate intracellular cAMP-pools and thereby contribute to the compartmentalization of cAMP-dependent effects. We explored the involvement of PDEs in limiting the ß(2) adrenoceptor-mediated positive inotropic (PIR) and lusitropic (LR) responses in sham-operated (Sham) and failing rat hearts. EXPERIMENTAL APPROACH: Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Rats developing heart failure were studied 6 weeks after surgery. Contractility was measured in left ventricular strips from failing and Sham hearts. cAMP was quantified by RIA. KEY RESULTS: In ventricular strips, stimulation of ß(2) -adrenoceptors with (-)-adrenaline (300 nM CGP20712A present) exerted a small PIR and LR. In Sham hearts, ß(2) -adrenoceptor-mediated as well as ß(1) -adrenoceptor-mediated PIR and LR were increased by selective inhibition of either PDE3 (1 µM cilostamide) or PDE4 (10 µM rolipram). In failing rat hearts, PDE3 inhibition enhanced PIR and LR to both ß(1) - and ß(2) -adrenoceptor stimulation while PDE4 inhibition had no effect on these responses despite a significant increase in cAMP levels. Combined PDE3/4 inhibition further enhanced the PIR and LR of ß(2) - and ß(1) -adrenoceptor activation both in Sham and failing hearts, compared with PDE3 inhibition alone. PDE4 enzyme activity was reduced in failing hearts. CONCLUSIONS AND IMPLICATIONS: Both PDE3 and PDE4 attenuated ß(2) - and ß(1) -adrenoceptor-mediated contractile responses in Sham hearts. In failing hearts, these responses are attenuated solely by PDE3 and thus even selective PDE3 inhibitors may provide a profound enhancement of ß-adrenoceptor-mediated responses in heart failure.

Natriuretic peptides increase beta1-adrenoceptor signalling in failing hearts through phosphodiesterase 3 inhibition.

AIMS: Whereas natriuretic peptides increase cGMP levels with beneficial cardiovascular effects through protein kinase G, we found an unexpected cardio-excitatory effect of C-type natriuretic peptide (CNP) through natriuretic peptide receptor B (NPR-B) stimulation in failing cardiac muscle and explored the mechanism. METHODS AND RESULTS: Heart failure was induced in male Wistar rats by coronary artery ligation. Contraction studies were performed in left ventricular muscle strips. Cyclic nucleotides were measured by radio- and enzyme immunoassay. Apoptosis was determined in isolated cardiomyocytes by Annexin-V/propidium iodide staining and phosphorylation of phospholamban (PLB) and troponin I was measured by western blotting. Stimulation of NPR-B enhanced beta1-adrenoceptor (beta1-AR)-evoked contractile responses through cGMP-mediated inhibition of phosphodiesterase 3 (PDE3). CNP enhanced beta1-AR-mediated increase of cAMP levels to the same extent as the selective PDE3 inhibitor cilostamide and increased beta1-AR-stimulated protein kinase A activity, as demonstrated by increased PLB and troponin I phosphorylation. CNP promoted cardiomyocyte apoptosis similar to inhibition of PDE3 by cilostamide, indicative of adverse effects of NPR-B signalling in failing hearts. CONCLUSION: An NPR-B-cGMP-PDE3 inhibitory pathway enhances beta(1)-AR-mediated responses and may in the long term be detrimental to the failing heart through mechanisms similar to those operating during treatment with PDE3 inhibitors or during chronic beta-adrenergic stimulation.

Appearance of a ventricular 5-HT4 receptor-mediated inotropic response to serotonin in heart failure.

BACKGROUND: Current pharmacological treatment of congestive heart failure (CHF) addresses changes in neurohumoral stimulation or cardiac responsiveness to such stimulation. Yet, undiscovered neurohumoral changes, adaptive or maladaptive, may occur in CHF and suggest novel pharmacological treatment. Serotonin [5-hydroxytryptamine (5-HT)] enhances contractility and causes arrhythmias through 5-HT(4) receptors in human atrium and ventricle but not through rat ventricular 5-HT(4) receptors. OBJECTIVE: We investigated whether CHF could induce ventricular responsiveness to serotonin. METHODS: Postinfarction CHF was induced in male Wistar rats by coronary artery ligation. Contractility was measured in left ventricular papillary muscles 6 weeks after infarction. Messenger RNA was quantified by RT-PCR and cAMP by RIA. RESULTS: Serotonin caused positive inotropic (-logEC(50)=7.5) and lusitropic effects in CHF but not Sham papillary muscles. The inotropic effect of 10 muM serotonin in CHF (31.3+/-2.2%) was of similar size as the effect of 10 muM isoproterenol (34.0+/-1.7%). The effects of serotonin were antagonised by GR113808 (0.5-5 nM), consistent with mediation through 5-HT(4) receptors. This was further supported by positive inotropic effects of the 5-HT(4)-selective partial agonist RS67506. Carbachol blunted the serotonin responses and serotonin increased ventricular and cardiomyocyte cAMP, consistent with coupling to G(s) and adenylyl cyclase. Quantitative RT-PCR revealed fourfold increased 5-HT(4(b)) mRNA expression in CHF vs. Sham ventricles. CONCLUSION: Functional ventricular 5-HT(4) receptors are induced by myocardial infarction and CHF of the rat heart. We propose that they are a model for ventricular 5-HT(4) receptors of human failing heart and may play a pathophysiological role in heart failure.

Functional serotonin 5-HT4 receptors in porcine and human ventricular myocardium with increased 5-HT4 mRNA in heart failure.

Serotonin (5-hydroxytryptamine, 5-HT) increases contractile force and elicits arrhythmias through 5-HT(4) receptors in porcine and human atrium, but its ventricular effects are unknown. We now report functional 5-HT(4) receptors in porcine and human ventricle. 5-HT(4) mRNA levels were determined in porcine and human ventricles and contractility studied in ventricular trabeculae. Cyclic AMP-dependent protein kinase (PKA) activity was measured in porcine ventricle. Porcine and human ventricles expressed 5-HT(4) receptor mRNA. Ventricular 5-HT(4(b)) mRNA was increased by four times in 20 failing human hearts compared with five donor hearts. 5-HT increased contractile force maximally by 16% (EC(50)=890 nM) and PKA activity by 20% of the effects of (-)-isoproterenol (200 microM) in ventricular trabeculae from new-born piglets in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine. In ventricular trabeculae from adult pigs (3-isobutyl-1-methylxanthine present) 5-HT increased force by 32% (EC(50)=60 nM) and PKA activity by 39% of (-)-isoproterenol. In right and left ventricular trabeculae from failing hearts, exposed to modified Krebs solution, 5-HT produced variable increases in contractile force in right ventricular trabeculae from 4 out of 6 hearts and in left ventricular trabeculae from 3 out of 3 hearts- range 1-39% of (-)-isoproterenol, average 8%. In 11 left ventricular trabeculae from the failing hearts of four beta-blocker-treated patients, pre-exposed to a relaxant solution with 0.5 mM Ca(2+) and 1.2 mM Mg(2+) followed by a switch to 2.5 mM Ca(2+) and 1 mM Mg(2+), 5-HT (1-100 microM, 3-isobutyl-1-methylxanthine present) consistently increased contractile force and hastened relaxation by 46% and 25% of (-)-isoproterenol respectively. 5-HT caused arrhythmias in three trabeculae from 3 out of 11 patients. In the absence of phosphodiesterase inhibitor, 5-HT increased force in two trabeculae, but not in another six trabeculae from 4 patients. All 5-HT responses were blocked by 5-HT(4) receptor antagonists. We conclude that phosphodiesterase inhibition uncovers functional ventricular 5-HT(4) receptors, coupled to a PKA pathway, through which 5-HT enhances contractility, hastens relaxation and can potentially cause arrhythmias.

Alpha(1)-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation.

The possible involvement of different kinases in the alpha(1)-adrenoreceptor (AR)-mediated positive inotropic effect (PIE) was investigated in rat papillary muscle and compared with beta-AR-, endothelin receptor- and phorbol ester-induced changes in contractility. The alpha(1)-AR-induced PIE was not reduced by the inhibitors of protein kinase C (PKC), MAPK (ERK and p38), phosphatidyl inositol 3-kinase, or calmodulin kinase II. However, PKC inhibition attenuated the effect of phorbol 12-myristate 13-acetate (PMA) on contractility. alpha(1)-AR-induced PIE was reduced by approximately 90% during inhibition of myosin light chain kinase (MLCK) by 1-(5-chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine (ML-9). Endothelin-induced PIE was also reduced by ML-9, but ML-9 had no effect on beta-AR-induced PIE. The Rho kinase inhibitor Y-27632 also reduced the alpha(1)-AR-induced PIE. The alpha(1)-AR-induced PIE in muscle strips from explanted failing human hearts was also sensitive to MLCK inhibition. alpha(1)-AR induced a modest increase in (32)P incorporation into myosin light chain in isolated rat cardiomyocytes. This effect was eliminated by ML-9. The PIE of alpha(1)-AR stimulation seems to be dependent on MLCK phosphorylation.