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.
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.
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.
BACKGROUND AND PURPOSELevosimendan is known as a calcium sensitizer, although it is also known to inhibit PDE3. We aimed to isolate each component and estimate their contribution to the increased cardiac contractility induced by levosimendan.
EXPERIMENTAL APPROACHContractile force was measured in electrically stimulated ventricular strips from explanted failing human hearts and left ventricular strips from normal male Wistar rats. PDE activity was measured in a two-step PDE activity assay on failing human ventricle.
KEY RESULTSLevosimendan exerted a positive inotropic effect (PIE) reaching maximum at 10 −5 M in ventricular strips from failing human hearts. In the presence of the selective PDE3 inhibitor cilostamide, the PIE of levosimendan was abolished. During treatment with a PDE4 inhibitor and a supra-threshold concentration of isoprenaline, levosimendan generated an amplified inotropic response. This effect was reversed by β-adrenoceptor blockade and undetectable in strips pretreated with cilostamide. Levosimendan (10 −6 M) increased the potency of β-adrenoceptor agonists by 0.5 log units in failing human myocardium, but not in the presence of cilostamide. Every inotropic response to levosimendan was associated with a lusitropic response. Levosimendan did not affect the concentration-response curve to calcium in rat ventricular strips, in contrast to the effects of a known calcium sensitizer, EMD57033 [5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one]. PDE activity assays confirmed that levosimendan inhibited PDE3 as effectively as cilostamide.
CONCLUSIONS AND IMPLICATIONSOur results indicate that the PDE3-inhibitory property of levosimendan was enough to account for its inotropic effect, leaving a minor, if any, effect to a calcium-sensitizing component.
AbbreviationsCRC, contraction-relaxation cycles; cTnC, cardiac troponin C; dF/dtmax, maximal development of force; Fmax, maximal developed force; PIE, positive inotropic effect; RT, relaxation time; TPF, time to peak force; TR80, time to 80% relaxation.
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