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.
Background and Purposes Myocardial C‐type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP‐induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca2+ ATPase2 (SERCA2) activity is essential for these responses. Experimental Approach Contractility, cGMP levels, Ca2+ transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction. Key Results CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser16 and troponin I (TnI) Ser23/24 phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp‐8‐Br‐Pet‐cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca2+ transient and increased SERCA2 activity in cardiomyocytes. The CNP‐elicited NIR and LR were not affected by the L‐type Ca2+ channel activator BAY‐K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte‐specific inactivation of the SERCA2 gene. Conclusions and Implications We conclude that CNP‐induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.
We previously found a negative inotropic (NIR) and positive lusitropic response (LR) to C-type natriuretic peptide (CNP) in the failing heart ventricle. In this study, we investigated and compared the functional responses to the natriuretic peptides (NPs), brain (BNP) and C-type natriuretic peptide (CNP), and relate them to cGMP regulation and effects on downstream targets. Experiments were conducted in left ventricular muscle strips and ventricular cardiomyocytes from Wistar rats with heart failure 6 weeks after myocardial infarction. As opposed to CNP, BNP did not cause an NIR or LR, despite increasing cGMP levels. The BNP-induced cGMP elevation was mainly and markedly regulated by phosphodiesterase (PDE) 2 and was only marginally increased by PDE3 or PDE5 inhibition. Combined PDE2, -3, and -5 inhibition failed to reveal any functional responses to BNP, despite an extensive cGMP elevation. BNP decreased, whereas CNP increased, the amplitude of the Ca 21 transient. BNP did not increase phospholamban (PLB) or troponin I (TnI) phosphorylation, Ca 21 extrusion rate constant, or sarcoplasmatic reticulum Ca 21 load, whereas CNP did. Both BNP and CNP reduced the peak of the L-type Ca 21 current. Cyclic GMP elevations by BNP and CNP in cardiomyocytes were additive, and the presence of BNP did not alter the NIR to CNP or the CNP-induced PLB and TnI phosphorylation. However, a small increase in the LR to maximal CNP was observed in the presence of BNP. In conclusion, different responses to cGMP generated by BNP and CNP suggest different compartmentation of the cGMP signal and different roles of the two NPs in the failing heart.
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