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We characterized the Ca2+-sensitizing and phosphodiesterase (PDE)-inhibitory potentials of levosimendan and enoximone to assess their contributions to the positive inotropic effects of these drugs. In guinea pig hearts perfused in the working-heart mode, the maximal increase in cardiac output (55%, P<0.05) was attained at 50 nM levosimendan. The corresponding value for enoximone (36%) was significantly smaller (P<0.05) and was observed at a higher concentration (500 nM). In permeabilized myocyte-sized preparations levosimendan evoked a maximal increase of 55.8+/-8% (mean+/-SEM) in isometric force production via Ca2+ sensitization (pCa 6.2, EC50 8.4 nM). Enoximone up to a concentration of 10 microM failed to influence the isometric force. The PDE-inhibitory effects were probed on the PDE III and PDE IV isoforms. Levosimendan proved to be a 1300-fold more potent and a 90-fold more selective PDE III inhibitor (IC50 for PDE III 1.4 nM, and IC50 for PDE IV 11 microM, selectivity factor approximately 8000) than enoximone (IC50 for PDE III 1.8 microM, and IC50 for PDE IV 160 microM, selectivity factor approximately 90). Hence, our data support the hypothesis that levosimendan exerts positive inotropy via a Ca2+-sensitizing mechanism, whereas enoximone does so via PDE inhibition with a limited PDE III versus PDE IV selectivity.

In order to clarify the mechanisms of the positive inotropic actions of levosimendan and its optical isomer, dextrosimendan, we compared their concentration-dependent effects in intact papillary muscles, permeabilized cardiomyocytes and in purified phosphodiesterase enzyme preparations of guinea-pig hearts. In papillary muscles twitch tension increased with EC 50 values of 60 nM and 2.8 mM for levosimendan and dextrosimendan, respectively. Hence, the two enantiomers exhibited a 47 times potency difference in their positive inotropic effects in a preparation where theoretically Ca 2π -sensitization and phosphodiesterase inhibition could both contribute to the positive inotropic effects. In guinea-pig cardiomyocytes, levosimendan and dextrosimendan increased isometric force production (at pCa 6.2) due to Ca 2π -sensitization with EC 50 values of 8.4 nM and 0.64 mM, respectively, with a similar relative potency difference of 76. A major difference appeared in their relative pharmacological potencies, however, when the inhibitory effects of the two enantiomers were assayed on phosphodiesterase III, purified from guinea pig left ventricle (i.e. the phosphodiesterase isoenzyme which is dominant in that tissue). Levosimendan was a 427 times more potent phosphodiesterase inhibitor than dextrosimendan, with IC 50 values of 7.5 nM, and 3.2 mM, respectively. Taken together, our data support the hypothesis that levosimendan and dextrosimendan exert their positive inotropic effects via a stereoselective Ca 2π -sensitizing mechanism and not via stereoselective inhibition of phosphodiesterase III in the myocardium.Levosimendan is a positive inotropic and vasodilatory drug developed for the treatment of acute de novo or decompensated congestive heart failure (Follath et al. 2002). The drug binds to the Ca 2π saturated cardiac isoform of troponin C (Pollesello et al. 1994;Sorsa et al. 2001Sorsa et al. & 2003 and this binding is stereoselective (Sorsa et al. 2004). A correlation between the binding to troponin C and the positive inotropic effect of levosimendan has been established (Haikala et al. 1995a; Levijoki et al. 2000). However, the molecule displays structural similarities with a family of inhibitors of phosphodiesterases, which leads to an alternative hypothesis for its cardiotonic effects that signifies a possible phosphodiesterase inhibitory action (Boknik et al. 1997) and consequent augmentation of the Ca 2π transient. In contrast to this hypothesis, however, several reports show that levosimendan need not increase the intracellular Ca 2π concentration for the development of a sensible positive inotropic effect (Lancaster & Cook 1997;Hasenfuss et al. 1998;Brixius et al. 2002). To elucidate the pharmacological background of levosimendan-induced positive inotropy, the effects of levosimendan and of its active metabolite, OR-1896 as positive inotropes, Ca terase inhibitors were recently critically compared (Szilagyi et al. 2004). This study demonstrated well-pronounced similarities in the concentration dependen...

Prolonged Ca2؉ stimulations often result in a decrease in contractile force of isolated, demembranated human ventricular cardiomyocytes, whereas intact cells are likely to be protected from this deterioration. We hypothesized that cytosolic protein kinase C (PKC) contributes to this protection. Prolonged contracture (10 min) of demembranated human cardiomyocytes at half-maximal Ca 2؉ resulted in a 37 ؎ 5% reduction of active force (p < 0.01), whereas no decrease (2 ؎ 3% increase) was observed in the presence of the cytosol (reconstituted myocytes). The PKC inhibitors GF 109203X and Gö 6976 (10 mol/liter) partially antagonized the cytosol-mediated protection (15 ؎ 5 and 9 ؎ 2% decrease in active force, p < 0.05). Quantitation of PKC isoform expression revealed the dominance of the Ca 2؉ -dependent PKC␣ over PKC␦ and PKC⑀ (189 ؎ 31, 7 ؎ 3, and 7 ؎ 2 ng/mg protein, respectively). Ca 2؉ stimulations of reconstituted human cardiomyocytes resulted in the translocation of endogenous PKC␣, but not PKC␤1, ␦, and ⑀ from the cytosol to the contractile system (PKC␣ association: control, 5 ؎ 3 arbitrary units; ؉Ca ). Our data suggest that PKC␣ translocates to the contractile system and anchors to TnI in a Ca 2؉ -dependent manner in the human heart, contributing to the maintenance of contractile force. Protein kinase C (PKC)3 is a family of serine/threonine kinases (1). Multiple PKC isozymes are often expressed in the same cell, mediating specific functions. Conventional and novel PKCs can be activated by lipids, like the endogenous diacylglycerol (DAG) or the exogenous phorbol ester PMA. It was reported decades ago that PMA activation of PKC leads to the translocation of PKC from the soluble to the particulate fraction (2). This observation has been confirmed by later works, and some of the binding proteins for activated PKC isozymes were identified (receptors for activated C kinases) (3, 4). Binding to its respective receptors for activated C kinase localizes each PKC isozyme in the vicinity of a subset of substrates and away from others, and hence this spatial organization may well explain the specificity of PKC isozymes in their intracellular signaling.It is of interest that from the many PKC isozymes expressed in the heart (5), PKC␣ is the single isozyme that translocates to the contractile system upon Ca 2ϩ stimulation in the rat heart (6), suggesting a unique physiological role for PKC␣ in the Ca 2ϩ -dependent regulation of myofibrillar contractility. As a matter of fact, PKC␣ has been implicated in models of ischemic heart failure, myocardial hypertrophy, hypertension, and atherosclerosis (7). In addition, PKC-dependent phosphorylation of myofibrillar proteins such as desmin (8), myosin light chain (9), troponin I (TnI), and troponin T (TnT) (10) has been documented with suggested functional consequences ranging from changes in mechanical integrity of the cardiac sarcomere to decreased actin-myosin ATPase activity and force generation.Long term activation of PKC is an essential step in ischemic preconditioning (11), a...

This study is the first pharmacological characterization of the novel chemical entity, ORM-3819 (L-6-{4-[N'-(4-Hydroxi-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one), focusing primarily on its cardiotonic effects. ORM-3819 binding to cardiac troponin C (cTnC) was confirmed by nuclear magnetic resonance spectroscopy, and a selective inhibition of the phosphodiesterase III (PDE III) isozyme (IC50=3.88±0.3 nM) was revealed during in vitro enzyme assays. The Ca(2+)-sensitizing effect of ORM-3819 was demonstrated in vitro in permeabilized myocyte-sized preparations from left ventricles (LV) of guinea pig hearts (ΔpCa50=0.12±0.01; EC50=2.88±0.14 µM). ORM-3819 increased the maximal rate of LV pressure development (+dP/dtmax) (EC50=8.9±1.7 nM) and LV systolic pressure (EC50=7.63±1.74 nM) in Langendorff-perfused guinea pig hearts. Intravenous administration of ORM-3819 increased LV+dP/dtmax (EC50=0.13±0.05 µM/kg) and improved the rate of LV pressure decrease (-dP/dtmax); (EC50=0.03±0.02 µM/kg) in healthy guinea pigs. In an in vivo dog model of myocardial stunning, ORM-3819 restored the depressed LV+dP/dtmax and improved % segmental shortening (%SS) in the ischemic area (to 18.8±3), which was reduced after the ischaemia-reperfusion insult (from 24.1±2.1 to 11.0±2.4). Our data demonstrate ORM-3819 as a potent positive inotropic agent exerting its cardiotonic effect by a cTnC-dependent Ca(2+)-sensitizing mechanism in combination with the selective inhibition of the PDE III isozyme. This dual mechanism of action results in the concentration-dependent augmentation of the contractile performance under control conditions and in the postischemic failing myocardium.