Piero Pollesello

Levosimendan increases diastolic coronary flow in isolated guinea-pig heart by opening ATP-sensitive potassium channels.

Levosimendan, a novel calcium sensitizer developed for the treatment of acute heart failure, is an inodilator that increases coronary flow. Because it was recently shown that levosimendan stimulates potassium current through KATP channels in isolated rat arterial cells, our aim was to assess whether the levosimendan-induced increase in coronary flow is due to the opening of the KATP channels in coronary smooth muscle. The effect of levosimendan on the diastolic coronary flow velocity (DCFV) was measured in the Langendorff perfused spontaneously beating guinea-pig heart in the absence and presence of glibenclamide. Pinacidil was used as a reference compound, and the protein kinase C inhibitor bisindolylmaleimide was used to study the dilatory effect of levosimendan when the KATP channels in smooth muscle are not inhibited by PKC-dependent phosphorylation. Levosimendan (0.01–1 &mgr;M) increased DCFV concentration-dependently and was noncompetitively antagonized by 0.1 &mgr;M glibenclamide, whereas pinacidil was inhibited competitively by glibenclamide. In the presence of glibenclamide the positive inotropic and chronotropic effects of levosimendan were unaltered. The effect of bisindolylmaleimide and levosimendan on DCFV was additive. The results indicate that levosimendan induced coronary vasodilation through the opening of the KATP channels. Levosimendan and pinacidil probably have different binding sites on the KATP channels. The additive effect of bisindolylmaleimide and levosimendan on the increase of DCFV suggests that the latter binds to the unphosphorylated form of the channel.

Levosimendan : molecular mechanisms and clinical implications: consensus of experts on the mechanisms of action of levosimendan

The molecular background of the Ca(2+)-sensitizing effect of levosimendan relates to its specific interaction with the Ca(2+)-sensor troponin C molecule in the cardiac myofilaments. Over the years, significant preclinical and clinical evidence has accumulated and revealed a variety of beneficial pleiotropic effects of levosimendan and of its long-lived metabolite, OR-1896. First of all, activation of ATP-sensitive sarcolemmal K(+) channels of smooth muscle cells appears as a powerful vasodilator mechanism. Additionally, activation of ATP-sensitive K(+) channels in the mitochondria potentially extends the range of cellular actions towards the modulation of mitochondrial ATP production and implicates a pharmacological mechanism for cardioprotection. Finally, it has become evident, that levosimendan possesses an isoform-selective phosphodiesterase-inhibitory effect. Interpretation of the complex mechanism of levosimendan action requires that all potential pharmacological interactions are analyzed carefully in the framework of the currently available evidence. These data indicate that the cardiovascular effects of levosimendan are exerted via more than an isolated drug-receptor interaction, and involve favorable energetic and neurohormonal changes that are unique in comparison to other types of inodilators.

Levosimendan is a mitochondrial KATP channel opener

Levosimendan, a new inodilator developed for the treatment of heart failure has been shown to have a vasodilatory effect via opening of K(ATP) channels in the plasma membrane of vascular smooth muscle cells. In this study, we investigated the effects of levosimendan on the mitochondrial K(ATP) channel. This compound did not influence mitochondrial transmembrane potential (DeltaPsi), and at up to 2.2 microM had no effect on the respiration rate of rat liver mitochondria, respiring on 5 mM succinate (+5 microM rotenone). A sensitive method was developed for assessing K(ATP) channel opening activity employing rat liver mitochondria, respiring only on endogenous substrates in the presence of 400 microM ATP and 1 microg oligomycin/mg mitochondrial protein. In this model, levosimendan (0.7-2.6 microM) decreased DeltaPsi by 6.5-40.4% (n=3, incubation time 15 min). This effect was dependent on the K+ concentration in the incubation medium and was abolished by the selective blocker of the mitochondrial K(ATP) channel-5-hydroxydecanoate (200 microM). Our results indicate that levosimendan opens mitochondrial K(ATP) channels.

Binding of Levosimendan, a Calcium Sensitizer, to Cardiac Troponin C

Levosimendan is an inodilatory drug that mediates its cardiac effect by the calcium sensitization of contractile proteins. The target protein of levosimendan is cardiac troponin C (cTnC). In the current work, we have studied the interaction of levosimendan with Ca2+-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca2+-loaded regulatory domain of recombinant cTnCC35S was observed. The changes in the NMR spectra of the N-domain of full-length cTnCC35S, due to the binding of levosimendan to the primary site, were indicative of a slow conformational exchange. In contrast, no binding of levosimendan to the regulatory domain of cTnCA-Cys, where all the cysteine residues are mutated to serine, was detected. Moreover, it was shown that levosimendan was in fast exchange on the NMR time scale with a secondary binding site in the C-domain of both cTnCC35S and cTnCA-Cys. The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca2+-saturated cTnC but show no domain-domain closure. The experiments were run in the absence of the reducing agent dithiothreitol and the preservative sodium azide (NaN3), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Inhibition of lipid peroxidation in isolated rat liver mitochondria by the general anaesthetic propofol

The effect of the general anaesthetic propofol (2,6-diisopropylphenol) on lipid peroxidation in rat liver mitochondria was assessed with the thiobarbituric acid (TBA) assay. Propofol was shown to inhibit the accumulation of TBA-reactive compounds after initiation of radical production by the addition of the ADP-Fe2+ complex. Analysis of kinetics showed that propofol caused a concentration-dependent delay as well as a decrease in the rate of the peroxidation process. 1H-NMR spectra of mitochondrial lipid extracts indicated that 95% of the added propofol remained intact after 30 min incubation under conditions of low oxidative stress. The ESR spectrum of propofol incubated in the presence of EDTA-Fe2+ and H2O2 as initiators of radical production showed a radical that was most likely a decomposition product of the primary phenoxy radical of propofol. It is concluded that (a) propofol acts as a chain reaction-breaking antioxidant by forming a stable radical and (b) propofol does not seem to be metabolized in mitochondria in vitro.

The contractile apparatus as a target for drugs against heart failure: interaction of levosimendan, a calcium sensitiser, with cardiac troponin c.

Cardiac failure is one of the leading causes of mortality in developed countries. As life expectancies of the populations of these countries grow, the number of patients suffering from cardiac insufficiency also increases. Effective treatments are being sought and recently a new class of drugs, the calcium sensitisers, was developed. These drugs cause a positive inotropic effect on cardio-myocytes by interacting directly with the contractile apparatus. Their mechanism of action is not accompanied by an increase in intracellular calcium concentration at therapeutic doses, as seen for the older generation of positive inotropic drugs, and thus does not induce calcium-related deleterious effects such as arrhythmias or apoptosis.Levosimendan is a novel calcium sensitiser which has been discovered by using cardiac troponin C (cTnC) as target protein. This drug has been proved to be a well-tolerated and effective treatment for patients with severe decompensated heart failure.This review describes the basic principles of muscle contraction, the main components of the contractile apparatus and their roles in the heart contraction. The regulatory proteins troponin C (cTnC), troponin I (cTnI), troponin T (cTnT), and tropomyosin (Tm) and their interactions are discussed in details. The concept of calcium sensitisation is thereafter explained and a few examples of calcium sensitisers and their putative mechanisms are discussed. Finally, the binding of levosimendan to cTnC and its mechanism of action are described and the results discussed under the light of the action of this drug in vitro and in vivo (Mol Cell Biochem 266: 87–107, 2004)

A role for the RISK pathway and KATP channels in pre‐ and post‐conditioning induced by levosimendan in the isolated guinea pig heart

Background and purpose: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre‐ and/or post‐conditioned using levosimendan (levosimendan pre‐conditioning (LPC) and levosimendan post‐conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial KATP channels are involved.

The cardioprotective effects of levosimendan : Preclinical and clinical evidence

Levosimendan, a drug used in the treatment of acute and decompensated heart failure, has positive inotropic and antistunning effects mediated by calcium sensitization of contractile proteins, and vasodilatory and antiischemic effects mediated via the opening of ATP-sensitive potassium channels in vascular smooth-muscle cells. Recently, it also has been shown to act on mitochondrial ATP-sensitive potassium (mitoKATP) channels, an action thought to protect the heart against ischemia-reperfusion damage. This finding has suggested a possible application for levosimendan in clinical situations in which preconditioning would be beneficial (eg, in pre- and perioperative settings in cardiac surgery). The demonstration that levosimendan can prevent or limit myocyte apoptosis via the activation of mitoKATP channels provides a potential mechanism whereby this agent might protect cardiac myocytes during episodes of acute heart failure. This finding may explain why short-term treatment with levosimendan may improve longer-term survival. The present article reviews the literature on the cardioprotective actions of levosimendan, with particular emphasis on its recently recognized effects on mitoKATP channels and the putative preconditioning effects of that action. A therapeutic approach to acute heart failure that includes a cardioprotective strategy could have a clinically meaningful benefit on disease progression beyond alleviation of symptoms.

Effects of Levosimendan and milrinone on oxygen consumption in isolated guinea-pig heart

Abstract: Levosimendan is a novel calcium sensitizer that increases contraction force without change in intracellular calcium ([Ca2+]i); milrinone is a phosphodiesterase inhibitor that exerts a positive inotropic effect by increasing [Ca2+]i. The effects of levosimendan and milrinone on oxygen consumption in the isolated guinea-pig heart were studied. Isolated guinea-pig hearts were paced (280 beats/min) and perfused according to the Langendorff technique. Levosimendan (0.01–1 μM) or milrinone (0.1–10 μM) were added cumulatively and changes from baseline for diastolic and systolic pressure (LVEDP and LVSP), contractility and relaxation (+dP/dt and −dP/dt), and coronary flow and oxygen consumption (CF and VO2) were calculated. Levosimendan was found to be 10 to 30 times more potent than milrinone as an inotropic agent. The effect on VO2 was markedly lower in levosimendan-perfused hearts than in milrinone-perfused hearts (P = 0.031 between the concentration-dependent effects of the two drugs). The maximum increase in VO2 was 10 ± 4% in the levosimendan group and 38 ± 15% in the milrinone group. The economy of the contraction was more advantageous in levosimendan-perfused hearts (P ≤0.005 vs. milrinone group on both VO2/+dP/dt and VO2/LVSP). It was concluded that levosimendan exerts a positive inotropic effect without disturbing the energy balance of the heart.

Effects of levosimendan on cardiac remodeling and cardiomyocyte apoptosis in hypertensive Dahl/Rapp rats

Progression of heart failure in hypertensive Dahl rats is associated with cardiac remodeling and increased cardiomyocyte apoptosis. This study was conducted to study whether treatment with a novel inotropic vasodilator compound, levosimendan, could prevent hypertension‐induced cardiac remodeling and cardiomyocyte apoptosis.