Levosimendan in intensive care and emergency medicine: literature update and expert recommendations for optimal efficacy and safety.

The inodilator levosimendan, in clinical use for over two decades, has been the subject of extensive clinical and experimental evaluation in various clinical settings beyond its principal indication in the management of acutely decompensated chronic heart failure. Critical care and emergency medicine applications for levosimendan have included postoperative settings, septic shock, and cardiogenic shock. As the experience in these areas continues to expand, an international task force of experts from 15 countries (Austria, Belgium, China, Croatia, Finland, France, Germany, Greece, Hungary, Italy, the Netherlands, Spain, Sweden, Switzerland, and the USA) reviewed and appraised the latest additions to the database of levosimendan use in critical care, considering all the clinical studies, meta-analyses, and guidelines published from September 2019 to November 2021. Overall, the authors of this opinion paper give levosimendan a "should be considered" recommendation in critical care and emergency medicine settings, with different levels of evidence in postoperative settings, septic shock, weaning from mechanical ventilation, weaning from veno-arterial extracorporeal membrane oxygenation, cardiogenic shock, and Takotsubo syndrome, in all cases when an inodilator is needed to restore acute severely reduced left or right ventricular ejection fraction and overall haemodynamic balance, and also in the presence of renal dysfunction/failure.

Levosimendan meta-analyses: Is there a pattern in the effect on mortality?

BACKGROUND: Levosimendan is an inodilator developed for treatment of acute heart failure and other cardiac conditions where the use of an inodilator is considered appropriate. Levosimendan has been studied in different therapeutic settings including acutely decompensated chronic heart failure, advanced heart failure, right ventricular failure, cardiogenic shock, septic shock, and cardiac and non-cardiac surgery. This variety of data has been re-analysed in 25 meta-analyses from 15 different international research groups, based on different rationales to select the studies included. METHODS: We here review all previously published meta-analyses on levosimendan to determine any common denominators for its effects on patient mortality. In addition, we also perform a comparative meta-analysis of the six phase II and III randomized double-blind trials which were taken into consideration by the regulatory authorities for the purpose of introducing levosimendan into the market. RESULTS: Irrespective of clinical setting or comparator, all meta-analyses consistently show benefits for levosimendan, with lower relative risk (or odds ratio) for patient mortality. In 3/25 of the meta-analyses these beneficial trends did not reach statistical significance, while in 22/25 significance was reached. The relative risk is consistent overall, and very similar to that obtained in our own meta-analysis that considered only the 'regulatory' studies. CONCLUSION: The existing meta-analyses, now based on a population of over 6000 patients, provide the general message of significant benefits for levosimendan in terms of patient mortality. The weight of evidence is now clearly in favour of usefulness/efficacy of levosimendan, with data from multiple randomized trials and meta-analyses.

Calcium sensitizers: What have we learned over the last 25 years?

The use of inotropes for correcting hemodynamic dysfunction in patients with congestive heart failure has been described over many decades. Drugs such as cardiac glycosides, cathecolamines, phosphodiestherase inhibitors, and calcium sensitizers have been in turn proposed. However, the number of new chemical entities in this therapeutic field has been surprisingly low, and the current selection of drugs is limited. One of the paradigm shifts in the discovery for new inotropes was to focus on 'calcium sensitizers' instead of 'calcium mobilizers'. This was designed to lead to the development of safer inotropes, devoid of the complications that arise due to increased intracellular calcium levels. However, only three such calcium sensitizers have been fully developed over the latest 30 years. Moreover, two of these, levosimendan and pimobendan, have multiple molecular targets and other pharmacologic effects in addition to inotropy, such as peripheral vasodilation. More recently, omecamtiv mecarbil was described, which is believed to have a pure inotropy action that is devoid of pleiotropic effects. When the clinical data of these three calcium sensitizers are compared, it appears that the less pure inotropes have the cutting edge over the purer inotrope, due to additional effects during the treatment of a complex syndrome such as acute congested heart failure. This review aims to answer the question whether calcium sensitization per se is a sufficient strategy for bringing required clinical benefits to patients with heart failure. This review is dedicated to the memory of Heimo Haikala, a true and passionate innovator in this challenging field.

Pharmaco-economics of levosimendan in cardiology: a European perspective.

INTRODUCTION: Heart failure places a significant economic burden on health care. Acute heart failure requires hospitalization and often frequent re-hospitalization in expensive wards where vasoactive rescue therapy is often added on top of standard medications. In these lean times, there is a growing need for cost-effective therapeutic options that supply superior support and in addition shorten the length of stay in hospital and reduce re-hospitalization rates. The inodilator levosimendan represents the latest addition to the vasoactive treatments of acute heart failure patients, and it appears to meet these expectations. Our aim was to answer the question whether the treatment efficacy of levosimendan - when selected as therapy for patients hospitalized for acute heart failure - brings savings to hospitals in various European countries representing different economies. METHODS AND RESULTS: We took a conservative approach and selected some a fortiori arguments to simplify the calculations. We selected seven European countries to represent different economies: Italy, Spain, Greece, Germany, Sweden, Finland and Israel. Data on the costs of medications and on the cost per day were collected and fed in a simple algorithm to detect savings. These saving varied from country to country, from a minimum of €0.50 in Germany to a maximum of €354.64 in Sweden. CONCLUSIONS: The use of levosimendan as a therapy for patients hospitalized for acute heart failure provides a net saving to hospitals driven by a reduction in the length of hospital stay. This finding is true in each of the countries considered in this study.

Comparison of the efficiency of Na+/Ca2+ exchanger or Na+/H+ exchanger inhibition and their combination in reducing coronary reperfusion-induced arrhythmias.

During ischaemia/reperfusion, the rise in [Na(+)](i), induced by simultaneous depression of the Na(+)/K(+)-ATPase and activation of the Na(+)/H(+) exchanger (NHE), shifts the Na(+)/Ca(2+) exchanger (NCX) into reverse transport mode, resulting in Ca(2+)(i)overload, which is a critical factor in enhancing the liability to cardiac arrhythmias. The inhibition of NHE, and recently NCX has been suggested to effectively protect the heart from reperfusion-induced arrhythmias. In this study, we investigated and compared the efficacy of individual or the simultaneous inhibition of the NHE and NCX against reperfusion-induced arrhythmias in Langendorff-perfused rat hearts by applying a commonly used regional ischaemia-reperfusion protocol. The NHE and NCX were inhibited by cariporide and SEA0400 or the novel, more selective ORM-10103, respectively. Arrhythmia diagrams calculated for the reperfusion period were analysed for the incidence and duration of extrasystoles (ESs), ventricular tachycardia (VT) and ventricular fibrillation (VF). NHE inhibition by cariporide was highly efficient in reducing the recorded reperfusion-induced arrhythmias. Following the application of SEA0400 or ORM-10103, the number and duration of arrhythmic periods were efficiently or moderately decreased. While both NCX inhibitors effectively reduced ESs, the most frequently triggered arrhythmias, they exerted limited or no effect on VTs and VFs. Of the NCX inhibitors, ORM-10103 was more effective. Surprisingly, the simultaneous inhibition of the NCX and NHE failed to significantly improve the antiarrhythmic efficacy reached by NCX blockade alone. In conclusion, although principal simultaneous NHE+NCX inhibition should be highly effective against all types of the recorded reperfusion-induced arrhythmias, NCX inhibitors, alone or in combination with cariporide, seem to be moderately suitable to provide satisfactory cardioprotection - at least in the present arrhythmia model. Since ORM-10103 and SEA0400 are known to effectively inhibit after-depolarisations, it is suggested that their efficacy and that of other NCX inhibitors may be higher and more pronounced in the predominantly Ca(2+)(i)-dependent triggered arrhythmias.

Preoperative and perioperative use of levosimendan in cardiac surgery: European expert opinion.

In cardiac surgery, postoperative low cardiac output has been shown to correlate with increased rates of organ failure and mortality. Catecholamines have been the standard therapy for many years, although they carry substantial risk for adverse cardiac and systemic effects, and have been reported to be associated with increased mortality. On the other hand, the calcium sensitiser and potassium channel opener levosimendan has been shown to improve cardiac function with no imbalance in oxygen consumption, and to have protective effects in other organs. Numerous clinical trials have indicated favourable cardiac and non-cardiac effects of preoperative and perioperative administration of levosimendan. A panel of 27 experts from 18 countries has now reviewed the literature on the use of levosimendan in on-pump and off-pump coronary artery bypass grafting and in heart valve surgery. This panel discussed the published evidence in these various settings, and agreed to vote on a set of questions related to the cardioprotective effects of levosimendan when administered preoperatively, with the purpose of reaching a consensus on which patients could benefit from the preoperative use of levosimendan and in which kind of procedures, and at which doses and timing should levosimendan be administered. Here, we present a systematic review of the literature to report on the completed and ongoing studies on levosimendan, including the newly commenced LEVO-CTS phase III study (NCT02025621), and on the consensus reached on the recommendations proposed for the use of preoperative levosimendan.

Repetitive use of levosimendan for treatment of chronic advanced heart failure: clinical evidence, practical considerations, and perspectives: an expert panel consensus.

BACKGROUND: The intravenous inodilator levosimendan was developed for the treatment of patients with acutely decompensated heart failure. In the last decade scientific and clinical interest has arisen for its repetitive or intermittent use in patients with advanced chronic, but not necessarily acutely decompensated, heart failure. Recent studies have suggested long-lasting favourable effects of levosimendan when administered repetitively, in terms of haemodynamic parameters, neurohormonal and inflammatory markers, and clinical outcomes. The existing data, however, requires further exploration to allow for definitive conclusions on the safety and clinical efficacy of repetitive use of levosimendan. METHODS AND RESULTS: A panel of 30 experts from 15 countries convened to review and discuss the existing data, and agreed on the patient groups that can be considered to potentially benefit from intermittent treatment with levosimendan. The panel gave recommendations regarding patient dosing and monitoring, derived from the available evidence and from clinical experience. CONCLUSIONS: The current data suggest that in selected patients and support out-of-hospital care, intermittent/repetitive levosimendan can be used in advanced heart failure to maintain patient stability. Further studies are needed to focus on morbidity and mortality outcomes, dosing intervals, and patient monitoring. Recommendations for the design of further clinical studies are made.

Levosimendan: current data, clinical use and future development.

Levosimendan is an inodilator indicated for the short-term treatment of acutely decompensated severe chronic heart failure, and in situations where conventional therapy is not considered adequate. The principal pharmacological effects of levosimendan are (a) increased cardiac contractility by calcium sensitisation of troponin C, (b) vasodilation, and (c) cardioprotection. These last two effects are related to the opening of sarcolemmal and mitochondrial potassium-ATP channels, respectively. Data from clinical trials indicate that levosimendan improves haemodynamics with no attendant significant increase in cardiac oxygen consumption and relieves symptoms of acute heart failure; these effects are not impaired or attenuated by the concomitant use of beta-blockers. Levosimendan also has favourable effects on neurohormone levels in heart failure patients. Levosimendan is generally well tolerated in acute heart failure patients: the most common adverse events encountered in this setting are hypotension, headache, atrial fibrillation, hypokalaemia and tachycardia. Levosimendan has also been studied in other therapeutic applications, particularly cardiac surgery - in which it has shown a range of beneficial haemodynamic and cardioprotective effects, and a favourable influence on clinical outcomes - and has been evaluated in repetitive dosing protocols in patients with advanced chronic heart failure. Levosimendan has shown preliminary positive effects in a range of conditions requiring inotropic support, including right ventricular failure, cardiogenic shock, septic shock, and Takotsubo cardiomyopathy.

ORM-10103, a novel specific inhibitor of the Na+/Ca2+ exchanger, decreases early and delayed afterdepolarizations in the canine heart.

BACKGROUND AND PURPOSE: At present there are no small molecule inhibitors that show strong selectivity for the Na(+) /Ca(2+) exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca(2+) currents and on the formation of early and delayed afterdepolarizations. EXPERIMENTAL APPROACH: Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes. KEY RESULTS: ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 µM), ORM-10103 did not significantly change the L-type Ca(2+) current or the maximum rate of depolarization (dV/dtmax ), indicative of the fast inward Na(+) current. At 10 µM ORM-10103 did not affect the amplitude or the dV/dtmax of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca(2+) current. ORM-10103 did not influence the Na(+) /K(+) pump or the main K(+) currents of canine ventricular myocytes, except the rapid delayed rectifier K(+) current, which was slightly diminished by the drug at 3 µM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 µM) in a concentration-dependent manner. CONCLUSIONS AND IMPLICATIONS: ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events.

Effects of calcium sensitizer OR-1986 on a cardiovascular mortality and myocardial remodelling in hypertensive Dahl/Rapp rats.

Calcium-sensitizing agents have been shown to improve cardiac function in patients suffering from acute decompensated heart failure, however, their long-term effects on cardiac remodeling and cardiovascular mortality are still largely unknown. In the present study we tested the hypothesis whether OR-1896, an active and long-lasting metabolite of calcium sensitizer levosimendan, prevents cardiovascular mortality and hypertension-induced myocardial remodelling in salt-sensitive Dahl/Rapp rats. OR-1896 was given orally to Dahl/Rapp SS rats on high-salt diet (NaCl 7% w/w) for 7 weeks at two different doses (0.5 and 0.05 mg/kg). OR-1896 prevented salt-induced cardiovascular mortality (survival rate 75 % in OR-1896 treated groups vs 38 % in untreated controls, p<0.01), ameliorated cardiac hypertrophy and improved systolic functions of the heart without major influence on systemic blood pressure. OR-1896 also ameliorated salt-induced increase in cardiac ANP mRNA expression and plasma BNP level. Salt-induced cardiac remodelling was associated with 4-fold increase in cardiac p16(INK4a) mRNA expression, a marker of cellular senescence. OR-1896 dose-dependently ameliorated cardiomyocyte senescence. Our findings suggest a therapeutic role for OR-1896 in the prevention of cardiac remodelling in salt-sensitive forms of hypertension. The present study also underscores the importance of cellular senescence in the pathogenesis of salt-induced hypertensive heart disease.

A role for the RISK pathway and K(ATP) 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 K(ATP) channels are involved. EXPERIMENTAL APPROACH: Isolated guinea pig hearts were treated with two 5 min cycles of levosimendan (0.1 microM) interspersed with vehicle perfusion, or two 5 min cycles of ischaemia/reperfusion, before coronary artery ligation (CAL) for 40 min at 36.5 degrees C. Hearts were treated with mitochondrial or sarcolemmal K(ATP) channel blockers before LPC or LPostC. For post-conditioning, hearts received three 30 s cycles of ischaemia/reperfusion or levosimendan/vehicle. Hearts were pretreated with levosimendan immediately before CAL (without washout). Cardiac function, infarct size and reperfusion injury salvage kinase activity was assessed. KEY RESULTS: LPC and LPostC halved the infarct size compared with controls (P<0.05). Treatment with K(ATP) channel blockers before LPC or LPostC reversed this decrease. Pretreating hearts with levosimendan increased activity of extracellular signal-regulated kinase (ERK) 42/44 on reperfusion and had the most marked infarct-lowering effect (P<0.05). CONCLUSIONS AND IMPLICATIONS: (1) Hearts could be pharmacologically pre- and post-conditioned with levosimendan; (2) levosimendan pretreatment is the most effective way to reduce infarct size, possibly by increasing ERK 42/44 activity; (3) benefits of LPC and LPostC were abolished by both K(ATP) channel blockers and (4) LPC may be useful before elective cardiac surgery, whereas LPostC may be used after acute coronary artery events.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 6-week-old salt-sensitive Dahl/Rapp rats received levosimendan (0.3 mg kg(-1) and 3 mg kg(-1) via drinking fluid) and high salt diet (NaCl 7%) for 7 weeks, Dahl/Rapp rats on low-salt diet served as controls. Blood pressure, cardiac functions by echocardiography, cardiomyocyte apoptosis by TUNEL technique, tissue morphology, myocardial expression of calcium cycling proteins, and markers of neurohumoral activation were determined. KEY RESULTS: Untreated Dahl/Rapp rats on high salt diet developed severe hypertension, cardiac hypertrophy and moderate systolic dysfunction. 38% of Dahl/Rapp rats (9/24) survived the 7-week-follow-up period. Cardiomyocyte apoptosis was increased by 6-fold during high salt diet. Levosimendan improved survival (survival rates in low- and high-dose levosimendan groups 12/12 and 9/12, p<0.001 and p=0.05, respectively), increased cardiac function, and ameliorated cardiac hypertrophy. Levosimendan dose-dependently prevented cardiomyocyte apoptosis. Levosimendan normalized salt-induced increased expression of natriuretic peptide, and decreased urinary noradrenaline excretion. Levosimendan also corrected salt-induced decreases in myocardial SERCA2a protein expression and myocardial SERCA2a/NCX-ratio. CONCLUSIONS AND IMPLICATIONS: Improved survival by the novel inotropic vasodilator levosimendan in hypertensive Dahl/Rapp rats is mediated, at least in part, by amelioration of hypertension-induced cardiac remodeling and cardiomyocyte apoptosis.

Interaction of levosimendan with cardiac troponin C in the presence of cardiac troponin I peptides.

The interaction between troponin C (TnC) and troponin I (TnI) is essential for the regulation of muscle contraction. There are several binding sites for TnI on TnC that are differentially occupied depending on the phase of the contraction/relaxation cycle. TnI and TnC interact in an antiparallel fashion with each other. The C-domain of cTnC and the N-domain region of cTnI(residues 33-70) always interact under physiological conditions, whereas the interaction between regulatory regions of TnC and TnI (residues 128-166) is calcium dependent. Previously, it has been shown that levosimendan, a calcium sensitizer used as a treatment for acute heart failure, can interact with both domains of isolated cTnC. To understand which interaction is relevant for the mechanism of calcium sensitization, we used a more complete troponin model obtained by complexing cTnI(32-79) and cTnI(128-180) with calcium-saturated cTnC(CS). The cTnI peptides bound to cTnC(CS) to form a 1:1:1 complex. The interaction of levosimendan with this complex was followed by 1H-(15)N heteronuclear correlation spectroscopy. It was clear that based on chemical shift changes, cTnI(32-79) blocked the levosimendan interaction sites on the C-domain, whereas cTnI(128-180) did not compete with levosimendan for the binding site on the N-domain. Hence, the effective binding site of levosimendan on cTnC resulting in the calcium-sensitizing effect is located in the regulatory domain (N-domain).

Levosimendan is a mitochondrial K(ATP) 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.

Improved survival with simendan after experimental myocardial infarction in rats.

This study compared the effects of simendan, a calcium sensitizer, with those of milrinone and enalapril on survival of rats with healed myocardial infarction. Seven days after ligation-induced myocardial infarction, the rats were randomized to control, milrinone, enalapril, or simendan groups. All compounds were administered via the drinking water for 312 days, at which time there was 80% mortality in the control group--the study's primary endpoint. The infarct sizes were similar across all groups. At endpoint, the mortality rates were: 63% (milrinone), 56% (enalapril) and 53% (simendan); the risk reductions were 25% (P = 0.04 vs. control) and 28% (P = 0.02 vs. control) with enalapril and simendan, respectively. Milrinone had no statistically significant effect on the survival rate. These findings suggest that, like enalapril, simendan improved survival in rats with healed myocardial infarction.

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 K(ATP) 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 K(ATP) 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 K(ATP) channels in smooth muscle are not inhibited by PKC-dependent phosphorylation. Levosimendan (0.01-1 microM) increased DCFV concentration-dependently and was noncompetitively antagonized by 0.1 microM 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 K(ATP) channels. Levosimendan and pinacidil probably have different binding sites on the K(ATP) 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.

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 Ca(2+)-saturated cTnC by heteronuclear NMR and small angle x-ray scattering. A specific interaction between levosimendan and the Ca(2+)-loaded regulatory domain of recombinant cTnC(C35S) was observed. The changes in the NMR spectra of the N-domain of full-length cTnC(C35S), 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 cTnC(A-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 cTnC(C35S) and cTnC(A-Cys). The small angle x-ray scattering experiments confirm the binding of levosimendan to Ca(2+)-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 (NaN(3)), since we found that levosimendan reacts with these chemicals, commonly used for preparation of NMR protein samples.

Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings.

Conformations of the regulatory domain of cardiac troponin C (cNTnC) were studied by means of residual dipolar couplings measured from samples dissolved in dilute liquid crystals. Changes in the main chain HN residual dipolar couplings revealed a conformational change in cNTnC due to the complexation with the second binding region (amino acids 148-163) of cardiac troponin I (cTnI). Formation of the complex is accompanied with a molecular realignment in the liquid crystal. The residual dipolar couplings measured for apo-cNTnC and the complex with TnI were in agreement with the values computed from the corresponding closed and open solution structures, whereas for the calcium-loaded conformation the correlation and quality factor were only modest. Ca2+-cNTnC may be subject to conformational exchange. The data support the model that cardiac troponin C functions as a calcium-dependent open-closed switch, such as the skeletal troponin C.

Further evidence for the cardiac troponin C mediated calcium sensitization by levosimendan: structure-response and binding analysis with analogs of levosimendan.

Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helixepsilon of the N-terminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium-induced conformational changes in native and point-mutated cTnC was studied. Cys84-->Ser, Asp87-->Lys and Asp88-->Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca(2+)titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers.

Calcium sensitivity enhancers.

Patients suffering from acute decompensated heart failure, immediately after infarction, or late in the progression of heart failure, need short-term, positive inotropic support in their therapy. To date, drugs acting through cAMP are used to increase the contractile force of the heart of such patients, although it is well-known that these kind of drugs may trigger arrhythmias and as a result may worsen the long-term prognosis of the patients. Recently, levosimendan, a new drug acting through calcium sensitization of contractile proteins, has shed new light on inotropic therapy, and, importantly, has reduced mortality in acute heart failure patients. For this reason, this review focuses on calcium sensitizers and the mechanisms behind calcium sensitization. The compounds which have been selected for detailed consideration are limited only to positive inotropic compounds that produce calcium sensitization of contractile proteins. In addition, this review elucidates the differences between various calcium sensitizing mechanisms, and mainly focuses on the relaxation of cardiac muscle.