Calcium-sensitizing Properties Research Articles

Computer-aided drug discovery approach finds calcium sensitizer of cardiac troponin.

In the fight against heart failure, therapeutics that have the ability to increase the contractile power of the heart are urgently needed. One possible route of action to improve heart contractile power is increasing the calcium sensitivity of the thin filament. From a pharmaceutical standpoint, calcium sensitizers have the distinct advantage of not altering cardiomyocyte calcium levels and thus have lower potential for side-effects. Small chemical molecules have been shown to bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium-sensitizing properties, possibly by stabilizing cTnC in an open conformation. Building on existing structural data of a known calcium sensitizer bound to cardiac troponin, we combined computational structure-based virtual screening drug discovery methods and solution NMR titration assays to identify a novel calcium sensitizer 4-(4-(2,5-dimethylphenyl)-1-piperazinyl)-3-pyridinamine (NSC147866) which binds to cTnC and the cTnC-cTnI147-163 complex. Its presence increases the affinity of switch peptide to cTnC by approximately a factor of two. This action is comparable to that of known levosimendan analogues.

A review of the pharmacology and clinical uses of pimobendan.

To review the pharmacology, research developments, and clinical uses of pimobendan Original research articles and clinical studies from 1984 to August 2011. Pimobendan is approved for use in dogs for the treatment of congestive heart failure (CHF) secondary to chronic valvular heart disease (CVHD) and dilated cardiomyopathy (DCM). Expert-based veterinary guidelines recommend the use of pimobendan in the management of acute, hospital-based therapy for patients with CHF attributable to CVHD. The use of pimobendan, an inodilator with phosphodiesterase 3 (PDE3) inhibitory and calcium-sensitizing properties, is regarded as a component of the standard of care in the management of dogs with CHF secondary to both DCM and CVHD. Further studies are warranted to confirm the safety and efficacy of pimobendan for the off-label use of this drug in asymptomatic CVHD, pulmonary arterial hypertension, asymptomatic myocardial diseases, CHF from all other causes and in cats with CHF.

Absence of mitochondrial activation during levosimendan inotropic action in perfused paced guinea pig hearts as demonstrated by modular control analysis

Levosimendan is a calcium sensitizer developed for the treatment of heart failure. It increases contractile force by enhancing the sensitivity of myofilaments to calcium. Besides this sensitizing effect, the drug has also been reported to show some inhibitory action on phosphodiesterase 3 (PDE3). The inotropic effects of levosimendan have been studied on guinea pig paced perfused hearts by using modular control analysis (MoCA) (Diolez P, Deschodt-Arsac V, Raffard G, Simon C, Santos PD, Thiaudiere E, Arsac L, Franconi JM. Am J Physiol Regul Integr Comp Physiol 293: R13-R19, 2007.), an integrative approach of heart energetics using noninvasive (31)P NMR. The aim was to evaluate quantitatively the respective effects of this drug on energy supply and demand modules. Under our experimental conditions, 0.7 muM levosimendan induced a 45% increase in paced heart output associated with a 7% decrease in phosphocreatine and a negligible increase in oxygen consumption. Because MoCA allows in situ study of the internal regulations in intact beating heart energetics, it was applied to describe quantitatively by which routes levosimendan exerts its inotropic action. MoCA demonstrated the absence of any significant effect of the drug on the supply module, which is responsible for the lower increase in oxygen consumption, compared with epinephrine, which increases the ratio between myocardial oxygen consumption and cardiac contraction. This result evidences that, under our conditions, a possible effect of levosimendan on PDE3 activity and/or intracellular calcium remains very low on mitochondrial activity and insignificant on integrated cardiac energetics. Thus, levosimendan inotropic effect on guinea pig heart depends almost entirely on the calcium-sensitizing properties leading to myofilament activation and the concomitant activation of energy supply by the decrease in PCr, therefore improving energetic efficiency of contraction.

Calcium sensitization as new principle of inotropic therapy in end-stage heart failure?

Due to shortage of donor hearts and increasing waiting-lists of patients with end-stage heart disease, new pharmacological principles for bridging therapies are necessary. The positive inotropic effects of cAMP-increasing drugs (e.g. catecholamines, phosphodiesterase-inhibitors) are diminished in the failing myocardium. Hence, we investigated the usefulness and mechanism of the two calcium sensitizers, levosimendan and CGP 48506 in preparations from end-stage failing human hearts since the exact mechanism of the positive inotropic effects is not yet clearly understood. Failing human hearts which required orthotopic heart transplantation due to idiopathic dilated cardiomyopathy were investigated. Contraction experiments were performed using muscle strips of ventricles. Calcium sensitization was investigated in skinned fibers and phosphodiesterase activity was measured in ventricular homogenate. In addition, cAMP levels were quantified in myocytes from guinea-pig hearts. In muscle stripes from failing human hearts levosimendan (10 micromol/l) increased the force of contraction only to 112.8 +/- 6.7% of predrug values. In contrast, CGP 48506 increased the force of contraction to 311 +/- 59% of predrug values at 100 micromol/l. The time to peak tension and time of relaxation were increased to 175 +/- 4% and 205 +/- 15% of control levels at 100 micromol/l. Skinned fibers from failing human hearts were sensitized to calcium with an EC50 of 10 micromol/l. Other mechanisms of action were excluded since CGP 48506 affected neither the activity of phosphodiesterase isoenzymes I-IV in failing human hearts, nor cAMP levels in guinea-pig cardiomyocytes. On the other hand, levosimendan (1 micromol/l) increased cAMP content from 6.3 +/- 0.3 to 8.1 +/- 0.3 pmol/mg protein. CGP 48506 is an inotropic agent with calcium-sensitizing properties in the human heart, that is devoid of inhibitory activity on human cardiac phosphodiesterase isoenzymes. It offers, therefore, a new form of positive inotropic therapy that can be useful for the bridging treatment of heart failure before transplantation. On the other hand, levosimendan is a calcium sensitizer showing less-effective inotropic effects accompanied by increased cAMP levels.

Coronary vasorelaxant effect of levosimendan, a new inodilator with calcium-sensitizing properties.

We examined the action of levosimendan, a new Ca2+-sensitizing inodilator, on isolated porcine coronary arteries. Vessel rings were studied in isometric myographs. Arterial cyclic adenosine monophosphate (cAMP) levels were determined by radioimmunoassay. Levosimendan (10(-7)-10(-3) M) completely relaxed arteries preconstricted by prostaglandin F2alpha (PGF2alpha) with a pD2 (-logEC50) value of 3.99 +/- 0.05 (n = 6-9 in all experiments). Pretreatment with levosimendan also prevented contraction induced by PGF2alpha. The vasorelaxation produced by levosimendan (10(-7)-10(-3) M) was not attenuated by removal of the endothelium. Levosimendan (10(-7)-10(-3) M) relaxed contractions induced by 30 mM K+ as well as 80 mM K+, whereas the K+ channel opener levcromakalim selectively relaxed contraction induced by 30 mM K+. Neither the cyclooxygenase inhibitor indomethacin nor the beta-adrenoceptor blocker propranolol influenced levosimendan-induced vasorelaxation. The Ca2+-entry blocker isradipine failed to relax arteries precontracted by endothelin-1 in Ca2+-free/EGTA medium. However, levosimendan (10(-7)-3 x 10(-3) M) completely relaxed endothelin-1-induced contractions in this medium. Levosimendan potentiated the relaxant effect of a cAMP-stimulating drug, isoprenaline, but also that of nitroglycerin and isradipine. At a maximal effective concentration, it increased arterial tissue contents of cAMP twofold. In conclusion, levosimendan produces coronary vasorelaxation by a mechanism that seems to be endothelium independent and not mediated by K+ channel opening, Ca2+-entry blockade, release of cyclooxygenase products, or beta-adrenoceptor stimulation. Accumulation of cAMP may possibly participate in vasorelaxation at high concentrations of levosimendan, but a cAMP-independent mechanism seems to be involved at lower concentrations.

Contrasting preload-dependent hemodynamic and neurohumoral effects of isomazole, a partial phosphodiesterase inhibitor and calcium sensitizer

Background: Currently evaluated positive inotropic agents that act predominantly through phosphodiesterase III-inhibiting properties, have been disappointing in the treatment of heart failure. Lack of efficacy as a result of diminished cellular cyclic adenosine monophosphate and vasodilating tolerance and side effects are prevalent. In contrast, calcium sensitization is preserved in heart failure and agents that combine phosphodiesterase-inhibiting and calcium-sensitizing properties may be more efficacious. Isomazole is such a novel agent with combined properties. This study investigated the acute hemodynamic and neurohormonal effects of intravenous isomazole (3 μg/kg/min for 30 minutes). Methods and Results: The effects of preexisting preload were evaluated in 18 patients with heart failure, New York Heart Association class II/III, and elevated (>15 mmHg, n = 11, group I) and normal (n = 7, group II) pulmonary wedge pressure at baseline. In the overall group, isomazole increased myocardial contractility and relaxation and decreased systemic resistance by 20%. Left and right filling pressures fell by 35–45%, accompanied by a 69% reduction in cardiac atrial natriuretic peptide release. In contrast, levels of arterial norepinephrine and renin both increased by 27%. Cardiac output increased in group I (23%), but fell in group II (18%), accompanied by a 51% increase in arterial norepinephrine. Cardiac atrial natriuretic peptide decreased in group I, but not in group II. Conclusions: Isomazole induced positive inotropic and lusitropic effects and arterial vasodilation in all patients. Cardiac pump function improved only in group I, accompanied by a reduction in sympathetic activity and renin-angiotensin and aldosterone levels and a more pronounced decrease in cardiac atrial natriuretic peptide release. In contrast, in patients with normal to low preload, the further reduction in preload led to a deterioration of pump function and increased sympathetic tone.

Effects of pimobendan on exercise tolerance and quality of life in patients with heart failure.

There has been an intensive search for safe and clinically effective inotropic agents for use as adjunctive therapy in patients with advanced heart failure. Pimobendan is a benzimidazole-pyridazinone derivative with calcium-sensitizing properties that increases myocardial contractile force without increasing intracellular calcium. This review summarizes the data from five controlled, randomized prospective trials of pimobendan that demonstrate significant improvements in exercise capacity and quality of life in patients with heart failure. The clinical benefits of pimobendan found in these trials contrast with the adverse experience noted previously with milrinone and enoximone. This may be related to the different mechanism of action of pimobendan or to a study design that permitted examination of a lower dosage. These cumulative data suggest that pimobendan may have a useful adjunctive role in heart failure and that further assessment of its effects on overall mortality is needed.

A quantitative comparison of functional and anti‐ischaemic effects of the phosphodiesterase‐inhibitors, amrinone, milrinone and levosimendan in rabbit isolated hearts

1. The functional and anti-ischaemic effects of the phosphodiesterase (PDE)-inhibitors, amrinone, milrinone and levosimendan, a new agent combining PDE-inhibitory with calcium-sensitizing properties, were investigated in rabbit isolated hearts (Langendorff, constant pressure: 70 cmH2O, Tyrode solution, Ca2+ 1.8 mmol l-1, 37 degrees C). Anti-ischaemic effects were studied in electrically-driven hearts (200 beats min-1). Acute regional ischaemia was induced by ligature of a branch of the circumflex coronary artery and quantified from epicardial NADH-fluorescence photography. 2. Cumulative concentration-response curves in spontaneously beating hearts in the presence of isoprenaline (10(-10) M), showed a higher inotropic and coronary vasodilator potency for levosimendan (EC50: 7 x 10(-7) M) compared to milrinone (EC50: 7.7 x 10(-6) M) or amrinone (EC50: 2 x 10(-5) M). Although the maximal coronary dilator activity was similar for the three agents, the maximal inotropic and chronotropic effects were lower for levosimendan than for amrinone or milrinone (P < 0.05). 3. In regionally ischaemic hearts, milrinone (10(-5) M) or levosimendan (5 x 10(-6) M) similarly enhanced the left ventricular pressure (+15-20%) (P < 0.05) and the global coronary flow (+40-50%) (P < 0.05). The epicardial NADH-fluorescence area was significantly diminished by milrinone or levosimendan (-20-30%) (P < 0.05) and there was no significant difference between the anti-ischaemic effects of either agent (P > 0.05). 4. It is concluded that amrinone and milrinone possess similar functional profiles in rabbit isolated hearts and a higher inotropic and chronotropic efficacy than levosimendan. At functionally equieffective concentrations, milrinone and levosimendan show similar anti-ischaemic effects, related to an improvement of myocardial perfusion. The calcium-sensitizing properties seem not to be relevant for cardioprotection by levosimendan at the concentration used.

Functional and antiischaemic effects of the phosphodiesterase inhibitor levosimendan in isolated rabbit hearts.

Several phosphodiesterase inhibitors have been reported to possess antiischaemic properties by improving myocardial oxygen demand/supply balance. Levosimendan is a new phosphodiesterase inhibitor with calcium-sensitizing properties. We investigated the functional and antiischaemic properties of levosimendan in isolated electrically-driven rabbit hearts (Langendorff, constant perfusion pressure: 70 cm H2O, Tyrode solution, Ca++ 1.8 mmol/l, 37 degrees, 200 beats/min.). Acute regional myocardial ischaemia was induced by ligature of a circumflex artery branch and quantified from epicardial NADH-fluorescence photography. The left ventricular pressure was similarly enhanced by levosimendan 10(-7) M or 5 x 10(-6) M (+15%) (P < 0.05). The global coronary flow was increased more markedly by levosimendan 5 x 10(-6) M (+35-50%) compared to 10(-7) M (+25%) (P < 0.05). The relative coronary flow (= global coronary flow/pressure-rate-product) was significantly enhanced only by levosimendan 5 x 10(-6) M (+20-35%) (P < 0.05), but not by levosimendan 10(-7) M (+10-15%) (P > 0.05). Epicardial NADH-fluorescence area and intensity were significantly diminished by levosimendan (-20%) (P < 0.05), and there was no significant difference between myocardial ischaemia reduction by levosimendan 10(-7) M and 5 x 10(-6) M (P > 0.05). Levosimendan is an inotrope with coronary dilator activity, showing antiischaemic effects in isolated rabbit hearts. These may be caused by (1) phosphodiesterase-inhibition and improvement of myocardial perfusion and/or (2) by oxygen-sparing effects, related to calcium sensitization of myofilaments, particularly at lower concentrations associated with a lower coronary dilator activity.

Inotropic agents with calcium-sensitizing properties: clinical and hemodynamic effects of pimobendan.

Inotropic agents with calcium-sensitizing properties: clinical and hemodynamic effects of pimobendan.

Cardiotonic agents. Synthesis and cardiovascular properties of novel 2-arylbenzimidazoles and azabenzimidazoles.

Novel 2-arylbenzimidazoles and azabenzimidazoles were synthesized, and their inotropic action was evaluated. Changes in left ventricular pressure, dP/dt max, were measured as an index of cardiac contractility. The structural features that impart optimal inotropic activity are presented. The most potent compounds were evaluated orally in conscious dogs with implanted Konigsberg pressure transducers. To investigate the mechanism of action, the most potent compounds were tested for their calcium-sensitizing properties and their potential for the inhibition of phosphodiesterase. Two compounds, 1 and 41, showed interesting in vitro and oral activity without side effects. They have a more potent calcium-sensitizing effect than MCI-154 and are under further investigation.

A model for human cardiac troponin C and for modulation of its Ca2+ affinity by drugs

Calcium sensitizers are drugs which increase force development in striated muscle by sensitizing myofilaments to Ca2+. This can happen by increasing Ca2+ affinity of the regulatory domain of Ca2+ binding protein troponin C. High resolution crystal structures of two calcium binding proteins, calmodulin (Babu et al.: J. Mol. Biol. 203:191-204, 1988) and skeletal troponin C (Satyshur et al.: J. Biol. Chem. 263:1628-1647, 1988; Herzber et al.: J. Mol. Biol. 203:761-779, 1988), have recently been published. This makes it possible to model in detail the calcium-sensitizing action of drugs on troponin C. In this study a model of human cardiac troponin C in three-calcium state has been constructed. When calcium is bound to calcium site II of cardiac troponin C an open conformation of the protein results, which has a hydrophobic pocket surrounded by a few polar side chains. Complexation of three drugs, trifluoperazine, bepridil, and pimobendan, to the hydrophobic pocket is studied using energy minimization techniques. Two different binding modes are found, which differ in the location of a strong electrostatic interaction. In analogy with the crystal structure of skeletal troponin C it is hypothezed that in cardiac troponin C an interaction occurs between Gln-50 and Asp-88, which has a long-range effect on calcium binding. The binding modes of drugs, where a strong interaction with Asp-88 exists, can effectively prevent the interaction between Asp-88 and Gln-50 in the protein, and are proposed to be responsible for the calcium-sensitizing properties of the studied drugs.