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

Abstract

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 ACys, 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.The number of patients suffering from heart failure is increasing along with the aging of population. Calcium sensitizers have been proposed as a treatment for congestive heart failure since they exert a positive inotropic effect without increasing the intracellular calcium concentration (1). Levosimendan, a potent calcium sensitizer that improves the force development of the muscle contraction without increasing the cytosolic Ca 2ϩ ion concentration (2), was discovered using troponin C as target protein.Troponin C (TnC) 1 is responsible for the contraction trigger in the muscle. It belongs to the family of calcium binding EF-hand proteins and consists of two domains. The N-terminal half (NTnC) is responsible for the calcium-dependent regulation of the contraction, and the C-terminal half is a structural domain always loaded with divalent cations under physiological conditions. Troponin C interacts with troponin I (TnI), and this interaction is modulated by the binding of calcium. Studies of skeletal troponin C, a homologous protein, show that a hydrophobic patch is exposed in the open conformation of the calcium-loaded regulatory domain, which is a binding site for TnI (3). This has also been proposed to be a potential binding site for calcium sensitizers (4, 5). Contrary to skeletal troponin C, the binding of Ca 2ϩ to cTnC does not induce an opening of the conformation. Consequently there is, in vitro, no exposure of a hydrophobic region (6 -9). The simultaneous binding of cardiac troponin I and Ca 2ϩ to cNTnC, however, opens the structure of the N-terminal domain (10, 11). This structural and functional difference between TnC in skeletal and cardiac muscle is still to be clarified.Levosimendan has been...

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

“…Levosimendan treatment enhances cardiac efficiency without an increase in oxygen demand associated with enhanced contractility induced by other agents, such as dobutamine, currently in clinical use (see review by Endoh [47]). Levosimendan increases myofilament Ca 2+ sensitivity by a molecular mechanism that appears to involve direct binding to the N-domain of Ca 2+ -saturated cTnC [49,63], although this was challenged by a study showing that levosimendan did not bind to cTnC or a cTnC/cTnI complex [64]. A later report has shown that the inability to observe levosimendan binding to cTnC was in part due to sample degradation and the use of a recombinant cTnC having C35 and C84 mutated to serines [63].…”

“…Levosimendan increases myofilament Ca 2+ sensitivity by a molecular mechanism that appears to involve direct binding to the N-domain of Ca 2+ -saturated cTnC [49,63], although this was challenged by a study showing that levosimendan did not bind to cTnC or a cTnC/cTnI complex [64]. A later report has shown that the inability to observe levosimendan binding to cTnC was in part due to sample degradation and the use of a recombinant cTnC having C35 and C84 mutated to serines [63]. It appears that C84 is necessary for the interaction of levosimendan and cTnC.…”

Targeting the sarcomere to correct muscle function

“…Levosimendan can bind directly to troponin C 42,43 , but subsequent studies showed that the binding of levosimendan and its active metabolite, OR-1896, to troponin C is very weak 44 , with a K d of ~1 mM 45,46 . It was postulated that the reactive malononitrile moiety of levosimendan may form a covalent bond to Cys84 of cardiac troponin C 47 , although this reactive group is not present in OR-1896. Thus, the physiological significance of the weak levosimendan-troponin C interaction is unclear 48,49 .…”

Predictive Value of Fragmented QRS in Response to Levosimendan Therapy in Patients with Heart Failure

“…Positive inotropic drugs are added to the conventional treatment when peripheral hypoperfusion signs are present. Levosimendan does not distinctively potentiate intracellular cyclic adenosine monophosphate and calcium levels; in contrast to other inotropic drugs, levosimendan stabilizes cross-bonds between actin and myosin, leading to enhanced contractility (8)(9)(10). Levosimendan stimulates adenosine triphosphate-sensitive potassium channels on vascular smooth muscles, resulting in arterial and venous vasodilatation.…”