The effects of various drugs on the myocardial inotropic response

212

249

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca 2؉ -binding properties compared with skeletal TnC and features a Ca 2؉ -binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca 2؉ -saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca 2؉ -bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca 2؉

Section: Transient Increases In Cytosolic Camentioning

confidence: 74%

Structure of Cardiac Muscle Troponin C Unexpectedly Reveals a Closed Regulatory Domain

Sia

Spyracopoulos

et al. 1997

212

249

“…In this respect, the cTnC F27W mutants behaved differently from hydrophobic core mutants of calbindin D 9K , in which variations in the Ca 2ϩ affinity were a consequence of the perturbations of the Ca 2ϩ dissociation rate (24). Furthermore, Ca 2ϩ sensitizing mutants in cTnC F27W behaved differently from analogous mutants of sTnC F29W and the intact CaM mutant with a Phe 19 3 Trp mutation, in which the increase in Ca 2ϩ affinity was largely due to a decrease in Ca 2ϩ dissociation rate (25,43). This unusual behavior of cTnC could perhaps be related to the fact that despite being a regulatory Ca 2ϩ sensor, the N-domain of cTnC undergoes only a minor conformational transition upon Ca 2ϩ binding.…”

Section: Using Acts To Estimate the Ca 2ϩ Association Rates To The N-mentioning

confidence: 94%

Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Tikunova

Davis

2004

150

As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C F27W ϳ2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6 -8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C F27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.

“…: 358 9 191 595 40; Fax: 358 9 191 595 41; E-mail: ilkka.kilpelainen@helsinki.fi. 1 The abbreviations used are: TnC, troponin C; cTnC, cardiac TnC; NTnC, N-terminal half of TnC; TnI, troponin I; cTnC C35S , cardiac troponin C with Cys-35 mutated to Ser; cTnC A-Cys , cardiac troponin C with both cysteine residues 35 and 84 mutated to Ser; cNTnC, N-terminal domain of cardiac troponin C; DTT, dithiothreitol; HSQC, heteronuclear single-quantum coherence; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; ni, number of time increments; nt, number of transients; MALDI-TOF, matrixassisted laser desorption ionization time-of-flight mass spectrometry; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. chromatography and by mass spectrometry to ensure that no degradation had occurred during the course of the sample preparation.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Sorsa

Heikkinen

Abbott

et al. 2001

133

109

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...