Troponin C Research Articles

Free-Energy Linkage between Folding and Calcium Binding in EF-Hand Proteins

Troponin is the singular Ca 2+-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca 2+-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1–90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca 2+ was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (Δ V and Δ G atm). In this study, we propose that part of the affinity for Ca 2+ is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca 2+ binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca 2+ in EF-hand proteins, which is crucial to function.

The Cardiac Ca 2+-Sensitive Regulatory Switch, a System in Dynamic Equilibrium

The Ca 2+-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca 2+, and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca 2+-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca 2+ or TnI) and deactivation after rapid chelation of Ca 2+. Time-resolved measurements, stopped-flow measurements, and Ca 2+-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca 2+-induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca 2+ is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca 2+—when regulatory Ca 2+ is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15°C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (Δ H = −33.4 kJ/mol), entropy change (Δ S = −0.110 kJ/mol/K), heat capacity change (Δ C p = −7.6 kJ/mol/K), the enthalpy of activation ( δ ‡ = 10.6 kJ/mol) and the effective barrier crossing attempt frequency ( ν adj = 1.8 × 10 4 s −1).

A Novel Mutant Cardiac Troponin C Disrupts Molecular Motions Critical for Calcium Binding Affinity and Cardiomyocyte Contractility

Troponin C (TnC) belongs to the superfamily of EF-hand (helix–loop–helix) Ca 2+-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca 2+-binding Site II of TnC, TnC (E59D,D75Y). Expression of recombinant TnC (E59D,D75Y) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC D75Y was able to recapitulate the TnC (E59D,D75Y) phenotype, whereas TnC E59D was functionally benign. Force-pCa relationships in TnC (E59D,D75Y) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC (E59D,D75Y) labeled with 2-[(4′-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca 2+ sensitivity and Ca 2+ binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca 2+-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca 2+-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.

Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers

In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed. Dissociation and equilibrium binding of apo-TnC (i.e., lacking divalent cations) under different conditions were monitored by a standard test for maximum tension (P (o)). After 10 min in low-Mg2+ relaxing solution, rTnC dissociation (i.e., tension loss) was 80% vs only 45% in rigor. In rigor, adding myosin subfragment 1 (S1) reduced dissociation approximately twofold, whereas stretching to reduce filament overlap increased dissociation to nearly the value for relaxed fibers. Dissociation of rTnC after addition of Pi or MgADP to form A.M.Pi or A.M.ADP cross-bridges was significantly greater than with rigor (A.M) bridges. The increase in P (o) during equilibration with different concentrations of rTnC showed that the affinity for rTnC binding to the thin filament increased progressively with stronger cross-bridges: rTnC concentrations for half-maximal reconstitution (K (0.5)) were 8.1, 3.7, 2.9, and 1.1 microM for A + M.ADP.Pi, A.M.Pi, A.M, and A.M + S1. Cross-bridges containing MgADP(-) (A.M.ADP) were also less effective than rigor bridges in promoting rTnC binding. We conclude that cross-bridge state and number both modulate TnC affinity for the thin filament and that the TnC C-domain is a central element in this pathway.

Volume and Free Energy of Folding for Troponin C C-Domain: Linkage to Ion Binding and N-Domain Interaction

Troponin C (TnC) is an 18-kDa acidic protein of the EF-hand family that serves as the trigger for muscle contraction. In this study, we investigated the thermodynamic stability of the C-domain of TnC in all its occupancy states (apo, Mg (2+)-, and Ca (2+)-bound states) using a fluorescent mutant with Phe 105 replaced by Trp (F105W/C-domain, residues 88-162) and (1)H NMR spectroscopy. High hydrostatic pressure was employed as a perturbing agent, in combination with urea or without it. On the basis of changes in Trp emission, the C-domain apo state was denatured by pressure (in the range of 1-1000 bar) in the absence of urea. The fluorescence data were corroborated by following the changes in the (1)H NMR signal of Histidine 128. Addition of Ca (2+) or Mg (2+) increased the C-domain stability so that complete denaturation was attained only by the combined use of high hydrostatic pressure and either 7-8 M or 1.5-2 M urea, respectively. The (1)H NMR spectra in the presence of Ca (2+) was typical of a highly structured protein and allowed us to follow the changes in the local environment of several amino-acid residues as a function of pressure at 4 M Urea. Different residues presented different volume changes, but those that are in the hydrophobic core portrayed values very similar to that obtained for tryptophan 105 as measured by fluorescence, indicating that it is indeed a good probe for the overall tertiary structure. From these experiments, we calculated the thermodynamic parameters (Delta G degrees atm and Delta V) that govern the folding of the C-domain in all its possible physiological states and constructed a thermodynamic cycle. Furthermore, a comparison of the volume and free-energy changes of folding of isolated C-domain with those of intact TnC (F105W) revealed that the N-domain has little effect on the structure of the C-domain, even in the presence of Ca (2+). The volume and free-energy diagrams reveal a landscape of different conformations from the less structured, denatured apo form to the highly structured, Ca (2+)-bound form. The large change in folding free energy of the C-domain that takes place when Ca (2+) binds may explain the much higher Ca (2+) affinity of sites III and IV, 2 orders of magnitude higher than the affinity of sites I and II.

Calcium induces electron transfer in NADPH oxidase 5

Superoxide generated by non‐phagocyte NADPH oxidases (NOXs), such as NOX5 and DUOX, is of growing importance for vascular physiology and pathology. NOX5 and DUOX enzymes consist of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. They appear to be regulated by self‐contained Ca2+ binding domains (EFD). The basis for their catalytic profiles and regulation is unclear. Here, we demonstrated that Ca2+ gates heme reduction NOX5 in anaerobic conditions. To better understand this interaction, we monitored its Ca2+ and terbium (Tb3+) bindings by fluorescent spectroscopy using wild type and mutants of the recombinant EFD. The titration of EFD yielded a [Ca2+]0.5 of 4.5 μM, which is similar to published results, but the Ca2+ bindings appears to be tighter and occur in a cooperative manner. About 10 μM Ca2+ is required to fully saturate EFD and completely activate NOX5 superoxide production. EFD binds terbium with a [Tb3+]0.5 of 20 μM. The site‐directed labeling on Cys107 of EFD with IAEDNS had the same response to the Ca2+ level as the non‐labeled EFD. It reveals an anisotropy value of 0.11 and will be used to gather the structural information of the EFD‐flavoprotein complex using FRET. NOX5 contains a consensus peptide sequence that interacts with Ca2+‐bound calmodulin (CaM), and it has been demonstrated that this CaM/Ca2+ binding increases its sensitivity for free Ca2+. We used Troponin C (TnC)‐CaM chimeras to pinpoint the structural‐functional relationship to understand how individual EF‐hands motifs in CaM affect the electron transfer in NOX5. We are currently performing similar studies in DUOX. This work was supported by Beginner‐Grant‐in‐Aid from the American Heart Association

Infrared spectroscopic study of the binding of divalent cations to Akazara scallop troponin C: The effect of the methylene side chain of glutamate residue

Akazara scallop striated adductor muscle troponin C (TnC) binds only one Ca2+ because the three EF-hand motifs are short of critical residues for the coordination of Ca2+. Fourier-transform infrared spectroscopy was applied to study coordination structures of M2+ (= Mg2+, Ca2+, Sr2+, and Ba2+) bound in an Akazara scallop TnC mutant (E142D) and the wild-type TnC C-lobe in D2O solution. The region of the COO- antisymmetric stretch provides information regarding the coordination modes of a COO- group to a metal ion. The side chain COO- group of Asp142 did not bind to Ca2+ in the bidentate coordination mode, suggesting that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC (Nara et al., Vib Spect 2006, 42, 188-191). The present study has shown that the absence of a methylene group is not compensated for by a larger metal ion such as Sr2+ or Ba2+. CD spectra showed that the secondary structures are conserved between M2+-free (apo), Mg2+-loaded, Ca2+-loaded, Sr2+-loaded, and Ba2+-loaded states, which was consistent with the results estimated from their amide I band patterns. The metal-ligand interaction at position 12 of site IV is discussed in comparison with the coordination mode of the side chain COO- group of the wild-type TnC C-lobe.

Molecular characterization and immunohistochemical localization of a novel troponin C during silkworm development

We have cloned and sequenced a novel Bombyx mori gene that encodes a protein having a high degree of homology with other known troponin C (TnC) proteins. The amino acid sequence, DX[DN]X[DSG]X(6)E, a highly conserved putative Ca(2+) -binding motif found in loops within the globular domains of previously identified TnC proteins, is also present in BmTnC. We have expressed and purified to homogeneity a His-tagged BmTnC fusion protein having a molecular weight of approximately 21.6 kDa. We have used this purified fusion protein to produce polyclonal antibodies against BmTnC for Western blot analyses. These analyses have revealed that BmTnC is expressed in the larval head, the Malpighian tubule, the epidermis, the testis, and the gut, as has been confirmed by immunohistochemistry. In addition, real-time reverse transcription/polymerase chain reaction has shown that BmTnC mRNA levels differ substantially among these tissues. Our findings indicate that BmTnC is selectively expressed in the muscular tissues of the silkworm, including portions of the head, the Malpighian tubule, the body wall, and the gut.

Ca2+ exchange with troponin C and cardiac muscle dynamics

Controversy abounds in the cardiac muscle literature over the rate-limiting steps of cardiac muscle contraction and relaxation. However, the idea of a single biochemical mechanism being the all-inclusive rate-limiting step for cardiac muscle contraction and relaxation may be oversimplified. There is ample evidence that Ca(2+) concentration and dynamics, intrinsic cross-bridge properties, and even troponin C (TnC) Ca(2+) binding and dissociation can all modulate the mechanical events of cardiac muscle contraction and relaxation. However, TnC has generally been thought to play no role in influencing cardiac muscle dynamics due to the idea that Ca(2+) exchange with TnC is very rapid. This definitely is the case for isolated TnC, but not for the more sophisticated biochemical systems of reconstituted thin filaments and myofibrils. This review will discuss the biochemical influences on Ca(2+) exchange with TnC and their physiological implications.

Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca2+ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca2+-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca2+-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca2+-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca2+-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca2+-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca2+ signal to TnI in Tn ternary complex.

Differential Proteome Analysis of Hagfish Dental and Somatic Skeletal Muscles

Hagfish, the plesiomorphic sister group of all vertebrates, are deep-sea scavengers. The large musculus (m.) longitudinalis linguae (dental muscle) is a specialized element of the feeding apparatus that facilitates the efficient ingestion of food. In this article, we compare the protein expression in hagfish dental and somatic (the m. parietalis) skeletal muscles via two-dimensional gel electrophoresis and mass spectrometry in order to characterize the former muscle. Of the 500 proteins screened, 24 were identified with significant differential expression between these muscles. The proteins that were overexpressed in the dental muscle compared to the somatic muscle were troponin C (TnC), glycogen phosphorylase, beta-enolase, fructose-bisphosphate aldolase A (aldolase A), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In contrast, myosin light chain 1 (MLC 1) and creatine kinase (CK) were over-expressed in the somatic muscle relative to the dental muscle. These results suggest that these two muscles have different energy sources and contractile properties and provide an initial representative map for comparative studies of muscle-protein expression in low craniates.

Calcium, troponin, calmodulin, S100 proteins: From myocardial basics to new therapeutic strategies

Ca2+ acts as global second messenger involved in the regulation of all aspects of cell function. A multitude of Ca2+-sensor proteins containing the specific Ca2+ binding motif (helix-loop-helix, called EF-hand) developed early in evolution. Calmodulin (CAM) as the prototypical Ca2+-sensor with four EF-hands and its family members troponin-C (TNC), myosin light chains, and parvalbumin originated by gene duplications and fusions from a CAM precursor protein in prokaryotes. Rapid and precise regulation of heart and skeletal muscle contraction is assured by integration of TNC in the contractile structure and CAM in the sarcolemmal L-type Ca2+ entry channel and in the sarcoplasmic Ca2+ release channel RYR. The S100 proteins as evolutionary latecomers occur only in the animal subphylum vertebrates. They are not involved in switching on and off key cell functions but rather operate as modulators. In the heart S100A1 modulates Ca2+ homeostasis, contractile inotropy, and energy production by interaction with the elements involved in these functions. The binding properties of different Ca2+-sensor proteins associated with specific regulatory and modulatory functions in muscle are discussed in detail. Some of these sensor proteins are critically involved in certain diseases and are now used in clinical diagnostics.

Functional and evolutionary relationships of troponin C

Striated muscle contraction is initiated when, following membrane depolarization, Ca(2+) binds to the low-affinity Ca(2+) binding sites of troponin C (TnC). The Ca(2+) activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.

The origin of passive force enhancement in skeletal muscle

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 +/- 2.9 nN/ microm(2) when stretched to an average sarcomere length of 3.4 microm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only approximately 25% of the passive force enhancement observed in intact myofibrils. Therefore, approximately 75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation.

Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

We investigated whether changing thin filament Ca(2+) sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (P(i)). We increased or decreased Ca(2+) sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnC(F27W) and F20QTnC(F27W). The rate of isometric contraction was assessed as the rate of force redevelopment (k(tr)) after a rapid release and restretch to the original length of the muscle. Both in the absence of added P(i) and in the presence of 2.5 mM added P(i) 1) Ca(2+) sensitivity of k(tr) was increased by V44QTnC(F27W) and decreased by F20QTnC(F27W) compared with control TnC(F27W); 2) k(tr) at submaximal Ca(2+) activation was significantly faster for V44QTnC(F27W) and slower for F20QTnC(F27W) compared with control TnC(F27W); 3) at maximum Ca(2+) activation, k(tr) values were similar for control TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W); and 4) k(tr) exhibited a linear dependence on force that was indistinguishable for all TnCs. In the presence of 2.5 mM P(i), k(tr) was faster at all pCa values compared with the values for no added P(i) for TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W). This study suggests that TnC Ca(2+) binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca(2+) activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca(2+) activation.

Microtiter plate monoclonal antibody epitope analysis of Ca 2+- and Mg 2+-induced conformational changes in troponin C

Spectroscopic methods such as circular dichroism and Förster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca 2+ regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca 2+-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg 2+-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca 2+–Mg 2+ exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure–function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure–function relationship studies.

The Structure of Lethocerus Troponin C: Insights into the Mechanism of Stretch Activation in Muscles

To gain a molecular description of how muscles can be activated by mechanical stretch, we have solved the structure of the calcium-loaded F1 isoform of troponin C (TnC) from Lethocerus and characterized its interactions with troponin I (TnI). We show that the presence of only one calcium cation in the fourth EF hand motif is sufficient to induce an open conformation in the C-terminal lobe of F1 TnC, in contrast with what is observed in vertebrate muscle. This lobe interacts in a calcium-independent way both with the N terminus of TnI and, with lower affinity, with a region of TnI equivalent to the switch and inhibitory peptides of vertebrate muscles. Using both synthetic peptides and recombinant proteins, we show that the N lobe of F1 TnC is not engaged in interactions with TnI, excluding a regulatory role of this domain. These findings provide insights into mechanically stimulated muscle contraction.

Crystallization and preliminary X-ray analysis of the Ca2+-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop

Troponin C (TnC) is the Ca(2+)-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca(2+) at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 A. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 A(3) Da(-1) and a solvent content of 33.0%.

Levosimendan in Cardiac Surgery: A Unique Drug for the Treatment of Perioperative Left Ventricular Dysfunction or Just Another Inodilator Searching for a Clinical Application?

Paul S. Pagel, MD, PhD The myofilament calcium (Ca ) sensitizers are a class of positive inotropic, vasodilating drugs (“inodilators”) that augment myocardial contractility by increasing the Ca sensitivity of the contractile apparatus without altering intracellular Ca concentration (1). Ca sensitizers (including levosimendan, pimobendan, sulmazole, EMD 57033, and MCI-154) have received considerable attention for the treatment of acute and chronic congestive heart failure because, unlike 1-adrenoceptor agonists or cardiac phosphodiesterase (PDE) III inhibitors that stimulate cyclic adenosine monophosphate (cAMP)mediated signaling and increase intracellular Ca concentration, these drugs do not adversely affect myocardial oxygen supply-demand relations (2), produce cardiotoxicity, or predispose to the development of arrhythmias (3). Levosimendan was developed over a decade ago, and based on a large body of accumulated experimental and clinical evidence, appears to be the most promising of these drugs. Levosimendan has already been approved for the treatment of acute exacerbation of chronic heart failure in several European countries following European Society of Cardiology guidelines (4,5). The drug is currently undergoing Phase III clinical trials in the United States (REVIVE study) to evaluate its utility for the acute or chronic management of heart failure, and has received “fast-track” status from the Food and Drug Administration. The mechanisms by which levosimendan enhances the inotropic state and produces vasodilation have been extensively studied (1). Briefly, levosimendan binds to the regulatory protein troponin C (TnC) (6) and stabilizes the Ca -bound conformation of TnC, thereby allowing unopposed interaction between actin and myosin filaments and enhancing the rate and extent of myocyte contraction (7). A unique feature of levosimendan-TnC binding is its dependence on intracellular Ca concentration that facilitates the interaction between TnC and Ca during systole, while simultaneously allowing Ca to dissociate from the protein during diastole (8). This Ca -dependence of TnC binding prevents deleterious abnormalities in relaxation that would otherwise be expected to occur (9). Preservation of lusitropic function is also facilitated by the PDE-inhibiting properties of levosimendan that occur at higher doses of the drug (10). Levosimendan-induced systemic, pulmonary, and coronary vasodilation occurs as a result of at least three distinct mechanisms. Levosimendan opens several types of potassium (K ) channels (including voltagedependent, ATP-sensitive, and Ca -activated forms) in conductance and resistance vessels, actions that reduce intracellular Ca concentration in vascular smooth muscle (11). Levosimendan induces Ca desensitization of the contractile apparatus in vascular smooth muscle that does not contain TnC independent of intracellular Ca concentration (12). PDE inhibition may also play a role in vasodilation produced by higher doses of the drug. Unlike other inotropic drugs, levosimendan may exert important antiischemic effects by virtue of its actions as a KATP channel opener. Levosimendan From the Anesthesia Service, the Clement J. Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin. Accepted for publication December 7, 2006. Address correspondence to Paul S. Pagel, MD, PhD, Clement J. Zablocki Veterans Affairs Medical Center, Anesthesia Service, 5000 W. National Ave., Milwaukee, WI 53295. Address e-mail to paul.pagel@med.va.gov. Copyright © 2007 International Anesthesia Research Society DOI: 10.1213/01.ane.0000256864.75206.6d

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

Troponin I (TnI) is a muscle-specific protein and plays an allosteric function in the Ca 2+ regulation of cardiac and skeletal muscle contraction. Expression of cloned cDNA in Escherichia coli is an essential approach to preparing human TnI and mutants for structural and functional studies. The expression level of cardiac TnI in E. coli is very low. To reduce the potential toxicity of cardiac TnI to the host cell, we constructed a bi-cistronic expression vector to co-express cardiac TnI and cardiac/slow troponin C (TnC), a natural binding partner of TnI and a protein that readily expresses in E. coli at high levels. The co-expression moderately increased the expression of cardiac TnI although a high amount of TnC protein was produced from the bi-cistronic mRNA. The use of an E. coli strain containing additional tRNAs for certain low bacterial usage eukaryotic codons improved the expression of cardiac TnI. Modifications of two 5′-regional codons that have predicted low usages in bacterial cells did not reproduce the improvement, indicating that not the 5′ but the overall codon usage restricts the translational efficiency of cardiac TnI mRNA in E. coli. However, deletion of the cardiac TnI-specific N-terminal 28 amino acids significantly improved the protein expression independent of the host cell tRNA modifications. The results suggest that the regulatory N-terminal domain of cardiac TnI is a dominant factor for the incompatibility in bacterial cells, supporting its role in modulating the overall molecular conformation.