Human cardiac troponin complex. Structure and functions

Troponin complex is a component of skeletal and cardiac muscle thin filaments. It consists of three subunits — troponin I, T, and C, and it plays a crucial role in muscle activity, connecting changes in intracellular Ca²⁺ concentration with generation of contraction. In spite of more than 40 years of studies, many aspects of troponin functioning are still not completely understood, and several models describing the mechanism of muscle contraction exist. Being a key factor in the regulation of cardiac muscle contraction, troponin complex is utilized in medicine as a target for some cardiotonic drugs used in the treatment of heart failure. A number of mutations in troponin subunits are associated with development of different types of cardiomyopathy. Moreover, for the last 25 years cardiac isoforms of troponin I and T have been widely used for immunochemical diagnostics of pathologies associated with cardiomyocyte death (myocardial infarction, myocardial trauma, and others). This review summarizes the existing evidence on the structure and function of troponin complex subunits, their role in the regulation of cardiac muscle contraction, and their clinical applications.     

TnI Structural Interface with the N-Terminal Lobe of TnC as a Determinant of Cardiac Contractility

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca²⁺ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca²⁺ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca²⁺ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca²⁺ transient. Free-energy perturbation calculations of cTnC-Ca²⁺ binding indicate no difference in cTnC-Ca²⁺ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility.     

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

Reversible Ca²⁺ binding to troponin is the primary on-off switch of the contractile apparatus of striated muscles, including the heart. Dominant missense mutations in human cardiac troponin genes are among the causes of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. Structural understanding of troponin action has recently advanced considerably via electron microscopy and molecular dynamics studies of the thin filament. As a result, it is now possible to examine cardiomyopathy-inducing troponin mutations in thin-filament structural context, and from that to seek new insight into pathogenesis and into the troponin regulatory mechanism. We compiled from consortium reports a representative set of troponin mutation sites whose pathogenicity was determined using standardized clinical genetics criteria. Another set of sites, apparently tolerant of amino acid substitutions, was compiled from the gnomAD v2 database. Pathogenic substitutions occurred predominantly in the areas of troponin that contact actin or tropomyosin, including, but not limited to, two regions of newly proposed structure and long-known implication in cardiomyopathy: the C-terminal third of troponin I and a part of the troponin T N terminus. The pathogenic mutations were located in troponin regions that prevent contraction under low Ca²⁺ concentration conditions. These regions contribute to Ca²⁺-regulated steric hindrance of myosin by the combined effects of troponin and tropomyosin. Loss-of-function mutations within these parts of troponin result in loss of inhibition, consistent with the hypercontractile phenotype characteristic of HCM. Notably, pathogenic mutations are absent in our dataset from the Ca²⁺-binding, activation-producing troponin C (TnC) N-lobe, which controls contraction by a multi-faceted mechanism. Apparently benign mutations are also diminished in the TnC N-lobe, suggesting mutations are poorly tolerated in that critical domain.     

Protective role of magnesium in cardiovascular diseases: A review

A considerable number of experimental, epidemiological and clinical studies are now available which point to an important role of Mg²⁺ in the etiology of cardiovascular pathology. In human subjects, hypomagnesemia is often associated with an imbalance of electrolytes such as Na⁺, K⁺ and Ca²⁺. Abnormal dietary deficiency of Mg²⁺ as well as abnormalities in Mg²⁺ metabolism play important roles in different types of heart diseases such as ischemic heart disease, congestive heart failure, sudden cardiac death, atheroscelerosis, a number of cardiac arrhythmias and ventricular complications in diabetes mellitus. Mg²⁺ deficiency results in progressive vasoconstriction of the coronary vessels leading to a marked reduction in oxygen and nutrient delivery to the cardiac myocytes.Numerous experimental and clinical data have suggested that Mg²⁺ deficiency can induce elevation of intracellular Ca²⁺ concentrations, formation of oxygen radicals, proinflammatory agents and growth factors and changes in membrane permeability and transport processes in cardiac cells. The opposing effects of Mg²⁺ and Ca²⁺ on myocardial contractility may be due to the competition between Mg²⁺ and Ca²⁺ for the same binding sites on key myocardial contractile proteins such as troponin C, myosin and actin.Stimulants, for example, catecholamines can evoke marked Mg²⁺ efflux which appears to be associated with a concomitant increase in the force of contraction of the heart. It has been suggested that Mg²⁺ efflux may be linked to the Ca²⁺ signalling pathway. Depletion of Mg²⁺ by alcohol in cardiac cells causes an increase in intracellular Ca²⁺, leading to coronary artery vasospasm, arrhythmias, ischemic damage and cardiac failure. Hypomagnesemia is commonly associated with hypokalemia and occurs in patients with hypertension or myocardial infarction as well as in chronic alcoholism.The inability of the senescent myocardium to respond to ischemic stress could be due to several reasons. Mg²⁺ supplemented K⁺ cardioplegia modulates Ca²⁺ accumulation and is directly involved in the mechanisms leading to enhanced post ischemic functional recovery in the aged myocardium following ischemia. While many of these mechanisms remain controversial and in some cases speculative, the beneficial effects related to consequences of Mg²⁺ supplementation are apparent. Further research are needed for the incorporation of these findings toward the development of novel myocardial protective role of Mg²⁺ to reduce morbidity and mortality of patients suffering from a variety of cardiac diseases.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

The determinants of contractility in the heart

The contraction-relaxation cycle of the heart represents the combined action of a variety of different components of the myocytes. For many years an ‘index’ of contractility has been sought as a means of describing and integrating the large amount of information available from the studies of heart muscle contraction. This review will undertake to show that dF/dt, recorded from the whole heart, and dT/dt, recorded in isometric studies of isolated heart muscle preparations, should not be considered as the ‘index’ of contractility. Examples will be presented in which an increasing dT/dt is paradoxically accompanied by a lower tension, while a decreasing dT/dt can occur concomitantly with an increased contractile tension. Arguments are further presented in support of the concept that Ca²⁺, in conjunction with troponin C, is the main determinant of cardiac contractility and that dT/dt reflects a dynamic equilibrium between free and troponin-bound Ca²⁺. Peak tension is thus the net result of overlapping events competing for Ca²⁺ during the latter part of contraction, that is, during Phase II of contraction as defined below. These suggestions are based upon the following considerations: (a) The Ca²⁺ pumps are active even during rest and serve to maintain low cytosolic Ca²⁺ levels, (b) As cytosolic Ca²⁺ concentration increases, Ca²⁺ pump activity also increases, (c) In addition, the Na⁺Ca²⁺ exchange is activated by elevated Ca²⁺ concentrations and serves to decrease cytosolic Ca²⁺ levels, (d) The net result is a decline in free Ca²⁺ concentration during Phase II and a reduction in the rate of cross-bridge formation until peak tension is reached. Thus, the Ca²⁺ handling elements of the myocyte serve as a finely tuned feedback device, regulating troponin C-Ca²⁺ interactions controlling the Ca²⁺ concentration of the cytosol and as a result, the actin and myosin interaction. Factors which influence the function of these elements will change the contractility of the heart.     

Targeted disruption of the cardiac troponin T gene causes sarcomere disassembly and defects in heartbeat within the early mouse embryo

Cardiac troponin T (cTnT) is a component of the troponin (Tn) complex in cardiac myocytes, and plays a regulatory role in cardiac muscle contraction by anchoring two other Tn components, troponin I (TnI) and troponin C, to tropomyosin (Tm) on the thin filaments. In order to determine the in vivo function of cTnT, we created a null cTnT allele in the mouse TNNT2 locus. In cTnT-deficient (cTnT^−/−) cardiac myocytes, the thick and thin filaments and α-actinin-positive Z-disk-like structures were not assembled into sarcomere, causing early embryonic lethality due to a lack of heartbeats. TnI was dissociated from Tm in the thin filaments without cTnT. In spite of loss of Tn on the thin filaments, the cTnT^−/− cardiac myocytes showed regular Ca²⁺-transients. These findings indicate that cTnT plays a critical role in sarcomere assembly during myofibrillogenesis in the embryonic heart, and also indicate that the membrane excitation and intracellular Ca²⁺ handling systems develop independently of the contractile system. In contrast, heterozygous cTnT^+/− mice had a normal life span with no structural and functional abnormalities in their hearts, suggesting that haploinsufficiency could not be a potential cause of cardiomyopathies, known to be associated with a variety of mutations in the TNNT2 locus.     

Calcium signaling phenomena in heart diseases: a perspective

Ca²⁺ is a major intracellular messenger and nature has evolved multiple mechanisms to regulate free intracellular (Ca²⁺)_i level in situ. The Ca²⁺ signal inducing contraction in cardiac muscle originates from two sources. Ca²⁺ enters the cell through voltage dependent Ca²⁺ channels. This Ca²⁺ binds to and activates Ca²⁺ release channels (ryanodine receptors) of the sarcoplasmic reticulum (SR) through a Ca²⁺ induced Ca²⁺ release (CICR) process. Entry of Ca²⁺ with each contraction requires an equal amount of Ca²⁺ extrusion within a single heartbeat to maintain Ca²⁺ homeostasis and to ensure relaxation. Cardiac Ca²⁺ extrusion mechanisms are mainly contributed by Na⁺/Ca²⁺ exchanger and ATP dependent Ca²⁺ pump (Ca²⁺-ATPase). These transport systems are important determinants of (Ca²⁺)_i level and cardiac contractility. Altered intracellular Ca²⁺ handling importantly contributes to impaired contractility in heart failure. Chronic hyperactivity of the β-adrenergic signaling pathway results in PKA-hyperphosphorylation of the cardiac RyR/intracellular Ca²⁺ release channels. Numerous signaling molecules have been implicated in the development of hypertrophy and failure, including the β-adrenergic receptor, protein kinase C, Gq, and the down stream effectors such as mitogen activated protein kinases pathways, and the Ca²⁺ regulated phosphatase calcineurin. A number of signaling pathways have now been identified that may be key regulators of changes in myocardial structure and function in response to mutations in structural components of the cardiomyocytes. Myocardial structure and signal transduction are now merging into a common field of research that will lead to a more complete understanding of the molecular mechanisms that underlie heart diseases. Recent progress in molecular cardiology makes it possible to envision a new therapeutic approach to heart failure (HF), targeting key molecules involved in intracellular Ca²⁺ handling such as RyR, SERCA2a, and PLN. Controlling these molecular functions by different agents have been found to be beneficial in some experimental conditions.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

SERCA2a in Heart Failure: Role and Therapeutic Prospects

Ca²⁺ is a key molecule controlling several cellular processes, from fertilization to cell death, in all cell types. In excitable and contracting cells, such as cardiac myocytes, Ca²⁺ controls muscle contractility. The spatial and temporal segregation of Ca²⁺ concentrations are central to maintain its concentration gradients across the cells and the cellular compartments for proper function. SERCA2a is a cornerstone molecule for maintaining a balanced concentration of Ca²⁺ during the cardiac cycle, since it controls the transport of Ca²⁺ to the sarcoplasmic reticulum (SR) during relaxation. Alterations of the activity of this pump have been widely investigated, emphasizing its central role in the control of Ca²⁺ homeostasis and consequently in the pathogenesis of the contractile defect seen with heart failure. This review focuses on the molecular characteristics of the pump, its role during the cardiac cycle and the prospects derived from the manipulation of SERCA2a for heart failure treatment.     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca²⁺ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca²⁺ binding in the NH₂-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca²⁺ slowed HDX up to 2 orders of magnitude within the NH₂-lobe and the NH₂-lobe-associated TnI switch helix, implying that Ca²⁺ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca²⁺ and actin. Ca²⁺-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca²⁺ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca²⁺-mediated regulation: the coiled-coil''s NH₂-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca²⁺-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca²⁺, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca²⁺ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features.     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca²⁺ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca²⁺ binding site in the NH₂ domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D₂O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D₂O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca²⁺ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca²⁺-saturated TnC NH₂ domain. More notably, Ca²⁺ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca²⁺ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.     

Calcium binding to cardiac troponin C

The binding of Ca²⁺ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca²⁺ concentration by means of a Ca²⁺-specific electrode. Cardiac troponin C contains three Ca²⁺-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg²⁺ which competes with the Ca²⁺.     

F?rster Resonance Energy Transfer Structural Kinetic Studies of Cardiac Thin Filament Deactivation

Cardiac thin filament deactivation is initiated by Ca²⁺ dissociation from troponin C (cTnC), followed by multiple structural changes of thin filament proteins. These structural transitions are the molecular basis underlying the thin filament regulation of cardiac relaxation, but the detailed mechanism remains elusive. In this study Förster resonance energy transfer (FRET) was used to investigate the dynamics and kinetics of the Ca²⁺-induced conformational changes of the cardiac thin filaments, specifically the closing of the cTnC N-domain, the cTnC-cTnI (troponin I) interaction, and the cTnI-actin interaction. The cTnC N-domain conformational change was examined by monitoring FRET between a donor (AEDANS) attached to one cysteine residue and an acceptor (DDPM) attached the other cysteine of the mutant cTnC(L13C/N51C). The cTnC-cTnI interaction was investigated by monitoring the distance changes from residue 89 of cTnC to residues 151 and 167 of cTnI, respectively. The cTnI-actin interaction was investigated by monitoring the distance changes from residues 151 and 167 of cTnI to residue 374 of actin. FRET Ca²⁺ titrations and stopped-flow kinetic measurements show that different thin filament structural transitions have different Ca²⁺ sensitivities and Ca²⁺ dissociation-induced kinetics. The observed structural transitions involving the regulatory region and the mobile domain of cTnI occurred at fast kinetic rates, whereas the kinetics of the structural transitions involving the cTnI inhibitory region was slow. Our results suggest that the thin filament deactivation upon Ca²⁺ dissociation is a two-step process. One step involves rapid binding of the mobile domain of cTnI to actin, which is kinetically coupled with the conformational change of the N-domain of cTnC and the dissociation of the regulatory region of cTnI from cTnC. The other step involves switching the inhibitory region of cTnI from interacting with cTnC to interacting with actin. The latter processes may play a key role in regulating cross-bridge kinetics.Cardiac muscle utilizes troponin to sense the concentration changes of myoplasmic Ca²⁺ and translate the transient Ca²⁺ signal into a cascade of events within the thin filament that ultimately leads to force generation or relaxation. The cardiac thin filament is composed of the heterotrimeric troponin complex and tropomyosin bound to the double helical actin filament (1, 2). The cardiac troponin is formed by three subunits: troponin C (cTnC),² troponin I (cTnI), and troponin T (cTnT). The subunit cTnC is the Ca²⁺-binding protein, cTnI binds actin and inhibits actomyosin ATPase in relaxed muscle, and cTnT anchors the troponin complex on the actin filament. A prominent feature of cardiac muscle regulation is the Ca²⁺-dependent dynamic interactions among the thin filament proteins and the multiple structural transitions at the interface between troponin and the actin filament. These structural transitions include opening/closing of the N-domain of cTnC (3, 4), changes in conformation of both the inhibitory region, and regulatory region of cTnI (5–7), switching of the inhibitory/regulatory regions of cTnI from interacting with actin to interacting with cTnC (8), and movement of tropomyosin on the actin surface (9), which permits cross-bridge cycling between actin and myosin. These Ca²⁺-induced structural transitions are the molecular basis of cardiac thin filament regulation. The strong cross-bridge formed between myosin heads and actin modulates the interactions among thin filament proteins and further affects thin filament regulation (10–12). This feedback has been identified as an important mechanism for the beat-to-beat regulation of cardiac output. However, the mechanism by which the thin filament regulation in cardiac muscle is fine tuned at a molecular level by cross-bridges remains to be determined.It has been suggested recently that the rate of myoplasmic Ca²⁺ removal does not rate limit contraction and relaxation of the muscle (13). For example, the mechanistic studies on cardiac trabeculae (14) and myofibrils (15, 16) suggest that Ca²⁺ binding to cTnC induced switching on of the thin filament regulatory unit well before force generation. In corroboration of the conclusion, de Tombe and co-workers (17) recently reported that changes in myofilament Ca²⁺ sensitivity do not affect the kinetics of myofibrillar contraction and relaxation, i.e. the cross-bridge cycling rate is independent of the dynamics of thin filament activation. This notion is consistent with findings from a recent study where Ca²⁺-induced conformational changes of cTnC were measured simultaneously with force development of myofibril (18). It was found that kinetics of the Ca²⁺-induced conformational change of cTnC was much faster than cross-bridge kinetics. However, one study using photolysis of caged Ca²⁺ reported that the rate of Ca²⁺-induced muscle contraction (k_Ca) was slower than the rate of force redevelopment (k_tr), suggesting the importance of the thin filament in regulating cross-bridge kinetics (19). These results raise questions as to how the thin filament regulation through Ca²⁺-cTnC interaction controls muscle contraction kinetics. If the kinetics of the cross-bridge formation and detachment determine the rate of cardiac muscle contraction and relaxation, what will be the regulatory role of thin filament in heart function? The fact is that a high percentage of cardiomyopathy mutations occur among the thin filament proteins, and some of these mutations can severely hinder the kinetics of heart contraction and relaxation (20). Without a link between Ca²⁺ regulation and dynamics of cross-bridge formation and detachment, it will be difficult to interpret the mechanism underlying how these mutations affect force development and relaxation in the diseased heart.Signal transduction of Ca²⁺ activation/deactivation along the thin filament involves multiple structural transitions of the thin filament proteins (21). Each structural transition may have different dynamics that can differ from Ca²⁺ exchange with cTnC. Therefore, the dynamics of these structural transitions within the thin filament may provide insight into the dynamic linkage between the Ca²⁺ binding to cTnC and the activation state of the cardiac thin filament. Time-resolved Förster resonance energy transfer (FRET), which can quantitate the distribution of inter-probe distances (22), provides a clear metric for study of Ca²⁺-induced structural changes (on Å scale) in the thin filament. FRET involves two fluorophores (one is the FRET donor and the other is an acceptor) attached to two different sites of proteins. Because FRET provides information on the conformational changes of proteins only around a specific region of interest, it is a unique approach for monitoring specific structural changes associated with the functional activities of the thin filament. Especially when combined with fast time-resolved techniques, FRET can provide dynamic and kinetic information associated with a specific structural transition in a multiple structural transition system (23–26).Accordingly, we focused our investigation on the relaxation kinetics of (a) cTnC N-domain closing, (b) cTnC-cTnI interaction, and (c) cTnI-actin interaction within the reconstituted thin filament upon Ca²⁺ removal from the regulatory binding site of cTnC. The kinetics of these structural transitions were measured using FRET stopped-flow to monitor structural changes associated with each transition in the reconstituted thin filament in the absence and presence of strongly bound myosin subfragment 1 (S1). Our results showed that all structural transitions occurred in two phases, one fast and the other slow. The fast phase transition accounted for more than two-thirds of the total FRET change, and the slow phase transition accounted for less than one-third of the total FRET change. Our study suggests that different structural transitions have different kinetics upon Ca²⁺ removal from cTnC. Structural transitions associated with the mobile domain and the regulatory region of cTnI occur at fast kinetic rates, whereas the structural transitions involving transversal movement of the inhibitory region of cTnI occur at slow rates.     

Solution Structure of Human Cardiac Troponin C in Complex with the Green Tea Polyphenol, (?)-Epigallocatechin 3-Gallate

Heart muscle contraction is regulated by Ca²⁺ binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca²⁺ is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (−)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123–130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787–800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca²⁺-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C·troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C·troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.Cardiovascular disease (CVD)² is the number one cause of morbidity and mortality in western culture. In the United States, ∼1 in 3 deaths in 2004 were caused by CVD (1). In heart failure, the ability of the heart to distribute blood throughout the body is perturbed, and there is a growing interest to develop drugs that directly regulate the response of the myofilament to Ca²⁺. Regulation of muscle contraction is triggered by Ca²⁺ binding to troponin. The troponin complex is situated at regular intervals along the thin filament, which is made up of two elongated polymers, f-actin and tropomyosin. The backbone of the thin filament is composed of actin molecules arranged in a double helix with tropomyosin wound around actin as a coiled-coil. Anchored at every seventh actin molecule is the heterotrimeric troponin complex, which consists of troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnC is the Ca²⁺-binding subunit of troponin and has four EF-hand helix-loop-helix motifs. TnI is the inhibitory subunit of troponin. It regulates the actin-myosin cross-bridge formation by flipping between TnC and actin in a Ca²⁺-dependent manner. At low levels of cytosolic Ca²⁺, TnI is bound to actin, causing tropomyosin to sterically block the binding of the actomyosin cross-bridges. On the other hand, when Ca²⁺ concentration is high, TnI translocates from actin to TnC inducing tropomyosin to change its orientation on actin so that the actin-myosin interaction may occur. The subunit TnT fetters the troponin complex to the thin filament by way of its association with TnI (for reviews on contraction see Refs. 2–5).The large number of structural studies on troponin and the thin filament has helped gain insight into the molecular mechanism of muscle contraction. TnC is a dumbbell-shaped protein that consists of terminal domains connected by an elongated flexible linker, as shown by solution NMR (6). The overall folds of the terminal domains of skeletal TnC (sTnC) and cardiac TnC (cTnC) are very similar (7–9). The apo state of the N-domain of sTnC (sNTnC) and cTnC (cNTnC) reveals that the domain is in a “closed” conformation, such that the hydrophobic core of the protein is buried (8, 10, 11). In the skeletal system, sNTnC “opens” when two Ca²⁺ ions bind (8, 10, 11). Alternatively, cNTnC contains only one functional Ca²⁺-binding site, and its global conformation does not change as significantly as in sNTnC (11). Nonetheless, Ca²⁺ binding promotes the association of the switch region of cTnI (residues 147–163) with cNTnC. cTnI-(147–163) forms an α-helix when associated with cNTnC and has been elucidated by NMR in the solution structure of cNTnC·Ca²⁺·cTnI-(147–163) (12) and by the x-ray crystallography structure of cTnC·3Ca²⁺·cTnI·-(31–210)·cTnT-(183–288) (13). The interaction of cTnI-(147–163) with cNTnC·Ca²⁺ is essential to draw the inhibitory (cTnI-(128–147)) and C-terminal (cTnI-(163–210)) regions of cTnI away from actin. cTnI-(128–147) is not visualized in the cardiac structure, probably due to disorder (13). In the skeletal crystal structure of sTnC·4Ca²⁺·sTnI-(1–182)·sTnT-(156–262), however, the inhibitory region of sTnI is visualized and makes electrostatic contacts with the central helix connecting the N- and C-terminal lobes of cTnC (14). The C-domain (CTnC) of both sTnC and cTnC has two functional binding sites for Ca²⁺ and remains largely unstructured without Ca²⁺ bound. The folding of this domain occurs in the presence of Ca²⁺ (15, 16). Throughout the relaxation-contraction cycle, cCTnC is Ca²⁺-saturated with both Ca²⁺-binding sites occupied (cCTnC·2Ca²⁺) and is associated with the anchoring region of cTnI (cTnI-(34–71)). The crystal structure of cTnC·3Ca²⁺·cTnI·-(31–210)·cTnT-(183–288) shows cTnI-(34–71) is α-helical when bound with cCTnC·2Ca²⁺(13). The interaction of cCTnC·2Ca²⁺ with cTnI-(34–71) is the primary site in which cTnC is tethered to the thin filament.In light of the importance of the Ca²⁺-dependent cTnI-cTnC interaction in the signaling of muscle contraction, the design of drugs that modulate this interaction would be useful in the treatment of heart disease. Compounds that treat CVD through modulation of the activity of cTnC are called Ca²⁺ sensitizers or desensitizers, depending on whether they positively or negatively influence its function. These drugs are safer than other currently prescribed medicines that alter the cytosolic Ca²⁺ homeostasis (such as milrinone and dobutamine), which may cause arrhythmia or death with prolonged usage.The potential therapeutic advantage of Ca²⁺ (de)sensitizers has led to the development of a number of compounds that target cTnC. Compounds have been identified that elicit their activity through binding either cNTnC or cCTnC. Levosimendan and pimobendan are examples of molecules that increase heart muscle contractility through binding to cNTnC. Conversely, the molecule W7 decreases contractility via its interaction with cNTnC. For recent reviews on the molecular mechanism of these compounds and others see Refs. 17–19. The discovery of small molecules that bind to cCTnC to elicit their Ca²⁺-sensitizing effects suggests that cCTnC is also a suitable target for the development of therapeutics. The Ca²⁺ sensitizer, EMD 57033, is approved for the treatment for heart failure in dogs and binds to cCTnC·2Ca²⁺(20). In the NMR structure of cCTnC·2Ca²⁺·EMD 57033, EMD 57033 is associated in the hydrophobic cavity of cCTnC·2Ca²⁺ (21). The interaction of EMD 57033 with cCTnC is stereospecific for the (+)-enantiomer and explains why the (−)-enantiomer is inactive (22). Because EMD 57033 has been shown to bind cCTnC·2Ca²⁺ concurrently with cTnI-(128–147) but not with cTnI-(34–71) (23), one postulate is that EMD 57033 acts as a Ca²⁺ sensitizer by weakening the interaction of cTnI-(34–71) with cCTnC·2Ca²⁺, thus increasing the propensity of cTnI-(128–147) to bind cCTnC·2Ca²⁺ in vivo. The dilated cardiomyopathy (DCM) mutation, G159D, of cCTnC has renewed interest in the role of the C-lobe for regulation in contraction. The mutation has been identified to decrease the sensitivity of the thin filament to Ca²⁺ (24). The source of the DCM phenotype of G159D might come from the modulation of the interaction of cCTnC·2Ca²⁺ with cTnI-(34–71) (25).Green tea (Camellia sinensis) is one of the most widely consumed beverages in the world, and several epidemiological studies have linked the consumption of tea with a decrease in CVD (26, 27). (−)-Epigallocatechin gallate (EGCg) is a polyphenol that exists abundantly in unfermented teas and has been identified as a modulator of heart contraction through its interaction with cTnC (28–30). Here we use NMR spectroscopy to elucidate the three-dimensional structure of the cCTnC·2Ca²⁺·EGCg complex. The solution structure reveals that EGCg binds at the hydrophobic core of cCTnC inducing a small structural “opening.” We also use two-dimensional NMR spectroscopy to monitor the binding of EGCg to cCTnC·2Ca²⁺ and cCTnC·2Ca²⁺·cTnI-(34–71). Because EGCg and cTnI-(34–71) can bind cCTnC concurrently, the inotropic effect of EGCg may stem from its modulation of the cTnI-(34–71)-cCTnC·2Ca²⁺ interaction. The solution structure of cCTnC·2Ca²⁺·EGCg provides insight into the mechanism in which EGCg might influence heart contraction. These results taken with previous research on the Ca²⁺ sensitizer EMD 57033 and the DCM mutation G159D bring into question the dogma that cNTnC is the exclusive site for regulation of contraction in cTnC.     

An in vitro study of the interaction of heart mitochondria with troponin-bound Ca2+

Rat heart mitochondria are able to extract a large fraction of the Ca²⁺ tightly bound to rabbit skeletal muscle troponin, or to the 18.300 daltons, Ca²⁺ receptor fragment of the troponin molecule (TN-C). The amount of Ca²⁺ removed may reach 100% in the case of TN-C- but substantially less with intact troponin. The reaction is fairly rapid, often reaching completion in seconds, and is inhibited by uncouplers and by the classical inhibitor of Ca²⁺ transport in mitochondria, ruthenium red.     

E-1020, a water soluble imidazopyridine,has direct effects on Ca2+-dependent force and ATP hydrolysis of canine and bovine cardiac myofilaments

E-1020 is a cardiotonic agent that acts as a cyclic-AMP phosphodiesterase inhibitor but also may have actions which alter myofilament response to Ca²⁺. To identify direct actions of E-1020 on cardiac contractile proteins, effects of E-1020 on myofibrillar Ca²⁺ dependent MgATPase and force generation in chemically skinned fiber bundles were measured. In bovine cardiac myofibrils, E-1020 (100 M) significantly increased myofilament Ca²⁺ sensitivity and Ca²⁺-dependent ATPase activity at submaximal pCa values. At pCa 6.75, E-1020 significantly increased ATPase activity in bovine (10–100 pM) and canine (1–100 pM) cardiac myofibrils but had no effect on rat cardiac myofibrils. Moreover, in one population of canine ventricular fiber bundles, E-1020 (0.0–10 M) significantly increased isometric tension at pCa 6.5 and 6.0, whereas in another population of bundles E-1020 had no effect on tension. In no case was resting (pCa 8.0) or maximal tension (pCa 4.5) increased by E-1020. Measurements of Ca²⁺ binding to canine ventricular skinned fiber preparations demonstrated that E-1020 does not alter the affinity of myofilament troponin C for Ca²⁺. We conclude that part of the mechanism by which E-1020 acts as an inotropic agent may involve alterations in the responsiveness of contractile proteins to Ca²⁺. The lack of effect of E-1020 on some preparations may be dependent on isoform populations of myofilament proteins.     

Effects of the cardiomyopathy-causing E244D mutation of troponin T on the structures of cardiac thin filaments studied by small-angle X-ray scattering

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca²⁺. Analysis by model calculation showed that upon Ca²⁺-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca²⁺-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.     

Cooperative Cross-Bridge Activation of Thin Filaments Contributes to the Frank-Starling Mechanism in Cardiac Muscle

Myosin cross-bridges play an important role in the regulation of thin-filament activation in cardiac muscle. To test the hypothesis that sarcomere length (SL) modulation of thin-filament activation by strong-binding cross-bridges underlies the Frank-Starling mechanism, we inhibited force and strong cross-bridge binding to intermediate levels with sodium vanadate (Vi). Force and stiffness varied proportionately with [Ca²⁺] and [Vi]. Increasing [Vi] (decreased force) reduced the pCa₅₀ of force-[Ca²⁺] relations at 2.3 and 2.0 μm SL, with little effect on slope (n_H). When maximum force was inhibited to ∼40%, the effects of SL on force were diminished at lower [Ca²⁺], whereas at higher [Ca²⁺] (pCa < 5.6) the relative influence of SL on force increased. In contrast, force inhibition to ∼20% significantly reduced the sensitivity of force-[Ca²⁺] relations to changes in both SL and myofilament lattice spacing. Strong cross-bridge binding cooperatively induced changes in cardiac troponin C structure, as measured by dichroism of 5′ iodoacetamido-tetramethylrhodamine-labeled cardiac troponin C. This apparent cooperativity was reduced at shorter SL. These data emphasize that SL and/or myofilament lattice spacing modulation of the cross-bridge component of cardiac thin-filament activation contributes to the Frank-Starling mechanism.