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The cyclic interaction of myosin and actin coupled ATP hydrolysis generates the mechanical force of muscle contraction. During this process, the system passes through several steps. One of these is thought to be identical to the stable rigor complex formed by myosin and actin in the absence of ATP. This cyclic interaction is regulated by changes in tropomyosin (Tm) and troponin (Tn) located on the actin filament in response alterations in intracellular Ca concentration (). Tm contains seven quasi-equivalent regions, each of which has a pair of putative actin-binding motifs. Tn comprises three different subunits, TnC, TnI, and TnT. TnI alone inhibits actomyosin ATPase activity which is removed on adding TnC, irrespective of Ca concentration. TnT is required for full Ca-regulation of the ATPase activity of a reconstituted system (). The globular part of the Tn complex (TnC, TnI and the C-terminal region of TnT) is located on residues 150–180 of Tm (), and the elongated part, composed of the N-terminal region, covers an extensive region of the C-terminal half of Tm. The binding of Ca to TnC induces a series of conformational changes in the other components of the thin filament. This allows the effective association of myosin with actin, thus producing force. Although numerous studies have characterized the interaction between these thin filament proteins, the molecular mechanism whereby the Ca-trigger is propagated from TnC to the rest of the thin filament is still not well understood.

We have measured the intersite distance, side-chain mobility and orientation of specific site(s) of troponin (Tn) complex on the thin filaments or in muscle fibres as well as in solution by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). We have examined the Ca-induced movement of the B and C helices relative to the D helix in a human cardiac (hc)TnC monomer state and hcTnC-hcTnI binary complex. An interspin distance between G42C (B helix) and C84 (D helix) was 18.4 Å in the absence of Ca. The distance between Q58C (C helix) and C84 (D helix) was 18.3 Å. Distance changes were observed by the addition of Ca and by the formation of a complex with TnI. Both Ca and TnI are essential for the full opening ∼3 Å of the N-domain in cardiac TnC.

We have determined the distances between C35 and C84 by measuring pulsed electron-electron double resonance (PELDOR) spectroscopy. The distances were 26.0 and 27.2 Å in the monomer state and in reconstituted fibres, respectively. The addition of Ca decreased the distance to 23.2 Å in fibres but only slightly in the monomer state, indicating that Ca binding to the N-lobe of hcTnC induced a larger structural change in muscle fibres than in the monomer state.

We also succeeded in synthesizing a new bifunctional spin labels that is firmly fixed on a central E-helix (94C–101C) of skeletal(sk)TnC to examine its orientation in reconstituted muscle fibres. EPR spectrum showed that this helix is disordered with respect to the filament axis.

We have studied the calcium structural transition in skTnI and tropomyosin on the filament by SDSL-EPR. The spin label at a TnI switch segment (C133) showed three motional states depending on Ca and actin. The data suggested that the TnI switch segment binds to TnC N-lobe in +Ca state, and that in −Ca state it is free in TnC-I-T complex alone while fixed to actin in the reconstituted thin filaments. In contrast, the side chain spin labels along the entire tropomyosin molecule showed no Ca-induced mobility changes.

Tropomyosin (Tm) is a 400 Å long coiled coil protein, and with troponin it regulates contraction in skeletal and cardiac muscles in a [Ca]-dependent manner. Tm consists of multiple domains with diverse stabilities in the coiled coil form, thus providing Tm with dynamic flexibility. This flexibility must play important roles in the actin binding and the cooperative transition between the calcium regulated states of the entire muscle thin filament. In order to understand the flexibility of Tm in its entirety, the atomic coordinates of Tm are needed. Here we report the two crystal structures of Tm segments. One is rabbit skeletal muscle α-Tm encompassing residues 176–284 with an N-terminal extension of 25 residues from the leucine zipper sequence of GCN4, which includes the region that interacts with the troponin core domain. The other is α-Tm encompassing residues 176–273 with N- and C-terminal extensions of the leucine zipper sequences. These two crystal structures imply that this molecule is a flexible coiled coil. First, Tm’s are not homogeneous and smooth coiled coils, but instead they undulate, with highly fluctuating local parameters specifying the coiled coil. Independent fluctuating showed by two crystal structures is important. Second, in the first crystal, the coiled coil is bent by 9 degrees in the region centered about Y214-E218-Y221, where the inter-helical distance has its maximum. On the other hand, no bend is observed at the same region in the second crystal even if its inter-helical distance has also its maximum. E218, an unusual negatively charged residue at the position in the heptad repeat, seems to play the key role in destabilizing the coiled coil with alanine destabilizing clusters.

There are two muscle tissues in the nematode : the pharynx for feeding and the body wall for locomotion. These correspond to cardiac and skeletal muscles in vertebrates, respectively. Study of the muscle genes of can be classified into three stages; first, mutant isolation and gene mapping, second, cloning and sequencing of the gene, and third, complete sequences of all genes. Many uncoordinated mutant animals have been isolated (; ; ) and the complete amino acid sequence of myosin heavy chain, twitchin, and paramyosin, (invertebrate specific core protein of thick filament), and were the first determined in any animals by analyzing the , and mutants, respectively (; ; ). Tropomyosin and troponin components are also present but as with actin and myosin heavy chain in the worm, there are some differences in gene structure and sequence compared to those in other animals (; ; ). Deficiencies of body wall troponin C or tropomyosin in cause the Pat (aralyzed rrest at embryonic wo-fold stage) phenotype (; ) and those of troponin T cause Mup (uscle osition abnormal) phenotype (). After determining the complete genome sequences of the nematode (The ), we can find out how isoforms are related to each other. Only one troponin C gene, , is expressed in the body wall muscles and the gene defect causes a developmental arrest of the animals ().

The Ca-regulation of scallop striated muscle contraction, a Ca-regulation mechanism that is linked to myosin, was first discovered by A. G. Szent-Györgyi and his colleagues. , In myosin-linked Ca-regulation, the Ca -receptive site is the essential light chain of myosin, and the ATPase of the scallop myofibrils has been found to be desensitized to Ca by removal of the regulatory light chain (RLC) of myosin in response to treatment with a divalent cation chelator (EDTA). At the same time, three components of troponin and tropomyosin have also been isolated from scallop striated muscle, and several of their biochemical properties have been investigated.– In this troponin-linked Ca-regulation, the concurrent presence of all three components of troponin (troponins C, I, and T; TnC, TnI, and TnT) and tropomyosin are necessary for the regulation of actomyosin ATPase activity.– The action of Ca on TnC ultimately induces actomyosin ATPase activity. Troponin-linked Ca -regulation is also desensitized by the removal of TnC in response to treatment with divalent cation chelators such as EDTA or CDTA. The mutual relation of these two types of Ca-regulations in scallop myofibrils was then investigated as follows. Desensitized scallop myofibrils were prepared by removing both RLC and TnC by treatment with a divalent cation chelator, CDTA, and the effects of reconstitution with RLC and/or TnC on the ATPase activity of the desensitized myofibrils were examined.

Twitches are the unitary contractile events in both heart and skeletal muscles, but twitch plasticity in terms of force and the kinetics of force development differs considerably in the two muscle types. In skeletal muscle, twitch contractions are relatively invariant as long as temperature is constant and the muscle is well rested. In contrast, twitches in heart muscle exhibit much greater dynamic range, such that both force and the kinetics of force development can vary tremendously on a beat-to-beat basis. These differences are in part due to muscle-specific differences in the delivery of Ca to the myoplasm during excitation-contraction coupling. In skeletal muscle, a single action potential elicits a transient increase in intracellular Ca sufficient to saturate thin filament regulatory sites on troponin-C. Because of this, force development and the ability to do work depend upon the duration of the Ca transient and therefore the time available for cross-bridge binding to actin, which in skeletal muscles can be prolonged by tetanic stimulation. In heart muscle, the increase in intracellular Ca during a twitch is typically insufficient to saturate thin filament sites, so that twitch force and work production are sub-maximal. In contrast to skeletal muscle, cardiac muscle cannot be tetanized under physiological conditions, but twitch force and power can be varied by regulating the delivery of Ca to the myoplasm and also by agonist-induced regulation of cross-bridge cycling kinetics.

Over the forty years since its discovery, there has been a profound transition in thinking with regard to the role of troponin in the control of cardiac function. This transition involved a change in perception of troponin as a passive molecular switch responding to membrane controlled fluctuations in cytoplasmic Ca to a perception of troponin as a critical element in signaling cascades that actively engage in control of cardiac function. Evidence demonstrating functionally significant developmental and mutant isoform switches and post-translational modifications of cardiac troponin complex proteins, troponin I (cTnI) and troponin T (cTnT) provided convincing evidence for a more complicated role of troponin in control of cardiac function and dynamics. The physiological role of these modifications of troponin is reviewed in this monograph and has also been reviewed elsewhere (; ; ; ). Our focus here is on studies related to modifications in troponin that appear important in the processes leading from compensated hypertrophy to heart failure. These studies reveal the potentially significant role of post-translational modifications of troponin in these processes. Another focus is on troponin as a target for inotropic agents. Pharmacological manipulation of troponin by small molecules remains an important avenue of approach for the treatment of acute and chronic heart failure ().

Cardiomyopathies are a group of cardiac disorders characterized by structural and functional abnormalities of the myocardium of unexplained aetiology. By convention idiopathic cardiomyopathies are divided into 4 different diagnostic entities: and (Figure 18.1). Recent investigations have revealed that the conditions in many cases are hereditary.–

Troponin plays a central role in the Ca regulation of contraction in vertebrate skeletal and cardiac muscles. It consists of three subunits with distinct structure and function, troponin T (TnT), troponin I (TnI), and troponin C (TnC), and their accurate and complex intermolecular interaction in response to the rapid rise and fall of Ca in cardiac and skeletal myocytes plays a key role in maintaining the normal cardiac pump function and body movement. Over past decade, a great number of mutations in human genes for the troponin subunits have been shown to cause striated muscle disorders.

The enzymatic assay of creatine kinase (CK) activity in serum was established in 1959 by Okinaka’s group including Dr. Ebashi. As a biomarker of muscle cell injury, the activity assay has been a main procedure to make the precise diagnosis among most markers. It is my honor and privilege to appreciate Dr. Setsuro Ebashi and his wife, Dr. Fumiko Ebashi, for their discovery of troponin (Tn) in skeletal muscle and cardiac muscle and their contribution to the clinical research of a variety of skeletal and cardiac muscle diseases or syndromes, as well as the biological significance.