Troponin-tropomyosin System Research Articles

Regulation of structure and function of sarcomeric actin filaments in striated muscle of the nematode Caenorhabditis elegans.

The nematode Caenorhabditis elegans has been used as a valuable system to study structure and function of striated muscle. The body wall muscle of C. elegans is obliquely striated muscle with highly organized sarcomeric assembly of actin, myosin, and other accessory proteins. Genetic and molecular biological studies in C. elegans have identified a number of genes encoding structural and regulatory components for the muscle contractile apparatuses, and many of them have counterparts in mammalian cardiac and skeletal muscles or striated muscles in other invertebrates. Applicability of genetics, cell biology, and biochemistry has made C. elegans an excellent system to study mechanisms of muscle contractility and assembly and maintenance of myofibrils. This review focuses on the regulatory mechanisms of structure and function of actin filaments in the C. elegans body wall muscle. Sarcomeric actin filaments in C. elegans muscle are associated with the troponin-tropomyosin system that regulates the actin-myosin interaction. Proteins that bind to the side and ends of actin filaments support ordered assembly of thin filaments. Furthermore, regulators of actin dynamics play important roles in initial assembly, growth, and maintenance of sarcomeres. The knowledge acquired in C. elegans can serve as bases to understand the basic mechanisms of muscle structure and function.

Molecular mechanisms of the regulation of ATPase cycle in striated muscle

AbstractNew data on the molecular mechanism of the regulation of ATPase cycle by troponin-tropomyosin system have been obtained in reconstructed muscle fibers by using the polarized fluorescence technique, which allowed us following the azimuthal movements of tropomyosin, actin subdomain-1 and myosin SH1 helix motor domain during the sequential steps of ATPase cycle. We found that tropomyosin strands "rolling" on thin filament surface from periphery to center at ATPase cycle increases the amplitudes of multistep changes in special arrangement of SH1 helix and subdomain-1 at force generation states. These changes seem to convey to actin monomers and to myosin "lever arm", resulting in enhance of the effectiveness of each cross-bridge work. At high-Ca^2+^ troponin, a shift of tropomyosin strands further to center at strong-binding states increases this effect. At low-Ca^2+^ troponin "freezes" tropomyosin and actin in states typical for weak-binding states, resulting in disturbing the teamwork of actin and myosin.

Exon Skipping in Cardiac Troponin T of Turkeys with Inherited Dilated Cardiomyopathy

Troponin T is a central component of the thin filament-associated troponin-tropomyosin system and plays an essential role in the Ca(2+) regulation of striated muscle contraction. The importance of the structure and function of troponin T is evident in the regulated isoform expression during development and the point mutations resulting in familial hypertrophic and dilated cardiomyopathies. We report here that turkeys with inherited dilated cardiomyopathy and heart failure express an unusual low molecular weight cardiac troponin T missing 11 amino acids due to the splice out of the normally conserved exon 8-encoded segment. The deletion of a 9-bp segment from intron 7 of the turkey cardiac troponin T gene may be responsible for the weakened splicing of the downstream exon 8 during mRNA processing. The exclusion of the exon 8-encoded segment results in conformational changes in cardiac troponin T, an altered binding affinity for troponin I and tropomyosin, and an increased calcium sensitivity of the actomyosin ATPase. Expression of the exon 8-deleted cardiac troponin T prior to the development of cardiomyopathy in turkeys indicates a novel RNA splicing disease and provides evidence for the role of troponin T structure-function variation in myocardial pathogenesis and heart failure.

Airway smooth muscle.

The greatest impetus to research in elucidating the fundamental biophysics and biochemistry of airway smooth muscle (ASM) has undoubtedly been provided by the need to understand how these are altered in asthma. Many of the biophysical and biochemical properties of this muscle have been reviewed before (Stephens, 1970; Stephens, 1977; Mulvaney, 1979; Souhrada and Loader, 1979; Stephens and Kroeger, 1980). They resemble those of striated muscle; however, even though mechanical properties are very similar, there are differences in biochemistry. For example, in smooth muscle, calcium-sensitive regulation of contraction is mediated by a calmodulin/myosin-light-chain kinase/phosphatase system, not by the familiar troponin-tropomyosin system (Gorecka et al., 1974; Mrwa and Ruegg, 1975; Dillon et al., 1981; Aksoy et al., 1982). Thus, the molecular mechanisms to be investigated in understanding disorders of increased smooth muscle contraction, which occur in allergic bronchospasm (Souhrada and Dickey, 1976), for example, may be quite different from those in striated muscle. Much of the following material is based on studies of canine tracheal smooth muscle (TSM) because there is evidence (Jenne et al., 1975) that it serves as a model for ASM-at least with respect to contractility down to the sixth generation of airways. Studies of isolated smooth muscle from smaller airways (Russell, 1978) are few and are based mainly on studies of lung strips (Lulich et al., 1976). Since then, we have developed a bronchial smooth muscle preparation (fifth generation) that allows precise study of those airways that are involved in allergic bronchospasm. Considerable work has been carried out on ASM from a variety of animal models of asthma. It should be pointed out that none of these reproduces the human disease exactly, and that they really should be identified as examples of nonspecific hyperreactivity. Be that as it may, the nonspecificity found in human patients in vivo and in animals (Peterson et al., 1971; Hargreave et al., 1980) suggests that the primary cause of asthma may reside at the muscle cell level. Whether it is the cell membrane, the excitation-contraction coupling apparatus, or the contractile machinery that is primarily involved, is not yet known with certainty.

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration–contractile response curve for external Ca 2+ was shifted to the left in a parallel manner, suggesting an increase in Ca 2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca 2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin–tropomyosin complex. Ca 2+-, K +-EDTA- or Mg 2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca 2+ sensitivity of the contractile apparatus mediated through troponin–tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.

Cardiac contractility: how calcium activates the myofilaments.

In both cardiac and skeletal muscle, the force-generating molecular motors (crossbridges) are turned on by increasing the intracellular free calcium level that regulates the troponin-tropomyosin system. However, calcium activation is a two-way process in the sense that activated crossbridges also affect the troponin-tropomyosin system. Here we review the mechanism of calcium action on myofilament proteins, particularly tropomyosin, that affects both the extent and the rate of force development and hence the contractility of the myocardium. At low myoplasmic Ca2+ concentrations tropomyosin is located at the edge of the thin filament, thereby interfering with the formation of strong actin-myosin linkages (blocked state). An increase in Ca2+ activity causes an azimuthal shift of tropomyosin around the filament (by about 30 degrees), thereby increasing the probability of low-force crossbridge interaction, a process which by cooperative effects induces further tropomyosin movement (by an additional 10 degrees) which results in the open state of the filament characterized by forceful crossbridge interaction. (This mechanism may be analogous to that in ligand-gated ion channels, where ligand binding increases the open probability of the pore.) The extent of activation then depends on the free Ca2+ concentration and on the calcium sensitivity of the thin filament that may be affected by protein phosphorylation, crossbridge attachment, the troponin isoform composition of the filament, and novel calcium-sensitizing drugs that act on the contractile or regulatory proteins and thus increase the force of the heart.

Regulation of actomyosin interactions in Limulus muscle proteins.

Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.

Airway smooth muscle.

The greatest impetus to research in elucidating the fundamental biophysics and biochemistry of airway smooth muscle (ASM) has undoubtedly been provided by the need to understand how these are altered in asthma. Many of the biophysical and biochemical properties of this muscle have been reviewed before (Stephens, 1970; Stephens, 1977; Mulvaney, 1979; Souhrada and Loader, 1979; Stephens and Kroeger, 1980). They resemble those of striated muscle; however, even though mechanical properties are very similar, there are differences in biochemistry. For example, in smooth muscle, calcium-sensitive regulation of contraction is mediated by a calmodulin/myosin-light-chain kinase/phosphatase system, not by the familiar troponin-tropomyosin system (Gorecka et al., 1974; Mrwa and Ruegg, 1975; Dillon et al., 1981; Aksoy et al., 1982). Thus, the molecular mechanisms to be investigated in understanding disorders of increased smooth muscle contraction, which occur in allergic bronchospasm (Souhrada and Dickey, 1976), for example, may be quite different from those in striated muscle. Much of the following material is based on studies of canine tracheal smooth muscle (TSM) because there is evidence (Jenne et al., 1975) that it serves as a model for ASM-at least with respect to contractility down to the sixth generation of airways. Studies of isolated smooth muscle from smaller airways (Russell, 1978) are few and are based mainly on studies of lung strips (Lulich et al., 1976). Since then, we have developed a bronchial smooth muscle preparation (fifth generation) that allows precise study of those airways that are involved in allergic bronchospasm. Considerable work has been carried out on ASM from a variety of animal models of asthma. It should be pointed out that none of these reproduces the human disease exactly, and that they really should be identified as examples of nonspecific hyperreactivity. Be that as it may, the nonspecificity found in human patients in vivo and in animals (Peterson et al., 1971; Hargreave et al., 1980) suggests that the primary cause of asthma may reside at the muscle cell level. Whether it is the cell membrane, the excitation-contraction coupling apparatus, or the contractile machinery that is primarily involved, is not yet known with certainty.

Removal of troponin C and desensitization of myosin B from ascidian smooth muscle by treatment with ethylene diamine tetraacetate.

On treatment with 10 mM EDTA at 30 degrees C, protein of 18,000 daltons was released from myofibrils, thin filaments and myosin B prepared from the smooth muscle of an ascidian, Halocynthia roretzi. This protein was purified from the EDTA extract of myofibrils by differential centrifugation, freeze-drying and gel-filtration. Based on its molecular weight, electrophoretic mobilities in the presence and absence of Ca2+ and other properties, it was identified as troponin C. By EDTA treatment, ascidian myosin B lost the Ca2+-sensitivity of Mg2+-ATPase, and EDTA-treated myosin B recovered the sensitivity by mixing with the EDTA extract of myosin B in the presence of Mg2+. Gel-electrophoretic patterns indicated that desensitization and resensitization of ascidian myosin B were accompanied by the removal and binding of troponin C. These results indicate that ascidian smooth muscle is regulated by a troponin-tropomyosin system, and desensitization induced by EDTA treatment is due to the removal of troponin C but not the release of the light chains of the myosin molecule. Based on these findings, we have established a simple method for the purification of troponin C from ascidian smooth muscle.

The Croonian lecture, 1979: Regulation of muscle contraction.

In this lecture I review briefly the history of the recognition of calcium ion as the sole regulatory factor of muscle contraction at the molecular level and how this led to the discovery of the troponin-tropomyosin system, which is the regulatory system of striated muscles of almost all deuterostomias and some protostomias. This is followed by a brief comment on the myosin-linked regulation, which plays a dominating role in many protostomian muscles. The regulatory mechanism in vertebrate smooth muscle is then discussed; the view is advanced that the leiotonin-tropomyosin system may be the only regulatory device for this muscle. Ca-binding components of troponin and smooth muscles of vertebrates are compared with modulator protein, an omnipresent Ca-binding protein of very conservative nature throughout evolution. Finally, the modes of action of Ca ion in different kinds of cell motility are discussed from an evolutionary point of view.

Cooperative effects of calcium on myofibrillar ATPase of normal and hypertrophied heart.

The ATPase of myofibrils from normal and hypertrophied rat heart was studied in relation to the Ca2+-dependence of the reaction. The experimental data were fitted to theoretical curves based on the Hill equation. A positive cooperativity (n = 1.6) was found for the activation curve of the ATPase from normal heart. For hypertrophied heart no significant change in either the cooperativity or the Ca2+-sensitivity of troponin was detected. This indicates that the troponin-tropomyosin system is not altered during pressure-overload hypertrophy. However, it should be noted that no precautions were taken to control the state of phosphorylation of contractile proteins during the preparation of myofibrils.

‘Freezing’ of Ca-regulated conformation of reconstituted thin filament of skeletal muscle by glutaraldehyde

REGULATION of skeletal muscle contraction by calcium ions is mediated by the troponin–tropomyosin system located in the groove of actin double strands1. Acting with tropomyosin, troponin depresses the ability of the actin filament to interact with the myosin molecule in the presence of ATP. Some structural changes in the thin filament associated with this depression have been detected by physical techniques2–8. When the actin filament is cross-linked with glutaraldehyde, it retains the ability to activate myosin ATPase9, and troponin and tropomyosin can no longer exert their regulatory effect on these cross-linked or ‘frozen’ actin filaments10. Using glutaraldehyde as cross-linking agent we have now been able to ‘freeze’ the activated (presence of calcium) and depressed (absence) states of the actin filament induced by the troponin–tropomyosin system: this suggests that the structural changes detected using physical methods are essential for regulation by calcium.

Elementary events in excitation-contraction coupling of the mammalian myocardium.

Excitation-contraction coupling of the mammalian myocardium is widely assumed to comprise the following chain of events: (1) Influx of Ca++ into the cell during the plateau phase of the action potential as well as Ca++ release from the subsarcolemmal cisternae of sarcoplasmic reticulum. (2) Activation of the contractile proteins by interaction of Ca++ with the troponin-tropomyosin system. (3) Relaxation due to reuptake of Ca++ by the sarcotubular system as well as by pumping of Ca++ out of the cell through the surface membrane and the transverse tubular system. (4) Movement of Ca++ in the longitudinal tubules back to the subsarcolemmal cisternae. A short comment is included about the effects of hypertrophy on the excitatory processes as well as on cellular structural constituents directly involved in excitation-contraction coupling.

Circular dichroism and difference spectra of members of the troponin-tropomyosin system in cetyltrimethylammonium bromide.

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The delta epsilonM values at their difference maxima were found to be 1300 at 292 nm for Ac-Trp-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studies by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.

Gizzard Troponin.

Native tropomyosin from the gizzard was separated into troponin and tropomyosin. The mode of action of the troponin-tropomyosin system of gizzard was shown to be distinclty different from that of skeletal muscle.

A discussion on recent developments in vertebrate smooth muscle physiology: concluding remarks.

I do not think I have been given a very easy task in being asked to sum up this session, especially as I have a feeling that it was hoped that I would be able to point to some firm conclusions about the matter most in dispute, namely whether the myosin in vertebrate smooth muscles is organized into rods or ribbons. However, I think it is apparent that conflicting points of view, which cannot be resolved on present experimental evidence, are held by different workers in the field, and that no amount of reasoning or expressions of opinion will settle the question. More evidence is needed. However, I think we should not lose sight of the fact that a very large measure of agreement does exist now on several very important basic facts about the structure of smooth muscles. First, vertebrate smooth muscle contains actin filaments virtually indistinguishable from those in striated muscles, together with a very similar troponin-tropomyosin system giving calcium sensitivity to the contractile system.

Calcium ions and muscle contraction.

The Ca ion has many functions in living organisms. In this article, the mode of action of the Ca ion in regulating the contractile processes of muscle through the troponin-tropomyosin system is briefly described.