Absence Of Troponin Research Articles

Negative Troponin I as a Predictor of Survival in Patients With Coronavirus Disease 2019.

Despite a large amount of evidence evaluating elevated troponin I levels and adverse clinical outcomes, little is known about the role of a normal (negative) troponin I during the first 24h of admission for risk stratification in patients with Coronavirus Disease 2019 (COVID-19). This study aims to evaluate the utility and negative predictive value of a serum troponin I level to predict in-hospital mortality. We retrospectively analyzed all adult patients (>18years of age) with COVID-19 admitted to an HCA Healthcare facility between March 2020 and March 2021 who had a troponin I level drawn at admission. Patients were initially stratified into two groups based on their cardiac troponin I value in the first 24h of admission (elevated vs negative). A total of 65,580 patients were included in the final analysis. A negative troponin I value was associated with lesser odds of death during admission (OR=0.32, 95% CI 0.31-0.34, p<0.01) and cardiac complications (OR=0.38, 95% CI 0.37-0.40, p<0.01). The negative predictive value of a negative troponin value for all-cause in-hospital mortality was 85.7%. Our study found a significant association between a negative troponin I value in the first 24h of admission and decreased odds of death during admission in patients with confirmed COVID-19 infection, in addition to decreased odds of cardiac complications but no significant difference in hospital length of stay. Therefore, the authors suggest that the absence of troponin I elevation may serve as an indicator of a more benign hospital course.

The ACTC M305L Hypertrophic Cardiomyopathy Mutation Results in Hypercontractility and Impaired Relaxation of Drosophila Muscles

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle characterized by ventricular wall thickening, hyperdynamic contractile properties, and impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. The alpha-cardiac actin ACTC M305L HCM mutation is located near the nucleotide-binding site and residues predicted to help confine tropomyosin to an inhibitory position along thin filaments. We generated several Drosophila models of the disorder to evaluate the effects of the lesion from the organ through molecular level. In all muscles tested, phenotypic disturbances correlated with mutant protein load. For example, relative to wildtype actin, when highly overexpressed in the fly heart, the variant significantly reduced cardiac output, prolonged systole, and restricted diastolic volumes. Preliminary force measurements from jump muscle fibers corroborated a dose-dependent, M305L actin-induced pathological effect. Moreover, we observed greater incremental decreases in flight ability with increasing amounts of mutant versus wildtype actin expression in indirect flight muscles (IFM). Flight was abolished when the mutant was highly overexpressed and destructive myosin-induced hypercontraction was evident, suggesting disrupted contractile regulation and poorly inhibited actomyosin associations. However, regulated in vitro motility parameters, including Vmax, nH, and pCa50, of thin filaments reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin-tropomyosin, were indistinguishable. Likewise, no overt differences in computationally-derived electrostatic interaction energy landscapes between M305L and wildtype actin-tropomyosin filaments were observed, suggesting unperturbed tropomyosin-positioning in the absence of troponin. Nonetheless, over sparsely populated beds of myosin, a significantly higher percentage of troponin-free M305L versus wildtype actin-tropomyosin filaments were motile, implying tropomyosin is less effective at blocking actomyosin associations on mutant filaments. Mispositioning of tropomyosin may contribute to the supranormal contraction and compromised relaxation observed in our in vivo models and, potentially, in human patients.

Abstract 12886: In-hospital and Early Post-discharge Troponin Elevations Among Patients Hospitalized for Heart Failure: Insights From the ASTRONAUT Trial

Introduction: Troponin level is commonly elevated among patients hospitalized for heart failure (HF) and may correlate with worse clinical outcomes. The prognostic significance of early post-discharge troponin level and persistent troponin elevation is unclear. Methods: The ASTRONAUT trial randomized 1,615 hospitalized HF patients with ejection fraction ≤40% to daily aliskiren or placebo, in addition to standard care. Troponin I was measured per protocol at baseline and 1-month in a core lab with elevated level defined as &gt;0.04 ng/mL. Outcomes were compared by presence/ absence of troponin I elevation at baseline and 1-month. Landmark analysis was performed to account only for events subsequent to troponin measurement. Results: Overall, 1,469 (91.0%) patients had troponin data at baseline, of which 609 (41.5%) had elevated levels (median 0.10 ng/mL). At 1 month, 1,301 (80.6%) patients had troponin data, of which 389 (29.9%) had elevated levels (median 0.10 ng/mL). Among patients with baseline elevated troponin with troponin data at 1 month, 313/518 (60.4%) had persistently elevated troponin level. After adjustment, elevated baseline troponin was not significantly associated with 1-year all-cause death or cardiovascular mortality/ HF hospitalization (Table). However, 1-month troponin elevation was independently predictive of 1-year clinical outcomes. The association between 1-month troponin elevation and outcomes was not influenced by presence/ absence of baseline troponin elevation (P for interaction ≥ 0.69). Conclusion: Troponin I was elevated in &gt;40% of hospitalized HF patients, and remained persistently elevated in &gt;60% of these patients early post-discharge. Elevated troponin I levels at 1-month follow-up, but not during hospitalization, were independently predictive of increased clinical events. Measurement of troponin I during the early post-discharge vulnerable phase should be considered as a practical means of risk stratification.

Characterization of paramyosin and thin filaments in the smooth muscle of acorn worm, a member of hemichordates.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.

Structure of the F-Actin-Tropomyosin Complex Revealed by Electron Cryomicroscopy

Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 A in complex with tropomyosin at a resolution of 6.5 A, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg2+ and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

Structure of the F-actin-tropomyosin complex.

Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7Å in complex with tropomyosin at a resolution of 6.5Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

A Periodic Pattern of Evolutionarily Conserved Basic and Acidic Residues Constitutes the Binding Interface of Actin-Tropomyosin

Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.

Troponin T is essential for sarcomere assembly in zebrafish skeletal muscle

In striated muscle, the basic contractile unit is the sarcomere, which comprises myosin-rich thick filaments intercalated with thin filaments made of actin, tropomyosin and troponin. Troponin is required to regulate Ca2+-dependent contraction, and mutant forms of troponins are associated with muscle diseases. We have disrupted several genes simultaneously in zebrafish embryos and have followed the progression of muscle degeneration in the absence of troponin. Complete loss of troponin T activity leads to loss of sarcomere structure, in part owing to the destructive nature of deregulated actin–myosin activity. When troponin T and myosin activity are simultaneously disrupted, immature sarcomeres are rescued. However, tropomyosin fails to localise to sarcomeres, and intercalating thin filaments are missing from electron microscopic cross-sections, indicating that loss of troponin T affects thin filament composition. If troponin activity is only partially disrupted, myofibrils are formed but eventually disintegrate owing to deregulated actin–myosin activity. We conclude that the troponin complex has at least two distinct activities: regulation of actin–myosin activity and, independently, a role in the proper assembly of thin filaments. Our results also indicate that sarcomere assembly can occur in the absence of normal thin filaments.

Tropomyosin: The Gatekeeper's View of the Actin Filament Revealed

Tropomyosin: The Gatekeeper's View of the Actin Filament Revealed

Atomic Model of F-Actin-Tropomyosin

The presence of tropomyosin on the surface of actin filaments governs the access and hence the interactions and activity of numerous actin-binding proteins in muscle and non-muscle thin filaments. Electron microscopy and fiber diffraction studies of native and reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position and pseudo-rotation of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin, unconstrained by troponin in striated muscle or other actin-binding proteins in smooth muscle or somatic cells, cannot be formulated, and thus the optimal interfacial contacts between actin and tropomyosin remain unknown. We have carried out a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions and pseudo-rotations of αα-tropomyosin on F-actin. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic amino acids on successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into EM reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the “broad” and “narrow” faces typifying tropomyosin's twisting superhelical coiled-coil to the twisting contours of the tropomyosin densities seen in F-actin-tropomyosin reconstructions. Our computational search indicates that, in the absence of troponin or other actin-binding proteins, tropomyosin occupies an optimal z-position and rotation in the surrounds of the closed C-state location on actin. However, no specific electrostatic minima were noted when tropomyosin was shifted computationally toward the open or blocked states on the troponin/myosin-free structure. The functional implications of the F-actin-tropomyosin model that has been determined will be discussed.

Troponin T is essential for sarcomere assembly in zebrafish skeletal muscle.

In striated muscle, the basic contractile unit is the sarcomere, which comprises myosin-rich thick filaments intercalated with thin filaments made of actin, tropomyosin and troponin. Troponin is required to regulate Ca(2+)-dependent contraction, and mutant forms of troponins are associated with muscle diseases. We have disrupted several genes simultaneously in zebrafish embryos and have followed the progression of muscle degeneration in the absence of troponin. Complete loss of troponin T activity leads to loss of sarcomere structure, in part owing to the destructive nature of deregulated actin-myosin activity. When troponin T and myosin activity are simultaneously disrupted, immature sarcomeres are rescued. However, tropomyosin fails to localise to sarcomeres, and intercalating thin filaments are missing from electron microscopic cross-sections, indicating that loss of troponin T affects thin filament composition. If troponin activity is only partially disrupted, myofibrils are formed but eventually disintegrate owing to deregulated actin-myosin activity. We conclude that the troponin complex has at least two distinct activities: regulation of actin-myosin activity and, independently, a role in the proper assembly of thin filaments. Our results also indicate that sarcomere assembly can occur in the absence of normal thin filaments.

Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

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

Heart muscle contraction is regulated by Ca(2+) binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca(2+) 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 Ca(2+)-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.

Conserved Asp-137 Imparts Flexibility to Tropomyosin and Affects Function

Tropomyosin (Tm) is an alpha-helical coiled-coil that controls muscle contraction by sterically regulating the myosin-actin interaction. Tm moves between three states on F-actin as either a uniform or a non-uniform semi-flexible rod. Tm is stabilized by hydrophobic residues in the "a" and "d" positions of the heptad repeat. The highly conserved Asp-137 is unusual in that it introduces a negative charge on each chain in a position typically occupied by hydrophobic residues. The occurrence of two charged residues in the hydrophobic region is expected to destabilize the region and impart flexibility. To determine whether this region is unstable, we have substituted hydrophobic Leu for Asp-137 and studied changes in Tm susceptibility to limited proteolysis by trypsin and changes in regulation. We found that native and Tm controls that contain Asp-137 were readily cleaved at Arg-133 with t 1/2 of 5 min. In contrast, the Leu-137 mutant was not cleaved under the same conditions. Actin stabilized Tm, causing a 10-fold reduction in the rate of cleavage at Arg-133. The actin-myosin subfragment S1 ATPase activity was greater for the Leu mutant compared with controls in the absence of troponin and in the presence of troponin and Ca2+. We conclude that the highly conserved Asp-137 destabilizes the middle of Tm, resulting in a more flexible region that is important for the cooperative activation of the thin filament by myosin. We thus have shown a link between the dynamic properties of Tm and its function.

Structural Kinetics of Cardiac Troponin C Mutants Linked to Familial Hypertrophic and Dilated Cardiomyopathy in Troponin Complexes

The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.

A BULK OF BUNDLES

Although the common notion `movement is life and stagnancy is death' is not entirely correct, we know that muscles are essential to animal life. They facilitate motility in search of sex and food, ensure the circulation of blood and air, and drive the functions of internal organs such as glands, intestines and bladders. Three different types of muscles are involved in these processes in vertebrates: smooth, cardiac and skeletal muscles. Skeletal muscles are composed of repetitive contractile units that are arranged in a highly ordered manner. Muscle fibres originate from progenitor cells, called myoblasts, which fuse during development into myotubes – muscle fibres' embryonic ancestors. Ultimately, muscle fibres are made up of sarcomeres, comprised of actin and myosin filaments, which are the muscle's ATP fuelled contractile elements. Development of such a complex structure can be described as an opera in two acts. The first act presents myoblast differentiation and fusion to form myotubes; the second act stages further differentiation steps including the assembly of the sarcomeres. But how is the complex cyto-architecture of muscle fibres achieved during development? What are the regulatory principles,and which factors are involved in sarcomere formation? Recent studies performed in insects have provided important answers to some of these questions, and a team of British scientists, led by John Sparrow, have recently revealed new insights into the molecular details of myofibril assembly and its relationship with the regulation of muscle contraction.In order to study myofibril assembly, Sparrow and his colleagues established a unique genetic system using Drosophila indirect flight muscles, which allows in vivo investigation of myogenesis. Aware of the muscle cell's developmental path, the team addressed two major issues: (1)does troponin I, which is part of a regulatory complex inhibiting muscle contraction in the absence of Ca2+, play a role in sarcomere formation, and (2) do actin–myosin interactions affect myofibril assembly?To answer these questions, the scientists analysed several Drosophila mutants with defects in the biosynthesis of sarcomeric proteins. When they investigated null mutants lacking either actin or myosin,they found that the insects had normal muscle fibre morphology, indicating that neither actin, nor myosin, is necessary for myoblast fusion or subsequent differentiation events. In other words, fibre morphogenesis does not require sarcomere assembly.By contrast, fibre morphogenesis and sarcomere formation do seem to depend on the interaction of sarcomeric proteins. When the scientists looked at a homozygote null mutant lacking functional troponin I, they found muscle degeneration with defects in sarcomere formation. Since troponin I is part of the muscle contraction regulatory complex, another question manifested itself. The team reasoned that in the absence of troponin I, unregulated muscle contractions caused by actin–myosin interactions must occur during assembly. Hence the question: are these interactions responsible for the mutant's phenotype? To answer this question, the British team crossed a heterozygote troponin I mutant with a fly strain expressing modified myosin that can assemble into filaments but cannot interact with actin filaments to produce force. Surprisingly, they found that muscle degeneration and the defects in sarcomere formation were suppressed, indicating the need for inhibition of muscle contractions by troponin I during development.Although many important questions of myogenesis are still unsolved, it is clear now that fibre morphogenesis and sarcomere formation are separate and independent processes requiring the inhibition of muscle contraction.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1-phosphate (S1P) can induce intracellular Ca(2+) mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca(2+)-independent mechanisms of cell contraction have been the focus of numerous studies on Ca(2+) sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho-dependent kinase mediate most of Ca(2+)-independent contractile responses. In the present study, we examined the potential role of Rho/Rho-kinase cascade activation in S1P-induced C2C12 cell contraction. First, we showed that depletion of Ca(2+), by pre-treatment with BAPTA, did not affect S1P-induced myoblastic contractility, whereas it abolished S1P-induced Ca(2+) transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P-stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist-stimulation in cells depleted of Ca(2+) and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y-27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKC alpha and PKC delta, respectively, failed to inhibit the agonist-induced myoblastic contraction. Single particle tracking of G-actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca(2+)-independent/Rho-Rho kinase-dependent pathways may exert an important role in S1P-induced myoblastic cell contraction.

Effects of Troponin T Mutations in Familial Hypertrophic Cardiomyopathy on Regulatory Functions of Other Troponin Subunits

We have previously shown that mutations in troponin T (TnT), which is associated with familial hypertrophic cardiomyopathy (HCM), cause an increase in the Ca(2+) sensitivity and a potentiation of cardiac muscle contraction. To gain further insight into the patho-physiological role of these mutations, four mutations (Arg92Gln, Phe110Ile, Glu244Asp, Arg278Cys) were introduced into recombinant human cardiac TnT, and the mutants were exchanged into isolated porcine cardiac myofibrils. The effects of mutations were tested on maximal ATPase activity, the inhibitory function of troponin I (TnI) in the absence of troponin C (TnC), and the neutralizing function of TnC. Arg92Gln, Phe110Ile, and Glu244Asp markedly impaired the inhibitory function of TnI. Arg278Cys also impaired the inhibitory function of TnI, but the effect was much smaller. Phe110Ile and Glu244Asp markedly enhanced the neutralizing function of TnC and potentiated the maximum ATPase activity. Arg92Gln and Arg278Cys only slightly enhanced the neutralizing function of TnC, and they conferred no potentiation on the maximum ATPase activity. These results indicate that mutations in TnT impair multiple processes of Ca(2+) regulation by troponin, and there are marked differences in the degree of impairment from mutation to mutation.

Prognostic value of cardiac troponin I re-elevation following percutaneous coronary intervention in high-risk patients with acute coronary syndromes

Troponin I is a predictive marker of short- and intermediate-term adverse cardiac events in patients with acute coronary syndromes (ACS). These high-risk patients may benefit from early percutaneous coronary intervention. However, whether additional myocardial injury, defined as postprocedural troponin I elevation, may be associated with adverse short- and intermediate-term outcomes has not been fully explored. Accordingly, we studied 132 consecutive patients with non–ST-elevation ACS (62% with non–Q-wave myocardial infarction) and elevated troponin I levels at admission (>0.15 ng/ml) who underwent percutaneous coronary intervention ≥48 hours after admission. Troponin I levels were routinely measured at 6 and 18 to 24 hours after intervention and patients were stratified according to the presence or absence of troponin I re-elevation, defined as postprocedural troponin I levels >1 times the admission levels. In-hospital and cumulative 6-month clinical outcomes were compared between groups. Patients with troponin I re-elevation (n = 51) were older (68 ± 13 vs 64 ± 12 years, p = 0.05) and had experienced prior myocardial infarction more frequently (92.5 vs 82.1, p = 0.09), but otherwise had similar baseline clinical characteristics. Patients with troponin I re-elevation had significantly higher in-hospital mortality (9.8% vs 0%, p = 0.016) and a higher 6-month cumulative death rate (24% vs 3.7%, p = 0.001). There was a trend for an increased 6-month myocardial infarction rate in patients with troponin I re-elevation (13.7% vs 3.7%, p = 0.11) and target vessel revascularization was similar between groups (16.7% vs 17.4%, p = 0.92). By multivariate analysis, troponin I re-elevation (odds ratio [OR] 6.2, p = 0.011) and diabetes mellitus (OR 5.7, p = 0.014) were the strongest independent predictors for increased 6-month cumulative mortality, whereas creatine kinase MB-fraction re-elevation had no prognostic value. We conclude that troponin I re-elevation after percutaneous coronary intervention in high-risk patients with ACS is associated with a substantial increase in mortality and reduced event-free survival at 6-month follow-up.