Component Of Thin Filaments Research Articles

Influence of native thin filament type on the regulation of atrial and ventricular myosin motor activity

Ca2+-mediated activation of thin filaments is a crucial step in initiating striated muscle contraction. To gain mechanistic insight into this regulatory process, thin filament (TF) components and myosin motors from diverse species and tissue sources are often combined in minimal in vitro systems. The contribution of tissue-specific TF composition with native myosin motors in generating contraction speed remains unclear. To examine TF-mediated regulation, we established a procedure to purify native TFs (nTF) and myosin motors (M-II) from the same cardiac tissue samples as low as 10 mg and investigated their influence on gliding speeds and Ca2+ sensitivity. The rabbit atrial and ventricular nTFs and M-II were assessed in in vitro nTF motility experiments under varying Ca2+ concentrations. The speed-pCa relationship yielded a maximum TF speed of 2.58 μm/s for atrial (aM-II) and 1.51 μm/s for ventricular myosin (vM-II), both higher than the respective unregulated actin filament gliding speeds. The Ca2+ sensitivity was different for both protein sources. After swapping the nTFs, the ventricular TFs increased their gliding speed on atrial myosin, while the atrial nTFs reduced their gliding speed on ventricular myosin. Swapping of the nTFs decreased the calcium sensitivity for both vM-II and aM-II, indicating a strong influence of the thin filament source. These studies suggest that the nTF-myosin combination is critical to understanding the Ca2+ sensitivity of the shortening speed. Our approach is highly relevant to studying precious human cardiac samples, i.e., small myectomy samples, to address the alteration of contraction speed and Ca2+ sensitivity in cardiomyopathies.

Mouse Models of Cardiomyopathies Caused by Mutations in Troponin C.

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).

CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle

The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.

Protein-Protein Docking Reveals Dynamic Interactions of Tropomyosin on Actin Filaments

Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a “divide and conquer” protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin “ligand” segments were rotated and translated over potential “target” binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

Structure of the Actin-Tropomyosin-TNT Complex

Thin filament-linked proteins actin, tropomyosin, and troponin are the major regulators of cardiac and skeletal muscle that activate contraction through calcium-mediated conformational changes. Single point mutations within the cardiac forms of these proteins cause hypertrophic (HCM) and dilated (DCM) cardiomyopathy, affecting more than 1 in every 500 people. In order to determine mechanisms of thin filament regulation and the initial mutation-associated insults that arise during disease development, it is necessary to define the molecular interactions of the thin filament components. The TnT tail (TnT1), and more specifically the N-terminal region (residues 1-156 in cardiac TnT isoform 6), tethers the troponin core domain (i.e. TnT2, TnC and TnI) to tropomyosin on the thin filament and thereby assumes the 40 nm tropomyosin periodicity. Additionally, TnT1 stabilizes the tropomyosin head-to-tail domain to form cables spanning the full filament. TnT1-156also contains more than one third of all thin filament mutations that cause HCM and DCM. Because no high-resolution structure of this region is available, residue-level interactions with tropomyosin as well as the positions of many HCM and DCM point mutants remain elusive. We are using cryo-electron microscopy to solve the high-resolution structure of the cardiac actin-tropomyosin-TnT1-156 complex. The complex has been fully optimized and 2-dimensional class averages display clear TnT density on actin-tropomyosin. 3-dimensional reconstructions reveal that TnT1-156 makes the tropomyosin appear broader and flatter, consistent with an α-helix binding alongside tropomyosin, rather than hovering over it. Given current cryo-EM optimization at nanometer resolution, we anticipate resolution to near-atomic levels, thus leading to the residue-level determination of the TnT-tropomyosin interaction and a more precise understanding of mutation-associated deficits.

Sensitization profile of cricket food-allergic or cricket tolerant patients in an entomophagous population in Niamey, Niger

Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a “divide and conquer” protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin “ligand” segments were rotated and translated over potential “target” binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

Sarcomere length-dependent effects on Ca2+-troponin regulation in myocardium expressing compliant titin.

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca2+-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca2+-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca2+-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca2+-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation of the myocardium.

High-resolution imaging of muscle attachment structures in Caenorhabditis elegans.

We used structured illumination microscopy (SIM) to obtain super-resolution images of muscle attachment structures in Caenorhabditis elegans striated muscle. SIM imaging of M-line components revealed two patterns: PAT-3 (β-integrin) and proteins that interact in a complex with the cytoplasmic tail of β-integrin and localize to the basal muscle cell membrane [UNC-112 (kindlin), PAT-4 (ILK), UNC-97 (PINCH), PAT-6 (α-parvin), and UNC-95], are found in discrete, angled segments with gaps. In contrast, proteins localized throughout the depth of the M-line (UNC-89 (obscurin) and UNC-98) are imaged as continuous lines. Systematic immunostaining of muscle cell boundaries revealed that dense body components close to the basal muscle cell membrane also localize at cell boundaries. SIM imaging of muscle cell boundaries reveal "zipper-like" structures. Electron micrographs reveal electron dense material similar in appearance to dense bodies located adjacent to the basolateral cell membranes of adjacent muscle cells separated by ECM. Moreover, by EM, there are a variety of features of the muscle cell boundaries that help explain the zipper-like pattern of muscle protein localization observed by SIM. Short dense bodies in atn-1 mutants that are null for α-actinin and lack the deeper extensions of dense bodies, showed "zipper-like" structures by SIM similar to cell boundary structures, further indicating that the surface-proximal components of dense bodies form the "zipper-like" structures at cell boundaries. Moreover, mutants in thin and thick filament components do not have "dot-like" dense bodies, suggesting that myofilament tension is required for assembly or maintenance of proper dense body shape.

Sarcomere Dysfunction in Nemaline Myopathy.

Nemaline myopathy (NM) is among the most common non-dystrophic congenital myopathies (incidence 1:50.000). Hallmark features of NM are skeletal muscle weakness and the presence of nemaline bodies in the muscle fiber. The clinical phenotype of NM patients is quite diverse, ranging from neonatal death to normal lifespan with almost normal motor function. As the respiratory muscles are involved as well, severely affected patients are ventilator-dependent. The mechanisms underlying muscle weakness in NM are currently poorly understood. Therefore, no therapeutic treatment is available yet.Eleven implicated genes have been identified: ten genes encode proteins that are either components of thin filament, or are thought to contribute to stability or turnover of thin filament proteins. The thin filament is a major constituent of the sarcomere, the smallest contractile unit in muscle. It is at this level of contraction – thin-thick filament interaction – where muscle weakness originates in NM patients.This review focusses on how sarcomeric gene mutations directly compromise sarcomere function in NM. Insight into the contribution of sarcomeric dysfunction to muscle weakness in NM, across the genes involved, will direct towards the development of targeted therapeutic strategies.

Sarcomere Lengthdependent Effects on the Ca2+-Troponin Regulation in Skinned Myocardial Fiber from Titin RBM20 Deletion Mice

The length dependent activation of the myofilaments has been considered the cellular basis underlying the Frank-Starling law of the heart trough dynamic and complex interplays between a multitude of thick- and thin-filament components. Among these components, both troponin and giant protein titin are likely key players, but the mechanism, by which the length dependent myofilament Ca2+ regulation is linked functionally to titin, is elusive. In this study we investigate the link using in situ FRET technique to monitor how the length dependent Ca2+-induced conformational change of the N-domain of cTnC within myocardium is modulated by alteration of titin compliance that directly determines the passive tension of myocardium. To achieve the objective, FRET donor and acceptor modified cTnC(13C/51C)AEDANS-DDPM was reconstituted into chemically-skinned myocardial fibers from wild-type mice and RBM20 deficient mice that expresses more compliant N2BA titin. The Ca2+-induced conformational change in cTnC under different conditions were quantitatively characterized using time-resolved FRET measurements. Our results from wild type myocardium showed that sarcomere length dependent enhancement of Ca2+-troponin regulation was found to be directly associated with strong crossbridge and it was diminished when the strong crossbridge was inhibited. When measurements were performed with skinned myocardial fibers from RBM20 deletion animals, the length dependent change was blunted. These results support that titin based passive tension development of myocardium is indeed involved in sarcomere length modulated Ca2+-troponin regulation and that the strong crossbridge plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation.

Identification, characterization, and expression of sarcomeric tropomyosin isoforms in zebrafish.

Tropomyosin is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. In vertebrates, except for fish, four TPM genes TPM1, TPM2, TPM3, and TPM4 are known. In zebrafish, there are six TPM genes that include the paralogs of the TPM1 (TPM1-1 and TPM1-2), the paralogs of the TPM4 gene (TPM4-1 and TPM4-2), and the two single copy genes TPM2 and TPM3. In this study, we have identified, cloned, and sequenced the TPM1-1κ isoform of the TPM1-1 gene and also discovered a new isoform TPM1-2ν of the TPM1-2. Further, we have cloned and sequenced the sarcomeric isoform of the TPM4-2 gene designated as TPM4-2α. Using conventional RT-PCR, we have shown the expression of the sarcomeric isoforms of TPM1-1, TPM1-2, TPM2, TPM3, TPM4-1, and TPM4-2 in heart and skeletal muscles. By qRT-PCR using both relative expression as well as the absolute copy number, we have shown that TPM1-1α, TPM1-2α, and TPM1-2ν are expressed mostly in skeletal muscle; the level of expression of TPM1-1κ is significantly lower compared to TPM1-1α in skeletal muscle. In addition, both TPM4-1α and TPM4-2α are predominantly expressed in heart. 2D Western blot analyses using anti-TPM antibody followed by Mass Spectrometry of the proteins from the antibody-stained spots show that TPM1-1α and TPM3α are expressed in skeletal muscle whereas TPM4-1α and TPM3α are expressed in zebrafish heart. To the best of our knowledge, this is by far the most comprehensive analysis of tropomyosin expression in zebrafish, one of the most popular animal models for gene expression study.

Expression of various sarcomeric tropomyosin isoforms in equine striated muscles.

In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM isoforms from horse heart and skeletal muscle. We have cloned and sequenced two sarcomeric isoforms of the TPM1 gene called TPM1α and TPM1κ, one sarcomeric isoform of the TPM2 and one of the TPM3 gene, TPM2α and TPM3α respectively. By qRT-PCR using both relative expression and copy number, we have shown that TPM1α expression compared to TPM1κ is very high in heart. On the other hand, the expression of TPM1α is higher in skeletal muscle compared to heart. Further, the expression of TPM2α and TPM3α are higher in skeletal muscle compared to heart. Using western blot analyses with CH1 monoclonal antibody we have shown the high expression levels of sarcomeric TPM proteins in cardiac and skeletal muscle. Due to the paucity of isoform specific antibodies we cannot specifically detect the expression of TPM1κ in horse striated muscle. To the best of our knowledge this is the very first report on the characterization of sarcmeric TPMs in horse striated muscle.

Recent Advances on the TPM4 Gene Expression in Humans

Tropomyosin, a coiled-coil dimeric protein, is a component of thin filaments that constitute myofibrils, the contractile apparatus of striated muscles. It is also a component of the actin filament network in non-muscle cells. In vertebrates, except for fish, there are four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, except for TPM4, the sarcomeric TPM isoform (s) for each TPM gene have been known for long time. Recently, we have cloned and sequenced the sarcomric isoform of the TPM4 gene designated as TPM4a. In addition, using qRT-PCR we have reported the expression of TPM4a in human striated muscles. Also, using 2D Western blot analyses followed by Mass spectra, we have reported the potential TPM4a protein expression in human cardiac tissues. However, much of the role of TPM4a in muscle contraction in human is yet to be elucidated. Although the role of the TPM4 gene in human diseases in not well documented, new information is emerging in this regard. For example, of the TPM4 isoforms TPM4g has been reported as a non-invasive biomarker in prenatal diagnosis of congenital heart defects; mutations in TPM4 have been implicated in Macrothombocytopenia in humans; differential expression of two TPM isoforms TPM4b and TPM4g in human breast cancer cells. However, the role of TPM4-ALK oncogenes in inflammatory myofibroblastic tumors in humans is well documented.

Molecular cloning, structural analysis, and tissue expression of the TNNT3 gene in Guizhou black goat

The vertebrate fast skeletal troponin T (TNNT3) protein is an important regulatory and structural component of thin filaments in skeletal muscle, which improves meat quality traits of livestock and poultry. In this study, the troponin T isoforms from adult goat (skeletal muscle mRNA) were identified. We isolated the full-length coding sequence of the goat TNNT3 gene (GenBank: KM042888), analyzed its structure, and investigated its expression in different tissues from different aged goats (10, 30, 90, 180, and 360days old). Real-time quantitative reverse transcription-polymerase chain reaction analyses revealed that Guizhou black goat TNNT3 was highly expressed in the biceps femoris muscle, abdominal muscle, and longissimus dorsi muscle (P<0.01), and lowly expressed in the cardiac muscle, masseter muscle, and rumen tissue (P>0.05). Western blotting confirmed that the TNNT3 protein was expressed in the muscle tissues listed above, with the highest level found in the longissimus dorsi muscle, and the lowest level in the masseter muscle. In the 10 to 360day study period the TNNT3 protein expression level was the highest when the goats were 30days old. A peptide, ASPPPAEVPEVHEEVH that may contribute to improved goat meat tenderness was identified. This study provides an insight into the molecular structure of the vertebrate TNNT3 gene.

Exon 12 of slow skeletal troponin t affects calcium sensitivity of force development and interactions with other thin filament components (1102.30)

Little information exists concerning the functional roles of the human slow skeletal troponin T isoforms (HSSTnT isoforms) in slow skeletal muscle. Three HSSTnT isoforms have been found in human slow skeletal muscle: HSSTnT1 (+ exons 5 and 12), HSSTnT2 (+5, ‐12), and HSSTnT3 (‐5, ‐12). Another potential isoform HSSTnT‐Hyp (‐5, +12) was recently found at the mRNA level. The objective of this study was to determine the physiological role of these SSTnT isoforms in slow skeletal muscle. To investigate these SSTnT isoforms several methods including skinned fiber mechanics, peptide spot blot, and mammalian two‐hybrid assays were utilized. Skinned rabbit slow soleus muscle fibers were displaced with HSSTnT1, 2, 3 or Hyp and reconstituted with the human slow skeletal troponin I (HSSTnI)/human cardiac troponin C (HCTnC) complex. The calcium sensitivity increased between SSTnT isoforms: isoform 1 (pCa50 = 5.74) &lt; Hyp isoform (pCa50 = 5.80) &lt; isoform 2 (pCa50 = 5.81) &lt; isoform 3 (pCa50 = 5.84). In a reconstituted skeletal muscle system, the HSSTnT 1‐3 isoforms had similar actomyosin ATPase activation or inhibition in the presence or absence of calcium. Peptide spot blot analysis revealed novel interactions between HSSTnT peptides and HCTnC, RSTm and HCTnI. Mammalian two‐hybrid studies showed that the alternatively spliced regions affect the interaction of HSSTnT with other thin filament components. These results suggest that the functional differences that occur between HSSTnT isoforms may be partially due to alternative splicing of exon 12. Overall, HSSTnT isoforms were found to have distinct functional properties in slow skeletal muscle regulation and are likely to be relevant for the understanding of skeletal muscle diseases.Grant Funding Source: UC Davis research funds and NIH

Mg2+ Dependent Modulation of Striated Muscle Myosin ATPase by Thin Filament Components

While it's been reported that non-muscle myosin such as myosin V can be regulated by Mg2+, striated muscle myosin modulation by Mg2+-concentration dependency, especially in relation to thin filament components, has not been well studied up to now. It has been suggested that ADP release (product release) from myosin V is affected by Mg2+ concentration. Therefore, Mg2+-dependent modulation of striated muscle myosin ATPase was explored in the presence of reconstituted muscle thin filament components, i.e. actin, tropomyosin and troponin. ATPase activity assays and Nuclear Magnetic Resonance (NMR) spectroscopy were employed to understand the mechanisms of ATP hydrolysis by striated muscle myosin and the role of Mg2+ in the enzymatic activity. An NMR technique called Water Ligand-Observed Gradient Spectroscopy (WaterLOGSY) was utilized to probe if the interaction of myosin with ADP (one of the products resulted from ATP hydrolysis) is different depending on Mg2+ concentration as well as the presence of muscle thin filament components. Our WaterLOGSY data demonstrated that this technique is a promising method to monitor ADP binding to myosin head S1 even in the presence of actin filaments and that there was a change in the appearance of peaks between with and without tropomyosin at a low Mg2+ concentration. This set of results indicates that the way by which myosin binds ADP is different depending on the states of thin filaments. Moreover, actomyosin S1 ATPase rate decreased as Mg2+ concentration increased. When tropomyosin was reconstituted in the thin filaments, the ATPase rate decreased more rapidly compared with actomyosin alone. Thus we conclude that Mg2+ plays an important role in striated muscle myosin ATPase and that regulatory components such as tropomyosin modulate Mg2+ dependency of myosin ATPase in the striated muscle.

Schistosome Muscles Contain Striated Muscle-Like Myosin Filaments in a Smooth Muscle-Like Architecture

Schistosomes are parasitic worms infecting 200 million humans worldwide. Schistosomiasis is treated with the drug praziquantel, which affects the parasite muscle, possibly by binding to myosin light chains. To understand the molecular functioning of Schistosome muscles, we have studied their cellular and molecular makeup. EM sections of adult worms reveal exclusively smooth muscle throughout the body wall (thick filaments not in register, dense bodies instead of Z-lines). We carried out 3D-reconstruction of negatively stained, relaxed Schistosome thick filaments. Surprisingly, the reconstruction was indistinguishable from those obtained previously for arthropod striated muscles, showing a 4-fold helical arrangement of myosin interacting-head motifs. Published data show that only one myosin heavy chain (MHC) gene is expressed in Schistosoma mansoni. By proteomic-Mass Spectrometry (MS) analysis of myosin from the adult parasite stage we showed that this was similar to a striated, not smooth, MHC. Analysis of published protein sequences supports this finding. A percentage-of-identity distance tree, and alignment of multiple diverse MHC sequences, showed two main branches: one for vertebrate and invertebrate striated muscles, and a smooth and nonmuscle branch. The Schistosome MHC lies in the striated muscle group, completely separate from the smooth/nonmuscle group. The in vitro motility assay showed movement of Schistosome thin filaments over unregulated tarantula striated myosin filaments independent of calcium and similar to rabbit F-actin motility, suggesting the absence of thin filament regulation. In agreement with this result, proteomic-MS analysis of Schistosome filament homogenates demonstrated the expression of the thin filament components tropomyosin and actin, but no signal for troponin was detected. We conclude that Schistosome muscles are hybrids, containing striated muscle-like thick filaments (with striated muscle MHC) and smooth muscle-like thin filaments (no troponin), arranged in a smooth muscle-like architecture.

The nebulin SH3 domain is dispensable for normal skeletal muscle structure but is required for effective active load bearing in mouse

Nemaline myopathy (NM) is a congenital myopathy with an estimated incidence of 150,000 live births. It is caused by mutations in thin filament components, including nebulin, which accounts for about 50% of the cases. The identification of NM cases with nonsense mutations resulting in loss of the extreme C-terminal SH3 domain of nebulin suggests an important role of the nebulin SH3 domain, which is further supported by the recent demonstration of its role in IGF-1-induced sarcomeric actin filament formation through targeting of N-WASP to the Z-line. To provide further insights into the functional significance of the nebulin SH3 domain in the Z-disk and to understand the mechanisms by which truncations of nebulin lead to NM, we took two approaches: (1) an affinity-based proteomic screening to identify novel interaction partners of the nebulin SH3 domain; and (2) generation and characterization of a novel knockin mouse model with a premature stop codon in the nebulin gene, eliminating its C-terminal SH3 domain (NebΔSH3 mouse). Surprisingly, detailed analyses of NebΔSH3 mice revealed no structural or histological skeletal muscle abnormalities and no changes in gene expression or localization of interaction partners of the nebulin SH3 domain, including myopalladin, palladin, zyxin and N-WASP. Also, no significant effect on peak isometric stress production, passive tensile stress or Young's modulus was found. However, NebΔSH3 muscle displayed a slightly altered force-frequency relationship and was significantly more susceptible to eccentric contraction-induced injury, suggesting that the nebulin SH3 domain protects against eccentric contraction-induced injury and possibly plays a role in fine-tuning the excitation-contraction coupling mechanism.

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.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.