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The troponin complex regulates the Ca(2+) activation of myofilaments during striated muscle contraction and relaxation. Troponin genes emerged 500–700 million years ago during early animal evolution. Troponin T (TnT) is the thin-filament–anchoring subunit of troponin. Vertebrate and invertebrate TnTs have conserved core structures, reflecting conserved functions in regulating muscle contraction, and they also contain significantly diverged structures, reflecting muscle type- and species-specific adaptations. TnT in insects contains a highly-diverged structure consisting of a long glutamic acid–rich C-terminal extension of ∼70 residues with unknown function. We found here that C-terminally truncated Drosophila TnT (TpnT–CD70) retains binding of tropomyosin, troponin I, and troponin C, indicating a preserved core structure of TnT. However, the mutant TpnT(CD70) gene residing on the X chromosome resulted in lethality in male flies. We demonstrate that this X-linked mutation produces dominant-negative phenotypes, including decreased flying and climbing abilities, in heterozygous female flies. Immunoblot quantification with a TpnT-specific mAb indicated expression of TpnT–CD70 in vivo and normal stoichiometry of total TnT in myofilaments of heterozygous female flies. Light and EM examinations revealed primarily normal sarcomere structures in female heterozygous animals, whereas Z-band streaming could be observed in the jump muscle of these flies. Although TpnT–CD70-expressing flies exhibited lower resistance to cardiac stress, their hearts were significantly more tolerant to Ca(2+) overloading induced by high-frequency electrical pacing. Our findings suggest that the Glu-rich long C-terminal extension of insect TnT functions as a myofilament Ca(2+) buffer/reservoir and is potentially critical to the high-frequency asynchronous contraction of flight muscles.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca(2+)-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca(2+) activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC–cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

Myosin is the principal component of the thick filaments that, through interactions with the actin thin filaments, mediates force production during muscle contraction. Myosin is a hexamer, consisting of two heavy chains, each associated with an essential (ELC) and a regulatory (RLC) light chain, which bind the lever-arm of the heavy chain and play important modulatory roles in striated muscle contraction. Nevertheless, a comprehensive assessment of the sequences of the ELC and RLC isoforms, as well as their post-translational modifications, in the heart remains lacking. Herein, utilizing top-down high-resolution mass spectrometry (MS), we have comprehensively characterized the sequences and N-terminal modifications of the atrial and ventricular isoforms of the myosin light chains from human and swine hearts, as well as the sites of phosphorylation in the swine proteins. In addition to the correction of disparities in the database sequences of the swine proteins, we show for the first time that, whereas the ventricular isoforms of the ELC and RLC are methylated at their N-termini, which is consistent with previous studies, the atrial isoforms of the ELC and RLC from both human and swine are Nα-methylated and Nα-acetylated, respectively. Furthermore, top-down MS with electron capture dissociation enabled localization of the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Collectively, these results provide new insights into the sequences and modifications of myosin light chain isoforms in the human and swine hearts, which will pave the way for a better understanding of their functional roles in cardiac physiology and pathophysiology.

The Warner-Bratzler shear force (WBSF) and myofibrillar fragmentation index (MFI) are complementary methodologies used to measure beef tenderness. Longissimus thoracis samples from the 20 most extreme bulls (out of 80 bulls set) for WBSF (tender (n = 10) and tough (n = 10)) and MFI (high (n = 10) and low (n = 10)) traits were collected to perform transcriptomic analysis using RNA-Sequencing. All analysis were performed through CLC Genomics Workbench. A total of 39 and 27 transcripts for WBSF and MFI phenotypes were DE, respectively. The possible DE novel mRNA isoforms, for WBSF and MFI traits, are myosin encoders (e.g. MYL1 and MYL6). In addition, we identified potential mRNA isoforms related to genes affecting the speed fibers degradation during the meat aging process. The DE novel transcripts are transcripted by genes with biological functions related to oxidative process, energy production and striated muscle contraction. The results suggest that the identified mRNA isoforms could be used as potential candidate to select animals in order to improve meat tenderness.

Inhibition of striated muscle contraction at resting Ca(2+) depends on the C-terminal half of troponin I (TnI) in thin filaments. Much focus has been on a short inhibitory peptide (Ip) sequence within TnI, but structural studies and identification of disease-associated mutations broadened emphasis to include a larger mobile domain (Md) sequence at the C-terminus of TnI. For Md to function effectively in muscle relaxation, tight mechanical coupling to troponin's core-and thus tropomyosin-is presumably needed. We generated recombinant, human cardiac troponins containing one of two TnI constructs: either an 8-amino acid linker between Md and the rest of troponin (cTnILink8), or an Md deletion (cTnI1-163). Motility assays revealed that Ca(2+)-sensitivity of reconstituted thin filament sliding was markedly increased with cTnILink8 (∼0.9 pCa unit leftward shift of speed-pCa relation compared to WT), and increased further when Md was missing entirely (∼1.4 pCa unit shift). Cardiac Tn's ability to turn off filament sliding at diastolic Ca(2+) was mostly (61%), but not completely eliminated with cTnI1-163. TnI's Md is required for full inhibition of unloaded filament sliding, although other portions of troponin-presumably including Ip-are also necessary. We also confirm that TnI's Md is not responsible for superactivation of actomyosin cycling by troponin.

In contrast with the past, the water buffalo is now not only a draft animal, but also an important food source of milk and meat. It is increasingly apparent that the water buffalo have huge potential for meat production, but its breeding needs to be investigated. Regarding the molecular mechanisms involved in the meat quality difference between the buffalo (Bubalus bulabis) and yellow cattle (Bos taurus), 12 chemical-physical characteristics related to the meat quality of longissimus thoracis muscles (LTM) have been compared at the age of 36 months. Intramuscular lipid and b* (yellowness) were greater in cattle than the buffalo, whereas a* (redness) was greater in the buffalo. Gene expression profiles were constructed by bovine genome array. A total of 8884 and 10,960 probes were detected in buffalo and cattle, respectively, with 1580 genes being differentially expressed. Over 400 probes were upregulated and nearly 1200 were downregulated in LTM of the buffalo, most being involved in ribosomal RNA (rRNA) processing, cholesterol homeostasis, regulation of transcription, response to hypoxia, and glycolysis. Quantitative real-time PCR was used to validate the microarray data. Enriched GO analyses of highly expressed genes in LTM showed that protein biosynthesis, striated muscle contraction, iron homeostasis, iron transport, glycolysis and glucose metabolism were similar between the buffalo and cattle. High protein content, low fat content and deep meat color of buffalo LTM may be closely associated with the increased expression of genes involved in cholesterol and iron homeostasis, while also reducing the expression of genes involved in ubiquitin-mediated proteolysis and protein oxidative phosphorylation. These results establish the groundwork for further studies on buffalo meat quality and will be beneficial in improving water buffalo breeding by molecular biotechnology.

The investigation of healthy and diseased muscle behavior via in silico analysis requires the modeling of biophysical processes on multiple spatial and temporal scales. Owing to the complexity of the phenomena in question, simultaneous simulations of all the processes across different scales are extremely computationally expensive. Therefore, many multi-scale models utilize simplified phenomenological models at the micro level. However, such models may not be able to predict transient contractile behavior accurately when the deformation is unsteady or non-uniform. To overcome these deficiencies of phenomenological models, we propose a novel multi-scale muscle model in which continuum muscle mechanics are modeled utilizing the finite element method, and the material characteristics of muscle tissues at the microscopic scale are defined by Huxley’s model of muscle contraction. Owing to the specific application of the sliding-filament theory coupled with the kinetic formulation of Gordon’s length-tension relationship, the proposed model can provide more precise simulations of muscle behavior under both isotonic and transient conditions. The proposed model is verified using both benchmark data and real-world examples, and the results are compared to corresponding predictions obtained using the FE-Hill model. Specific implementations of biophysical components at the muscle fiber scale are validated by comparing them to predictions obtained using a spatially explicit molecular model implemented on the MUSICO platform. To enable the execution of two-scale simulations in a reasonable timeframe, we utilize a custom-tailored parallelization platform called Mexie. The ability of the proposed model to describe tissue-scale motor system behavior and the efficiency of its parallel execution are demonstrated through simulations of tongue movement during the propulsive phase of human swallowing. In these simulations the tissue’s complex muscular structure is represented by a 2D finite element mesh. The proposed model provides tools for the scientific investigation of musculoskeletal disorders and facilitates the prospective development of clinical applications for characterizing neuromuscular disorders and monitoring disease progression during therapy.

Summary Ryanodine receptors (RyRs) release Ca 2+ to initiate striated muscle contraction. Three highly divergent regions (DRs) in the RyR protein sequence (DR1, DR2, and DR3) may confer isoform-specific functional properties to the RyRs. We used cell-based fluorescence resonance energy transfer (FRET) measurements to localize these DRs to the cryoelectron microscopic (cryo-EM) map of the skeletal muscle RyR isoform (RyR1). FRET donors were targeted to RyR1 using five different FKBP12.6 variants labeled with Alexa Fluor 488. FRET was then measured to the FRET acceptors, Cy3NTA or Cy5NTA, targeted to decahistidine tags introduced within the DRs. DR2 and DR3 were localized to separate positions within the "clamp" region of the RyR1 cryo-EM map, which is presumed to interface with Ca v 1.1. DR1 was localized to the "handle" region, near the regulatory calmodulin-binding site on the RyR. These localizations provide insights into the roles of DRs in RyR allosteric regulation during excitation contraction coupling.

Background The substantial contribution of genetic variants to inter-individual variance in antidepressant response makes genetic polymorphisms a unique opportunity to provide tailored antidepressant treatments. The present study combined a candidate approach of variants with strong previous evidence and a genome-wide approach in order to investigate the role of eight genes that appear optimal candidates for involvement in antidepressant response on the basis of their biological function and previous literature suggesting a pleiotropic effect across several psychiatric traits. Methods Three samples of patients with major depressive disorder (n=357, 218 and 96) were genotyped for 44 SNPs in genes involved in neurotransmission (CACNA1C, CACNB2, ANK3), neural differentiation, synaptic plasticity, adhesion processes, structural organization (GRM7, TCF4, ITIH3, SYNE1) and glucocorticoid signaling (FKBP5). Phenotypes were response/remission at week 4 and Treatment-Resistant Depression (TRD: non response/non remission to at least two antidepressants). STAR⁎D genome-wide data were used to replicate findings for response/remission (Level 1, n=1409) and TRD (Level 2, n=620). Standard quality control and genotype imputation were performed and pathways including the most promising candidate genes for involvement in TRD were investigated in STAR⁎D Level 2. Top pathway(s) were investigated also using machine learning (R cran Caret package). At SNP-level results with p Results Several FKBP5 SNPs (rs3800373, rs1360780, rs9470080) showed consistent associations with response, remission or TRD across at least two samples. Results involving CACNA1C SNPs (rs2283326, rs10848635, rs1006737) had contradictory direction of association across samples. ANK3 rs1049862 AA genotype showed a consistent association with a more favorable outcome in two samples. In STAR⁎D pathway analysis for the top candidate genes did not identify significant results after permutation. The best pathway associated with TRD included the CACNA1C gene (GO:0006942, regulation of striated muscle contraction, corrected p=0.15). Neural networks and gradient boosted machine showed that independent SNPs in this pathway predicted TRD with a mean accuracy of 0.73, sensitivity of 0.83 and specificity of 0.56. Discussion According to the present results and previous literature, FKBP5 polymorphisms should be considered for inclusion in pharmacogenetic tests for the prediction of antidepressant response and TRD. The contribution of CACNA1C polymorphisms was unclear, but further investigation is supported by previous literature and interesting findings for GO:0006942 pathway that includes several genes coding for ion channels that are expressed in the central nervous system as well as other genes relevant for excitatory mechanisms.

Troponin is well known as a Ca 2+ -dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin–myosin interaction at low Ca 2+ concentrations, and Ca 2+ at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca 2+ -dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca 2+ . Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

The regulation of vertebrate striated muscle contraction involves a number of different molecules, including the thin-filament accessory proteins tropomyosin and troponin that provide Ca 2+ -dependent regulation by controlling access to myosin binding sites on actin. Cardiac myosin binding protein C (cMyBP-C) appears to modulate this Ca 2+ -dependent regulation and has attracted increasing interest due to links with inherited cardiac diseases. A number of single amino acid mutations linked to clinical diseases occur in the N-terminal region of cMyBP-C, including domains C0 and C1, which previously have been shown to bind to F-actin. This N-terminal region also has been shown to both inhibit and activate actomyosin interactions in vitro . Using electron microscopy and three-dimensional reconstruction, we show that C0 and C1 can each bind to the same two distinctly different positions on F-actin. One position aligns well with the previously reported binding site that clashes with the binding of myosin to actin, but would force tropomyosin into an “on” position that exposes myosin binding sites along the filament. The second position identified here would not interfere with either myosin binding or tropomyosin positioning. It thus appears that the ability to bind to at least two distinctly different positions on F-actin, as observed for tropomyosin, may be more common than previously considered for other actin binding proteins. These observations help to explain many of the seemingly contradictory results obtained with cMyBP-C and show how cMyBP-C can provide an additional layer of regulation to actin–myosin interactions. They also suggest a redundancy of C0 and C1 that may explain the absence of C0 in skeletal muscle.

Myomesin plays an important structural and functional role in the M-band of striated muscles. The C-terminal domain 13 of myomesin dimerises and forms antiparallel strands which cross-link neighboring Myosin filaments and titin in the M-line of the sarcomeres. These interactions stabilise the contractile apparatus during striated muscle contraction. Since myomesin is an important component of the M-band we screened the myomesin gene for genetic variants in patients with hypertrophic cardiomyopathy (HCM). We identified the missense mutation V1490I in domain 12 of myomesin in a family with inherited HCM. Analytical ultracentrifugation experiments, circular dichroism spectra, and surface plasmon resonance spectroscopy of myomesin fragments were carried out to investigate the effects of the mutation V1490I on structure and function of myomesin domains 11-13 and 12-13. Both the wild type and mutated myomesin domains My11-13 revealed similar secondary structures and formed stable dimers. Mutated myomesin domains My11-13 and My12-13 dimers revealed a reduced thermal stability and a significantly decreased dimerisation affinity, showing disturbed functional properties of V1490I mutated myomesin. However, monomeric myomesin domains My11-12, i.e. without dimerisation domain 13 showed no difference in thermal stability between wild type and V1490I mutated myomesin. In conclusion, the V1490I mutation associated with HCM lead to myomesin proteins with abnormal functional properties which affect dimerisation properties of myomesin domain 13. These effects may contribute to the pathogenesis of HCM.

In order to study the molecular mechanism involved in meat quality differences especially tenderness variance caused by castration in Qinchuan cattle males, this study investigated the gene expression profile of Longissimus thoracis (LT) muscle and screened differentially expressed genes in LT muscle from both intact male and castrated male Qinchuan cattle at 36 months of age utilising Bovine Genome Array. Significance Analysis of Microarrays (SAM) was used to identify the differentially expressed genes, Go (Gene Ontology) and pathway analyses were conducted on which by a free Web-based Molecular Annotation System 2.0 (MAS 2.0). Approximately 11,000 probe sets representing 10,000 genes were detected in LT muscle of 36-month-old Qinchuan cattle. After SAM analysis of the microarray data, 142 genes were shown to be differentially expressed. These genes were predominantly involved in collagen fibril organization and synthesis, regulation of cell growth and development and striated muscle contraction. The significant pathways involved mainly included ECM–receptor (extracellular matrix-receptor) interaction, cell communication and focal adhesion. Quantitative real-time PCR (qRT-PCR) was performed to validate some differentially expressed genes identified by microarray. These patterns of gene expression may contribute to understanding the molecular mechanism of better meat quality of beef derived from castrates than from intact males.

Troponin T (TnT) and troponin I (TnI) are two evolutionarily and functionally linked subunits of the troponin complex that regulates striated muscle contraction. We previously reported a single amino acid substitution in the highly conserved TnT-binding helix of cardiac TnI (cTnI) in wild turkey hearts in concurrence with an abnormally spliced myopathic cardiac TnT (cTnT) (Biesiadecki, B. J., Schneider, K. L., Yu, Z. B., Chong, S. M., and Jin, J. P. (2004) J. Biol. Chem. 279, 13825-13832). To investigate the functional effect of this cTnI mutation and its potential value in compensating for the cTnT abnormality, we developed transgenic mice expressing the mutant cTnI (K118C) in the heart with or without the deletion of the endogenous cTnI gene to mimic the homozygote and heterozygote of wild turkeys. Double and triple transgenic mice were created by crossing the cTnI-K118C lines with transgenic mice overexpressing the myopathic cTnT (exon 7 deletion). Functional studies of ex vivo working hearts found that cTnI-K118C alone had a dominantly negative effect on diastolic function and blunted the inotropic responses of cardiac muscle to beta-adrenergic stimuli without abolishing the protein kinase A-dependent phosphorylation of cTnI. When co-expressed with the cTnT mutation, cTnI-K118C corrected the significant depression of systolic function caused by cTnT exon 7 deletion, and the co-existence of exon 7-deleted cTnT minimized the diastolic abnormality of cTnI-K118C. Characterization of this naturally selected pair of mutually rescuing mutations demonstrated that TnI-TnT interaction is a critical link in the Ca(2+) signaling and beta-adrenergic regulation in cardiac muscle, suggesting a potential target for the treatment of troponin cardiomyopathies and heart failure.

Over the 40 years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca(2+)-dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca(2+)-sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca(2+) concentration is not perturbed, preserving the regulation of other Ca(2+)-based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C-troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.

Troponin (Tn), in association with tropomyosin (Tm), plays a central role in the calcium regulation of striated muscle contraction. Fluorescence resonance energy transfer (FRET) between probes attached to the Tn subunits (TnC, TnI, TnT) and to Tm was measured to study the spatial relationship between Tn and Tm on the thin filament. We generated single-cysteine mutants of rabbit skeletal muscle alpha-Tm, TnI and the beta-TnT 25-kDa fragment. The energy donor was attached to a single-cysteine residue at position 60, 73, 127, 159, 200 or 250 on TnT, at 98 on TnC and at 1, 9, 133 or 181 on TnI, while the energy acceptor was located at 13, 146, 160, 174, 190, 209, 230, 271 or 279 on Tm. FRET analysis showed a distinct Ca(2+)-induced conformational change of the Tm-Tn complex and revealed that TnT60 and TnT73 were closer to Tm13 than Tm279, indicating that the elongated N-terminal region of TnT extends beyond the beginning of the next Tm molecule on the actin filament. Using the atomic coordinates of the crystal structures of Tm and the Tn core domain, we searched for the disposition and orientation of these structures by minimizing the deviations of the calculated FRET efficiencies from the observed FRET efficiencies in order to construct atomic models of the Tn-Tm complex with and without bound Ca(2+). In the best-fit models, the Tn core domain is located on residues 160-200 of Tm, with the arrowhead-shaped I-T arm tilting toward the C-terminus of Tm. The angle between the Tm axis and the long axis of TnC is approximately 75 degrees and approximately 85 degrees with and without bound Ca(2+), respectively. The models indicate that the long axis of TnC is perpendicular to the thin filament without bound Ca(2+), and that TnC and the I-T arm tilt toward the filament axis and rotate around the Tm axis by approximately 20 degrees upon Ca(2+) binding.

Transient receptor potential ion channel of the vanilloid type 1 (TRPV1)-dependent pathway, consisting of capsaicin-sensitive tachykininergic primary afferent and myenteric nitrergic neurons, has been suggested to mediate the inhibitory effect of capsaicin on vagally mediated striated muscle contractions in the rat esophagus. In a recent study, similar but also different effects of capsaicin and piperine on TRPV1 were demonstrated. Therefore, this study aimed to compare the effects of these two drugs on vagally induced contractions in the mouse esophagus. Capsaicin and piperine inhibited vagally induced contractions of a thoracic esophageal segment in a concentration-dependent manner. Ruthenium red (10 μM; a non-selective blocker of transient receptor potential cation channels) and SB-366791 (10 μM; a novel selective antagonist of TRPV1) blocked the inhibitory effect of capsaicin but not that of piperine. Piperine inhibited the vagally mediated contractions in esophagi of adult mice neonatally injected with capsaicin, while capsaicin failed to do so. Desensitization of TRPV1 in the mouse esophagus by in vitro pretreatment with capsaicin failed to affect the inhibitory effect of piperine, whereas the piperine effect was cross-desensitized by capsaicin pretreatment in rat and hamster esophagi. Additionally, a tachykinin NK 1 receptor antagonist, L-732,138 (1 μM), as well as a nitric oxide synthase inhibitor, N G-nitro- l -arginine methyl ester ( l -NAME 200 μM), blocked the inhibitory effect of capsaicin but not that of piperine. Taken together, the results suggest that piperine inhibits the vagally mediated striated muscle contraction in the mouse esophagus through its action on a TRPV1-dependent pathway as well as a TRPV1-independent site.

Zebrafish have become a prominent animal model of human disease because of their genetic tractability and rapid development (Lieschke & Currie, 2007). Several muscle diseases have been studied using zebrafish, including muscular dystrophy (Johnson et al., 2013), hypertrophic and dilated cardiomyopathy, nemaline myopathy (Sehnert et al., 2002; Asnani & Peterson, 2014; Telfer et al., 2012) and distal arthrogryposis (Ha et al., 2013). Here we show that zebrafish myofilaments and their assemblies have molecular structures similar to those of higher vertebrates, demonstrating their usefulness in elucidating structural changes due to disease mutations. EM and 3D reconstruction have shown that striated muscle contraction is regulated by Ca2+-induced movement of tropomyosin on thin filaments, which uncovers myosin-binding sites on actin. Using negative staining EM and single particle reconstruction we find that tropomyosin is well resolved in zebrafish native thin filaments and undergoes the same Ca2+-induced movement as seen in other species. Zebrafish thin filaments are therefore good models for studying the impact of thin filament mutations (actin, troponin, tropomyosin, nebulin) on thin filament structure and regulation. Thick filaments are also readily isolated from zebrafish muscle and their 3D reconstruction is similar to that of mammalian filaments (Gonzalez-Sola et al., 2014). We have isolated intact filament assemblies (myofibrils and A-segments) from zebrafish and find that these also closely resemble those of other vertebrates, including mammals, clearly revealing MyBP-C and M-line periodic organization. These are therefore good models for elucidating the structural impact of mutations in myosin, MyBP-C and M-line proteins. We conclude that the structures of zebrafish skeletal muscle thick and thin filaments and their native assemblies closely resemble those of higher vertebrates, making them excellent models for studying the molecular impact of disease-causing mutations on filament structure.

Striated muscle contraction is regulated by an interaction network connecting the effects of troponin, Ca(2+), and myosin-heads to the azimuthal positioning of tropomyosin along thin filaments. Many missense mutations, located at the actin-tropomyosin interface, however, reset the regulatory switching mechanism either by weakening or strengthening residue-specific interactions, leading to hyper- or hypo-contractile pathologies. Here, we compute energy landscapes for the actin-tropomyosin interface and quantify contributions of single amino acid residues to actin-tropomyosin binding. The method is a useful tool to assess effects of actin and tropomyosin mutations, potentially relating initial stages of myopathy to alterations in thin filament stability and regulation. Landscapes for mutant filaments linked to hyper-contractility provide a simple picture that describes a decrease in actin-tropomyosin interaction energy. Destabilizing the blocked (relaxed)-state parallels previously noted enhanced Ca(2+)-sensitivity conferred by these mutants. Energy landscapes also identify post-translational modifications that can rescue regulatory imbalances. For example, cardiomyopathy-associated E62Q tropomyosin mutation weakens actin-tropomyosin interaction, but phosphorylation of neighboring S61 rescues the binding-deficit, results confirmed experimentally by in vitro motility assays. Unlike results on hyper-contractility-related mutants, landscapes for tropomyosin mutants tied to hypo-contractility do not present a straightforward picture. These mutations may affect other components of the regulatory network, e.g., troponin-tropomyosin signaling.

The troponin (Tn) complex regulates the thin filament of striated muscle by transducing [Ca2+] fluctuations into conformational changes. These changes propagate to tropomyosin (Tm), which then assumes a new disposition with respect to actin, reversibly exposing actin's binding sites for the thick filament motor-ATPase (myosin). To date, the structural biology of thin filament regulation has been studied in the context of two equilibrium states corresponding to high (contraction-activated) and low (contraction-inhibited) sarcomeric [Ca2+]. New electron micrographic reconstructions of the thin filament have resolved Tn, actin, and Tm in high and low [Ca2+] states, integrating high-resolution structures of the Tn core, actin, and Tm. The resultant picture of thin filament regulation does not resolve all of the functionally significant portions of troponin I (TnI) or troponin C (TnC). Those regions of Tn have been shown (using NMR relaxation spectroscopy) to undergo conformational fluctuations, rationalizing the absence of these regions from micrograph-based reconstructions. The disordered portions of Tn are, to date, being interpreted within a canonical structure-activity paradigm. Here we present a new mechanism for the regulation of Tn having explicit descriptions of the kinetic pathways of activation and inhibition. Our thesis is that the intrinsic disorder of TnI is mechanistically significant. As the coupling of folding to binding has been shown to confer an inherent kinetic advantage (known as flycasting activity), our thesis accounts for TnI's conformational heterogeneity and known structure-activity relationships in a parsimonious fashion. We integrate recent NMR structures of the C-terminus of TnI and NMR observations of the conformational dynamics of the Tn complex into high-resolution models of the thin filament. Ways of evaluating the mechanism are discussed. The novel conceptual framework presented here prompts new hypotheses regarding the mechanism of pH sensitivity and of pathogenic mutations in troponin.

Most studies of regulation of vertebrate striated muscle contraction have focussed on activation of the thin filament via calcium binding to the troponin-tropomyosin complex. There is accumulating evidence, however, that thick filament activation is an additional regulatory mechanism. Under relaxing conditions, the two heads of a myosin can interact to form an inactive asymmetric off-state, called the interacting-heads motif (IHM). The off-state inhibits cross-bridge formation and cycling by blocking actin binding in one head and ATPase activity in the other. Docking of the IHM onto the thick filament backbone results in the super-relaxed state (SRX) observed in muscle fibres, where the myosin ATPase is strongly inhibited. What regulates formation and disruption of the IHM is largely uncharacterised. Myosin binding protein C, also known as C-protein, is a key sarcomere component, bound at sites spaced by 43 nm in the cross-bridge regions of thick filaments. The arrangement, mode of action, and interactions of C-protein with myosin are poorly understood. In the heart, phosphorylation of C-protein increases the rate of contraction and its ablation disrupts the SRX. To investigate the interaction of human cardiac C-protein with β-cardiac heavy meromyosin (HMM), we have imaged the complex by electron microscopy and single particle averaging. Cardiac HMM in the IHM motif has the characteristic blocked and inhibited head conformations. The fraction of HMM in the IHM motif is increased when dephosphorylated C-protein is bound, suggesting it stabilises the IHM. Characterization of the complex should provide insight into the regulatory and structural roles of the cardiac C-protein/myosin interaction in heart.

Striated muscle contraction is tightly regulated by the binding and release of calcium to troponin, which permits tropomyosin to move across actin exposing myosin binding sites. The process of activation is cooperative; myosins propagate the binding of further myosins. However unlike other cooperative systems that are limited by the constituent number of subunits, regulated actin filaments (RTFs) are continuous and therefore the cooperative unit size (regulatory unit) needs to be functionally defined. To provide molecular details of this cooperative activation process we devised an assay in which RTFs are suspended between pedestals above a microscope slide surface. GFP tagged myosin-S1 (FL-myosin-S1) binding to these RTFs was used to report activation. By measuring the binding of FL-myosin-S1 as a function of [ATP], [Ca2+] and [myosin] we were able to determine the mechanism of activation. Global fitting of these data revealed excellent agreement to a model described by a Ca2+ followed by a myosin induced activation switch, akin to that previously reported (McKillop and Geeves 1993). Our results indicate that there is little cooperativity in Ca2+ binding (nH ∼ 1.15) and the regulatory unit size is ∼ 11. Dynamic observation of FL-myosin-S1 binding and release offers further insight into activation. In sub-optimal activation conditions FL-myosin-S1 binds in clusters that remain active and are capable of diffusing along the RTF. The unbiased nature of their diffusion suggests that there is no propensity for new FL-myosin-S1s to bind and release from the pointed over the barbed end of an active cluster. In addition, we detect binding events consistent with stepwise loading of myosins; however their probability of detachment appears greater than expected for a stochastic process. We discuss the possibility that this coordinated release may be modulated by Tm.

In vitro motility assays with surface-adsorbed myosin motor fragments have given important insights into the molecular physiology and pathology of striated muscle contraction and inspired nanotechnological applications e.g. lab-on-a-chip devices. However, to date neither precise localized control of the motor density nor introduction of nanoscale three-dimensional geometrical constraints has been possible. This hampers studies of cooperative phenomena and realization of three-dimensional (3D) transport systems. Here, we take critical steps to overcome these limitations, using aluminium oxide coated gallium phosphide nanowires as scaffolds for heavy meromyosin (HMM) adsorption. The wires (diameter: 100-200 nm; height < 5 μm) were either positioned vertically in regular arrays or horizontally on surfaces after etching the GaP core to give hollow nanowires. Upon ATP addition, actin filaments were propelled by HMM on top of the arrays, with filaments spanning inter-wire distances up to 1 μm. The filaments also moved up and down vertical nanowires as detected using sub-wavelength light guiding properties of the nanowires. Motility on top of nanowire arrays holds potential for studies of cooperative phenomena e.g. local enhancement of myosin binding along actin filaments upon actomyosin interactions. Here we tested whether the low velocity for long filaments seen at uniformly low motor densities on flat surfaces may be attributed to loss of cooperative enhancement of myosin head binding locally to actin close to an existing actomyosin cross-bridge. Velocity data showing 1/3 the velocity with 1 μm compared to 300 nm inter-wire spacing, in both cases with ∼90 HMM molecules per 150 nm wide wire tip (giving high local motor density), argue against this idea. We further demonstrate HMM propelled transport through hollow nanowires of 80 nm inner diameter. Uses of hollow nanowires in fundamental studies of actomyosin and in nanotechnological applications will be considered.

Troponin T (TnT) is an essential protein in the transduction of the Ca 2+ -binding signal that triggers striated muscle contraction. Functional diversity among various TnT isoforms found in cardiac and skeletal muscles has been correlated with the sequence heterogeneity at the amino (N-) and the carboxyl (C-) terminal regions. The most striking difference between cardiac TnT (cTnT) and skeletal TnT (sTnT) is that cTnT has an extended N-terminus, which is rich in negatively charged amino acids. To investigate the role of this region in cTnT, we deleted the first 76 amino acids in rat cTnT (cTnT 77–289 ) by site-directed mutagenesis. We exchanged the native troponin complex in rat cardiac myofibrillar preparations and detergent skinned cardiac fiber bundles by treatment with excess cTnT or cTnT 77–289 . After reconstituting the cTnT 77–289 containing myofibrils with cardiac troponin I–cardiac troponin C (cTnI–cTnC), the MgATPase activity was 70% of the cTnT treated myofibrils in the relaxed state and 83% of the cTnT treated myofibrils in the maximal Ca 2+ -activated state. These observations were supported by force measurements in which cTnT and cTnT 77–289 were exchanged into skinned fiber bundles. Prior to reconstitution with cTnI–cTnC, the Ca 2+ -independent maximal force developed by the cTnT 77–289 containing fiber was 45% of the force developed by the cTnT containing fiber. After reconstituting with cTnI–cTnC, the Ca 2+ -activated maximal force of the cTnT 77–289 containing fiber was 62% of the force developed by the cTnT containing +cTnI–cTnC reconstituted fiber. In both assays, no significant changes in the normalized Ca 2+ -activity relation or in co-operativity were observed. Fluorescence experiments using pyrene-labeled Tm demonstrated that the binding of cTnT 77–289 to Tm was 3–4 fold stronger than that of cTnT. Our results suggest that strong interactions between cTnT 77–289 and Tm stabilize cardiac myofilaments in a sub-maximally activated state. Our findings also indicate that the N-terminus of cTnT is essential for maximal activation of cardiac myofilaments.

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96–116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95–116, 95–119 or 95–124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117–124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1–153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95–153 are particularly important for anchoring, and residues 95–119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.

Striated muscle contraction is driven by cyclical interaction between myosin-containing thick and actin-containing thin filaments and is regulated by the tropomyosin-troponin complex. In the absence of calcium, troponin constrains tropomyosin in a blocking position where it shields myosin binding sites on actin. Calcium binding to troponin enables azimuthal movement of tropomyosin, which exposes myosin binding sites, and relieves contractile inhibition.Previous models indicated that K326 and K328 on actin form electrostatic contacts with E181 of tropomyosin in an inhibitory state (Li, et al. 2011), and that K328 also contacts E286 of myosin S1 during contraction (Behrmann, et al. 2012). A recent proteomic study showed that K326 and K328 are acetylated in guinea pig cardiac thin filaments (Foster, et al. 2013). Since this post-translational modification would negate the positively charged lysines, and potentially ablate vital ionic interactions between actin and tropomyosin or myosin, we predicted K326 and K328 acetylation would alter muscle performance. We tested this hypothesis in vivo by expressing K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin in Drosophila muscle. Polarized light microscopy of K328Q and K326Q/K328Q indirect flight muscle revealed a severe disturbance to fiber structure, and flight tests confirmed complete loss in flight ability relative to control. Flies harboring only the K326Q mutation, however, had similar fiber structure to control, but nonetheless displayed a significant decrease in flight performance. These results suggest that K326 and K328 play important roles in maintaining proper muscle function. We are currently investigating the effects of the pseudo-acetylated actin residues on Drosophila hearts. Overall, our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in the physiological context of muscle.

Tropomyosin, a coiled-coil dimer, stabilizes actin filaments and is central to the control of calcium-regulated striated muscle contraction. Striated muscle-specific alpha-tropomyosin is the predominant isoform in cardiac muscle, with low levels of beta-tropomyosin restricted to fetal development in the mouse. To understand the functional role of various tropomyosin isoforms during myofilament activation and regulation in the intact sarcomere, we generated transgenic mice that overexpress striated muscle-specific beta-tropomyosin in the adult heart. Our earlier results succinctly demonstrate that overexpression of beta-tropomyosin in the hearts of transgenic mice decreases endogeneous alpha-tropomyosin levels while altering diastolic function of the myocardium. To explore further the significance of altering the alpha- to beta-tropomyosin isoform ratio in developing murine myocardium, we generated transgenic mice which express beta-tropomyosin at high levels in the heart. The data show that higher levels of beta-tropomyosin expression are lethal with death ensuing between 10-14 days postnatally. A detailed histological analysis demonstrates that the hearts of these mice exhibit several pathological abnormalities, including thrombus formation in the lumen of both atria and in the subendocardium of the left ventricle. Other changes include atrial enlargement and fibrosis, and diffuse myocytolysis, Physiological analyses using ventricular muscle strip preparations from these mice reveal that both myocardial contraction and relaxation parameters are severely impaired. Thus, these results firmly demonstrate an essential difference in tropomyosin isoform function in physiologically regulating cardiac performance.

Troponin I (TnI) is a constituent protein of the troponin complex located on the thin filament of striated muscle that provides a calcium-sensitive switch for striated muscle contraction. Unlike other contractile proteins, the cardiac isoform of troponin I (TnIc) is expressed only in cardiac muscle and therefore offers a model for cardiac-specific expression. It is also subject to developmental regulation with increased expression occurring at the time of birth. Here we describe the isolation and characterization of the human TnIc gene (HGMW-approved symbol TNNI3) and its promoter. The gene comprises eight exons contained within 6.2 kb of genomic DNA. The proximal promoter and 1.1-kb 5'-flanking region were sequenced, and several putative cis-acting elements that are conserved between the human and the mouse TnIc genes were identified. In addition, multiple copies of a 37-bp chromosome 19-specific mini-satellite sequence were identified within this region. Following transfection, 2300 bp of 5' sequence is active in both cardiac myocytes and skeletal muscle cells but is inactive in fibroblasts, indicating that it can drive expression but is insufficient to confer cardiac specificity.

Troponin (Tn), containing three subunits: Ca2+ binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT), plays a crucial role in the Ca2+ regulation of vertebrate striated muscle contraction. These three subunits function by interacting with each other and with the other thin filament proteins. Previous studies suggested that the primary role of TnT is to anchor the TnI.TnC complex to the thin filament, primarily through its interactions with TnI and tropomyosin. We propose here a new role for TnT. Our results indicate that, when TnT is combined with the TnI.TnC complex, there is an activation of actomyosin ATPase that is Ca(2+)-dependent. To determine whether the latter results from a direct effect of TnC on TnT or indirectly from an effect of TnC on TnI which is transmitted to TnT, we prepared a deletion mutant (deletion of residues 1-57) of TnI, TnId57 (Sheng et al. (1992) J. Biol. Chem. 267, 25407-25413), which interacts with TnC but not TnT. Both wild type (TnI.TnC.TnT) and mutant (TnId57.TnC.TnT) Tn complexes demonstrated equivalent activity in the Ca2+ regulation of actomyosin-S1 ATPase activity. Similarly, both TnI and TnId57 could equally reconstitute TnI-depleted skinned muscle fibers. Therefore, since TnId57 does not interact with TnT, these results suggest that TnT reconstitutes native Ca2+ sensitivity via direct interaction with TnC. Thus Ca2+ binding to TnC would have a dual role: 1) release of the ATPase inhibition by TnI and 2) activation of the ATPase through interaction with TnT.

In past decades, several investigators have characterized deficiencies in striated muscle contraction in murine models of Duchenne muscular dystrophy. We have identified in multiple studies potential beneficial therapeutic combinations of ACE inhibitors and anti-mineralocorticoids. There has been an almost exclusive focus on the force deficits in tetanic contractions of extensor digitorum longus (EDL) and diaphragm muscles from these models, as well as their susceptibility to fatigue from repeated contractions. However the force of contraction is not of sole importance. The kinetics of twitch contractions and baseline tension both are critically important parameters that can impact dysfunction. Here we show EDL and diaphragm muscles from dystrophin deficient (mdx) mice have significantly slower kinetic parameters of contraction and relaxation than their wild-type counterparts. We have also found that treatment with Lisinopril and Spironolactone can significantly alter these parameters, and in some cases, back to near-wild type values. Additionally, we investigated kinetic changes in another dystrophin deficient model that is also haplosufficient for compensating utrophin treated with Eplerenone. By calculating the impulse of twitch contractions, we can not only determine work being performed, but also the magnitude of previously mentioned kinetic parameters on this computation. We illustrate that the kinetic information in contractile measurements can further elucidate our understanding of both underlying pathology, as well as serve as quantitative markers of treatment effectiveness.

Striated muscle contraction is governed by the release of Ca2+ from the sarcoplasmic reticulum via the sarcomeric calcium sensor, the troponin complex. A trimer consisting of troponins T, I, and C, the complex undergoes calcium-dependent conformational changes that regulate the accessibility of myosin binding sites along actin filaments. We used a high throughput screen to identify compounds that activate the ATPase activity of skinned fast skeletal myofibrils; optimization of the initial hit compounds has resulted in compounds with improved potency and medicinal chemical properties. The most advanced exemplar of this chemical series, CK-2017357, shifts the calcium sensitivity of detergent-skinned fast skeletal myofibrils by >10-fold in a concentration dependent manner. This compound specifically activates fast skeletal myofibrils, with no effect on either slow skeletal or cardiac myofibrils. A reconstituted sarcomere assay using combinations of fast skeletal, slow skeletal, and cardiac components demonstrates that the activity of CK-2017357 requires the presence of fast skeletal troponin. Isothermal titration calorimetry indicates the compound binds directly to fast skeletal troponin with a sub-micromolar dissociation constant, while experiments with the fluorescent calcium chelator Quin-2 demonstrate that CK-2017357 slows calcium dissociation from troponin. Consistent with this ability to stabilize the calcium-troponin complex, CK-2017357 increases sub-maximal force development in vitro and in vivo, suggesting this mechanism may increase power or strength in diseases where muscle function is compromised due to injury, disease or age.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. In the present study, we investigated the sites of interaction between the N-terminal regulatory domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57) that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone (BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis. The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121. Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a complex with TnI, TnC adopts a more compact conformation than in the crystal structure.

Troponin I (TnI) is a myofibrillar protein involved in the calcium-mediated regulation of striated muscle contraction. Three isoforms of TnI are known and each is expressed in a muscle fiber-type-specific manner. TnI-fast and TnI-slow are expressed exclusively in fast-twitch and slow-twitch skeletal muscle myofibers, respectively, while a third isoform, TnI-card, is expressed in both the atrium and the ventricle of the heart. An explanation of the myofiber-type-restricted expression of the troponin I multigene family will further aid in understanding how various types of striated muscle fibers are established. To initiate the study of TnI isoform gene expression, we have isolated a full-length cDNA representing the human slow-twitch skeletal muscle isoform of troponin I. Sequence comparisons demonstrate that the TnI-slow protein is highly conserved between species. Therefore, the cDNA was used as a probe to investigate the tissue-specific and developmental regulation of the TnI-slow gene in both rodent and human myogenic cells. TnI-slow message appears to be restricted to muscle tissue containing slow-twitch skeletal muscle myofibers. TnI-slow gene expression is induced in differentiated cultures of primary human muscle cells and several (but not all) myogenic cell lines. In addition, a human-specific probe prepared from the 3′ untranslated region of the cDNA has been used to probe a panel of human/mouse somatic cell hybrid lines, resulting in the assignment of the human TnI-slow gene to the q12→qter region of chromosome 1. The locus is designated TNNI1.

Muscle contraction results from a series of molecular events that involve transient interactions between myosin-containing thick and actin-containing thin filaments. The high affinity of myosin for actin suggests that without regulation, muscle would remain in a continuous state of contraction. Regulation of striated muscle contraction is primarily achieved by Ca2+-dependent modulation of myosin crossbridge cycling on actin by the thin filament (TF) troponin-tropomyosin complex. Alterations in various subunits of the complex trigger contractile dysregulation and myopathy. For example, point mutations located over a span of ten amino acids (130-39) of human cardiac troponin T (cTnT) are associated with distinct cardiomyopathic responses. The Drosophila up101 (E88K) mutation localizes to the end of this well-conserved region of TnT. Here we examined the consequences of the lesion on distinct muscle types from the fly. Cardiac performance and morphology were assessed using direct immersion DIC optics, high-speed video imaging and motion analysis. Relative to controls, up101 hearts displayed a phenotype reminiscent of human restrictive cardiomyopathy. End-diastolic and end-systolic dimensions and percent fractional shortening were significantly reduced. Furthermore systolic intervals were significantly prolonged. This suggests TF dysregulation initiates excessive periods of force production and diastolic dysfunction. To characterize the fundamental cause of cardiac remodeling, we isolated TFs from the indirect flight muscles (IFM). The constitutively expressed up101 mutation results in flightlessness due to severe IFM hypercontraction. Electron microscopy and three-dimensional reconstruction of IFM TFs revealed Ca+2-dependent tropomyosin movement, typical of control filaments, was not a general feature of the TnT mutants. The vast majority of Ca+2-free mutant TFs exhibited tropomyosin associated with successive actin monomers over actin subdomains 3 and 4, distal to known myosin binding sites. Thus, up101 TnT likely promotes TF dysinhibition and ubiquitous myopathic remodeling.

Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 is localized in the muscle nucleus, and either its full-length or COOH-terminus leads to muscle cell apoptosis. Here, we further explored the mechanism by which it enters the nucleus and promotes cytotoxicity and investigated its role in nuclear signaling pathway. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK). Deleting this domain or substituting lysine and arginine residues (KLAAQK) resulted in a dramatic loss of TnT3 nuclear and nucleolar localization. In contrast, the GATAKGKVGGRWK domain-DsRed construct localized exclusively in the cytoplasm, indicating that a nuclear exporting sequence is possibly localized at the COOH-terminus. Additionally, we identified a classical DNA-binding Leucine Zipper Domain (LZD). Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. shRNA knocking down of TnT3 in C2C12 myotubes down-regulated the expression of calcium channel α1s (CaVα1s) but not β1a subunit. Luciferase activity analysis further verified that knocking down TnT3 in C2C12 muscle cells inhibited while overexpression of TnT3-DsRed in mouse muscle in vivo increased the Cacna1 promoter activity. Additionally, chromatin immunoprecipitation using TnT3 antibody cocktails pulled down Cacna1 promoter fragments from C2C12 myotubes. In contrast, pre-rRNA processing in the nucleolus was not affected by TnT3 depletion. We conclude that TnT3 contains both a nuclear localization signal and a DNA binding domain, which may mediate its nuclear signaling in muscle cell apoptosis and gene expression regulation.

Activation of striated muscle contraction is a cooperative process initiated by calcium binding to troponin C (TnC). This is a solid-phase signal transduction process involving 3 states of thin filament: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interaction, and an fully activated open or M state. Here we evaluated the influence of ionic strength on Tn dissociation in relationship to myofibril thin filament state. The data we obtained showed that ionic strength had a significant influence on Tn dissociation with low ionic strength increasing Tn dissociation rate from non-overlap region (B or C state) and high ionic strength increasing Tn dissociation rate from overlap region (M state) of myofibril thin filament in both absence and presence of calcium. When ionic strength was decreased to ≤ 50 mM, Tn dissociation from non-overlap region became even faster than overlap region in the absence of calcium, suggesting that actin-tropomyosin in the non-overlap region actually changed from the inhibited B state to the C state at low ionic strength. Also, low ionic strength increased myofibril ATPase activity in the absence of calcium and the calcium-sensitivity of ATPase activity. Based on this and our current studies, we propose a refined kinetic scheme for Tn dissociation from myofibril thin filament, which suggests that the regulatory dissociation of TnI from actin-tropomyosin and association to TnC is not necessarily calcium coupled. Low ionic strength favors the B to C state equilibrium towards the C state (Head et al., Eur. J. Biochem, 227: 694) by favoring TnI dissociation from actin and possibly association with TnC in the absence of calcium. This well explains reported inconsistence between ATPase activity and S1 binding to actin under low ionic strength.

Many studies have been conducted analyzing the manometric properties of patients with achalasia, but the striated portion of the esophagus has never been analyzed and is often overlooked. We retrospectively reviewed 120 manometric tracings (20 achalasia, 100 controls) performed between 1994 and 1997 and excluded tracings from patients with chronic cough and nutcracker esophagus. The data were assessed for age, sex, symptoms, duration of symptoms, lower esophageal sphincter pressure, gastroesophageal gradient, upper esophageal sphincter pressure, smooth muscle contraction amplitude and duration, striated muscle contraction amplitude and duration, length from upper esophageal sphincter to maximal striated muscle contraction, and esophageal length. The maximum striated muscle contraction amplitude was significantly decreased in achalasia patients with a median amplitude of 45 mm Hg (range 12-95) vs 76 mm Hg (range 30-210) in the control group (P = 0.002). Although the wave forms were similar, the maximum striated muscle contraction duration and the distance from the upper esophageal sphincter in achalasia patients was not significantly different from controls. The length of the esophagus was significantly longer in achalasia patients with a median value of 25 cm (range 21-30) vs 21 cm (range 17-26) in the control group (P < 0.001). Patients with achalasia have significantly lower maximum striated muscle contraction amplitudes and longer esophagi, but the duration of the contractions and the configuration of the wave forms are not different.

The esophagus is a region with three functional zones: (1) the upper esophageal sphincter; (2) the esophageal body; and (3) the lower esophageal sphincter. Control mechanisms within the central nervous system and peripherally serve to integrate these functional zones in a region where voluntary and involuntary control mechanisms and the activity of two different types of muscle are intimately coordinated. The distal 50 to 60 per cent of the esophagus in humans is entirely smooth muscle. Extrinsic control for esophageal motor function resides in a brainstem swallowing center with an afferent reception system, an efferent system of motor neurones, and a complex organizing or internuncial system of neurones. Sensory information from the esophagus is carried in the vagus nerves, but sensory pathways are also present in sympathetics entering the spinal cord. The vagus nerve receiving fibers both from the nucleus ambiguus and the dorsal motor nucleus innervates the striated and smooth muscle esophagus, respectively, including the sphincters. There is a myenteric nerve plexus in both the striated and smooth muscle segments. In the smooth muscle esophagus, there are two important effector neurones, an excitatory cholinergic neurone, and a nonadrenergic, noncholinergic (NANC) inhibitory neurone. The striated muscle contraction is directed and coordinated by sequential excitation through vagal fibers programmed by the central control mechanism. There are at least four potential control mechanisms for peristalsis in the smooth muscle esophagus: efferent motor fibers programmed by the swallowing center fire sequentially during peristalsis; the intramural neural mechanism can be excited to produce peristalsis near the onset of stimulation or with a delay after termination of stimulation; there is evidence for myogenic propagation of a peristaltic contraction. In humans, swallow-induced peristalsis is cholinergic and appears to result primarily from sequencing and activation of the intramural excitatory cholinergic neurones. Both central and peripheral levels of control are highly integrated to focus on the excitatory cholinergic neurones. It is likely that under normal circumstances, the central control mechanism exerts the dominant influence on these neurones for initiation and coordination of peristalsis in the smooth muscle esophagus. In humans, resting tone in the lower esophageal sphincter is predominantly cholinergic, but this tone is regulated by a balance between many excitatory and inhibitory influences. The relaxation on swallowing is caused by active inhibition of the muscle through NANC inhibitory neurones and cessation of tonic neural excitation to the

Most current textbooks of cell biology and histology use the steric blocking model to describe the protein mechanism by which vertebrate striated muscle contraction is regulated. Evidence accumulated in the past decade, however, reveals the regulation of muscle contraction to be far more complex than this model predicts.

The role of oxygen-derived free radicals in the local control of blood flow (BF) was evaluated in the microcirculation of the hamster cremaster muscle superfused with the enzymatic radical scavengers superoxide dismutase (SOD, 10 μg/ml) and catalase (CAT, 20 μg/ml). A bioassay of the cheek pouch microcirculation, using a previously described fluorescent microscopic technique, verified the in vivo activity of SOD and CAT. Increased superfusate O2 content to 95% O2 was correlated with an arteriolar vasoconstriction of approximately 50% and a 70% reduction in estimated BF, neither of which was affected by SOD-CAT treatment. These data suggest that free radicals do not mediate the vasoactive effect of O2. SOD-CAT did not alter vascular reactivity because a twofold vasodilation and a fourfold BF increase after topical adenosine application and a fivefold vasoconstriction after topical norepinephrine were unaffected by enzyme treatment. A twofold vasodilation accompanied striated muscle contraction, but there was no effect of SOD-CAT. Assuming SOD and CAT are not excluded from the striated muscle interstitial space, we conclude that the release of superoxide radicals into the extracellular space does not participate in local microvascular control of cremaster muscle BF.

The mechanical and thermal properties of Huxley's 1957 sliding filament model for striated muscle contraction are reconsidered, with emphasis on general relationships between two-state models and muscle behaviour. New empirical forms for the rate functions f(x) and g(x) are proposed, which give an improved fit to experiments. A specific procedure, based on the shortening heat, for determining model parameters is described and applied to various choices of rate functions. The change in slope of the tension-velocity curve observed by Katz is shown to require at least one discontinuity in the binding rate f(x), and a general formula is given. The two-state model also gives a symmetric cusp in the ATP-rate vs velocity curve at v = 0. The theory is extended to time-varying situations and applied to transient responses following isometric activation by tetanus (including latency relaxation), and unloaded and after-loaded contractions. The early burst of heat production can also be fitted by assigning appropriate values to the heats of binding and dissociation.

Several properties of the “static” tension-length diagram of striated muscle fibers are interpreted on the assumption that on excitation there is a cross-bonding of contractile elements previous to the longitudinal shortening. An attempt is also made to reconcile the Fenn effect with the notion that the free energy released by metabolism is used to extend the contractile units. This suggested reconciliation is based on the cross-bonding assumption and on the notion that in a tetanus a contractile unit is undergoing a succession of contraction-relaxation cycles.

Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 A) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles. We also show that any increase of intensity on the 59 A and 51 A layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.

Purified troponin prepared according to published procedures was separated into four major protein fractions when chromatographed on DEAE-Sephadex in 6 m urea. The main components in Fractions 1 to 4 had approximate molecular weights of 14,000, 24,000, 35,000, and 21,000, respectively, as determined by sodium dodecyl sulfate gel electrophoresis. Reconstitution experiments using all possible combinations indicated that Fractions 2, 3, and 4 restored the Ca++ requirement for the ATPase activity of actomyosin in the presence of tropomyosin. Recovery of activity could only be achieved when the required components were mixed prior to removal of urea. This suggests that one or more of these proteins may refold improperly on removal of urea unless the other components are present.