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Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at

Striated muscle contraction involves sliding of actin thin filaments along myosin thick filaments, controlled by calcium through thin filament activation. In relaxed muscle, the two heads of myosin interact with each other on the filament surface to form the interacting-heads motif (IHM). A key question is how both heads are released from the surface to approach actin and produce force. We used time-resolved synchrotron X-ray diffraction to study tarantula muscle before and after tetani. The patterns showed that the IHM is present in live relaxed muscle. Tetanic contraction produced only a very small backbone elongation, implying that mechanosensing—proposed in vertebrate muscle—is not of primary importance in tarantula. Rather, thick filament activation results from increases in myosin phosphorylation that release a fraction of heads to produce force, with the remainder staying in the ordered IHM configuration. After the tetanus, the released heads slowly recover toward the resting, helically ordered state. During this time the released heads remain close to actin and can quickly rebind, enhancing the force produced by posttetanic twitches, structurally explaining posttetanic potentiation. Taken together, these results suggest that, in addition to stretch activation in insects, two other mechanisms for thick filament activation have evolved to disrupt the interactions that establish the relaxed helices of IHMs: one in invertebrates, by either regulatory light-chain phosphorylation (as in arthropods) or Ca 2+ -binding (in mollusks, lacking phosphorylation), and another in vertebrates, by mechanosensing.

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction., The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation is located near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here the authors assessed M305L actin in vivo, in vitro, and in silico to characterize emergent pathological properties and define the mechanistic basis of disease.

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.

Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.

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.

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which enables tropomyosin to expose myosin-binding sites on actin. Myosin binding holds tropomyosin in an open position, exposing more myosin-binding sites on actin, leading to cooperative activation. At lower calcium levels, troponin and tropomyosin turn off the thin filament; however, this is antagonised by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation, we used single-molecule imaging of green fluorescent protein (GFP)-tagged myosin-S1 (S1-GFP) to follow the activation of RTF tightropes. In sub-maximal activation conditions, RTFs are not fully active, enabling direct observation of deactivation in real time. We observed that myosin binding occurs in a stochastic step-wise fashion; however, an unexpectedly large probability of multiple contemporaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated active process.

Since the discovery of muscle in the 19th century, myosins as molecular motors have been extensively studied. However, in the last decade, a new functional super-relaxed (SRX) state of myosin has been discovered, which has a 10-fold slower ATP turnover rate than the already-known non-actin-bound, disordered relaxed (DRX) state. These two states are in dynamic equilibrium under resting muscle conditions and are thought to be significant contributors to adaptive thermogenesis in skeletal muscle and can act as a reserve pool that may be recruited when there is a sustained demand for increased cardiac muscle power. This report provides an evolutionary perspective of how striated muscle contraction is regulated by modulating this myosin DRX↔SRX state equilibrium. We further discuss this equilibrium with respect to different physiological and pathophysiological perturbations, including insults causing hypertrophic cardiomyopathy, and small-molecule effectors that modulate muscle contractility in diseased pathology.

Treatment options for denervated skeletal muscle atrophy are limited, in part because the underlying molecular mechanisms are not well understood. Unlike previous transcriptomics studies conducted in rodent models of peripheral nerve injury, in the present study, we performed high-throughput sequencing with denervated atrophic biceps muscle and normal (non-denervated) sternocleidomastoid muscle samples obtained from four brachial plexus injury (BPI) patients. We also investigated whether Ficus carica L. (FCL.) extract can suppress denervated muscle atrophy in a mouse model, along with the mechanism of action. We identified 1471 genes that were differentially expressed between clinical specimens of atrophic and normal muscle, including 771 that were downregulated and 700 that were upregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed genes were mainly enriched in the GO terms “structural constituent of muscle,” “Z disc,” “M band,” and “striated muscle contraction,” as well as “Cell adhesion molecules,” “Glycolysis/Gluconeogenesis,” “Peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway,” and “P53 signaling pathway.” In experiments using mice, the reduction in wet weight and myofiber diameter in denervated muscle was improved by FCL. extract compared to saline administration, which was accompanied by downregulation of the proinflammatory cytokines interleukin (IL)-1β and IL-6. Moreover, although both denervated groups showed increased nuclear factor (NF)-κB activation and PPARα expression, the degree of NF-κB activation was lower while PPARα and inhibitor of NF-κB IκBα expression was higher in FCL. extract-treated mice. Thus, FCL. extract suppresses denervation-induced inflammation and attenuates muscle atrophy by enhancing PPARα expression and inhibiting NF-κB activation. These findings suggest that FCL. extract has therapeutic potential for preventing denervation-induced muscle atrophy caused by peripheral nerve injury or disease.

Pharmacology research & perspectives 8(5), - (2020). doi:10.1002/prp2.663, Published by Wiley, Chichester [u.a.]

Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, "relaxed" muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing "relaxed" population, and a slower-hydrolyzing "super relaxed" (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.

Striated muscle contraction is initiated by Ca2+ binding to, and activating, thin filament regulatory units (RU) within the sarcomere, which then allows myosin cross-bridges from the opposing thick filament to bind actin and generate force. The amount of overlap between the filaments dictates how many potential cross-bridges are capable of binding, and thus how force is generated by the sarcomere. Myopathies and atrophy can impair muscle function by limiting cross-bridge interactions between the filaments, which can occur when the length of the thin filament is reduced or when RU function is disrupted. To investigate how variations in thin filament length and RU density affect ensemble cross-bridge behavior and force production, we simulated muscle contraction using a spatially explicit computational model of the half-sarcomere. Thin filament RUs were disabled either uniformly from the pointed end of the filament (to model shorter thin filament length) or randomly throughout the length of the half-sarcomere. Both uniform and random RU ‘knockout’ schemes decreased overall force generation during maximal and submaximal activation. The random knockout scheme also led to decreased calcium sensitivity and cooperativity of the force-pCa relationship. We also found that the rate of force development slowed with the random RU knockout, compared to the uniform RU knockout or conditions of normal RU activation. These findings imply that the relationship between RU density and force production within the sarcomere involves more complex coordination than simply the raw number of RUs available for myosin cross-bridge binding, and that the spatial pattern in which activatable RU are distributed throughout the sarcomere influences the dynamics of force production.

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.

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.

Based on the framework of sliding-filament theory and on the cross-bridges dynamics, a mathematical model for the simulation of the force response and length change of individual myofibril is presented. The myofibril is modeled as a group of segments placed in series, each segment represents a half-sarcomere with active and elastic properties. A multiple-state cross-bridge formalism relates the half Sarcomere force to the chemical kinetics of ATP hydrolysis. The corresponding system of nonlinear nonlocal partial differential equations of the model is analyzed. A numerical approach is introduced and some numerical tests are performed. The proposed in-silico model enables the study of biologically relevant process in the muscle contraction process, also in the case of muscular diseases, with reasonable computational effort.

Many genetic mutations in sarcomeric proteins, including the cardiac myosin regulatory light chain (RLC) encoded by the MYL2 gene, have been implicated in familial cardiomyopathies. Yet, the molecular mechanisms by which these mutant proteins regulate cardiac muscle mechanics in health and disease remain poorly understood. Evidence has been accumulating that RLC phosphorylation has an influential role in striated muscle contraction and, in addition to the conventional modulation via Ca2+ binding to troponin C, it can regulate cardiac muscle function. In this review, we focus on RLC mutations that have been reported to cause cardiomyopathy phenotypes via compromised RLC phosphorylation and elaborate on pseudo-phosphorylation rescue mechanisms. This new methodology has been discussed as an emerging exploratory tool to understand the role of phosphorylation as well as a genetic modality to prevent/rescue cardiomyopathy phenotypes. Finally, we summarize structural effects post-phosphorylation, a phenomenon that leads to an ordered shift in the myosin S1 and RLC conformational equilibrium between two distinct states.

As an important secretory organ, skeletal muscle has drawn attention as a potential target tissue for type 2 diabetic mellitus (T2DM). Recent peptidomics approaches have been applied to identify secreted peptides with potential bioactive. However, comprehensive analysis of the secreted peptides from skeletal muscle tissues of db/db mice and elucidation of their possible roles in insulin resistance remains poorly characterized. Here, we adopted a label-free discovery using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology and identified 63 peptides (42 up-regulated peptides and 21 down-regulated peptides) differentially secreted from cultured skeletal muscle tissues of db/db mice. Analysis of relative molecular mass (Mr), isoelectric point (pI) and distribution of Mr vs pI of differentially secreted peptides presented the general feature. Furthermore, Gene ontology (GO) and pathway analyses for the parent proteins made a comprehensive functional assessment of these differential peptides, indicating the enrichment in glycolysis/gluconeogenesis and striated muscle contraction processes. Intercellular location analysis pointed out most precursor proteins of peptides were cytoplasmic or cytoskeletal. Additionally, cleavage site analysis revealed that Lysine (N-terminal)-Alanine (C-terminal) and Lysine (N-terminal)-Leucine (C-terminal) represents the preferred cleavage sites for identified peptides and proceeding peptides respectively. Mapped to the precursors' sequences, most identified peptides were observed cleaved from creatine kinase m-type (KCRM) and fructose-bisphosphate aldolase A (Aldo A). Based on UniProt and Pfam database for specific domain structure or motif, 44 peptides out of total were positioned in the functional motif or domain from their parent proteins. Using C2C12 myotubes as cell model in vitro, we found several candidate peptides displayed promotive or inhibitory effects on insulin and mitochondrial-related pathways by an autocrine manner. Taken together, this study will encourage us to investigate the biologic functions and the potential regulatory mechanism of these secreted peptides from skeletal muscle tissues, thus representing a promising strategy to treat insulin resistance as well as the associated metabolic disorders.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease characterized by cortical and spinal motor neuron degeneration, some inherited cases of which are caused by mutations in the gene coding for copper-zinc superoxide dismutase-1 (SOD1). The SOD1G93A mutant model mouse, which expresses large amounts of mutant SOD1, develops adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis. We used the Tandem Mass Tag technique to investigate the proteome profile of hippocampus, cerebral cortex, and medulla oblongata of the SOD1G93A mutant model mice as compared with that of wild-type (WT) mice. Fifteen proteins were significantly increased or decreased (i.e., changed) in all three tissues. Gene ontology analysis revealed that the changed proteins were mainly enriched in negative regulation of reactive oxygen species, myosin complex and copper ion binding. In the Striated Muscle Contraction Pathway, most of the identified proteins were decreased in the SOD1G93A mice compared with the WT mice. Myosin-1 (MYH1), fructose-2,6-bisphosphatase TIGAR (TIGAR), and sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) were significantly reduced in mutant vs WT mice, as confirmed by Western blot analysis. Since myosins and tropomyosins are specific for synapse function and drive actin dynamics in the maturation of dendritic spines, changes in these proteins may contribute to perturbations of brain neuronal circuitry in addition to spinal motor neuron disease.

Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in the sarcomere and generate pulling forces. This process is regulated by calcium, and it can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations (e.g., β-adrenergic stimulation), and pharmacological interventions. Therefore, it is important to have a methodology to robustly determine the impact of these perturbations and statistically evaluate their effects. Here, we present an approach to measure the equilibrium constants that govern muscle activation, estimate uncertainty in these parameters, and statistically test the effects of perturbations. We provide a MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials that make this approach accessible. The hypothesis testing and error estimation approaches described here are broadly applicable, and the provided tools work with other types of data, including cellular measurements. To demonstrate the utility of the approach, we apply it to elucidate the biophysical mechanism of a mutation that causes familial hypertrophic cardiomyopathy. This approach is generally useful for studying muscle diseases and therapeutic interventions that target muscle contraction.

Striated muscle contraction occurs when myosin thick filaments bind to thin filaments in the sarcomere and generate pulling forces. This process is regulated by calcium, and it can be perturbed by pathological conditions (e.g., myopathies), physiological adaptations (e.g., β-adrenergic stimulation), and pharmacological interventions. Therefore, it is important to have a methodology to robustly determine the mechanism of these perturbations and statistically evaluate their effects. Here, we present an approach to measure the equilibrium constants that govern muscle activation, estimate uncertainty in these parameters, and statistically test the effects of perturbations. We provide a MATLAB-based computational tool for these analyses, along with easy-to-follow tutorials that make this approach accessible. The hypothesis testing and error estimation approaches described here are broadly applicable, and the provided tools work with other types of data, including cellular measurements. To demonstrate the utility of the approach, we apply it to determine the biophysical mechanism of a mutation that causes familial hypertrophic cardiomyopathy. This approach is generally useful for studying the mechanisms of muscle diseases and therapeutic interventions that target muscle contraction.

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.

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.

Striated muscle contraction is regulated by Ca(2+)-dependent modulation of myosin cross-bridge binding to F-actin by the thin filament troponin (Tn)-tropomyosin (Tm) complex. In the absence of Ca(2+), Tn binds to actin and constrains Tm to an azimuthal location where it sterically occludes myosin binding sites along the thin filament surface. This limits force production and promotes muscle relaxation. In addition to Tn-actin interactions, inhibitory Tm positioning requires associations between other thin filament constituents. For example, the actin “A-triad”, composed of residues K326, K328, and R147, forms numerous, highly favorable electrostatic contacts with Tm that are critical for establishing its inhibitory azimuthal binding position. Here, we review recent findings, including the identification and interrogation of modifications within and proximal to the A-triad that are associated with disease and/or altered muscle behavior, which highlight the surface feature’s role in F-actin-Tm interactions and contractile regulation.

Doxorubicin (DOX) is a chemotherapeutic agent of which the medical use is limited due to cardiotoxicity. While acute cardiotoxicity is reversible, chronic cardiotoxicity is persistent or progressive, dose-dependent and irreversible. While DOX mechanisms of action are not fully understood yet, 3 toxicity processes are known to occur in vivo: cardiomyocyte dysfunction, mitochondrial dysfunction and cell death. We present an in vitro experimental design aimed at detecting DOX-induced cardiotoxicity by obtaining a global view of the induced molecular mechanisms through RNA-sequencing. To better reflect the in vivo situation, human 3D cardiac micro tissues were exposed to physiologically-based pharmacokinetic (PBPK) relevant doses of DOX for 2 weeks. We analysed a therapeutic and a toxic dosing profile. Transcriptomics analysis revealed significant gene expression changes in pathways related to "striated muscle contraction" and "respiratory electron transport", thus suggesting mitochondrial dysfunction as an underlying mechanism for cardiotoxicity. Furthermore, expression changes in mitochondrial processes differed significantly between the doses. Therapeutic dose reflects processes resembling the phenotype of delayed chronic cardiotoxicity, while toxic doses resembled acute cardiotoxicity. Overall, these results demonstrate the capability of our innovative in vitro approach to detect the three known mechanisms of DOX leading to toxicity, thus suggesting its potential relevance for reflecting the patient situation. Our study also demonstrated the importance of applying physiologically relevant doses during toxicological research, since mechanisms of acute and chronic toxicity differ.

White striping (WS) is one of the most common myopathies identified in broiler chickens leading to substantial production losses, where the incidence reaches 12% in commercial chickens. It occurs primarily in heavier chickens being a modification of the breast muscle characterized by the presence of pale parallel streaks in the same orientation of the muscle fibers. Since the WS etiology remains unclear, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of WS in affected and unaffected chicken breast muscles. A total of 11,177 genes were expressed in the pectoralis major muscle. Out of those, 1,441 genes were differentially expressed (FDR ≤ 0.01) between the two analyzed groups, being, respectively, 772 genes upregulated and 669 downregulated in the WS affected group. A total of 36 significantly overrepresented GO terms related to WS myopathy were enriched, and the most relevant biological processes were activation of immune system, angiogenesis, hypoxia, cell death, and striated muscle contraction. The unbalance of those biological processes may trigger the occurrence of the WS phenotype in broilers. The possible lack of capillary blood supply homogeneously in the muscle triggers the hypoxia, following the activation of glycolysis, calcium signaling and apoptosis related genes facilitating the tissue damage and WS incidence.

Acute ischemic stroke is a significant cause of high morbidity and mortality in the aging population globally. However, current therapeutic strategies for acute ischemic stroke are limited. Atherosclerotic plaque is considered an independent risk factor for acute ischemic stroke. To identify biomarkers for carotid atheromatous plaque, bioinformatics analysis of the gene microarray data of plaque and intact tissue from individuals was performed. Differentially expressed genes (DEGs) were identified using the Multtest and Limma packages of R language, including 56 downregulated and 69 upregulated DEGs. Enriched microRNA (miRNA or miR) DEGs networks were generated using WebGestalt software and the STRING databases, and the miRNAs were validated using serum from acute ischemic stroke patients with reverse transcription quantitative PCR (RT‑qPCR). Four confirmed differentially expressed miRNAs (miR‑9, ‑22, ‑23 and ‑125) were associated with 28 upregulated DEGs, and 7 miRNAs (miR‑9, ‑30, ‑33, ‑124, ‑181, ‑218 and ‑330) were associated with 25 downregulated DEGs. Gene ontology (GO) function suggested that the confirmed miRNA‑targeted DEGs predominantly associated with signal transduction, the circulatory system, biological adhesion, striated muscle contraction, wound healing and the immune system. The confirmed miRNA‑targeted genes identified serve as potential therapeutic targets for acute ischemic stroke.

To determine whether a capsaicin-sensitive local neural circuit constitutively modulates vagal neuromuscular transmission in the esophageal striated muscle or whether the neural circuit operates in a stimulus-dependent manner, we compared the motility of esophageal preparations isolated from intact rats with those in which capsaicin-sensitive neurons had been destroyed. Electrical stimulation of the vagus nerve trunk evoked contractile responses in the esophagus isolated from a capsaicin-treated rat in a manner similar to those in the esophagus from a control rat. No obvious differences were observed in the inhibitory effects of D-tubocurarine on intact and capsaicin-treated rat esophageal motility. Destruction of the capsaicin-sensitive neurons did not significantly affect latency, time to peak and duration of a vagally evoked twitch-like contraction. These findings indicate that the capsaicin-sensitive neural circuit does not operate constitutively but rather is activated in response to an applied stimulus.

Summary Introduction The minimum criterion for the diagnosis of hypertrophic cardiomyopathy (HCM) is thickening of the left ventricular wall, typically in an asymmetrical or focal fashion, and it requires no functional deficit. Using this criterion, we identified a family with four affected individuals and a single unrelated individual essentially with restrictive cardiomyopathy (RCM). Mutations in genes coding for the thin filaments of cardiac muscle have been described in RCM and HCM with ‘restrictive features’. One such gene encodes for cardiac troponin I (TNNI3), a sub-unit of the troponin complex involved in the regulation of striated muscle contraction. We hypothesised that mutations in TNNI3 could underlie this particular phenotype, and we therefore screened TNNI3 for mutations in 115 HCM probands. Methods Clinical investigation involved examination, echocardiography, chest X-ray and an electrocardiogram of both the index cases and close relatives. The study cohort consisted of 113 South African HCM probands, with and without known founder HCM mutations, and 100 ethnically matched control individuals. Mutation screening of TNNI3 for disease-causing mutations were performed using high-resolution melt (HRM) analysis. Results HRM analyses identified three previously described HCM-causing mutations (p.Pro82Ser, p.Arg162Gln, p.Arg170Gln) and a novel exonic variant (p.Leu144His). A previous study involving the same amino acid identified a p.Leu144Gln mutation in a patient presenting with RCM, with clinical features of HCM. We observed the novel p.Leu144His mutation in three siblings with clinical RCM and varying degrees of ventricular hypertrophy. The isolated index case with the de novo p.Arg170Gln mutation presented with a similar phenotype. Both mutations were absent in a healthy control group. Conclusion We have identified a novel disease-causing p.Leu144His mutation and a de novo p.Arg170Gln mutation associated with RCM and focal ventricular hypertrophy, often below the typical diagnostic threshold for HCM. Our study provides information regarding TNNI3 mutations underlying RCM in contrast to other causes of a similar presentation, such as constrictive pericarditis or infiltration of cardiac muscle, all with marked right-sided cardiac manifestations. This study therefore highlights the need for extensive mutation screening of genes encoding for sarcomeric proteins, such as TNNI3 to identify the underlying cause of this particular phenotype.

Muscle contraction results from cyclic interactions between the contractile proteins myosin and actin, driven by the turnover of adenosine triphosphate (ATP). Despite intense studies, several molecular events in the contraction process are poorly understood, including the relationship between force-generation and phosphate-release in the ATP-turnover. Different aspects of the force-generating transition are reflected in the changes in tension development by muscle cells, myofibrils and single molecules upon changes in temperature, altered phosphate concentration, or length perturbations. It has been notoriously difficult to explain all these events within a given theoretical framework and to unequivocally correlate observed events with the atomic structures of the myosin motor. Other incompletely understood issues include the role of the two heads of myosin II and structural changes in the actin filaments as well as the importance of the three-dimensional order. We here review these issues in relation to controversies regarding basic physiological properties of striated muscle. We also briefly consider actomyosin mutation effects in cardiac and skeletal muscle function and the possibility to treat these defects by drugs.

Troponin is a heterotrimeric Ca2+-binding protein that has a well-established role in regulating striated muscle contraction. However, mounting evidence points to novel cellular functions of troponin, with profound implications in cancer, cardiomyopathy pathogenesis and skeletal muscle aging. Here, we highlight the non-canonical roles and aberrant expression patterns of troponin beyond the sarcomeric milieu. Utilizing bioinformatics tools and online databases, we also provide pathway, subcellular localization, and protein-protein/DNA interaction analyses that support a role for troponin in multiple subcellular compartments. This emerging knowledge challenges the conventional view of troponin as a sarcomere-specific protein exclusively involved in muscle contraction and may transform the way we think about sarcomeric proteins, particularly in the context of human disease and aging.

SUMMARY Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder., In Brief Viswanathan et al. demonstrate that the conserved actin A-triad, composed of K326, K328, and R147, normally biases tropomyosin to a position that impedes actomyosin associations along resting striated muscle thin filaments. The proximally located actin A295S hypertrophic cardiomyopathy mutation distorts A-triad-tropomyosin associations, which promotes contractile disinhibition, hypercontraction, and disease pathogenesis.

Normal pregnancy requires adaptations of the maternal vasculature. During preeclampsia these adaptations are not well established, which may be related to maternal hypertension and proteinuria. The effects of preeclampsia on the maternal vasculature are not yet fully understood. We aimed to evaluate gene expression in aortas of pregnant rats with experimental preeclampsia using a genome wide microarray. Aortas were isolated from pregnant Wistar outbred rats with low-dose LPS-induced preeclampsia (ExpPE), healthy pregnant (Pr), non-pregnant and low-dose LPS-infused non-pregnant rats. Gene expression was measured by microarray and validated by real-time quantitative PCR. Gene Set Enrichment Analysis was performed to compare the groups. Functional analysis of the aorta was done by isotonic contraction measurements while stimulating aortic rings with potassium chloride. 526 genes were differentially expressed, and positive enrichment of “potassium channels”, “striated muscle contraction”, and “neuronal system” gene sets were found in ExpPE vs. Pr. The potassium chloride-induced contractile response of ExpPE aortic rings was significantly decreased compared to this response in Pr animals. Our data suggest that potassium channels, neuronal system and (striated) muscle contraction in the aorta may play a role in the pathophysiology of experimental preeclampsia. Whether these changes are also present in preeclamptic women needs further investigation.

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.

Troponin, a Ca(2+)-dependent regulator of striated muscle contraction, has been characterized in vertebrates, protochordates (amphioxus and ascidian), and many invertebrate animals that are categorized in protostomes, but it has not been detected in echinoderms, such as sea urchin and sea cucumber, members of subphylum Eleutherozoa. In this study, we examined the muscle of a species of isocrinid sea lilies, a member of subphylum Pelmatozoa, that constitute the most basal group of extant echinoderms to clarify whether troponin is lacking from the early evolution of echinoderms. Native thin filaments were released from the muscle homogenates in a relaxing buffer containing ATP and EGTA, a Ca(2+)-chelator, and were collected by ultra-centrifugation. Actin and tropomyosin, but not a troponin-like protein, were detected in the filament preparation. The filaments increased Mg(2+)-ATPase activity of rabbit skeletal muscle myosin irrespective of the presence or absence of Ca(2+). The results indicate that Ca(2+)-sensitive factor, troponin, is lacking in the thin filaments of sea lily muscle as in those of the other echinoderms, sea urchin and sea cucumber. On the other hand, a paramyosin-like protein that is absent from chordates was detected in sea lily muscle as in the muscles of the other echinoderms and invertebrate animals of protostomes.

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 C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Troponin T (TnT) plays a major role in striated muscle contraction. We recently demonstrated that the fast skeletal muscle TnT3 isoform 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. Amino acid truncation and substitution showed that its COOH-terminus contains a dominant nuclear/nucleolar localization sequence (KLKRQK) and the basic lysine and arginine residues might play an important role in the nuclear retention and nucleolar enrichment of KLKRQK-DsRed fusion proteins. 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 in this region. Additionally, we identified a classical DNA-binding Leucine Zipper Domain (LZD) which is conserved among TnT isoforms and species. Deletion of LZD or KLKRQK sequence significantly reduced cell apoptosis compared to full-length TnT3. We conclude that TnT3 contains both a nuclear localization signal and a DNA binding domain, which may mediate nuclear/nucleolar signaling and muscle cell apoptosis.

Troponin T (TnT) is the tropomyosin (Tm)-binding and thin filament-anchoring subunit of troponin and plays a central role in striated muscle contraction. A nonsense mutation in exon 11 of the TNNT1 gene encoding slow skeletal muscle troponin T (ssTnT) truncating the polypeptide chain at Glu(180) causes a lethal recessive nemaline myopathy (NM) in the Amish (ANM). More TNNT1 NM mutations have been reported recently with similar recessive phenotypes. A nonsense mutation in exon 9 causes truncation at Ser(108), and a splicing site mutation causes truncation at Leu(203). Another splicing site mutation causes an internal deletion of the 39 exon 8-encoded amino acids. We engineered and characterized these ssTnT mutants to demonstrate that the Ser(108) truncation exhibits a Tm binding affinity lower than that of the ANM Glu(180) truncation, indicating a partial loss of Tm-binding site 1. Despite the presence of Tm-binding sites 1 and 2, ssTnT truncated at Leu(203) binds Tm with decreased affinity, consistent with its recessive NM phenotype and the requirement of troponin complex formation for high-affinity binding of TnT to Tm. The exon 8-deleted ssTnT has a partial loss of Tm-binding site 1 but retains high-affinity Tm-binding site 2. However, exon 8-deleted ssTnT exhibits a dramatically diminished Tm binding affinity, indicating a long-range conformational effect of this middle region deletion. Predicted from the TnT structure-function relationship, removal of the N-terminal variable region partially rescued this negative impact. These novel findings lay a foundation for understanding the pathogenesis of TNNT1 myopathies and provide insights into the development of targeted treatment.

End-to-end connections between adjacent tropomyosin molecules along the muscle thin filament allow long-range conformational rearrangement of the multicomponent filament structure. This process is influenced by Ca(2+) and the troponin regulatory complexes, as well as by myosin crossbridge heads that bind to and activate the filament. Access of myosin crossbridges onto actin is gated by tropomyosin, and in the case of striated muscle filaments, troponin acts as a gatekeeper. The resulting tropomyosin-troponin-myosin on-off switching mechanism that controls muscle contractility is a complex cooperative and dynamic system with highly nonlinear behavior. Here, we review key information that leads us to view tropomyosin as central to the communication pathway that coordinates the multifaceted effectors that modulate and tune striated muscle contraction. We posit that an understanding of this communication pathway provides a framework for more in-depth mechanistic characterization of myopathy-associated mutational perturbations currently under investigation by many research groups.

Striated muscle contraction is regulated primarily through the action of tropomyosin and troponin that are bound to actin. Activation requires Ca(2+) binding to troponin and/or binding of high affinity myosin complexes to actin. Mutations within components of the regulatory complex may lead to familial cardiomyopathies and myopathies. In several cases examined, either physiological or pathological changes in troponin alter the distribution among states of actin-tropomyosin-troponin that differ in their abilities to stimulate myosin ATPase activity. These observations open possibilities for managing disorders of the troponin complex. Furthermore, analyses of mutant forms of troponin give insights into the regulation of striated muscle contraction.

Actin-myosin interactions provide the driving force underlying each heartbeat. The current view is that actin-bound regulatory proteins play a dominant role in the activation of calcium-dependent cardiac muscle contraction. In contrast, the relevance and nature of regulation by myosin regulatory proteins (for example, myosin light chain-2 [MLC2]) in cardiac muscle remain poorly understood. By integrating gene-targeted mouse and computational models, we have identified an indispensable role for ventricular Mlc2 (Mlc2v) phosphorylation in regulating cardiac muscle contraction. Cardiac myosin cycling kinetics, which directly control actin-myosin interactions, were directly affected, but surprisingly, Mlc2v phosphorylation also fed back to cooperatively influence calcium-dependent activation of the thin filament. Loss of these mechanisms produced early defects in the rate of cardiac muscle twitch relaxation and ventricular torsion. Strikingly, these defects preceded the left ventricular dysfunction of heart disease and failure in a mouse model with nonphosphorylatable Mlc2v. Thus, there is a direct and early role for Mlc2 phosphorylation in regulating actin-myosin interactions in striated muscle contraction, and dephosphorylation of Mlc2 or loss of these mechanisms can play a critical role in heart failure.

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.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10-12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

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.

ca2+ sensitivity of striated muscle contraction is modulated by a number of factors, the mechanism by which force is altered by Ca2+ appears to be complex. If we focus on the level of the regulatory, heterotrimeric troponin complex (Tn = TnC·TnI·TnT), not only an altered Ca2+ affinity of troponin

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.