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The molecular motor myosin is post‐translationally modified in its globular head, its S2 hinge, and its thick filament domain during human skeletal muscle aging. To determine the importance of such modifications, we performed an integrative analysis of transgenic Drosophila melanogaster expressing myosin containing post‐translational modification mimic mutations. We determined effects on muscle function, myofibril structure, and myosin biochemistry. Modifications in the homozygous state decreased jump muscle function by a third at 3 weeks of age and reduced indirect flight muscle function to negligible levels in young flies, with severe effects on flight muscle myofibril assembly and/or maintenance. Expression of mimic mutations in the heterozygous state or in a wild‐type background yielded significant, but less severe, age‐dependent effects upon flight muscle structure and function. Modification of the residue in the globular head disabled ATPase activity and in vitro actin filament motility, whereas the S2 hinge mutation reduced actin‐activated ATPase activity by 30%. The rod modification diminished filament formation in vitro. The latter mutation also reduced proteostasis, as demonstrated by enhanced accumulation of polyubiquitinated proteins. Overall, we find that mutation of amino acids at sites that are chemically modified during human skeletal muscle aging can disrupt myosin ATPase, myosin filament formation, and/or proteostasis, providing a mechanistic basis for the observed muscle defects. We conclude that age‐specific post‐translational modifications present in human skeletal muscle are likely to act in a dominant fashion to affect muscle structure and function and may therefore be implicated in degeneration and dysfunction associated with sarcopenia. [ABSTRACT FROM AUTHOR]

Considering the occurrence of serious heart failure in a gene knockout mouse of PIP5Kγ and in congenital abnormal cases in humans in which the gene was defective as reported by others, the present study attempted to localize PIP5Kγ in the heart during prenatal stages. It was done on the basis of the supposition that phenotypes caused by gene mutation of a given molecule are owed to the functional deterioration of selective cellular sites normally expressing it at significantly higher levels in wild mice. PIP5Kγ‐immunoreactivity was the highest in the heart at E10 in contrast to almost non‐significant levels of the immunoreactivity in surrounding organs and tissues such as liver. The immunoreactivity gradually weakened in the heart with the prenatal age, and it was at non‐significant levels at newborn and postnatal stages. Six patterns in localization of distinct immunoreactivity for PIP5Kγ were recognized in cardiomyocytes: (1) its localization on the plasma membranes and subjacent cytoplasm without association with short myofibrils and (2) its localization on them as well as short myofibrils in association with them in cardiomyocytes of early differentiation at E10; (3) its spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E10; (4) rare occurrences of such spot‐like localization along long myofibrils in cardiomyocytes of advanced differentiation at E14; (5) its localization at Z‐bands of long myofibrils; and (6) its localization at intercellular junctions including the intercalated discs in cardiomyocytes of advanced differentiation at E10 and E14, especially dominant at the latter stage. No distinct localization of PIP5Kγ‐immunoreactivity of any patterns was seen in the heart at E18 and P1D. The present finding suggests that sites of PIP5Kγ‐appearance and probably of its high activity in cardiomyocytes are shifted from the plasma membranes through short myofibrils subjacent to the plasma membranes and long myofibrils, to Z‐bands as well as to the intercalated discs during the mid‐term gestation. It is further suggested that PIP5Kγ is involved in the differentiation of myofibrils as well as intercellular junctions including the intercalated discs at later stages of the mid‐term gestation. Failures in its involvement in the differentiation of these structural components are thus likely to cause the mid‐term gestation lethality of the mutant mice for PIP5Kγ. [ABSTRACT FROM AUTHOR]

Bali cattle (Bos javanicus) are a local breed from Indonesia with the potential for beef production, but its meat has been reported to be tough. This indicates that there is a need to develop various methods that can be used for assessment to improve the quality of the meat. One such method is muscle scoring (MS), which is often used to predict the amount of meat in the carcass from live animals, but the muscle microstructure contributing to the quality is often neglected. Therefore, this study aims to evaluate the correlation between the muscle score of Bali cattle and the quality of muscle microstructure in terms of myofibril morphometric and connective tissue composition using a histochemistry approach. The population consisted of 31 Bali cattle, and MS was performed by evaluating the muscle line using a range of 1 to 15. The three individuals with the highest scores (Hi) and the three with the lowest (Lo) were sampled for semitendinosus muscle microstructural observation. The results showed that cattle in the Hi group had a higher surface area of myofibril and sarcomere length, but had a significantly lower relative percentage of intramuscular collagen compared to others in the Lo group (p < 0.05). Generally, muscle score had a positive correlation with body weight, surface area of myofibril and sarcomere length. In all cattle, the fibres identified were similar, where the thick types were dominant in the epimysium and perimysium, followed by the thin variants. Furthermore, the endomysium prominently contained non‐fibrous or other types of fibre. This study can provide the basic data that can be used as a pilot for future studies of muscle score assessment in Indonesian cattle. [ABSTRACT FROM AUTHOR]

The maintenance of skeletal muscle mass plays a fundamental role in health and issues associated with quality of life. Mechanical signals are one of the most potent regulators of muscle mass, with a decrease in mechanical loading leading to a decrease in muscle mass. This concept has been supported by a plethora of human- and animal-based studies over the past 100 years and has resulted in the commonly used term of 'disuse atrophy'. These same studies have also provided a great deal of insight into the structural adaptations that mediate disuse-induced atrophy. For instance, disuse results in radial atrophy of fascicles, and this is driven, at least in part, by radial atrophy of the muscle fibers. However, the ultrastructural adaptations that mediate these changes remain far from defined. Indeed, even the most basic questions, such as whether the radial atrophy of muscle fibers is driven by the radial atrophy of myofibrils and/or myofibril hypoplasia, have yet to be answered. In this review, we thoroughly summarize what is known about the macroscopic, microscopic, and ultrastructural adaptations that mediated disuse-induced atrophy and highlight some of the major gaps in knowledge that need to be filled. [ABSTRACT FROM AUTHOR]

The skeletal muscle of teleost fish is remarkable in its capacity for indeterminate growth and phenotypic plasticity to function across different environmental conditions. Generation of the sarcomeric proteins that comprise the muscle myofibrils is the single biggest contributor to protein synthesis in fish muscle. In the past decade, numerous studies have shown sarcomeric proteins, especially those of the troponin complex and myosins, exhibit altered expression in exceptionally rapidly growing fish muscle. This includes cases associated with inherent inter-individual variation in growth rates, including hybrid vigour, and induction during rapid compensatory growth. The purpose of this review is to present the evolutionary mechanisms (i.e. whole genome and gene duplication) responsible for the expanded genomic array of sarcomeric proteins found in teleost fish. On this backdrop, the parallels between then changes in sarcomeric gene expression linked with growth and those associated with thermal acclimation (an example of phenotypic plasticity) are discussed. The plasticity associated with optimizing muscle performance during thermal acclimation appears closely coupled with indeterminate growth, and I speculate on some of the endocrine factors (e.g. thyroid hormone signaling) where the two are likely to converge. Together, this review provides a timely synthesis of the determinants of sarcomeric protein expression in fish, and highlights areas ripe for future investigation. This review also emphasizes that, to facilitate comparisons between different species and across various contexts of muscle growth and plasticity, it is imperative that upcoming studies on genes encoding sarcomeric proteins in teleost fish adopt a consistent and evolutionarily-informed universal nomenclature. [ABSTRACT FROM AUTHOR]

Summary: The alkaline ionised water (AIW) could inactivate food‐borne microorganisms, ensuring the vegetables and fruits security. However, the application of AIW in preserving fish is still unknown. Therefore, the present study aimed to explore the effects of AIW on grass carp fillets during storage at 4 °C for 12 days. The results of total volatile basic nitrogen (TVB‐N), thiobarbituric acid reactive substances (TBARS), colour and the sensory attributes showed that the AIW‐treated grass carp fillets exhibited better shelf life. As for the microbial composition, AIW treatment for 5 min significantly inhibited the growth of the predominant bacteria, such as Staphylococcus and Pseudomonas. According to the result from microstructure, the neat and even structure of myofibrillar proteins (MPs) was conducive maintained the fish quality. Overall, the results demonstrated that AIW treatment exerted a protective effect of inactivating spoilage microorganisms and preserving the quality of grass carp fillets. [ABSTRACT FROM AUTHOR]

The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body wt/day) treatment for 5 days, which decreased GSH content to ~10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N = 17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR. [ABSTRACT FROM AUTHOR]

Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc. [ABSTRACT FROM AUTHOR]

Plectin, a highly versatile cytolinker protein, is crucial for myofiber integrity and function. Accordingly, mutations in the human gene (PLEC) cause several rare diseases, denoted as plectinopathies, with most of them associated with progressive muscle weakness. Of several plectin isoforms expressed in skeletal muscle and the heart, P1d is the only isoform expressed exclusively in these tissues. Using high-resolution stimulated emission depletion (STED) microscopy, here we show that plectin is located within the gaps between individual α-actinin-positive Z-disks, recruiting and bridging them to desmin intermediate filaments (Ifs). Loss of plectin in myofibril bundles led to a complete loss of desmin Ifs. Loss of Z-disk-associated plectin isoform P1d led to disorganization of muscle fibers and slower relaxation of myofibrils upon mechanical strain, in line with an observed inhomogeneity of muscle ultrastructure. In addition to binding to α-actinin and thereby providing structural support, P1d forms a scaffolding platform for the chaperone-assisted selective autophagy machinery (CASA) by directly interacting with HSC70 and synpo2. In isoform-specific knockout (P1d-KO) mouse muscle and mechanically stretched plectin-deficient myoblasts, we found high levels of undigested filamin C, a bona fide substrate of CASA. Similarly, subjecting P1d-KO mice to forced swim tests led to accumulation of filamin C aggregates in myofibers, highlighting a specific role of P1d in tension-induced proteolysis activated upon high loads of physical exercise and muscle contraction. [ABSTRACT FROM AUTHOR]

This study was undertaken to evaluate the impact of proteases inactivation on the textural properties of yellow-feathered chicken (YFC) meat and explore the underlying mechanism driving these changes. The results showed that firmness and shelf life of the YFC meat were significantly improved by the proteases inactivation pretreatment in a dose-dependent manner. Specially, the 40% and 60% residual protease activities were identified to yield textures comparable to the hot-fresh YFC meat. Hydration and water retention of the proteases inactivated YFC meat were also found to be enhanced. Further analysis of myofibrillar protein (MP) revealed that cross-linkages of MP were induced by proteases inactivation with 40% and 60%, evidenced by higher levels of disulfide, carbonyl-amine bonds and di-tyrosine. Additionally, the chemical changes in amino acid residues led to the sequestration of tryptophan residues and the formation of β-sheet structures, accompanied by increased turbidity and particle size of MP. Moreover, water holding capacity, lightness, digestibility and strength of the MP gel were found to be improved. The results would provide insights into potential application of proteases inactivation as a strategy to improve YFC textural quality. • Textural quality of cold-stored yellow-feathered chicken was improved and shelf life was extended. • Proteases inactivation enhanced moisture retention and water stability. • Cross-linkage via disulfide and carbonyl bond in myofibrillar protein (MP) was promoted. • Proteases inactivation induced the burial of tryptophan residues and formation of β-sheet in MP. • Water holding capacity, lightness, digestibility, and strength of MP gel were improved. [ABSTRACT FROM AUTHOR]

Simple Summary: Myosin is a molecular motor protein that is critical for using stored energy to yield contraction in both skeletal muscle and in the heart. Mutations in myosin can result in inherited human hypertrophic cardiomyopathy, wherein the heart walls become thickened. This results in inability to adequately pump enough oxygenated blood to meet bodily demands, and can lead to abnormal heart beating patterns and the possibility of sudden death. Typically, over-active myosin can be causative for this disease. Here, we used the common fruit fly to study one of these cardiomyopathy-causing mutations by expressing the mutant protein in the indirect flight muscle of the fly. Surprisingly, we found that the mutation and associated mutations decrease myosin and muscle function instead of enhancing it. We show that at least some of these defects result from improper interactions within the myosin protein. This suggests that appropriate communications within myosin are critical for its function and further indicates that certain aspects of indirect flight muscle myosin are functionally distinct from those of myosin in the human heart. By understanding the intricate interactions within the myosin motor protein that are important for its normal role, therapeutics for improving mutant myosin function can be developed. The R249Q mutation in human β-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a β-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent β-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the β-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture. [ABSTRACT FROM AUTHOR]

Skeletal muscle consists of slow and fast myofibers in which different myosin isoforms are expressed. Approximately 300 myosins form a single-thick filament in the myofibrils, where myosin is continuously exchanged. However, endogenous slow and fast myosin dynamics have not been fully understood. To elucidate those dynamics, here we generated mice expressing green fluorescence protein-tagged slow myosin heavy chain (GFP-Myh7) and Kusabira Orange fluorescence protein-tagged fast myosin heavy chain (KuO-Myh1). First, these mice enabled us to distinguish between GFP- and KuO-myofibers under fluorescence microscopy: GFP-Myh7 and KuO-Myh1 were exclusively expressed in slow myofibers and fast myofibers, respectively. Next, to monitor endogenous myosin dynamics, fluorescence recovery after photobleaching (FRAP) was conducted. The mobile fraction (Mf) of GFP-Myh7 and that of KuO-Myh1 were almost constant values independent of the regions of the myofibers and the muscle portions where the myofibers were isolated. Intriguingly, proteasome inhibitor treatment significantly decreased the Mf in GFPMyh7 but not in KuO-Myh1 myofibers, indicating that the response to a disturbance in protein turnover depended on muscle fiber type. Taken together, the present results indicated that the mice we generated are promising tools not only for distinguishing between GFP- and KuO-myofibers but also for studying the dynamics of endogenous myosin isoforms by live-cell fluorescence imaging. [ABSTRACT FROM AUTHOR]

Meat is the richest source of protein and according to meat sources, the protein is composed of different types of amino acids which are completely dependent upon the protein types. Myofibril is basically muscle protein that's widely present in different types of fish and low quantity is present in different meat (camel). Myofibril is made up of thick and thin myofilaments chemically. Actin, tropomyosin, troponin, and tropomyosin are thin myofilaments on the other hand myosin is only the thick myofilament. It is helpful in the formation of strong skeleton muscle and can also stronger and formation the cardiac layers (epicardium, myocardium and endocardium). Technologically, it can be used in the formation of different food products for the purpose of developing diet supplements. This review discussed different sources and chemical properties of myofibril protein and also its potential role toward the cardiac health. Myofibril's thick and thin filaments contribute in the construction of skeletal muscle and enhance the heart's protective coverings. The literature has been collected from Science Direct, Google Scholar and Web of Science. Myofibrils are made up of thick and thin filaments that can help in different human body functions. In the future, more research is necessary that discuss the close relation between myofibril protein and cardiac layers. [ABSTRACT FROM AUTHOR]

New Findings: What is the main observation in this case?The main observation of this case report is that blood flow‐restricted exercise can cause myofibrils to have an aberrant wave‐like appearance that is accompanied by irregular pockets of sarcoplasm in the intermyofibrillar space, while traditional forms of damage to the Z‐discs and contractile elements are not as apparent.What insights does it reveal?Our findings indicate that blood flow restriction‐mediated fluid pooling might cause alterations in skeletal muscle ultrastructure after exercise that might be directly related to myofibre swelling. The acute effects of blood flow‐restricted (BFR) exercise training on skeletal muscle ultrastructure are poorly understood owing to inconsistent findings and the use of largely imprecise systemic markers for indications of muscle damage. The purpose of this study was to compare myofibrillar ultrastructure before and 30 min after normal and BFR resistance exercise using transmission electron microscopy in a single individual to evaluate the feasibility of this more nuanced approach. One apparently healthy male with 13 years of resistance exercise completed six sets of both BFR [30% of one‐repetition maximum (1‐RM)] and normal non‐occluded (70% of 1‐RM) unilateral angled leg press on the contralateral leg, as a control, after assessment of 1‐RM 72 h before. Vastus lateralis muscle biopsies were collected before and 30 min after each exercise session. The lengths and widths of 250 sarcomeres and the sarcoplasmic area were assessed via 20 individual transmission electron photomicrographs. Analysis revealed that BFR training (1.769 ± 0.12 μm) increased sarcomere length when compared with normal exercise (1.64 ± 0.17 μm; P < 0.001), without differences in sarcomere width between conditions (BFR, 0.90 ± 0.26 μm; normal, 0.93 ± 0.27 μm; P = 0.172). Furthermore, there were no significant interaction (P = 0.168) or condition effects between BFR (25.98 ± 4.17%) and normal (27.3 ± 6.49%) resistance exercise for sarcoplasmic area (P = 0.229). Exercise also increased sarcoplasmic area within the myofibril (pre‐exercise, 24.42 ± 5.13%; postexercise, 28.95 ± 5.92%) for both conditions (P = 0.001). This case study demonstrates a unique BFR training‐induced alteration in myofibril ultrastructure that appeared wave like and was accompanied by intracellular abnormalities that appeared to be fluid pockets of sarcoplasm disrupting the surrounding myofibrils. [ABSTRACT FROM AUTHOR]

Myosin, the most abundant myofibrillar protein in skeletal muscle, functions as a motor protein in muscle contraction. Myosin polymerizes into the thick filaments in the sarcomere where approximately 50% of embryonic myosin (Myh3) are replaced within 3 h (Ojima K, Ichimura E, Yasukawa Y, Wakamatsu J, Nishimura T, Am J Physiol Cell Physiol 309: C669‐C679, 2015). The sarcomere structure including the thick filament is maintained by a balance between protein biosynthesis and degradation. However, the involvement of a protein degradation system in the myosin replacement process remains unclear. Here, we show that the muscle‐specific ubiquitin ligase Ozz regulates replacement rate of Myh3. To examine the direct effect of Ozz on myosin replacement, eGFP‐Myh3 replacement rate was measured in myotubes overexpressing Ozz by fluorescence recovery after photobleaching. Ozz overexpression significantly decreased the replacement rate of eGFP‐Myh3 in the myofibrils, whereas it had no effect on other myosin isoforms. It is likely that ectopic Ozz promoted myosin degradation through increment of ubiquitinated myosin, and decreased myosin supply for replacement, thereby reducing myosin replacement rate. Intriguingly, treatment with a proteasome inhibitor MG132 also decreased myosin replacement rate, although MG132 enhanced the accumulation of ubiquitinated myosin in the cytosol where replaceable myosin is pooled, suggesting that ubiquitinated myosin is not replaced by myosin in the myofibril. Collectively, our findings showed that Myh3 replacement rate was reduced in the presence of overexpressed Ozz probably through enhanced ubiquitination and degradation of Myh3 by Ozz. [ABSTRACT FROM AUTHOR]

Background: Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available. Methods: A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy. Results: At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure. Conclusions: A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages. [ABSTRACT FROM AUTHOR]

Sarcomere, compartmentalization, concentration, subcellular localization, CUTie sensor, cyclic nucleotide, myofibril, nanodomain Its diffusion is limited by phosphodiesterases (PDEs), which convert it to AMP.[1] The holotetrameric protein kinase A (PKA) constitutes a primary effector of cAMP. A 30 nm ruler is added to facilitate the comparison with panel A. (C) Cartoon representations of skeletal and cardiac muscle troponin systems colored as in B. The protein segment proposed as AKAP in the cardiac TnT is shown with all-atoms representation, and the helix-breaking proline residue in the skeletal TnT is highlighted. Keywords: sarcomere; cyclic nucleotide; compartmentalization; concentration; subcellular localization; CUTie sensor; myofibril; nanodomain EN sarcomere cyclic nucleotide compartmentalization concentration subcellular localization CUTie sensor myofibril nanodomain 1 3 3 10/21/22 20220901 NES 220901 Intracellular signaling is often conceptualized as a series of processes triggered by increasing/decreasing concentrations of small molecules. [Extracted from the article]

The force--length relation is one of the most defining features of muscle contraction, and yet a topic of debate in the literature. The sliding filament theory predicts that the force produced by muscle fibres is proportional to the degree of overlap between myosin and actin filaments, producing a linear descending limb of the active force--length relation. However, several studies have shown forces that are larger than predicted, especially at long sarcomere lengths (SLs). Studies have been conducted with muscle fibres, preparations containing thousands of sarcomeres that make measurements of individual SL challenging. The aim of this study was to evaluate force production and sarcomere dynamics in isolated myofibrils and single sarcomeres from the rabbit psoas muscle to enhance our understanding of the theoretically predicted force--length relation. Contractions at varying SLs along the plateau (SL = 2.25--2.39 µm) and the descending limb (SL > 2.39 µm) of the force--length relation were induced in sarcomeres and myofibrils, and different modes of force measurements were used. Our results show that when forces are measured in single sarcomeres, the experimental force--length relation follows theoretical predictions. When forces are measured in myofibrils with large SL dispersions, there is an extension of the plateau and forces elevated above the predicted levels along the descending limb. We also found an increase in SL non-uniformity and slowed rates of force production at long lengths in myofibrils but not in single sarcomere preparations. We conclude that the deviation of the descending limb of the force--length relation is correlated with the degree of SL non-uniformity and slowed force development. [ABSTRACT FROM AUTHOR]

Objectives: Cardiac damage in patient with diphtheritic myocarditis is reported as the leading cause of mortality. Diphtheria toxin (DTx) is a well-known bacterial toxin inducing various cytotoxic effects. Mainly, catalytic fragment inhibits protein synthesis, induces cytotoxicity, and depolymerizes actin filaments. In this study, we aimed to demonstrate the extent of myofibrillar damage under DTx treatment to porcine cardiac tissue samples. Methods: Tissue samples were incubated with DTx for 1-3 h in culture conditions. To analyze whole toxin (both fragments) distribution, conjugation of DTx with FITC was performed. Measurements were carried out with fluorescence spectrophotometer before and after dialysis. Immunofluorescence microscopy was used to show localization of DTx-FITC (15 nM) on cardiac tissue incubated for 2 h. Ultrastructural characterization of cardiac tissue samples treated with DTx (15 or 150nM) wasperformed with transmission electronmicroscopy. Results: DTx exerts myofibrillar disorganization. Myofilament degeneration, mitochondrial damage, vacuolization, and abundant lipid droplets were determined with 150 nM of DTx treatment. Conclusions: This finding is an addition to depolymerization of actin filaments as a result of the DTx-actin interactions in in vitro conditions, indicating that myofilament damage can occur with DTx directly besides protein synthesis inhibition. Ultrastructural results support the importance of filamentous actin degeneration at diphtheritic myocarditis. [ABSTRACT FROM AUTHOR]

Obscure puffer fish (Takifugu obscurus) flesh, often eaten as raw fish fillet, is susceptible to softening. To investigate the mechanisms for the softening of flesh and changes in firmness, the proliferation of total microbial flora, Enterobacteriaceae, Pseudomonas spp., and lactic acid bacteria (LAB), total volatile basic nitrogen (TVBN), trimethylamine (TMA), and pH were periodically assayed during storage at 0, 4, and 8ºC. Myofibril extraction and separation of puffer fish flesh was carried out and the length of the separated myofibrils was measured periodically using an optical microscope during the entire storage time. Transmission electron microscopy was also used to inspect the changes in myofibrillar characteristics of the fish flesh at the beginning and end of the storage period. The results indicated that lower chilled temperature can contribute to reduced proliferation of microbial flora and less variation in TVBN, TMA, and pH and further maintain the integrity of myofibrils and retain firmness of puffer fish flesh during storage. [ABSTRACT FROM AUTHOR]

• Intramuscular connective tissue (IMCT) was mainly degraded in first 10 days. • IMCT degradation was mainly attributed to decorin. • IMCT was hydrolyzed by endogenous collagenase rather than cathepsins. • IMCT and myofibrils (MF) contributed equally to fish texture maintenance. • IMCT failed to protect MF in the process of texture degradation. Although intramuscular connective tissue (IMCT) is low in fish, its impact on texture cannot be ignored due to its special location. Therefore, this study was aimed to investigate the contribution of IMCT degradation to fish softening and its mechanism induced by endogenous proteases. Results showed that IMCT honeycomb-like structure collapsed entirely on the 10th day of ice storage, along with a decrease of shear force by 36.5%. Meanwhile, IMCT and myofibrils (MF) degradation accelerated softening by 25.1% and 15.3% during 10 days of ice storage, respectively. Next, IMCT deterioration was indicated to be highly correlated with decorin degradation (0.956**), followed by elastin (0.928**) and collagen (0.904**). Ulteriorly, endogenous collagenase was shown to degrade IMCT crucial components, while endogenous cathepsins had little effect. In conclusion, this study confirmed that IMCT played an essential role in maintaining fish texture and was mainly degraded by endogenous collagenase. [ABSTRACT FROM AUTHOR]

[Display omitted] • The strength of salting and oxidation interactively affected water binding of gels. • The collapse of the gel structure under oxidation was mitigated at 3% salt. • The gel-oriented crystallization of MP at 5% salt was •OH concentration dependent. Free radical grafting and oxidative modification show superiority in myofibrillar protein (MP) aggregation patterns during salting process, but their consequent formation mechanisms of protein hydration network require further evaluation. Herein, we explored the effect of salt-curing (0, 1, 3 and 5 %) on MP protein polymer substrate, water-protein interaction, crystallization events and thermal stability under H 2 O 2 /ascorbate-based hydroxyl radical (•OH)-generating system (HRGS) (1, 10, 20 mM H 2 O 2). Results showed that moderate salting (≤3%) favored the water binding of MP gels during the oxidation course. Accordingly, the maximum thermal stability (T m) of MP gels was obtained at 3 % salting could be greatly attributed to the protein chain solubilization and refolding process. However, 5 % salt synergized with •OH oxidation intensified diffraction peak 2 (the most striking diffraction feature). Microstructural analysis validated a maximum compactness of MP gel following brining with 5 % salt at potent oxidation strength (20 mM H 2 O 2). This study maybe promises efficient strategy to the myogenetic fibril products and biomaterials. [ABSTRACT FROM AUTHOR]

• Vacuum impregnation was effective in introducing coatings to fillets. • The gelatin-GSE coating inhibited protein oxidation during chilled storage. • The gelatin-GSE coating maintained the morphology of myofibrils. • The gelatin-GSE group showed better quality and freshness than the control group. The present study studied the effects of fish gelatin (FG) incorporated with grape seed extract (GSE) through vacuum impregnation (VI) on refrigerated tilapia (Oreochromis niloticus) fillets over 12 days. The VI of FG-GSE significantly improved the quality of the fish by decreasing drip loss, texture changes, and microbial survival. It also delayed protein oxidation by inhibiting the formation of disulphide bonds and carbonyl groups, and maintaining a higher sulfhydryl content and Ca2+-ATPase activity. Regarding myofibril degradation, FG-GSE maintained their secondary structure by increasing the ratio of α-helices and β-sheets (70.88–75.51%). Atomic force microscopy further revealed that the FG-GSE coating preserved the myofibril nanostructure by maintaining their length, width, and height. Overall, the synergistic effects of VI with 3% FG and 0.9% GSE suggested a promising approach for fillet preservation. [ABSTRACT FROM AUTHOR]

Skeletal muscle consists of bundles of myofibers containing millions of myofibrils, each of which is formed of longitudinally aligned sarcomere structures. Sarcomeres are the minimum contractile unit, which mainly consists of four components: Z‐bands, thin filaments, thick filaments, and connectin/titin. The size and shape of the sarcomere component is strictly controlled. Surprisingly, skeletal muscle cells not only synthesize a series of myofibrillar proteins but also regulate the assembly of those proteins into the sarcomere structures. However, authentic sarcomere structures cannot be reconstituted by combining purified myofibrillar proteins in vitro, therefore there must be an elaborate mechanism ensuring the correct formation of myofibril structure in skeletal muscle cells. This review discusses the role of myosin, a main component of the thick filament, in thick filament formation and the dynamics of myosin in skeletal muscle cells. Changes in the number of myofibrils in myofibers can cause muscle hypertrophy or atrophy. Therefore, it is important to understand the fundamental mechanisms by which myofibers control myofibril formation at the molecular level to develop approaches that effectively enhance muscle growth in animals. [ABSTRACT FROM AUTHOR]

This study investigated the role of three proteases in myofibril degradation. Tilapia muscle was soaked in MDL-28170, E-64, and AC-DEVD-CHO, specific inhibitors of calpain, cathepsin, and caspase, respectively, for 14 days to determine the in vivo degradation of myofibril. Desmin and troponin T were significantly inhibited after treatment with inhibitors compared to the control (p <.05). For in vitro analyses, myofibril was incubated with recombinant active μ-calpain, cathepsin B, and caspase-3. Desmin was significantly reduced when incubated with recombinant proteases (p <.05). However, troponin T showed significant degradation only in the μ-calpain and cathepsin B treatment groups (p <.05). Our results reveal μ-calpain and cathespin B have similar degradation patterns, while caspase-3 has partly similar degradation for myofibril in vivo and in vitro. This suggests that μ-calpain and cathespin B are the main proteases responsible for post-mortem myofibril degradation, while caspase-3 is minor. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effect of protein oxidation on microbial, free amino acids (FAAs) and biogenic amines (BAs) in Chinese dry sausage during processing. Three different batches of Chinese dry sausages were examined: control batch, pro-oxidation batch and anti-oxidation batch. The experimental results revealed that anti-oxidation batch showed the lowest degree of protein oxidation compared with control batch, while the results of the pro-oxidation batch were just opposite. With the increasing protein oxidation, estimated by protein carbonyls and sulfhydryls, microbial content and free amino acid content increased, while BA content decreased. The protein carbonyls were positively correlated with total FAAs (P < 0.01) and total BA content (P < 0.05), while sulfhydryls content was negatively correlated with total FAA content (P < 0.01), total viable counts (P < 0.01), and total BA content (P < 0.01). In conclusion, reduced protein oxidation contributes to improved quality of Chinese dry sausage. This research provided a new strategy to improve the quality of meat product manufacture. [ABSTRACT FROM AUTHOR]

Highlights • Calcium ions improved the gel strength of acid-treated surimi gels to 494.56 g. • The citric acid-added surimi gels had an 85.85% lower lipid content. • Calcium promoted the formation of disulphide and ionic bonds in the gel matrix. • Atomic force microscopy images visualised the myofibril proteins' morphology. • Calcium ion treatment decreased the height of the gelled proteins by 83.33%. Abstract The effect and mechanisms of calcium chloride and citric acid addition on the acid method of processing tilapia protein isolates and surimi gels were studied. The alkaline method shows better gel quality than the acid method with lower yield and high lipid content. The addition of calcium chloride increased the breaking force to 494.56 g and citric acid helped reducing the lipid content by 85.85%, resulting in a higher yield of 75.36%. Gels with calcium maintained low levels of expressible moisture (2.30%), indicating a well-organised gel matrix. The decrease in -SH content and increase in ionic bonds content suggested increased formation of disulphide bonds and ionic bonds. Oscillatory dynamic measurement indicated that more heat resistant bonds were formed. The morphology of myofibril proteins studied by atomic force microscopy (AFM) showed that the height of the proteins in the calcium-added gels decreased 79.4%, which suggested that the protein structure was "suppressed". [ABSTRACT FROM AUTHOR]

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type. [Display omitted] • MF30 enhances myofibril contraction, exhibits positive cooperative binding to the unfolded myosin, and competes with MyBPC. • MF20 does not impact myofibril contraction and may potentially compete with MyBPC. • Expansion microscopy significantly improves image resolution by physically expanding the sample. [ABSTRACT FROM AUTHOR]

Abstract: Bromelain was used to tenderize golden pomfrets (Trachinotus blochii). The enzyme kinetic model was x = 2.447 × ln [ 1 + ( 1332.21 × E 0 S 0 − 1.74 ) t ] , which indicated that the degree of hydrolysis (DH, x) was dependent on hydrolysis time (t), the initial concentration of myofibril (S0) and bromelain (E0). The relationship between the overall hydrolysis rate (v), S0, E0, and t is demonstrated as: v = ( 16.50 ( E 0 S 0 ) − 1.33 ) S 0 exp { − 2.447 ln [ 1 + ( 1332.21 E 0 S 0 − 1.74 ) t 2 ] }. Sample of 0.40% E0/S0 was further used to study the effects of hydrolysis time on the changes of proteins, peptides, free amino acids (FAA), and protein nanostructure. SDS‐PAGE result showed that myosin heavy chain was degraded dramatically from 22.88% before treatment to 12.03% after 2 min bromelain treatment. Meanwhile, bromelain did not exhibit activity towards actin, trypomyosin, myosin light chain, and troponin C. A general increase of amino acids indicated the increased DH and the preferential cleavage sites of bromelain in the descending order of lysine, glutamic acid, glycine, ornithine, methionine sulfoxide, and alanine. Atomic force microscope images showed that the strip‐like structure of myofibril was considerably degraded by bromelain, and the granulation of protein after 20 min indicated possible self‐assembling of protein hydrolysate. Confocal laser scanning microscopy further confirmed the degradation of myofibril proteins and formation of protein aggregates. Practical Application: Meat of golden pomfrets is tough, thus not idea for fish balls or fish cakes. Tenderization is essential to achieve desired texture and consumer acceptance, especially for this fish meat with intrinsic hard texture. Bromelain can be extracted from pineapple processing waste. Enzymatic kinetics was studied to instruct industry to control the tenderness of the processed fish meat. The microstructural and mechanism study elucidate the process, thus could be applied to improve the quality of the seafood products correspondingly. [ABSTRACT FROM AUTHOR]

Near-infrared (NIR) spectra contain abundant data, heterospectral two-dimensional correlation (H2D-CS) analysis offers a good way to interpret these data. For the first time, H2D-CS was used to correlate the NIR hyperspectral imaging (HSI) data with mid-infrared spectra and to identify feature-related wavebands for developing models for monitoring the oxidative damage of pork myofibrils during frozen storage. The HSI images were acquired at frozen state without thawing and the oxidative damage of myofibrils was assessed by carbonyl content. Results showed that the simplified PLSR model based on H2D-CS identified feature wavebands obtained determination coefficient in prediction (R 2 P ) of 0.896 and root mean square error in prediction (RMSEP) of 0.177 nmol/mg protein, which was better than the partial least square regression (PLSR) model based on full wavebands (R 2 P  = 0.856, RMSEP = 0.209 nmol/mg protein). Therefore, H2D-CS was effective in selecting feature-related wavebands of NIR HSI. [ABSTRACT FROM AUTHOR]

The study investigated the possibility of using hyperspectral imaging (HSI) in the spectral range of 1000–2200 nm to characterize myofibrils cold structural deformation degrees of frozen pork samples. The HSI images of pork samples frozen under different freezing rates were acquired in the frozen state without thawing. The myofibrils cold structural deformation degrees were evaluated by surface hydrophobicity (S 0 ANS) and Ca 2+ -ATPase activity. Spectral angle mapping (SAM) algorithm was used for the first time to extract the spectral information for regression. Compared with the optimized partial least square regression (PLSR) models based on selected wavebands by successive projections algorithm (SPA), the optimized PLSR models developed based on the spectral angles calculated by the SAM algorithm achieved comparable or even better performance with R 2 P of 0.896 for S 0 ANS and 0.879 for Ca 2+ -ATPase activity, respectively. The implications of the frozen meat spectrum were also analyzed in the current study. [ABSTRACT FROM AUTHOR]

In striated muscles, approximately 300 myosin molecules form a single thick filament in myofibrils. Each myosin is continuously displaced by another myosin to maintain the thick filament structure. Our previous study using a fluorescence recovery after photobleaching ( FRAP) technique showed that the myosin replacement rate is decreased by inhibition of protein synthesis, but myosin is still exchangeable. This result prompted us to examine whether myosin in the cytoplasm is involved in myosin replacement in myofibrils. To address this, FRAP was measured in green fluorescent protein ( GFP)-tagged myosin heavy chain 3 (Myh3) expressing myotubes that were treated with streptolysin-O ( SLO), which forms pores specifically in the plasma membrane to induce leakage of cytoplasmic proteins. Our biochemical data demonstrated that the cytoplasmic myosin content was reduced in SLO-permeabilized semi-intact myotubes. Furthermore, FRAP experiments showed a sluggish substitution rate of GFP-Myh3 in SLO-permeabilized myotubes. Taken together, these results demonstrate that the myosin substitution rate is significantly reduced by a decreased amount of myosin in the cytoplasm and that cytoplasmic myosin contributes to myosin replacement in myofibrils. [ABSTRACT FROM AUTHOR]

An enzymatic method to tenderise golden pomfret (GP, Trachinotus blochii) flesh by marinating with bromelain (BML) solution was developed to produce GP fish balls with a texture similar to those of yellowtail fusilier (YF, Caesio cuning). Treatment with BML reduced the hardness, chewiness and gel strength significantly but increased the resilience of GP fish balls. As a result, 0.4% (enzyme-substrate ratio, w/ w) BML-treated GP fish balls had the same texture properties as YF fish balls. Meanwhile, changes in myofibrillar proteins in the fish balls were determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and atomic force microscopy. The results indicated that BML degraded myosin light chain and troponin T effectively, without affecting actin. BML treatment generated protein fragments with significantly smaller sizes, thereby improving the tenderness of the flesh. The length, width and height of the myofibrils from 0.4% BML-treated fish ball were 6.42, 1.52 and 1.48 μm, respectively, which were not significantly different from the myofibrils of YF fish balls, indicating that the similar nanostructure determined the comparable texture properties. Gas chromatography-mass spectrometry analysis showed that 0.4% BML decreased the amount of hexanal, hexadecane, 1-octen-3-ol and 2,6,10,14-tetramethylpentadecane but increased the ratio of heptadecane from 18.14 to 38.23% in the treated fish balls, making the flavour of 0.4% BML-treated GP fish balls similar to that of YF balls. Overall, the results suggest that 0.4% BML-tenderised GP could be a promising alternative to YF to produce quality fish balls. [ABSTRACT FROM AUTHOR]

Myofibrils made up of actin, myosin, and associated proteins generate the contractile force in muscle, and, consequently, mutations in these proteins may lead to heart failure. Septins are a conserved family of small GTPases that associate with actin filaments, microtubules, and cellular membranes. Despite the importance of septins in cytoskeleton organization, their role in cardiomyocyte organization and function is poorly characterized. Here, we show that septin 7 is expressed in both embryonic and adult zebrafish hearts and elucidate the physiological significance of sept7b, the zebrafish ortholog of human septin 7, in the heart in embryonic and larval zebrafish. Knockdown of sept7b reduced F-actin and α-cardiac actin expression in the heart and caused disorganization of actin filaments. Electron microscopy of sept7bdepleted larvae showed disorganization of heart myofibrils and partial detachment from Z-disks. Functional studies revealed that knockdown of sept7b leads to reduced ventricular dimensions, contractility, and cardiac output. Furthermore, we found that depletion of sept7b diminished the expression of retinaldehyde dehydrogenase 2, which catalyzes the synthesis of retinoic acid necessary for heart morphogenesis. We further observed that the sept7b and retinoic acid signaling pathways converge to regulate cardiac function. Together, these results specify an essential role for sept7b in the contractile function of the heart. [ABSTRACT FROM AUTHOR]

The effect of fish gelatin addition on mass transfer, nutrient loss, texture and nanostructure of fish balls was investigated. Mass transfer models were built and the root-mean-square-errors were 0.1432, 0.3178 and 0.1000 for exponential, power-law and linear models, respectively. After gelatin addition, the mass transfer coefficient/model parameter and moisture content increased, and the hardness and chewiness of fish balls decreased. Myofibrils were imaged using atomic force microscope (AFM). The length of the myofibrils was greater than 15 μm before and after boiling for 10 min; however, they decreased to around 14 and 11 μm after 20 and 30 min boiling, indicating degradation of myofibrils. Meanwhile, there was no significant difference among different groups, suggesting that the added gelatin did not affect the nanostructure of the fish balls. Furthermore, increasing gelatin addition resulted in fewer water-soluble proteins and peptides in the boiling water. The results suggest that added gelatin increased the mass transfer coefficient/model parameter by increasing the moisture content and decreasing the nutrient loss. It also improved the texture by decreasing the hardness and chewiness, and did not affect the nanostructure of fish ball myofibrils. [ABSTRACT FROM AUTHOR]

Key points When a skeletal muscle is stretched while it contracts, the muscle produces a relatively higher force than the force from an isometric contraction at the same length: a phenomenon referred to as residual force enhancement., Residual force enhancement is puzzling because it cannot be directly explained by the classical force-length relationship and the sliding filament theory of contraction, the main paradigms in the muscle field., We used custom-built instruments to measure residual force enhancement in skeletal myofibrils, and, for the first time, in cardiac myofibrils., Our data report that residual force enhancement is present in skeletal muscles, but not cardiac muscles, and is regulated by the different isoforms of the titin protein filaments., Abstract When a skeletal muscle contracts isometrically, the muscle produces a force that is relative to the final isometric sarcomere length (SL). However, when the same final SL is reached by stretching the muscle while it contracts, the muscle produces a relatively higher force: a phenomenon commonly referred to as residual force enhancement. In this study, we investigated residual force enhancement in rabbit skeletal psoas myofibrils and, for the first time, cardiac papillary myofibrils. A custom-built atomic force microscope was used in experiments that stretched myofibrils before and after inhibiting myosin and actin interactions to determine whether the different cardiac and skeletal titin isoforms regulate residual force enhancement. At SLs ranging from 2.24 to 3.13 μm, the skeletal myofibrils enhanced the force by an average of 9.0%, and by 29.5% after hindering myosin and actin interactions. At SLs ranging from 1.80 to 2.29 μm, the cardiac myofibrils did not enhance the force before or after hindering myosin and actin interactions. We conclude that residual force enhancement is present only in skeletal muscles and is dependent on the titin isoforms. [ABSTRACT FROM AUTHOR]

Muscle contraction is commonly associated with the cross-bridge and sliding filament theories, which have received strong support from experiments conducted over the years in different laboratories. However, there are studies that cannot be readily explained by the theories, showing 1) a plateau of the force-length relation extended beyond optimal filament overlap, and forces produced at long sarcomere lengths that are higher than those predicted by the sliding filament theory; 2) passive forces at long sarcomere lengths that can be modulated by activation and Ca2+, which changes the force-length relation; and 3) an unexplained high force produced during and after stretch of activated muscle fibers. Some of these studies even propose “new theories of contraction.” While some of these observations deserve evaluation, many of these studies present data that lack a rigorous control and experiments that cannot be repeated in other laboratories. This article reviews these issues, looking into studies that have used intact and permeabilized fibers, myofibrils, isolated sarcomeres, and half-sarcomeres. A common mechanism associated with sarcomere and half-sarcomere length nonuniformities and a Ca2+-induced increase in the stiffness of titin is proposed to explain observations that derive from these studies. [ABSTRACT FROM AUTHOR]

The periodically arranged thin filaments within the striated myofibrils of skeletal and cardiac muscle have precisely regulated lengths, which can change in response to developmental adaptations, pathophysiological states, and genetic perturbations. We have developed a user-friendly, open-source ImageJ plugin that provides a graphical user interface (GUI) for super-resolution measurement of thin filament lengths by applying Distributed Deconvolution (DDecon) analysis to periodic line scans collected from fluorescence images. In the workflow presented here, we demonstrate thin filament length measurement using a phalloidin-stained cryosection of mouse skeletal muscle. The DDecon plugin is also capable of measuring distances of any periodically localized fluorescent signal from the Z- or M-line, as well as distances between successive Z- or M-lines, providing a broadly applicable tool for quantitative analysis of muscle cytoarchitecture. These functionalities can also be used to analyse periodic fluorescence signals in nonmuscle cells. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effect of S. baltica on texture and flavour of golden pomfret were investigated. • S. baltica decreased fish hardness, and the storage and loss moduli of proteins. • MHC, MyBP-C and actin were degraded by S. baltica. • S. baltica produced dimethyl sulfide, 2-methyl-butanal and 3-methyl-butanal. Shewanella baltica has a high spoilage ability to decompose nutrients in fish. To investigate the role of S. baltica in fish protein and flavour during spoilage, the texture, rheology, protein patterns and volatile compounds of golden pomfret inoculated with S. baltica during 10-day storage were tested. During storage, S. baltica reduced the hardness of fish sticks by 29.73–49.24 %. Compared to the control (G 0 ′: 20.27 ± 2.15 kPa), inoculated samples showed lower moduli (G 0 ′: 16.71 ± 0.82–17.50 ± 1.80 kPa). Their myosin heavy chains, myosin-binding protein C and actin were decomposed into smaller proteins, which was validated by the lower intensities of molecules with M w 160–176 kDa. Furthermore, S. baltica generated volatile spoilage markers, including dimethyl sulfide, 2-methyl-butanal and 3-methyl-butanal. This study reveals the mechanism of fish texture and flavour changes induced by S. baltica , and provides insights into controlling bacterial spoilage of seafood. [ABSTRACT FROM AUTHOR]

Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis , distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5′-coding sequence of Xenopus adprhl1 . Over-expression of full length (40 kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity. [ABSTRACT FROM AUTHOR]

Cells that constitute fully differentiated tissues are characterised by an architecture that makes them perfectly suited for the job they have to do. This is especially obvious for cardiomyocytes, which have an extremely regular shape and display a paracrystalline arrangement of their cytoplasmic components. This article will focus on the two major cytoskeletal multiprotein complexes that are found in cardiomyocytes, the myofibrils, which are responsible for contraction and the intercalated disc, which mediates mechanical and electrochemical contact between individual cardiomyocytes. Recent studies have revealed that these two sites are also crucial in sensing excessive mechanical strain. Signalling processes will be triggered that## lead to changes in gene expression and eventually lead to an altered cardiac cytoarchitecture in the diseased heart, which results in a compromised function. Thus, understanding these changes and the signals that lead to them is crucial to design treatment strategies that can attenuate these processes. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel. [ABSTRACT FROM AUTHOR]

Contractile perturbations downstream of Ca 2+ binding to troponin C, the so-called sarcomere-controlled mechanisms, represent the earliest indicators of energy remodeling in the diseased heart [1]. Central to cellular energy “sensing” is the adenosine monophosphate-activated kinase (AMPK) pathway, which is known to directly target myofilament proteins and alter contractility [2–6]. We previously showed that the upstream AMPK kinase, LKB1/MO25/STRAD, impacts myofilament function independently of AMPK [5]. Therefore, we hypothesized that the LKB1 complex associated with myofilament proteins and that alterations in energy signaling modulated targeting or localization of the LKB1 complex to the myofilament. Using an integrated strategy of myofilament mechanics, immunoblot analysis, co-immunoprecipitation, mass spectroscopy, and immunofluorescence, we showed that 1) LKB1 and MO25 associated with myofibrillar proteins, 2) cellular energy stress re-distributed AMPK/LKB1 complex proteins within the sarcomere, and 3) the LKB1 complex localized to the Z-Disk and interacted with cytoskeletal and energy-regulating proteins, including vinculin and ATP Synthase (Complex V). These data represent a novel role for LKB1 complex proteins in myofilament function and myocellular “energy” sensing in the heart. [ABSTRACT FROM AUTHOR]

An “invariant proline” separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138–43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro . Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc 5 , an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue. [ABSTRACT FROM AUTHOR]

Coating of gelatin and chitosan can improve fish fillet’s quality, but the mechanism is not clear. Chitosan/gelatin coatings significantly prevented deterioration of golden pomfret fillet at 4 °C. Chitosan with 7.2% gelatin group showed the best effect on preserving the length of myofibril, which remained greater than 15 μm at day 17 of storage, while for control, chitosan and chitosan combined with 3.6% gelatin group, it was 5.03, 10.04 and 9.02 μm, respectively. The MALDI-TOF MS result revealed that the coatings slowed down the protein deterioration of fillet. On days 13 and 17, the myosin light chain and myoglobin in control group degraded, while the two proteins still existed in chitosan/gelatin coated groups. Overall, the chitosan with 7.2% gelatin coating had the best effect on preserving fillet’s quality during storage. The coating may exert its protective effect via inhibiting myofibril degradation within fillet. [ABSTRACT FROM AUTHOR]

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles. [ABSTRACT FROM AUTHOR]

The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific.We then generate Tmod4-/- mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4-/- muscle fibers leads to thin filament elongation by ~15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts. [ABSTRACT FROM AUTHOR]

Highly organized thick filaments in skeletal muscle cells are formed from ~300 myosin molecules. Each thick-filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching and found that myofibrillar myosin is actively replaced with an exchange half-life of ~3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C, which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin, as well as preexisting cytosolic myosin, contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the nonmyofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells. [ABSTRACT FROM AUTHOR]

Background When an activated muscle is rapidly stretched, force rises and peaks while muscle lengthens. The peak force is normally called critical-force (P c ). The mechanism behind this increase in force is not well understood, but it has been associated with crossbridges operating in different states. Methods Myofibrils were attached between a cantilever and a micro-needle, and activated with Ca 2+ or MgADP. During activation, the myofibrils were stretched by 3% SL o at 10 SL o ·s −1 . A crossbridge model was developed to better understand the effects of MgADP in myofibrils activation. Results Despite a similar stretch magnitude, MgADP activation produced a higher P c (1.37 ± 0.07 P/P o ) than Ca 2+ activation (P c = 1.23 ± 0.03 P/P o ). These results suggest that myofibrils activated with MgADP become stiffer than myofibrils activated with Ca 2+ . Conclusions MgADP induces a fraction of crossbridges to form a “rigor-like” state that precedes ADP release, and that may not contribute to isometric forces. Such interpretation was strengthened by the results obtained with the developed crossbridge model, which showed that MgADP bias crossbridges into the rigor-like state. This state would be crucial to initiate a cooperative activation of crossbridges and actin, and to resist to unbinding from actin when the myofibrils are stretched. Significance Our results suggest a new mechanism contributing for force output during stretch, which underlies basic mechanisms of muscle contraction. [ABSTRACT FROM AUTHOR]

Tetanized muscle myofibrils are often modeled as one-dimensional chains where springs represent half-sarcomeres (HS). The force-length relation for individual HSs (isometric tetanus) is known to have a 'descending limb', a segment with an apparently negative stiffness. Despite the potential mechanical instability on the descending limb, the isometric tetanus is usually interpreted as describing an affine deformation. At the same time, active stretching during tetanus around the descending limb is known to produce non-affine sarcomere patterns. In view of this paradox, the question whether the mechanical behavior of a myofibril can be interpreted as a response of a single contractile unit has been a subject of considerable controversy over the last 50 years. In this paper we question the claim that the isometric tetanus describes homogeneous configurations of the HS chain. To distinguish between the multitudes of non-affine equilibrium states available to this mechanical system, we propose to use the concept of a stored mechanical energy. While the notion of energy is natural from a mechanical point of view, physiologists have resisted it so far on the grounds that the contractile elements are active. We discuss how this objection can be overcome and show that the appropriately defined stored energy of a tetanized myofibril with N contractile units has exponentially many local minima. We then argue that the ruggedness of the ensuing energy landscape is responsible for the experimentally observed history dependence and hysteresis in the mechanical response of a tetanized muscle near the descending limb. A nonlocal extension of the chain model, accounting for surrounding tissues, shows that both the ground states and the marginally stable states are fine mixtures of short and long HSs. These mixtures are homogeneous at the macro-scale and inhomogeneous at the micro-scale and we show that the negative overall slope of the step-wise tetanus can coexist with a positive instantaneous stiffness. A salient feature of the nonlocal model is that the variation of the degree of non-uniformity with elongation follows a complete devil's staircase. [ABSTRACT FROM AUTHOR]

In skeletal muscle cells, myofibrillar proteins are highly organized into sarcomeres in which thick filaments interdigitate with thin filaments to generate contractile force. The size of thick filaments, which consist mainly of myosin molecules, is strictly controlled. However, little is known about the mechanisms by which myosin molecules assemble into thick filaments. Here, we assessed the ability of each domain of myosin heavy chain (Myh) to form thick filaments. We showed that exogenously expressed subfragment 2 ( S2) + light meromyosin ( LMM) of Myh was efficiently incorporated into thick filaments in muscle cells, although neither solely expressed S2 nor LMM targeted to thick filaments properly. In nonmuscle COS7 cells, S2+ LMM formed more enlarged filaments/speckles than LMM. These results suggest that Myh filament formation is induced by S2 accompanying LMM. We further examined the effects of Myh C-terminus on thick filament assembly. C-terminal deletion mutants were incorporated not into entire thick filaments but rather into restricted regions of thick filaments. Our findings suggest that the elongation of myosin filaments to form thick filaments is regulated by S2 as well as C-terminus of LMM. [ABSTRACT FROM AUTHOR]