Myofibrillar Structure Research Articles

WITHDRAWN: Beef tenderness evaluation using early post-mortem muscle nanostructure.

Ahead of Print article withdrawn by publisher.

Dietary curcumin decreased lipid oxidation and enhanced the myofibrillar protein structure of the duck (Anas Platyrhynchos) breast muscle when subjected to storage

Effects of dietary curcumin given to ducks on chemical composition, lipid and protein oxidation and changes of the protein structural properties of the duck breast muscle when subjected to storage (7-day at −18 °C then 12 h at 4 °C) were investigated. A total of 600 1-day-old male ducks were randomly assigned into four groups: the control group (T0, 0 mg/kg dietary curcumin) and curcumin groups (T300, 300 mg/kg; T400, 400 mg/kg and T500, 500 mg/kg dietary curcumin). On the day 70 of age, six ducks were selected and slaughtered and ducks breast muscle were stored at −18 °C then 12 h at 4 °C in each group. There were no significant effects of dietary curcumin on the chemical composition of the duck meat. Dietary curcumin significantly decreased the PV value, TBARS value and carbonyls group content, whereas enhanced the structure (free amino group, secondary structure) and decreased the fluorescence spectroscopy of the protein of the duck breast muscle when subjected to storage. In conclusion, dietary curcumin given to ducks decreased the lipid and protein oxidation, enhanced protein structure of the duck breast muscle when subjected to storage, whereas the dietary curcumin inclusion of 400–500 mg/kg would be more efficient.

Growth Kinetics and Spoilage Potential of Co-culturing Acinetobacter johnsonii and Pseudomonas fluorescens from Bigeye Tuna (Thunnus obesus) During Refrigerated Storage.

A. johnsonii and P. fluorescens are the well-known specific spoilage organisms in aquatic products and the study of the interactions between A. johnsonii and P. fluorescens are limited. This study aims to evaluate the growth kinetics, spoilage potential and interactions ofA.johnsonii andP. fluorescens isolated from spoiled bigeye tuna (Thunnus obesus) by inoculating into sterile fish slices and stored at 4°C for 6days. The growth kinetics of A. johnsonii and P. fluorescens were fitted with Baranyi and Roberts model. The chemical indexes (total volatile base nitrogen (TVB-N), trimethylamine (TMA), pH, proteolytic activity and protein content) of each inoculated block of bigeye tuna were increased during refrigerated storage. Moreover, the higher contents of chemical indexes were observed in co-culture with A. johnsonii and P. fluorescens compared with single culture of A. johnsonii and P. fluorescens. In addition, atomic force microscopy (AFM) observation of co-culturing A. johnsonii and P. fluorescens inoculation into sterile fish slices revealed damage of myofibrillar protein structures and the protein degradation. Based on these parameters, a rapid method to evaluate spoilage potential of A. johnsonii and P. fluorescens was positively correlated with TVB-N value, TMA value and pH value (P < 0.05) by the correlation coefficient. Consequently, spoilage potential of microorganisms became stronger evaluated in a mixed culture than single culture. This paper provides insight for a detection method of interactions of A. johnsonii and P. fluorescens during refrigerated storage.

Myofibrillar Protein Structure and Gel Properties of Trichiurus Haumela Surimi Subjected to High Pressure or High Pressure Synergistic Heat

The changes in gel properties and myofibrillar protein structure of Trichiurus haumela surimi subjected to high pressure (300–450 MPa; 5 min) or high pressure synergistic heat (90 °C; 30 min) treatment were investigated. The results showed that high pressure below 400 MPa could improve the gel properties. The water holding capacity of the 350 MPa group increased by 27.40% compared with the control group. Moreover, the gel strength of surimi gel induced by high pressure of 300 MPa and 350 MPa increased by 38.08% and 40.00%, respectively, and continually increased by 17.86% and 21.00% after further heating. Both of high pressure and heating affected the composition of myofibrillar protein and partially degraded the actin and myosin heavy chain. High pressure reduced the contents of α-helix and β-turn, but increased β-sheet and random coil in the myofibrillar protein. Subsequent heat treatment converted the partial β-sheet into the α-helix structure. In addition, in the gels of surimi subjected to high pressure, the secondary structure of myofibrillar protein was significantly correlated with the whiteness, gel strength, and texture characteristics of the gels. Collectively, the results supported an optimum pressure of 300–350 MPa for the induction of hairtail surimi gel.

Increased Microtubule Density and Altered Directionality Occur Commensurate with Myofibrillar Malformation in Dystrophic and Aged Skeletal Muscle

Increased microtubule (MT) density, and its level of post-translational modification by detyrosination, are consequences of skeletal muscle disease/dysfunction. Our work in muscular dystrophy, and new work in aging, align these MT changes to increased cytoskeletal stiffness and dysregulated excess in mechano-activated reactive oxygen species (ROS) and calcium (Ca2+) signals that contribute to contraction-induced injury. Our demonstration that a targeted reduction in MT density or its level of detyrosination effectively reduces contraction-induced injury implied the suppression of excess contraction elicited ROS and Ca2+ signaling as a parsimonious explanation. However, evidence that myofibers with structural malformations that also arise in these conductions to promote muscle damage, led us to question whether these structural alterations co-segregate with MT alterations. To this end, our recent work has focused on characterizing morphological changes to myofiber structure in relation to the structure and properties of the MT network. Using an automated imaging approach, we profiled myofiber, sarcomere, and cytoskeletal structure to quantify the coincidence of altered myofibrillar structure, altered MT density, and shifts in MT directionality. At areas of myofibrillar malformation, we find increased MT density and concomitant alterations in MT directionality. Given that MTs are essential to the maintenance of sarcomere structure, we hypothesize a relationship between disease-dependent MT alterations and the genesis of altered myofiber and sarcomere structure. We further hypothesize that the suppression of contraction injury, by targeting MTs, may have been due to their effect at these structures.

Mitochondrial bioenergetics links inflammation and cardiac contractility in endotoxemia.

There is current awareness about the central role of mitochondrial dysfunction in the development of cardiac dysfunction in systemic inflammatory syndromes, especially in sepsis and endotoxemia. The aim of this work was to elucidate the mechanism that governs the link between the severity of the systemic inflammatory insult and mitochondrial function, analysing the consequences on heart function, particularly in cardiac contractile state. Female Sprague-Dawley rats were subjected to low-grade endotoxemia (i.p. injection LPS 0.5mgkg-1 body weight) and severe endotoxemia (i.p. injection LPS 8mgkg-1 body weight) for 6h. Blood NO, as well as cardiac TNF-α and IL-1β mRNA, were found increased as the severity of the endotoxemia increases. Cardiac relaxation was altered only in severe endotoxemia, although contractile and lusitropic reserves were found impaired in both treatments in response to work-overload. Cardiac ultrastructure showed disorientation of myofibrillar structure in both endotoxemia degrees, but mitochondrial swelling and cristae disruption were only observed in severe endotoxemia. Mitochondrial ATP production, O2 consumption and mitochondrial inner membrane potential decreases were related to blood NO levels and mitochondrial protein nitration, leading to diminished ATP availability and impairment of contractile state. Co-treatment with the NOS inhibitor L-NAME or the administration of the NO scavenger c-PTIO leads to the observation that mitochondrial bioenergetics status depends on the degree of the inflammatory insult mainly determined by blood NO levels. Unravelling the mechanisms involved in the onset of sepsis and endotoxemia improves the interpretation of the pathology, and provides new horizons for novel therapeutic targets.

A mutation in the filamin c gene causes myofibrillar myopathy with lower motor neuron syndrome: a case report

BackgroundMyofibrillar myopathies (MFMs) are a genetically heterogeneous group of muscle disorders. Mutations in the filamin C gene (FLNC) have previously been identified in patients with MFM. The phenotypes of FLNC-related MFM are heterogeneous.Case presentationThe patient was a 37-year-old male who first experienced weakness in the distal muscles of his hand, which eventually spread to the lower limbs and proximal muscles. Serum creatine kinase levels were moderately elevated. Obvious neuropathic changes in the electromyographic exam and edema changes in lower distal limb magnetic resonance imaging were observed. Histopathological examination revealed the presence of abnormal protein aggregates and angular atrophy in some muscle fibers. Ultrastructural analysis showed inordinate myofibrillar structures and dissolved myofilaments. DNA sequencing analysis detected a heterozygous missense mutation (c.7123G > A, p.V2375I) in the immunoglobulin (Ig)-like domain 21 of FLNC.ConclusionsFLNC mutation c.7123G > A, p.V2375I in the immunoglobulin (Ig)-like domain 21 can be associated with distal myopathy with typical MFM features and lower motor neuron syndrome. Although electromyographic examination of our patient showed obvious neuropathic changes, MFM could not be excluded. Therefore, genetic testing is necessary to make an accurate diagnosis.

The effects of high pressure on the myofibrillar structure and meat quality of marinating Tan mutton

AbstractLamb leg meat (LLM) was marinated using solution of sodium chloride (NaCl) concentrations (0.04 g/g) for 15 min under high pressure (HP) treatment (100–400 MPa, 25°C) to investigate meat characteristics, volatile components and microstructure. HP treatment increased process yields during marinating, especially at 200 MPa, the shearing force and hardness reached their minimum value, which indicated that HP treatment (200 MPa) can effectively improve the tenderness of LLM. The electron microscopy revealed that HP modified the myofibrillar structure in the leg muscles. With the rise of pressure, the lightness (L*) value increased significantly. The red/green hue (a*) reached the maximum value at 200 MPa. HP influenced the formation of some key volatile compounds in LLM. LLM pressurized at 200–400 MPa had lower levels of furfural than control group, LLM pressurized at 200 MPa had higher levels of N‐nonanal and N‐octanal, lower levels of palmitic acid, but oleic acid and stearic acid was not detected. Only at 200–400 MPa, 2,3‐octanedione and 2‐octanone were detected in HP‐treated LLM.Practical applicationsHigh‐pressure (HP) is an effective technology as a pretreatment to postslaughter meat. Advantages include opportunities to improve tenderness and other characteristics of processed meat products by appropriate use of this technique. This study showed that application of high‐pressure premarinated treatment (200 MPa, 15 min, 25°C and 0.04 g/g salt content) could effectively improve the muscle fiber structure of lamb leg, several major volatile substances and tenderness. This work verifies the potential of HP for improving LLM characteristics and volatile components. These results can be beneficial for the applications of pressurized mutton in the mutton industry.

Effect of soluble soybean polysaccharides on freeze-denaturation and structure of myofibrillar protein of bighead carp surimi with liquid nitrogen freezing

This paper investigated the synergistic effect of 3% soluble soybean polysaccharides (SSPS) and liquid nitrogen freezing (−80 °C) on the freezing process and protein denaturation of bighead carp surimi. Freezing curve showed that liquid nitrogen freezing could significantly minimize the elapsed time of maximum-ice-crystal formation zone. Both liquid nitrogen freezing and SSPS were useful in preventing protein denaturation of surimi during 12-week frozen storage. Protein denaturation results indicated that SSPS-LNfreezing surimi11SSPS-added surimi frozen by liquid nitrogen freezing had the highest protein solubility, Ca2+-ATPase activity and total sulfhydryl content. SDS-PAGE indicated that SSPS and liquid nitrogen freezing could effectively inhibit the decrease of myosin heavy chain concentration after 12 weeks of frozen storage. Raman spectra showed that tryptophan and tyrosine were exposed to polar microenvironment, the ɑ-helix and β-sheet turned into random coil and β-turn, and the conformation of disulfide bond changed from trans-gauche-trans (t-g-t) to gauche-gauche-trans (g-g-t). Either SSPS or liquid nitrogen freezing could mitigate these changes during frozen storage and a synergistic effect emerged on preventing myofibrillar protein denaturation and protein structure change. The combination of SSPS with liquid nitrogen freezing could be applied to freeze bighead carp surimi.

Hydrolysates from rainbow trout (Oncorhynchus mykiss) processing by-products: Properties when added to fish mince with different freeze-thaw cycles

Rainbow trout (Oncorhynchus mykiss) mixed processing by-product homogenates were subjected to 8 mM CaCl2-5 mM citric acid (1:5 w/v) pretreatments followed by hydrolysis with Alcalase (W-CaCi-RTBH). These hydrolysates decreased the loss of total sulfhydryl groups, protein solubility and protein carbonyl formation in a fish mince after 6 freeze-thaw cycles (P ≤ 0.05). The thermal properties of myosin heavy chains and actin were maintained as shown using differential scanning calorimetry (DSC) (P ≤ 0.05). Based on low field NMR T2 relaxation, the fastest relaxation component (T21) showed no changes with addition of W-CaCi-RTBH into a fish mince after 6 freeze-thaw cycles, indicating no changes for the major pool of water molecules within the myofibrillar protein structure (P ≥ 0.05). After 6 freeze-thaw cycles, W-CaCi-RTBH had better antifreeze activity when combined with sucrose – sorbitol (4%) (P ≤ 0.05). Therefore, W-CaCi-RTBH could potentially be an additive in frozen fish mince.

Chimeric Mice with Deletion of Cfl2 that Encodes Muscle-Type Cofilin (MCF or Cofilin-2) Results in Defects of Striated Muscles, Both Skeletal and Cardiac Muscles.

Cofilin, a member of the ADF/cofilin family, is an actin-binding protein which is widely distributed among eukaryotic organisms and involved in actin filament dynamics in a variety of cell types. In mammalian striated muscles, muscle-type cofilin (MCF or cofilin-2) is predominantly expressed. Previous investigations have shown that MCF plays an essential role in the regulation of assembly of contractile apparatus in skeletal muscle, but its role in cardiac muscle has remained unclear. In the present study, in order to further clarify the role of MCF in organization of myofibrillar structure in vivo, we generated chimeric mice with a combination of MCF-deficient cells that were generated by Cfl2-knockout (Cfl2-/-) and wild type cells containing MCF, and examined the effect of MCF deficiency on striated muscles, especially on the fine structures of contractile apparatus in cardiac muscle. We found that mice chimeric for MCF deficient cells exhibited structural defects in their skeletal muscles as previously reported. Histological analysis showed that MCF deficiency leads to degradation of myofibers and promotion of muscle regeneration. Electron microscopic observation of cardiac muscle of the chimeric mice showed coexistence of the cells with normal sarcomeres and those with disorganized myofibrils in a chimeric pattern. In these cofilin-deficient cells, myofilaments were scattered in the cytoplasm and myofibrillar structures were severely disrupted. These results provide strong evidence for that MCF plays a critical role in the formation and the maintenance of myofibril structure not only in skeletal muscle but also in cardiac muscle.

Effects of hydroxyl radical induced oxidation on water holding capacity and protein structure of jumbo squid (Dosidicus gigas) mantle

SummaryProtein oxidation is considered as an important issue in food preservation process. In the present study, the potential influence of protein oxidation on water holding capacity and protein structure of jumbo squid (Dosidicus gigas) mantle was investigated. After the hydroxyl radical oxidation, it was found that the carbonyl, surface hydrophobicity and dityrosine content of myofibrillar protein significantly increased (P &lt; 0.05), while the content of total sulphydryl decreased significantly (P &lt; 0.01). Meanwhile, the fluorescence intensity of squid was weakened, and the maximum absorption peak of fluorescence red shift as the H2O2 concentration increased. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) showed that not only the protein cross‐linking but also degradation could have occurred. The content of α‐helix decreased, the content of β‐sheet, β‐turn and the unordered structures increased after oxidation. In addition, oxidation resulted in a decrease in water holding capacity. Taken together, oxidation resulted in the damage of the myofibrillar structure, the increase in muscle loss rate and the decrease in water holding capacity.

Effects of microwave and water bath heating on the interactions between myofibrillar protein from beef and ketone flavour compounds

AbstractThis paper presents the effects of microwave on the structure changes of myofibrillar protein compared with traditional water bath heating method. Under microwave or water bath heating conditions, α‐helix conformation of myofibrillar protein was converted into β‐turn, β‐sheet and randomly coiled structures gradually. T he fluorescence intensity of myofibrillar protein exhibited a marked red shift of the maximum absorption wavelength after microwave or water bath treatment. Total sulfhydryls groups and surface hydrophobicity contents were increased with progressing microwave or water bath treatment time. Results indicated that the thermal effects of microwave dominated the structure changes of myofibrillar protein, and microwaving also had an apparent influence on myofibrillar protein structure compared with water bath heating due to its nonthermal effects, and thus affecting the retention and release of ketone flavour compounds.

Tamoxifen prolongs survival and alleviates symptoms in mice with fatal X-linked myotubular myopathy

X-linked myotubular myopathy (XLMTM, also known as XLCNM) is a severe congenital muscular disorder due to mutations in the myotubularin gene, MTM1. It is characterized by generalized hypotonia, leading to neonatal death of most patients. No specific treatment exists. Here, we show that tamoxifen, a well-known drug used against breast cancer, rescues the phenotype of Mtm1-deficient mice. Tamoxifen increases lifespan several-fold while improving overall motor function and preventing disease progression including lower limb paralysis. Tamoxifen corrects functional, histological and molecular hallmarks of XLMTM, with improved force output, myonuclei positioning, myofibrillar structure, triad number, and excitation-contraction coupling. Tamoxifen normalizes the expression level of the XLMTM disease modifiers DNM2 and PI3KC2B, likely contributing to the phenotypic rescue. Our findings demonstrate that tamoxifen is a promising candidate for clinical evaluation in XLMTM patients.

Optical prediction of single muscle fiber force production using a combined biomechatronics and\xa0second harmonic generation imaging approach

Skeletal muscle is an archetypal organ whose structure is tuned to match function. The magnitude of order in muscle fibers and myofibrils containing motor protein polymers determines the directed force output of the summed force vectors and, therefore, the muscle’s power performance on the structural level. Structure and function can change dramatically during disease states involving chronic remodeling. Cellular remodeling of the cytoarchitecture has been pursued using noninvasive and label-free multiphoton second harmonic generation (SHG) microscopy. Hereby, structure parameters can be extracted as a measure of myofibrillar order and thus are suggestive of the force output that a remodeled structure can still achieve. However, to date, the parameters have only been an indirect measure, and a precise calibration of optical SHG assessment for an exerted force has been elusive as no technology in existence correlates these factors. We engineered a novel, automated, high-precision biomechatronics system into a multiphoton microscope allows simultaneous isometric Ca2+-graded force or passive viscoelasticity measurements and SHG recordings. Using this MechaMorph system, we studied force and SHG in single EDL muscle fibers from wt and mdx mice; the latter serves as a model for compromised force and abnormal myofibrillar structure. We present Ca2+-graded isometric force, pCa-force curves, passive viscoelastic parameters and 3D structure in the same fiber for the first time. Furthermore, we provide a direct calibration of isometric force to morphology, which allows noninvasive prediction of the force output of single fibers from only multiphoton images, suggesting a potential application in the diagnosis of myopathies.

Effect of low-dose ethanol consumption on expression of nuclear factor-κB in diabetic rats with myocardial injury

To investigate the effect of low-dose ethanol on the expression of nuclear factor-κB (NF-κB) in diabetic rats with myocardial injury. Rat models of diabetes were established by an intraperitoneal injection of 55 mg/kg streptozotocin (STZ). After successful modeling, the rats were given 2.5% ethanol (daily dose of 20 mg/kg) for 1 week, followed by 5% ethanol (daily dose of 39.45 mg/kg) for another 7 weeks. Normal rats without STZ injection and diabetic rats without ethanol treatment serve as the normal control and diabetic model groups, respectively. The ventricular function of the rats was determined using echocardiography. The plasma levels of interleukin-1 (IL-1) and IL-4 were detected in the rats, and the expressions of 4-HNE, NF-κB and IKK proteins in the left anterior myocardium was evaluated using immunohistochemistry or Western blotting; the ultrastructural changes of the myocardium were observed using transmission electron microscopy. Compared with the normal control group, the diabetic rats showed significantly lowered systolic and diastolic functions of the left ventricle, increased plasma level of IL-1 and myocardial 4-HNE expression (P &lt; 0.01), decreased plasma level of plasma IL-4 (P &lt; 0.01), and increased myocardial expressions of NF-κB and IKK proteins (P &lt; 0.01). Transmission electron microscopy revealed myofibrillar rupture, incomplete myofibrillar structure and mitochondrial damage in the cardiac myocytes in the diabetic rats. Compared with the diabetic rats, the rats with low-dose ethanol treatment exhibited improved systolic and diastolic functions of the left ventricle, milder myocardial myofibrillar and mitochondrial damages, and significantly lowered plasma IL-1 level and myocardial expressions of 4-HNE, NF-κB and IKK (P &lt; 0.01), and increased plasma IL-4 level (P &lt; 0.01). NF-κB expression is increased in the myocardium of diabetic rats with myocardial injury, and low-dose ethanol consumption lowers myocardial expression of NF-κB in diabetic rats, suggesting the involvement of NF-κB signaling pathway in the protective effect of low-dose ethanol against myocardial injury in diabetes mellitus.

Actinidin pretreatment and sous vide cooking of beef brisket: Effects on meat microstructure, texture and in vitro protein digestibility

The application of actinidin to beef brisket followed by thermal inactivation (by sous vide cooking) was studied for its effects on textural attributes, microstructure and protein digestibility under simulated gastric conditions. The optimal processing of meat was achieved by injecting 5% of a 3 mg/mL solution of commercial actinidin extract (Actazin™ from Anagenix Ltd.) into brisket steaks, followed by vacuum tumbling and cooking under sous vide conditions at 70 °C for 30 min. This cooking time is considerably less than sous vide cooking times normally used in the food service industry. The actinidin-treated meat had no change in pH, colour and cook loss, but showed improved sensory scores for tenderness, juiciness and flavour compared with the untreated meat. Transmission electron micrographs showed considerable breakdown of the myofibrillar structure, particularly around the Z-discs. An enhanced initial rate of muscle protein breakdown under simulated gastric conditions was observed using SDS-PAGE, demonstrating positive effects of the actinidin treatment on meat protein digestibility.

Proteomic changes of the porcine skeletal muscle in response to chronic heat stress.

Heat stress (HS) has an adverse effect on meat quality; however, the underlying molecular mechanisms altering meat quality due to muscle responses to stress remain unclear. Sixteen castrated male crossbreeds between Landrace × Yorkshire sows and Duroc boars (79.00 ± 1.50 kg body weight) were exposed to either thermal neutral (22 °C, n = 8) or HS (30 °C, n = 8) conditions for 3 weeks. Subsequently, the longissimus dorsi (LD) muscle of all pigs was assayed for meat quality parameters and proteome analysis. HS decreased post mortem (24 h) pH and intramuscular fat, changed ultimate L*, a* and b* values and increased drip loss and shear force. Proteome analysis of the LD was conducted by two-dimensional gel electrophoresis and mass spectrometry. A total of 23 differentially expressed proteins were identified, of which three were verified by western blotting analysis. The identified proteins were involved in six types of biological process: carbohydrate metabolism, myofibrillar and cytoskeleton structure, stress response, antioxidant and detoxification, calcium binding and cellular apoptosis. Interestingly, HS induced higher levels of heat shock protein, antioxidants and calcium binding proteins, which are involved in the mechanisms of defense and homeostasis. The results indicate that HS-induced changes in the expression of myofibrillar proteins, glucose and energy metabolism-related proteins, heat shock protein and antioxidant enzymes might, at least partly, contribute to increase in meat tenderness. These findings will provide the foundation for developing future mitigating solutions and preventative therapies to reduce the detrimental effects of chronic HS on muscle function, metabolism and meat quality. © 2017 Society of Chemical Industry.

CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease by haploinsufficiency.

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10.

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the β-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects.