Myosin Filaments Research Articles

Improving the solubility of myofibrillar proteins in water by destroying and suppressing myosin molecular assembly via glycation

Filamentous myosin is a self-assembling polymer that prevents myofibrillar proteins (MPs) from functioning in low ionic strength media. This study was aimed at investigating if glycation has the potential to improve the solubility of MPs in water. MPs were conjugated with monosaccharides, oligosaccharides, and polysaccharides under wet reaction conditions at 37 °C. The conjugation was verified by SDS-PAGE, FT-IR and amino acid analyses. MPs conjugated with dextran (DX) exhibited a higher solubility and dispersion stability in water, which corresponded to smaller particle size and more uniform distribution (P < 0.05). According to secondary and tertiary structure analyses, the loss of α-helix structures and unfolding of the MPs appear to be the main reasons for MP solubilization. Additionally, according to the zeta-potential, confocal laser scanning microscopy, and atomic force microscopy observation results, glycation can provide electrostatic repulsion or steric hindrance to disintegrate existing filamentous myosin aggregates and inhibit further self-assembly behavior.

Friction in Myocardial Anoxia Leads to Negative Excess Entropy Production, Self-Organization, and Dissipative Structures.

Contraction of the heart is caused by actin filaments sliding along myosin filaments. This generates a frictional force inducing wear of the contractile apparatus. We postulated that this process could be exacerbated when the heart was submitted to severe anoxia. Anoxia induced dramatic abnormalities in the molecular properties of actin-myosin crossbridges. We applied the formalism of far-from-equilibrium thermodynamics to the left ventricular papillary muscles (LVPMs) of mammalian rat hearts which had been subjected to a prolonged anoxia (3 h). We showed that when subjected to prolonged anoxia, the heart operated far-from-equilibrium as evidenced by the non-linearity between thermodynamic force (F/T: Frictional force/Kelvin temperature) and thermodynamic flow (v0: myofilament sliding velocity). The rate of entropy production (EPR) was the product of (F/T) and v0. The excess entropy production (EEP) was equal to = vo; (S: entropy). The tribological system remained stable when EEP was positive and became unstable when EEP became negative, thus characterizing instability of the system and reflecting the occurrence of self-organization and possibly dissipative structures. After 3 h anoxia, re-oxygenation induced significant reversibility. About 20% of the myosin heads did not recover despite re-oxygenation. These results may be of importance in the context of heart transplantation where the delay between the time of sampling from the donor and the time of the graft installation in the recipient should be as short as possible.

Vitamin E Suppresses Aortic Ultrastructural Alterations Induced by Toxic Doses of Monosodium Glutamate

An association between certain food additives and chronic diseases is reported. Current study determined whether administering toxic doses of the food additive monosodium glutamate (MSG) into rats can induce aortopathy in association with the oxidative stress and inflammatory biomarkers upregulation and whether the effects of MSG overdose can be inhibited by vitamin E. MSG at a dose of (4 mg/kg; orally) that exceeds the average human daily consumption by 1000x was administered daily for 7 days to the rats in the model group. Whereas, rats treated with vitamin E were divided into two groups and given daily doses of MSG plus 100 mg/ kg vitamin E or MSG plus 300 mg/kg vitamin E. On the eighth day, all rats were culled. Using light and electron microscopy examinations, a profound aortic injury in the model group was observed demonstrated by damaged endothelial layer, degenerated smooth muscle cells (SMC) with vacuoles and condensed nuclei, vacuolated cytoplasm, disrupted plasma membrane, interrupted internal elastic lamina, clumped chromatin, and damaged actin and myosin filaments. Vitamin E significantly protected aorta tissue and cells as well as inhibited MSG-induced tissue malondialdehyde (MDA), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). The highest used vitamin E dosage was more effective. Additionally, a significant correlation was observed between the aortic injury degree and tissue MDA, TNF-α, IL-6, and superoxide dismutase (SOD) levels (p=0.001). Vitamin E effectively protects against aortopathy induced by toxic doses of MSG in rats and inhibits oxidative stress and inflammation.

Interactions between unfolding/disassembling behaviors, proteolytic subfragments and reversible aggregation of oxidized skeletal myosin isoforms at different salt contents

Myosin filament plays a critical role in water-trapping and thermodynamic regulation during processing of brined muscle foods. The redox state and availability of proteolytic/antioxidant enzymes affected by salt may change the ion-binding capacity of myosin consequently contributing to swelling and rehydration. Thus, this study investigated the impact of different salt content (0%, 1%, 2%, 3%, 4%, 5% NaCl) and oxidation in vitro (10 mM H2O2/ascorbate-based hydroxyl radical (OH)-generating system) on the oxidative stability, solubility/dispersion capacity, chymotrypsin digestibility, aggregation site and the microrheological properties of isolated porcine myosin. The result showed that, brining at 2% salt exposed more sulfhydryl groups and inhibited the formation of disulfide bond, whereby smaller dispersed structure (diameter within 10–50 nm) and higher Ca2+-ATPase activity of the denatured myosin were observed. Accordingly, gel electrophoresis showed that myosin S1 and HMM subunits were highly oxidized and susceptible to reversible assembles. Despite enhanced hydrophobic interactions between swelled myosin at 3% salt content, ≥4% salt greatly promoted the exposure/polarization of tryptophan and cross-linking structures, mainly occurring at myosin S2 portion. The results of micro-rheology proved that oxidized myosin formed a tighter heat-set network following rehydration at high ion strength (≥4% salt), suggesting an increased inter-droplet resistance and macroscopic viscosity. This work is expected to give some useful insights into improved texture and functionality of engineered muscle foods.

Superresolution microscopy reveals actomyosin dynamics in medioapical arrays.

Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, superresolution approaches (grazing incidence structured illumination, GI-SIM, and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in Drosophila. In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved-some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue, show that medioapical arrays are tightly apposed to the plasma membrane and are continuous with meshworks of lamellar F-actin. Medioapical arrays thereby constitute modified cell cortex. In concert with other tagged array components, superresolution imaging of live specimens will offer new understanding of cortical architecture and function.

Orchestrating nonmuscle myosin II filament assembly at the onset of cytokinesis.

Contractile forces in the actomyosin cortex are required for cellular morphogenesis. This includes the invagination of the cell membrane during division, where filaments of nonmuscle myosin II (NMII) are responsible for generating contractile forces in the cortex. However, how NMII heterohexamers form filaments in vivo is not well understood. To quantify NMII filament assembly dynamics, we imaged the cortex of Caenorhabditis elegans embryos at high spatial resolution around the time of the first division. We show that during the assembly of the cytokinetic ring, the number of NMII filaments in the cortex increases and more NMII motors are assembled into each filament. These dynamics are influenced by two proteins in the RhoA GTPase pathway, the RhoA-dependent kinase LET-502 and the myosin phosphatase MEL-11. We find that these two proteins differentially regulate NMII activity at the anterior and at the division site. We show that the coordinated action of these regulators generates a gradient of free NMII in the cytoplasm driving a net diffusive flux of NMII motors toward the cytokinetic ring. Our work highlights how NMII filament assembly and disassembly dynamics are orchestrated over space and time to facilitate the up-regulation of cortical contractility during cytokinesis.

Interacting-heads motif explains the X-ray diffraction pattern of relaxed vertebrate skeletal muscle

Electron microscopy (EM) shows that myosin heads in thick filaments isolated from striated muscles interact with each other and with the myosin tail under relaxing conditions. This “interacting-heads motif” (IHM) is highly conserved across the animal kingdom and is thought to be the basis of the super-relaxed state. However, a recent X-ray modeling study concludes, contrary to expectation, that the IHM is not present in relaxed intact muscle. We propose that this conclusion results from modeling with a thick filament 3D reconstruction in which the myosin heads have radially collapsed onto the thick filament backbone, not from absence of the IHM. Such radial collapse, by about 3–4 nm, is well established in EM studies of negatively stained myosin filaments, on which the reconstruction was based. We have tested this idea by carrying out similar X-ray modeling and determining the effect of the radial position of the heads on the goodness of fit to the X-ray pattern. We find that, when the IHM is modeled into a thick filament at a radius 3–4 nm greater than that modeled in the recent study, there is good agreement with the X-ray pattern. When the original (collapsed) radial position is used, the fit is poor, in agreement with that study. We show that modeling of the low-angle region of the X-ray pattern is relatively insensitive to the conformation of the myosin heads but very sensitive to their radial distance from the filament axis. We conclude that the IHM is sufficient to explain the X-ray diffraction pattern of intact muscle when placed at the appropriate radius.

The Solubility and Structures of Porcine Myofibrillar Proteins under Low-Salt Processing Conditions as Affected by the Presence of L-Lysine.

This study aimed to investigate the presence of L-lysine (Lys) on the solubility and structures of myofibrillar proteins (MFPs) at different ionic strengths. Porcine MFPs were incubated at 4 °C with various levels of ionic strengths (0.15, 0.3, or 0.6 M NaCl) with or without the presence of 20 or 40 mM Lys. After 24 h of incubation, MFP solubility and turbidity were determined, and the particle size distribution, circular dichroism spectra, and intrinsic tryptophan fluorescence of MFP were analyzed to obtain their secondary and tertiary structure. Results showed that the solubilization effects of Lys on MFPs are dependent on the ionic strength. Particularly, the presence of Lys could improve MFP solubility at 0.3 M, which resembles salt-reducing processing conditions. Concomitantly, the secondary and tertiary structures were observed to change as a result of the varying ionic strengths and the addition of Lys, including myofibril swelling, dissociation of myosin filaments, uncoiling of α-helix, and unfolding of the tertiary structure. The possible mechanisms underlying the solubilization effects of Lys on MFPs at low ionic strengths are discussed from the perspective of protein structural changes.

Second Harmonic Generation Properties in Chiral Sarcomeres of Drosophila Larval Muscles

Polarimetric second harmonic generation (SHG) microscopy is a label-free ultrastructural imaging technique that is applied to study molecular organization in striated muscle structure. Wild type OreR and UNC45 knockdown Drosophila melanogaster somatic larva body wall muscles are used to elucidate the influence of myosin organization on SHG imaging response. Structural changes due to varying myosin content are studied with linear polarization-in polarization-out (PIPO) SHG microscopy. PIPO SHG microscopy revealed changes in the striated pattern of somatic muscles and a reduced signal intensity correlated with reduced order of myosin filaments. The achiral and chiral susceptibility component ratios R- and C-ratios, respectively, were deduced. R-ratio values were higher in the UNC45 knockdown larva mutant compared to control, suggesting that in addition to myosin, another protein structure provide basis for a second harmonic response. The SHG active protein structure is located in the region of isotropic bands of the striated muscles. The measured C-ratio from the muscle fibers appeared to be small. Nonetheless, the wide distribution of C-ratio values of myosin filaments could be observed in the wild-type OreR muscles. Overall, this technique provides insight on how second harmonic properties change with myosin filament structure.

Interaction of the M-domain of cardiac myosin binding protein-C with the native cardiac thin filament

Cardiac muscle contraction depends on precise interactions between thin (actin) and thick (myosin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels as well as other sarcomeric proteins, such as cardiac myosin binding protein C (cMyBP-C) and myosin. Recent studies have shown the first four N-terminal domains (NTDs) of cMyBP-C (i.e. C0, C1, M and C2), are effective modulators of the TF activity. Their collective activation of the TF is regulated by the phosphorylation of the M domain but the mechanism is not fully understood.

Cooling intact and demembranated trabeculae from rat heart releases myosin motors from their inhibited conformation.

Myosin filament-based regulation supplements actin filament-based regulation to control the strength and speed of contraction in heart muscle. In diastole, myosin motors form a folded helical array that inhibits actin interaction; during contraction, they are released from that array. A similar structural transition has been observed in mammalian skeletal muscle, in which cooling below physiological temperature has been shown to reproduce some of the structural features of the activation of myosin filaments during active contraction. Here, we used small-angle x-ray diffraction to characterize the structural changes in the myosin filaments associated with cooling of resting and relaxed trabeculae from the right ventricle of rat hearts from 39°C to 7°C. In intact quiescent trabeculae, cooling disrupted the folded helical conformation of the myosin motors and induced extension of the filament backbone, as observed in the transition from diastole to peak systolic force at 27°C. Demembranation of trabeculae in relaxing conditions induced expansion of the filament lattice, but the structure of the myosin filaments was mostly preserved at 39°C. Cooling of relaxed demembranated trabeculae induced changes in motor conformation and filament structure similar to those observed in intact quiescent trabeculae. Osmotic compression of the filament lattice to restore its spacing to that of intact trabeculae at 39°C stabilized the helical folded state against disruption by cooling. The myosin filament structure and motor conformation of intact trabeculae at 39°C were largely preserved in demembranated trabeculae at 27°C or above in the presence of Dextran, allowing the physiological mechanisms of myosin filament-based regulation to be studied in those conditions.

Exploring the super-relaxed state of myosin in myofibrils from fast-twitch, slow-twitch, and cardiac muscle

Muscle myosin heads, in the absence of actin, have been shown to exist in two states, the relaxed (turnover ∼0.05 s−1) and super-relaxed states (SRX, 0.005 s−1) using a simple fluorescent ATP chase assay (Hooijman, P. et al (2011) Biophys. J.100, 1969–1976). Studies have normally used purified proteins, myosin filaments, or muscle fibers. Here we use muscle myofibrils, which retain most of the ancillary proteins and 3-D architecture of muscle and can be used with rapid mixing methods. Recording timescales from 0.1 to 1000 s provides a precise measure of the two populations of myosin heads present in relaxed myofibrils. We demonstrate that the population of SRX states is formed from rigor cross bridges within 0.2 s of relaxing with fluorescently labeled ATP, and the population of SRX states is relatively constant over the temperature range of 5 °C–30 °C. The SRX population is enhanced in the presence of mavacamten and reduced in the presence of deoxy-ATP. Compared with myofibrils from fast-twitch muscle, slow-twitch muscle, and cardiac muscles, myofibrils require a tenfold lower concentration of mavacamten to be effective, and mavacamten induced a larger increase in the population of the SRX state. Mavacamten is less effective, however, at stabilizing the SRX state at physiological temperatures than at 5 °C. These assays require small quantities of myofibrils, making them suitable for studies of model organism muscles, human biopsies, or human-derived iPSCs.

Using Synchrotron Radiation Imaging Techniques to Elucidate the Actions of Hexarelin in the Heart of Small Animal Models.

The majority of the conventional techniques that are utilized for investigating the pathogenesis of cardiovascular disease in preclinical animal models do not permit microlevel assessment of in situ cardiomyocyte and microvascular functions. Therefore, it has been difficult to establish whether cardiac dysfunction in complex multiorgan disease states, such as heart failure with preserved ejection fraction and pulmonary hypertension, have their origins in microvascular dysfunction or rather in the cardiomyocyte. Herein, we describe our approach of utilizing synchrotron radiation microangiography to, first, ascertain whether the growth hormone secretagogue (GHS) hexarelin is a vasodilator in the coronary circulation of normal and anesthetized Sprague-Dawley rats, and next investigate if hexarelin is able to prevent the pathogenesis of right ventricle (RV) dysfunction in pulmonary hypertension in the sugen chronic hypoxia model rat. We show that acute hexarelin administration evokes coronary microvascular dilation through GHS-receptor 1a and nitric oxide, and through endothelium-derived hyperpolarization. Previous work indicated that chronic exogenous administration of ghrelin largely prevented the pathogenesis of pulmonary hypertension in chronic hypoxia and in monocrotaline models. Unexpectedly, chronic hexarelin administration prior to sugen chronic hypoxia did not prevent RV hypertrophy or RV cardiomyocyte relaxation impairment. Small-angle X-ray scattering revealed that super relaxed myosin filaments contributed to diastolic dysfunction, and that length-dependent activation might contribute to sustained contractility of the RV. Thus, synchrotron-based imaging approaches can reveal novel insights into cardiac and coronary functions in vivo.

Mitochondrial Network Configuration Influences Sarcomere and Myosin Filament Structure in Striated Muscles

Mitochondrial Network Configuration Influences Sarcomere and Myosin Filament Structure in Striated Muscles

Geometric frustration in the myosin superlattice of vertebrate muscle.

Geometric frustration results from an incompatibility between minimum energy arrangements and the geometry of a system, and gives rise to interesting and novel phenomena. Here, we report geometric frustration in a native biological macromolecular system---vertebrate muscle. We analyse the disorder in the myosin filament rotations in the myofibrils of vertebrate striated (skeletal and cardiac) muscle, as seen in thin-section electron micrographs, and show that the distribution of rotations corresponds to an archetypical geometrically frustrated system---the triangular Ising antiferromagnet. Spatial correlations are evident out to at least six lattice spacings. The results demonstrate that geometric frustration can drive the development of structure in complex biological systems, and may have implications for the nature of the actin--myosin interactions involved in muscle contraction. Identification of the distribution of myosin filament rotations with an Ising model allows the extensive results on the latter to be applied to this system. It shows how local interactions (between adjacent myosin filaments) can determine long-range order and, conversely, how observations of long-range order (such as patterns seen in electron micrographs) can be used to estimate the energetics of these local interactions. Furthermore, since diffraction by a disordered system is a function of the second-order statistics, the derived correlations allow more accurate diffraction calculations, which can aid in interpretation of X-ray diffraction data from muscle specimens for structural analysis.

Mechanobiology of muscle and myofibril morphogenesis.

Muscles generate forces for animal locomotion. The contractile apparatus of muscles is the sarcomere, a highly regular array of large actin and myosin filaments linked by gigantic titin springs. During muscle development many sarcomeres assemble in series into long periodic myofibrils that mechanically connect the attached skeleton elements. Thus, ATP-driven myosin forces can power movement of the skeleton. Here we review muscle and myofibril morphogenesis, with a particular focus on their mechanobiology. We describe recent progress on the molecular structure of sarcomeres and their mechanical connections to the skeleton. We discuss current models predicting how tension coordinates the assembly of key sarcomeric components to periodic myofibrils that then further mature during development. This requires transcriptional feedback mechanisms that may help to coordinate myofibril assembly and maturation states with the transcriptional program. To fuel the varying energy demands of muscles we also discuss the close mechanical interactions of myofibrils with mitochondria and nuclei to optimally support powerful or enduring muscle fibers.

Supranutritional Maternal Organic Selenium Supplementation during Different Trimesters of Pregnancy Affects the Muscle Gene Transcriptome of Newborn Beef Calves in a Time-Dependent Manner.

Selenium (Se) is an essential micronutrient for growth and immune function in beef cattle. We previously showed that supranutritional maternal organic Se supplementation during late pregnancy improves immune function in their newborn calves; however, the effects of maternal organic Se-supplementation on fetal programming during different pregnancy stages have yet to be elucidated. Herein, we investigated the effects of supranutritional maternal organic Se-supplementation in different pregnancy trimesters on their beef calf’s genome-wide transcriptome profiles. Within 12 to 48 h of birth, whole blood and Longissimus dorsi (LD) muscle biopsies were collected from calves born to 40 crossbred Angus cows that received, except for the control group (CTR), Se-yeast boluses (105 mg of Se/wk) during the first (TR1), second (TR2), or third (TR3) trimester of gestation. Whole-blood Se concentrations of newborn calves increased from CTR, TR1, TR2 to TR3, whereas muscle Se concentrations of newborn calves were only increased in TR3 group. We identified 3048 unique differentially expressed genes (DEGs) across all group comparisons (FDR ≤ 0.05 and |log2FC| ≥ 1.5). Furthermore, we predicted 237 unique transcription factors that putatively regulate the DEGs. Independent of supplementation trimester, supranutritional maternal organic Se supplementation downregulated genes involved in adaptive immunity in all trimesters. Dependent on supplementation trimester, genes involved in muscle development were upregulated by TR3 Se supplementation and downregulated by TR1 Se-supplementation, and genes involved in collagen formation were downregulated by TR2 Se-supplementation. Supranutritional maternal organic Se supplementation in the last trimester of pregnancy resulted in upregulation of myosin and actin filament associated genes, potentially allowing for optimal muscle function and contraction. Our findings suggest a beneficial effect of supranutritional maternal organic Se supplementation during late gestation on Se-status and muscle development and function of newborn calves.

Micron-scale supramolecular myosin arrays help mediate cytoskeletal assembly at mature adherens junctions.

Epithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms that build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized, and highly organized actomyosin cytoskeleton at the zonula adherens, combining genetic and pharmacologic approaches with superresolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins, or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micron-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, overlying disorganized actin filaments. This suggested that myosin arrays might bundle actin at mature junctions. Consistent with this idea, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization and prevented actin bundling and polarization. We obtained similar results in Caco-2 cells. These results suggest a novel role for myosin self-assembly, helping drive actin organization to facilitate cell shape change.

Regulation of myosin light-chain phosphorylation and its roles in cardiovascular physiology and pathophysiology.

The regulation of muscle contraction is a critical function in the cardiovascular system, and abnormalities may be life-threatening or cause illness. The common basic mechanism in muscle contraction is the interaction between the protein filaments myosin and actin. Although this interaction is primarily regulated by intracellular Ca2+, the primary targets and intracellular signaling pathways differ in vascular smooth muscle and cardiac muscle. Phosphorylation of the myosinregulatory light chain (RLC) is a primary molecular switch for smooth muscle contraction. The equilibrium between phosphorylated and unphosphorylated RLC is dynamically achieved through two enzymes, myosin lightchain kinase, a Ca2+-dependent enzyme, and myosin phosphatase, which modifies the Ca2+ sensitivity of contractions. In cardiac muscle, the primary target protein for Ca2+ is troponin C on thin filaments; however, RLC phosphorylation also plays a modulatory role in contraction. This review summarizes recent advances in our understanding of the regulation, physiological function, and pathophysiological involvement of RLC phosphorylation in smooth and cardiac muscles.

Splicing factor Srsf5 deletion disrupts alternative splicing and causes noncompaction of ventricular myocardium

SummaryThe serine/arginine-rich (SR) family of splicing factors plays important roles in mRNA splicing activation, repression, export, stabilization, and translation. SR-splicing factor 5 (SRSF5) is a glucose-inducible protein that promotes tumor cell growth. However, the functional role of SRSF5 in tissue development and disease remains unknown. Here, Srsf5 knockout (Srsf5−/−) mice were generated using CRISPR-Cas9. Mutant mice were perinatally lethal and exhibited cardiac dysfunction with noncompaction of the ventricular myocardium. The left ventricular internal diameter and volume were increased in Srsf5−/− mice during systole. Null mice had abnormal electrocardiogram patterns, indicative of a light atrioventricular block. Mechanistically, Srsf5 promoted the alternative splicing of Myom1 (myomesin-1), a protein that crosslinks myosin filaments to the sarcomeric M-line. The switch between embryonic and adult isoforms of Myom1 could not be completed in Srsf5-deficient heart. These findings indicate that Srsf5-regulated alternative splicing plays a critical role during heart development.