Myosin Content Research Articles

Remodeling of the rat distal colon in diabetes: function and ultrastructure.

This study seeks to define and explain remodeling of the distal colon in the streptozotocin (STZ)-treated rat model of diabetes through analysis of resting and active length dependence of force production, chemical composition, and ultrastructure. Compared with untreated controls, the passive stiffness on extension of the diabetic muscle is high, and active force produced at short muscle lengths is amplified but is limited by an internal resistance to shortening. The latter are accounted for by a significant increase in collagen type 1, with no changes in types 3 and 4. In the diabetic colon, ultrastructural studies show unique, conspicuous pockets of collagen among muscle cells, in addition to a thickened basement membrane and an extracellular space filled with collagen fibers and various fibrils. Measurements of DNA and total protein content revealed that the diabetic colon underwent hypertrophy, along with a proportional increase in actin and myosin contents, with no change in the actin-to-myosin ratio. Active force production per cross-sectional area was not different in the diabetic and normal muscles, consistent with the proportionality of changes in contractile proteins. The stiffness and the limit to shortening of the diabetic colon were significantly reduced by treatment with the glycation breaker alagebrium chloride (ALT-711), with no change in collagen contents. Functionally, this study shows that, in diabetes, the production of collagen type 1 and glycation increase stiffness, which limits distensibility on filling and limits shortening and expulsion of contents, both of which can be alleviated by treatment with ALT-711.

The Relationship Between Activity Pattern and Muscle Adaptation in Skeletal Muscle

Muscle is highly plastic in terms of size (maximum force), speed, maximum power, and endurance. Well-controlled studies in animals have shown that the adult skeletal muscle fiber has a remarkable ability to modify its gene expression so that with long-term substantial changes in the daily activity pattern the contractile phenotype can be modified across the whole spectrum of fiber type found in control muscle. The contractile phenotype in this context includes the isoform content of myosin and therefore the maximum velocity of shortening, the mitochondrial content and therefore the specific force and aerobic capacity (endurance), and the calcium handling proteins and therefore the speed of activation and relaxation. With voluntary exercise in human subjects, similar responses are observed, although the degree of transformation is restricted by the practical limitations of exercise dosing to changes in mitochondrial activity and muscle size rather than the more profound changes in contractile protein isoform that can be induced with artificial activation over a substantial proportion of the day.

Solubilisation of myosin in a solution of low ionic strength l-histidine: Significance of the imidazole ring

Myosin, a major muscle protein, can be solubilised in a low ionic strength solution containing l-histidine (His). To elucidate which chemical constituents in His are responsible for this solubilisation, we investigated the effects of 5mM His, imidazole (Imi), l-α-alanine (Ala), 1-methyl-l-histidine (M-his) and l-carnosine (Car) on particle properties of myosin suspensions and conformational characteristics of soluble myosin at low ionic strength (1mM KCl, pH 7.5). His, Imi and Car, each containing an imidazole ring, were able to induce a myosin suspension, which had small particle size species and high absolute zeta potential, thus increasing the solubility of myosin. His, Imi and Car affected the tertiary structure and decreased the α-helix content of soluble myosin. Therefore, the imidazole ring of His appeared to be the significant chemical constituent in solubilising myosin at low ionic strength solution, presumably by affecting its secondary structure.

Two-Phase Acto-Cytosolic Fluid Flow in a Moving Keratocyte: A 2D Continuum Model.

The F-actin network and cytosol in the lamellipodia of crawling cells flow in a centripetal pattern and spout-like form, respectively. We have numerically studied this two-phase flow in the realistic geometry of a moving keratocyte. Cytosol has been treated as a low viscosity Newtonian fluid flowing through the high viscosity porous medium of F-actin network. Other involved phenomena including myosin activity, adhesion friction, and interphase interaction are also discussed to provide an overall view of this problem. Adopting a two-phase coupled model by myosin concentration, we have found new accurate perspectives of acto-cytosolic flow and pressure fields, myosin distribution, as well as the distribution of effective forces across the lamellipodia of a keratocyte with stationary shape. The order of magnitude method is also used to determine the contribution of forces in the internal dynamics of lamellipodia.

Role of Heavy Meromyosin in Heat-Induced Gelation in Low Ionic Strength Solution Containing L-Histidine.

The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing L-histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without L-histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing L-histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l-histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat-induced globular masses of HMM in low ionic strength solution containing L-histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing L-histidine, resulting in formation of the unique gel.

Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia.

Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass.

MicroRNA Transcriptome Profile Analysis in Porcine Muscle and the Effect of miR-143 on the MYH7 Gene and Protein.

Porcine skeletal muscle fibres are classified based on their different physiological and biochemical properties. Muscle fibre phenotype is regulated by several independent signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) signalling pathways. MicroRNAs are non-coding small RNAs that regulate many biological processes. However, their function in muscle fibre type regulation remains unclear. The aim of our study was to identify miRNAs that regulate muscle fibre type during porcine growth to help understand the miRNA regulation mechanism of fibre differentiation. We performed Solexa/Illumina deep sequencing for the microRNAome during 3 muscle growth stages (63, 98 and 161 d). In this study, 271 mature miRNAs and 243 pre-miRNAs were identified. We detected 472 novel miRNAs in the muscle samples. Among the mature miRNAs, there are 23 highest expression miRNAs (over 10000 RPM), account for 85.3% of the total counts of mature miRNAs., including 10 (43.5%) muscle-related miRNAs (ssc-miR-133a-3p, ssc-miR-486, ssc-miR-1, ssc-miR-143-3p, ssc-miR-30a-5p, ssc-miR-181a, ssc-miR-148a-3p, ssc-miR-92a, ssc-miR-21, ssc-miR-126-5p). Particularly, both ssc-miR-1 and ssc-miR-133 belong to the MyomiRs, which control muscle myosin content, myofibre identity and muscle performance. The involvement of these miRNAs in muscle fibre phenotype provides new insight into the mechanism of muscle fibre regulation underlying muscle development. Furthermore, we performed cell transfection experiment. Overexpression/inhibition of ssc-miR-143-3p in porcine skeletal muscle satellite cell induced an/a increase/reduction of the slow muscle fibre gene and protein (MYH7), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate MYH7 through the HDAC4-MEF2 pathway.

Contractile properties of the myotomal muscle of sheepshead, Archosargus probatocephalus

Swimming in fishes is powered by myotomal red, white and pink skeletal muscle. Slow swimming is powered by the red (slow-twitch muscle), fast speeds are achieved by the white (fast-twitch) muscle and pink muscle apparently serves an intermediate function. In recent years, the physiological properties and molecular composition of red (slow) and white (fast) muscle fibers have been well studied, while the intermediate pink muscle, which falls in a thin sheet between the superficial red muscle and deeper white muscle, has received less attention. The goal of this study is to determine the contractile properties of red, pink, and white muscle and to establish the molecular basis of fiber type variations in contractile properties in a sheepshead (Archosargus probatocephalus). Isometric and isovelocity muscle mechanics experiments demonstrated a general pattern of increasing contractile speed from red to pink to white muscle, although red and pink muscle did not differ significantly for most contraction kinetics variables. As myosin heavy chain (MyHC) is the most important structural protein found in the muscle fibers, MyHC content was examined through immunohistochemistry. Myosin antibodies suggest a gradient in myosin content corresponding to differences in muscle contraction kinetics.

The Kinetics of Fesselin (Avian Synaptopodin 2) Binding to Smooth Muscle Myosin is Dependent on Calcium-Calmodulin

Fesselin (or avian synaptopodin 2) is an actin binding protein that nucleates actin filament formation (Leinweber et al. 1999; Beall et al. 2001) and bundles actin filaments (Schroeter et al. 2013). The nucleating activity is inhibited by Ca2+-calmodulin (Schroeter et al. 2004). Fesselin also binds myosin with an affinity of 2 x 106 M−1 at 50 mM ionic strength (Schroeter & Chalovich 2005). That binding increases the length and width of myosin filaments, and reduces the rate of dissociation of myosin filaments and actin-myosin complexes by ATP (Kingsbury et al. 2013). Fesselin labeled with IANBD produced a fluorescence increase upon binding to smooth muscle myosin or S1. The apparent rate constant for the fluorescence change increased with increasing concentrations of myosin or S1 in a hyperbolic fashion (105 mM ionic strength, 10 deg C). Binding to myosin began with the rapid formation of a low affinity intermediate followed by a second step having an observed rate constant (k2 + k-2) of 30/sec. Several observations suggested that the binding of fesselin to myosin was affected by calmodulin and calcium. The apparent rate constants for both processes in the binding of IANBD-fesselin to S1 were 2-fold faster in the absence of calcium. Binding of IANBD fesselin to intact myosin was complex in the presence of calcium with a rapid increase in fluorescence followed by a slower decrease; that behavior was not observed in the presence of calcium but absence of calmodulin. Ca2+-calmodulin may play roles in the formation of actin filaments, myosin filaments and actin-myosin complexes through fesselin.

Actomyosin Dynamics in 3D Traction Force Generation

The generation and coordination of cellular traction forces plays important roles in cell adhesion, cell migration, and extracellular matrix (ECM) remodeling, and hence in the development and repair of biological tissues. Historically, models for how cells generate and sense mechanical force have been derived from observations of cells grown on hard, two-dimensional (2D) surfaces. However, the cytoskeletal geometry of cells embedded in porous, 3D environments is inherently different than those found in 2D. Here we seek to understand the spatio-temporal regulation of cellular traction forces in a 3D environment. We embed primary human fibroblasts in a fluorescently labeled fibrin matrix, and use multicolor, time-lapse confocal microscopy to simultaneously image matrix deformation induced by cellular traction and the dynamics of the acto-myosin cytoskeleton and paxillin-rich cell-matrix adhesions. We observe significant traction forces transduced to the matrix through dynamic actomyosin-rich protrusions. Flux analysis of myosin concentrations in the protrusions reveals recruitment of myosin towards the protrusion tip during extension, and localization of myosin farther from the tip during retraction. Moreover, we observe both retrograde and anterograde movement of F-actin and myosin during cell traction generation, as well as similar bi-directional dynamics for paxillin-rich adhesions. Our data suggests a model of force generation and mechanotransduction different from the canonical continuous lamellipodial retrograde flow implicated in 2D cell migration. Ongoing work examines the role of signal transduction pathways in regulating cellular force generation, with the end goal of understanding how cells couple force generation and mechanotransduction in fully 3D environments.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization.

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

Ultraslow myosin molecular motors of placental contractile stem villi in humans.

Human placental stem villi (PSV) present contractile properties. In vitro mechanics were investigated in 40 human PSV. Contraction of PSV was induced by both KCl exposure (n = 20) and electrical tetanic stimulation (n = 20). Isotonic contractions were registered at several load levels ranging from zero-load up to isometric load. The tension-velocity relationship was found to be hyperbolic. This made it possible to apply the A. Huxley formalism for determining the rate constants for myosin cross-bridge (CB) attachment and detachment, CB single force, catalytic constant, myosin content, and maximum myosin ATPase activity. These molecular characteristics of myosin CBs did not differ under either KCl exposure or tetanus. A comparative approach was established from studies previously published in the literature and driven by mean of a similar method. As compared to that described in mammalian striated muscles, we showed that in human PSV, myosin CB rate constants for attachment and detachment were about 103 times lower whereas myosin ATPase activity was 105 times lower. Up to now, CB kinetics of contractile cells arranged along the long axis of the placental sheath appeared to be the slowest ever observed in any mammalian contractile tissue.

Constriction model of actomyosin ring for cytokinesis by fission yeast using a two-state sliding filament mechanism.

We developed a model describing the structure and contractile mechanism of the actomyosin ring in fission yeast, Schizosaccharomyces pombe. The proposed ring includes actin, myosin, and α-actinin, and is organized into a structure similar to that of muscle sarcomeres. This structure justifies the use of the sliding-filament mechanism developed by Huxley and Hill, but it is probably less organized relative to that of muscle sarcomeres. Ring contraction tension was generated via the same fundamental mechanism used to generate muscle tension, but some physicochemical parameters were adjusted to be consistent with the proposed ring structure. Simulations allowed an estimate of ring constriction tension that reproduced the observed ring constriction velocity using a physiologically possible, self-consistent set of parameters. Proposed molecular-level properties responsible for the thousand-fold slower constriction velocity of the ring relative to that of muscle sarcomeres include fewer myosin molecules involved, a less organized contractile configuration, a low α-actinin concentration, and a high resistance membrane tension. Ring constriction velocity is demonstrated as an exponential function of time despite a near linear appearance. We proposed a hypothesis to explain why excess myosin heads inhibit constriction velocity rather than enhance it. The model revealed how myosin concentration and elastic resistance tension are balanced during cytokinesis in S. pombe.

The solubility and conformational characteristics of porcine myosin as affected by the presence of l-lysine and l-histidine

The influence of l-lys and l-his on the solubility, surface hydrophobicity, sulphydryl content and conformational characteristics of porcine myosin solubilised in high (0.6M), physiological (0.15M) and low (1mM) ionic strength solutions were explored. The solubility of myosin was increased in the presence of l-his and/or l-lys in all ionic strength solutions used. The presence of l-his and l-lys caused increases in the surface hydrophobicity and reactive sulphydryl content (p<0.05). Circular dichroism revealed a significant decrease of α-helical content with an increase of random coils, β-turns and β-sheets in the presence of l-his and/or l-lys. These results demonstrate that the introduction of l-lys and l-his causes the unfolding of myosin, resulting in loss of α-helical structure, which is followed by increases in random coils, β-turns and β-sheets, which exposes buried hydrophobic and sulphydryl groups to the myosin surface, ultimately increasing the solubility of porcine myosin.

Myofilament dysfunction contributes to impaired myocardial contraction in the infarct border zone.

After myocardial infarction, a poorly contracting nonischemic border zone forms adjacent to the infarct. The cause of border zone dysfunction is unclear. The goal of this study was to determine the myofilament mechanisms involved in postinfarction border zone dysfunction. Two weeks after anteroapical infarction of sheep hearts, we studied in vitro isometric and isotonic contractions of demembranated myocardium from the infarct border zone and a zone remote from the infarct. Maximal force development (Fmax) of the border zone myocardium was reduced by 31 ± 2% versus the remote zone myocardium (n = 6/group, P < 0.0001). Decreased border zone Fmax was not due to a reduced content of contractile material, as assessed histologically, and from myosin content. Furthermore, decreased border zone Fmax did not involve altered cross-bridge kinetics, as assessed by muscle shortening velocity and force development kinetics. Decreased border zone Fmax was associated with decreased cross-bridge formation, as assessed from muscle stiffness in the absence of ATP where cross-bridge formation should be maximized (rigor stiffness was reduced 34 ± 6%, n = 5, P = 0.011 vs. the remote zone). Furthermore, the border zone myocardium had significantly reduced phosphorylation of myosin essential light chain (ELC; 41 ± 10%, n = 4, P < 0.05). However, for animals treated with doxycycline, an inhibitor of matrix metalloproteinases, rigor stiffness and ELC phosphorylation were not reduced in the border zone myocardium, suggesting that doxycycline had a protective effect. In conclusion, myofilament dysfunction contributes to postinfarction border zone dysfunction, myofilament dysfunction involves impaired cross-bridge formation and decreased ELC phosphorylation, and matrix metalloproteinase inhibition may be beneficial for limiting postinfarct border zone dysfunction.

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber.

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.

Non-weight bearing-induced muscle weakness: the role of myosin quantity and quality in MHC type II fibers.

We tested the hypothesis that non-weight bearing-induced muscle weakness (i.e., specific force) results from decreases in myosin protein quantity (i.e., myosin content per half-sarcomere and the ratio of myosin to actin) and quality (i.e., force per half-sarcomere and population of myosin heads in the strong-binding state during muscle contraction) in single myosin heavy chain (MHC) type II fibers. Fisher-344 rats were assigned to weight-bearing control (Con) or non-weight bearing (NWB). The NWB rats were hindlimb unloaded for 2 wk. Diameter, force, and MHC content were determined in permeabilized single fibers from the semimembranosus muscle. MHC isoform and the ratio of MHC to actin in each fiber were determined by gel electrophoresis and silver staining techniques. The structural distribution of myosin from spin-labeled fiber bundles during maximal isometric contraction was evaluated using electron paramagnetic resonance spectroscopy. Specific force (peak force per cross-sectional area) in MHC type IIB and IIXB fibers from NWB was significantly reduced by 38% and 18%, respectively. MHC content per half-sarcomere was significantly reduced by 21%. Two weeks of hindlimb unloading resulted in a reduced force per half-sarcomere of 52% and fraction of myosin strong-binding during contraction of 34%. The results suggest that reduced myosin and actin content (quantity) and myosin quality concomitantly contribute to non-weight bearing-related muscle weakness.

Paracrine effects of cancer on skeletal muscle: myosin loss and mitochondrial dysfunction (1163.10)

Cancer cachexia is a severe skeletal muscle wasting syndrome directly responsible for 30% of all cancer deaths. Cancer exerts paracrine effects on skeletal muscle that trigger myosin loss and metabolic dysfunction. Clinical trials using cyclooxygenase 2 (COX2) inhibitors to treat cachexia had some success. Therefore, we hypothesized that tumor‐induced COX2 expression contributes to myosin loss and mitochondrial dysfunction. We incubated C2C12 myotubes with control or Lewis lung carcinoma conditioned medium (LLCCM) for 48hr. We measured protein content by western blot, and oxygen consumption rate (OCR) of intact myotubes using a Seahorse XF24. Protein content is presented as means±SD in arbitrary units (n=6/group, t test). OCR is presented in pmoles O2/min (n=8/group, repeated measures ANOVA). LLCCM treatment decreased myosin content by 70%, from 7.2±2.7 to 2.1±1 AU (p&lt;0.01), while COX2 content increased 75%, from 1.9 ±0.1 to 3.3 ±0.2 (p&lt;0.001). There was no significant effect on mitochondrial complex content, p &gt;0.05 for complexes I, III, IV and V. LLCCM decreased basal OCR by 30% (p&lt;0.003), but did not alter leak or maximal uncoupled OCR. We found that LLCCM treatment induced myosin loss and impaired mitochondrial function in C2C12 myotubes, correlating with increased COX2 content. These data support our original hypothesis, and suggest that interventions that target COX2 may be useful for cancer cachexia.Grant Funding Source: Supported by Support: NIH AR055974 (FHA).

Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle

Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either (i) uniformly, from the Z-line end of thick-filaments, or (ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulations also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere.

Myosin denaturation in “Burnt” Bluefin tuna meat

A quantitative conversion of tuna meat into muscle homogenate made it possible to study myosin denaturation in Bluefin tuna meat. Myosin denaturation was accessed by measuring Ca2+-ATPase activity, salt-solubility with and without Mg-ATP, monomeric myosin content, and amount of subfragment-1 (S-1) and rod produced by chymotryptic digestion. Commercially available tuna used in this study showed a pH around 5.4–5.7. Myosin in the meat lost the salt-solubility measured in the absence of Mg-ATP; however, such myosin showed full salt-solubility when released from actin in the presence of Mg-ATP. Incubation of tuna meat at 30 °C for up to 90 min caused obvious myosin denaturation. However, the tuna meat dialyzed against neutral pH buffer showed practically no myosin denaturation by the same heating. It was suggested that exposure to lowered pH to around 5.5 and increased temperature of 30 °C led myosin denaturation. Myosin denaturation in the “Burnt” Bluefin tuna sample was analyzed. A significant myosin denaturation was observed with the part showing the “Burning” symptom, the inner part of the tuna meat near the spine. Myosin in that part showed almost no Ca2+-ATPase activity, no salt-solubility even with Mg-ATP, no recovery of monomeric myosin, and almost no production of S-1 by chymotryptic digestion. However, myosin denaturation was not detectable for the meat taken from outer parts of the same tuna near the skin with normal appearance. It was demonstrated that “Burning” of tuna meat occurring in the deep part of the body is accompanied by myosin denaturation. The above results suggested that insufficient cooling of the deep part of body would be the reason for “Burning” of tuna meat.