Rigor State Research Articles

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

Are there any differences on the quality attributes of pre - and post-rigor chevon?

The present study was conducted to find out whether pre-rigor and post-rigor state has significant impact on quality attributes of chevon. For this 10 Longissimus dorsi muscles from five Black Bengal goats were collected. Fresh chevon were considered as pre-rigor sample and other five samples were preserved at 4ºC counted as post-rigor samples. Independent t-test was used for analyses the data. The findings demonstrated that among physicochemical characteristics, rigor state had no discernible (p>0.05) impact on water holding capacity (WHC) and cooking loss (CL). However, post-rigor chevon drip loss was substantially higher (p<0.05) than pre-rigor chevon drip loss whereas pH value was significantly (p<0.05) higher in pre-rigor chevon. Higher CIE a*, b*, Hue angle values were found in post-rigor than pre-rigor chevon. Post-rigor chevon had substantially better (p<0.05) flavor, tenderness, texture and general acceptability. On the contrary, pre-rigor chevon's TVC, TCC and TYMC microbial count values were all substantially higher (p<0.05) than those of the post-rigor chevon. So, it can be sum up that, there are notable differences between pre-rigor and post-rigor chevon in a number of physicochemical and sensory properties. Despite having less drip loss of pre-rigor chevon, post-rigor chevon is more palatable and less contaminated by microbes.

The effect of modified atmosphere packaging at an early postmortem stage on lamb meat quality during subsequent aging.

This study explores the influence of modified atmosphere packaging (MAP) on fresh lamb meat quality with respect to gas concentration, rigor state, and post-mortem aging time. A comparison was done for the quality characteristics of lamb Longissimus thoracis lumborum chops that had been packaged separately in air, 75%O2 +25%CO2 MAP or 50%O2 +50%CO2 MAP at 1, 6, and 24h post-mortem and then stored for 6, 12, 24, 72, and 144h post-mortem, and the quality of lamb chops had been evaluated at each post-mortem period separately. Chops packaged at 1 and 6h post-mortem in MAP had reduced pH decline, less purge loss, and enhanced redness at early post-mortem storage times. Lamb color stability was evidently greater in 75%O2 +25%CO2 MAP than in 50%O2 +50%CO2 MAP during the early storage period when a* and R630/R580 were taken into account. Shear force values were lowest in lambs packaged at 1h post-mortem with 75%O2 +25%CO2 MAP at 12h post-mortem and then increased until 72h post-mortem, suggesting that rigor has been delayed by such a high O2 MAP. Thus, fresh lamb quality was maintained most effectively when packaged at 1h post-mortem in 75%O2 +25%CO2 MAP for consumption at 12h post-mortem. The exact mechanisms and optimization of MAP based on Chinese retail conditions should be considered in future studies. PRACTICAL APPLICATION: In this study, three slaughter patterns in the meat industry involving boning immediately after dressing (hot-boning) and chilling for a short period (warm-boning) or overnight (cold-boning) are considered, as well as the behavior of non-immediate consumption after purchase. Modified atmosphere packaging provides an effective preservation of early post-mortem muscles with enhanced color stability, water holding capacity, and texture during refrigerated storage. This could provide new insights into how to process lamb muscles in the early post-mortem period to improve and stabilize lamb quality.

Characterizing the functional impact of embryonic myosin mutations in stem cell-derived skeletal muscle.

Divergent contractile properties of different myosins suggest that each isoform performs distinct functions in embryonic and adult skeletal muscles. To better understand the role of embryonic myosin heavy chain 3 (MYH3) in regulating skeletal muscle pattern formation, growth and regeneration, our group investigated the functional defects caused by multiple mutations. Our team generated human induced pluripotent stem cell (iPSC) lines bearing T178I or R672C mutations in MYH3. Human iPSCs bearing these mutations were differentiated into skeletal muscle and evaluated for differences in the structure, maturation and functional performance of the sarcomere. Isolated myofibril mechanics showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to controls. Myofibril preparations were also used to compare ATP binding rates and showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. When incorporated into 3D engineered tissues, these changes in MYH3 mechanics resulted in slower relaxation kinetics and an incomplete relaxation of the tissue in response to repetitive tetanic stimulation, which may explain the emergence of developmental contractures in patients possessing MYH3 mutations. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the β-sheet and SH-Helix than in controls. Mutations also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

A molecular scale investigation of the mechanisms of contractile dysfunction for the hypertrophic cardiomyopathy MYH7 G256E mutation.

Genetic sequencing enables the pinpointing of specific genetic mutations correlated with striated muscle diseases. However, such associations do not describe mechanistic relationships between specific mutations and clinical phenotypes. To address this, we use CRISPR-edited human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to probe how mutations in β-myosin heavy chain (MHC) lead to cardiac muscle disease. Preliminary data show that the specific force generation and activation rate of mutant G256E myofibrils is greater than isogenic control myofibrils. During relaxation, the initial, slow phase kinetics were decreased, indicating slower cross-bridge detachment rate. Additionally, stopped-flow mant-ATP assays on isolated myofibrils showed slower ATP binding for the G256E mutation, supporting delayed relaxation observed in myofibrils. Overall, these data suggest a hypercontractile phenotype with impaired early relaxation for the G256E mutation. To understand the molecular structure basis of these effects we have used molecular dynamics simulations of wild type and mutant β-MHC. In post-rigor (M.ATP) and rigor state simulations, we observed reduced stability in the transducer region of β-myosin, due to weakened hydrogen bonds between neighboring β-sheet strands as well as significant changes in local contacts. Pairing these in silico data with our in vitro data, our results suggest that the G256E mutation affects the transducer region of myosin such that it alters communication between the nucleotide binding pocket and the actin binding surface during the acto-myosin chemo-mechanical cycle, leading to hypercontractility and slowed relaxation.

Embryonic myosin mutations alter the mechanics and maturation of HiPSC derived skeletal muscle.

Distal arthrogryposis (DA) is a skeletal muscle disorder characterized by joint contractures predominantly localized in the distal extremities. DA associated syndromes like Freeman-Sheldon syndrome (FSS) are linked to mutations in the MYH3 gene that encodes the embryonic skeletal muscle myosin. To study the mutation and its effect on developing muscle, we generated human induced pluripotent stem cell (hiPSC) lines bearing T178I or R672C MYH3 mutations. hiPSC were differentiated into skeletal muscle and evaluated for differences in the maturation of the contractile unit and its functional performance. Preliminary myofibril mechanics measurements showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to control myofibrils. Myofibril preparations were also used to compare ATP binding rates across conditions. Preliminary results showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. Mutations in MYH3 were also associated with alterations in myosin isoform content, with homozygous R672C having reduced MYH3 levels in day 7 cells. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the beta-sheet and SH-helix than in controls. Mutation also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

Insights into the gel and electronic sense characteristics of meat batters made from Funiu white goat and Oula sheep meat in different rigor states

To study the effect of rigor state on physicochemical characteristics of Funiu white goat and Oula sheep meat batters. In this study, the effect of rigor state of Funiu white goat and Oula sheep silverside muscle on meat batter quality and physicochemical properties was determined. Results indicated that meat batters obtained from post-rigor meat had less water holding capacity and lower immobilized water than that obtained from pre-rigor meat. The gel network of meat batters produced with Funiu white goat muscle appeared denser and smoother compared with Oula sheep meat batters. Furthermore, meat batters produced with pre-rigor meat had higher hardness compared to that produced with post-rigor meat. Differences in response values were found for CPS (sensitivity to sweet taste) of meat batters made with meat in the three rigor states. Collectively, both Funiu white goat and Oula sheep meat in pre-rigor state were more suitable to minced meat product production compared to post-rigor meat based on gel properties.

Molecular mechanisms of cardiac actomyosin transforming from rigor state to post-rigor state.

Sudden cardiac death contributed to half of all deaths from cardiovascular diseases. The mechanism of the kinetic cycle of cardiac myosin is crucial for heart protection and drug development. The state change in the myosin kinetic cycle from the rigor state to the post-rigor state is fundamental to explain binding and dissociation. Here, we used β-cardiac myosin in the rigor and post-rigor states to model the actomyosin complexes. Molecular dynamics simulations, electrostatic analysis, and energetic analysis of actomyosin complexes were performed in this work. The results showed that there are fewer interactions and lower electrostatic binding strength in the post-rigor state than in the rigor state. In the post-rigor state, there were higher free binding energy, fewer salt bridges, and fewer hydrogen bonds. The results showed a lower binding affinity in the post-rigor state than in the rigor state. The decrease in the binding affinity provided important conditions for dissociation of the myosin from the actin filament. Although previous studies focused mostly on the binding process, this study provides evidence of dissociation, which is even more important in the myosin kinetic cycle. This research on the mechanism of myosin kinetic cycles provides a novel direction for future genetic disease studies.

Differences in eating quality and electronic sense of meat samples as a function of goat breed and postmortem rigor state

Understanding the pre- and post-rigor meat quality characteristics is crucial for the development of featured goat meat products. To evaluate the quality characteristics of goat meat in different rigor states, knuckle muscle in pre-rigor, rigor, and post-rigor states of Taihang and Huanghuai goats were used to analyze chemical characteristics, water mobility, and electronic sense. Results indicated that Taihang and Huanghuai goat meat samples were rich in protein and essential amino acids, especially Lys. Pre-rigor goat meat samples had lower content of free moisture and higher water holding capacity than post-rigor goat meat samples. Furthermore, Taihang and Huanghuai goat meat samples exhibited different odour characteristics. Aromatic constituents content of post-rigor goat meat samples were greater than those of pre-rigor goat meat samples. The sweetness and bitterness of post-rigor goat meat samples were higher than those of pre-rigor goat meat samples. In conclusion, Taihang and Huanghuai goat meat samples have different quality characteristics, which were regulated by rigor state through different texture and flavor profiles.

Combined PEF, CO2 and HP application to chilled coho salmon and its effects on quality attributes under different rigor conditions

Effects of pulsed electric fields (PEF: 1–2 kV/cm, 20 μs) used alone and combined with carbon dioxide (CO2: 70%) and high hydrostatic pressure (HP: 150 MPa, 5 min) on the physicochemical properties and microbiological shelf life of coho salmon under pre- and post-rigor conditions were studied during refrigerated storage (25 days). The PEF + CO2 + HP treatments reduced color changes in pre-rigor more than in post-rigor salmon. Texture changes of pre- and post-rigor salmon differed between untreated and treated salmon. Combined treatments also effectively reduced the protease and lipase activity of pre-rigor salmon. Collagenase activity of post-rigor salmon remained low in all treatments. Principal component analysis showed a clear difference between pre- and post-rigor states in the evaluated parameters; however, microbiological shelf life increased with PEF + CO2 regardless of the rigor condition. In conclusion, combined PEF, CO2, and HP treatments can improve salmon quality during refrigerated storage and thus extend its microbial shelf life. Industrial relevanceThe current demand for processed seafood that is similar to fresh products has created a new area of development in non-thermal processing technologies. Refrigeration and freezing have been applied for many years to extend the shelf life of seafood products, but there is deterioration regardless of the preservation method. Therefore, the combination of non-thermal processing technologies (PEF + CO2 + HP) could be applied to a large variety of seafood industries. Seafood products could be treated by PEF combined with CO2 and HP to further evaluate quality attributes under different rigor conditions during chilled storage and estimate the shelf life of the processed product.

Cryo-EM structure of F-actin decorated by HMM in rigor state

Cryo-EM structure of F-actin decorated by HMM in rigor state

Longitudinal vibration interferes with cross-bridge attachment and prevents muscle fibre shrinkage under PSE-like conditions

The impact of longitudinal vibration on cross-bridge attachments between myofilaments was investigated in single fibres and intact muscle. Sinusoidal length vibration (frequency 50 Hz, amplitude 5% of fibre length) reduced active force by 40% when fibres were activated by elevation of [Ca2+], but did not alter the force when fibres were in rigor state. When vibrated for 30 min in rigor at pH 5.5 and 38 °C (PSE conditions), the lateral shrinkage of the fibres was significantly reduced, suggesting a potential positive influence of vibration on water-holding capacity. In whole muscle incubated at 38 °C until 8 h post mortem, the progress of rigor onset was accessed by measuring the increase in muscle stiffness. Vibration applied 3-5 h post mortem postponed rigor development, but did not have significant influence on water-holding capacity compared with non-vibrated conditions. In conclusion, the results suggest that muscle vibration can be a future technique to delay rigor development and prevent muscle fibre shrinkage and PSE development after slaughter.

The actomyosin interface contains an evolutionary conserved core and an ancillary interface involved in specificity

Plasmodium falciparum, the causative agent of malaria, moves by an atypical process called gliding motility. Actomyosin interactions are central to gliding motility. However, the details of these interactions remained elusive until now. Here, we report an atomic structure of the divergent Plasmodium falciparum actomyosin system determined by electron cryomicroscopy at the end of the powerstroke (Rigor state). The structure provides insights into the detailed interactions that are required for the parasite to produce the force and motion required for infectivity. Remarkably, the footprint of the myosin motor on filamentous actin is conserved with respect to higher eukaryotes, despite important variability in the Plasmodium falciparum myosin and actin elements that make up the interface. Comparison with other actomyosin complexes reveals a conserved core interface common to all actomyosin complexes, with an ancillary interface involved in defining the spatial positioning of the motor on actin filaments.

ADP-Bound Actomyosin State Exhibits Lower Stiffness than the Rigor State

ADP-Bound Actomyosin State Exhibits Lower Stiffness than the Rigor State

Estimating freshness of ice storage rainbow trout using bioelectrical impedance analysis.

This study aimed to evaluate the freshness of ice stored rainbow trout by bioelectrical impedance measurements. Rigor mortis, ATP‐related components, K‐value, and hardness of rainbow trout muscle during storage were monitored along with impedance. The results showed that the progress of rigor mortis was accompanied by an increase in impedance. Impedance kept decreasing even in rigor state, and during the gradual resolution of rigor mortis with impedance change upon storage of fish was biphasic (r = −0.944, p < .01). Thus, when impedance decreased close to the lowest value, K‐value was only about 61.57 ± 0.52%, but still exhibited a high pertinence (r = −0.959, p < .01). A gradual decrease of the hardness of fish muscle upon storage of fish showed a close correlation (r = 0.981, p < .01) with impedance decrease. These results suggested that the impedance measurement has a great potential for predicting the freshness of the rainbow trout during ice storage.

Structural and Computational Insights into a Blebbistatin-Bound Myosin•ADP Complex with Characteristics of an ADP-Release Conformation along the Two-Step Myosin Power Stoke.

The motor protein myosin drives a wide range of cellular and muscular functions by generating directed movement and force, fueled through adenosine triphosphate (ATP) hydrolysis. Release of the hydrolysis product adenosine diphosphate (ADP) is a fundamental and regulatory process during force production. However, details about the molecular mechanism accompanying ADP release are scarce due to the lack of representative structures. Here we solved a novel blebbistatin-bound myosin conformation with critical structural elements in positions between the myosin pre-power stroke and rigor states. ADP in this structure is repositioned towards the surface by the phosphate-sensing P-loop, and stabilized in a partially unbound conformation via a salt-bridge between Arg131 and Glu187. A 5 Å rotation separates the mechanical converter in this conformation from the rigor position. The crystallized myosin structure thus resembles a conformation towards the end of the two-step power stroke, associated with ADP release. Computationally reconstructing ADP release from myosin by means of molecular dynamics simulations further supported the existence of an equivalent conformation along the power stroke that shows the same major characteristics in the myosin motor domain as the resolved blebbistatin-bound myosin-II·ADP crystal structure, and identified a communication hub centered on Arg232 that mediates chemomechanical energy transduction.

The Structure of Acto-Myosin.

After several decades studying different acto-myosin complexes at lower and intermediate resolution- limited by the electron microscope instrumentation available then- recent advances in imaging technology have been crucial for obtaining a number of excellent high-resolution 3D reconstructions from cryo electron microscopy. The resolution level reached now is about 3-4Å, which allows unambiguous model building of filamentous actin on its own as well as that of actin filaments decorated with strongly bound myosin variants. The interface between actin and the myosin motor domain can now be described in detail, and the function of parts of the interface (such as, e.g., the cardiomyopathy loop) can be understood in a mechanistical way. Most recently, reconstructions of actin filaments decorated with different myosins, which show a strongly bound acto-myosin complex also in the presence of the nucleotide ADP, have become available. The comparison of these structures with the nucleotide-free Rigor state provide the first mechanistic description of force sensing. An open question is still the initial interaction of the motor domain of myosin with the actin filament. Such weakly interacting states have so far not been the subject of microscopical studies, even though high-resolution structures would be needed to shed light on the initial steps of phosphate release and power stroke initiation.

Factors Affecting the Digestibility of Beef and Consequences for Designing Meat-Centric Meals

The impact of the following on beef digestibility was determined by static in vitro methods: (1) age of cattle; (2) muscle rigor state, ultimate pH, and mincing/particle size; (3) muscle/meat cut; (4) organ meats; and (5) meat accompaniments. Results indicate that beef is more digestible from older compared to younger cattle; prerigor compared to postrigor meat; higher compared to lower ultimate pH meat; cuts with lower compared to higher collagen contents; finely compared to coarsely minced/ground meat; and organ (liver and kidney) compared to muscle meat. Beef digestibility is enhanced when cooked with mushroom and pumpkin and reduced with starchy foods such as rice and potatoes. The outcomes of this study provide a base for the scientific design of meals with beef as a central ingredient and digestibility as the main functionality of interest.

Changes in physicochemical characteristics and oxidative stability of pre- and post-rigor frozen chicken muscles during cold storage.

The objective of this study was to investigate the effects of rigor state on physicochemical characteristics and the oxidative stability of chicken leg and breast muscles as a function of freezing time. Breast and leg muscles were excised from 24 broiler chickens at 30min or 1.5h postmortem (PM), frozen overnight at - 75°C immediately, and then stored at - 20°C for 90days to measure the meat quality traits. Results showed that longer freezing led to deterioration of meat quality with higher deterioration for post-rigor frozen muscles. Pre-rigor frozen muscles had higher pH, water holding capacity (around 90%), and sarcomere length with a lower thaw and cook loss than post-rigor frozen muscles. The Warner-Bartzler shear force (WBSF) values for chicken leg and breast muscles were insignificant (except pre-rigor leg muscles which had significantly higher WBSF value only at 90thday of storage). The lightness (L*) value increased significantly with increasing storage for all samples. Post-rigor muscles had significantly higher TBARS values (0.62mg MDA/kg) than the pre-rigor muscles. The leg muscles had better physicochemical characteristics compared to breast muscles, except for the cook loss. Therefore, immediate freezing (prior to onset of rigor) could be an effective way to minimize the quality deterioration of frozen chicken muscles.

Atomically Detailed Simulation of the Powerstroke in Myosin II by Milestoning

The interaction between actin and myosin II plays an important role in a variety of cellular functions. In particular, myosin II is involved in muscle contraction, which is attributed to the sliding of thin filament actin past the thick myosin II filaments. Past studies on the structure of myosin have linked severe pathologies to defects in myosin, making it important to understand the mechanism of the system. In this study, we focus our analysis on the powerstroke of the myosin II cross bridge cycle, which is the active process of muscle contraction. To do this, we use an algorithm called Milestoning which partitions the dynamics into a sequence of trajectories between “milestones” along the reaction pathway. The structure of myosin II bound to actin in the rigor state was used as a starting point, and a structure for the bound pre-powerstroke state was developed using existing published structures for the unbound pre-powerstroke state as well as experimental data gathered about the movement of myosin II during the powerstroke. With both the beginning and final states of the powerstroke, we can interpolate between these structures to build intermediate states along the reaction pathway. A total of 97 all-atom structures along the pathway of the powerstroke were developed to serve as guides and “anchors” along the reaction pathway. Milestoning short trajectories between cells defined by the anchors will allow for the computation thermodynamics and kinetics of the myosin II powerstroke. This work will lead to a significant improvement in our understanding of the complete powerstroke mechanism, which will in turn facilitate future research on the effects of structural defects in myosin II on powerstroke function and muscle contraction.