Transition Of Myosin Research Articles

Vitamin C Drives Reentrant Actin Phase Transition: Biphasic Exocytosis Regulation Revealed by Single-Vesicle Electrochemistry.

Unveiling molecular mechanisms that dominate protein phase dynamics has been a pressing need for deciphering the intricate intracellular modulation machinery. While ions and biomacromolecules have been widely recognized for modulating protein phase separations, effects of small molecules that essentially constitute the cytosolic chemical atmosphere on the protein phase behaviors are rarely understood. Herein, we report that vitamin C (VC), a key small molecule for maintaining a reductive intracellular atmosphere, drives reentrant phase transitions of myosin II/F-actin (actomyosin) cytoskeletons. The actomyosin bundle condensates dissemble in the low-VC regime and assemble in the high-VC regime in vitro or inside neuronal cells, through a concurrent myosin II protein aggregation-dissociation process with monotonic VC concentration increase. Based on this finding, we employ in situ single-cell and single-vesicle electrochemistry to demonstrate the quantitative modulation of catecholamine transmitter vesicle exocytosis by intracellular VC atmosphere, i.e., exocytotic release amount increases in the low-VC regime and decreases in the high-VC regime. Furthermore, we show how VC regulates cytomembrane-vesicle fusion pore dynamics through counteractive or synergistic effects of actomyosin phase transitions and the intracellular free calcium level on membrane tensions. Our work uncovers the small molecule-based reversive protein phase regulatory mechanism, paving a new way to chemical neuromodulation and therapeutic repertoire expansion.

Exploring the Connection Between Relaxed Myosin States and the Anrep Effect.

The Anrep effect is an adaptive response that increases left ventricular contractility following an acute rise in afterload. Although the mechanistic origin remains undefined, recent findings suggest a two-phase activation of resting myosin for contraction, involving strain-sensitive and posttranslational phases. We propose that this mobilization represents a transition among the relaxed states of myosin-specifically, from the super-relaxed (SRX) to the disordered-relaxed (DRX)-with DRX myosin ready to participate in force generation. This hypothesis offers a unified explanation that connects myosin's SRX-DRX equilibrium and the Anrep effect as parts of a singular phenomenon. We underscore the significance of this equilibrium in modulating contractility, primarily studied in the context of hypertrophic cardiomyopathy, the most common inherited cardiomyopathy associated with diastolic dysfunction, hypercontractility, and left ventricular hypertrophy. As we posit that the cellular basis of the Anrep effect relies on a two-phased transition of myosin from the SRX to the contraction-ready DRX configuration, any dysregulation in this equilibrium may result in the pathological manifestation of the Anrep phenomenon. For instance, in hypertrophic cardiomyopathy, hypercontractility is linked to a considerable shift of myosin to the DRX state, implying a persistent activation of the Anrep effect. These valuable insights call for additional research to uncover a clinical Anrep fingerprint in pathological states. Here, we demonstrate through noninvasive echocardiographic pressure-volume measurements that this fingerprint is evident in 12 patients with hypertrophic obstructive cardiomyopathy before septal myocardial ablation. This unique signature is characterized by enhanced contractility, indicated by a leftward shift and steepening of the end-systolic pressure-volume relationship, and a prolonged systolic ejection time adjusted for heart rate, which reverses post-procedure. The clinical application of this concept has potential implications beyond hypertrophic cardiomyopathy, extending to other genetic cardiomyopathies and even noncongenital heart diseases with complex etiologies across a broad spectrum of left ventricular ejection fractions.

Abstract P1026: Structural And Biochemical Effects Of Missense MyBP-C Mutations On The Myofilament In Human Hypertrophic Cardiomyopathy

Significance: While the disease mechanisms of truncating MyBP-C variants in HCM have been well defined, the mechanisms by which missense MyBP-C variants cause HCM are largely unknown. Recent studies show that unlike truncating variants that push myosin into an active high energy state due to their overall reduction in MyBP-C, missense MyBP-C properly incorporates into the sarcomere without affecting protein abundance. The consequences of missense MyBP-C on myosin have not been explored and may have implications for the treatment of HCM patients with missense MyBP-C variants. Results: We performed small angle X-ray diffraction (XrD) on human HCM tissue from patients with missense MyBP-C, and healthy control hearts. When healthy tissue was moved from relaxing pCa 8 to activating pCa 5 the I 1,1 /I 1,0 equatorial intensity ratio increased (p<0.001) indicating myosin is moving away from the backbone and adopting an active conformation. However, in missense hearts I 1,1 /I 1,0 is lower than healthy hearts at pCa 8 meaning more myosin is localized to the backbone. This is contrary to other HCM mutations which are proposed to push myosin to an active conformation under relaxing conditions. I 1,1 /I 1,0 does not increase at pCa 5 (p>0.05) indicating that at activating Ca myosin remains in the inactive conformation suggesting the thick filament Ca response is impaired in missense hearts. Ongoing Experiments: We are planning to perform XrD on more samples and Mant-ATP assays to determine if the structural inactivation of myosin in missense tissue also results in myosin remaining in the low energy super relaxed state (SRX). We expect the structural state to correlate with the biochemical state meaning missense hearts will demonstrate an equivalent percentage of SRX myosin compared to control hearts. Conclusions: Our finding that the transition of myosin to a structurally active conformation does not occur in human MyBP-C missense hearts distinguishes these variants from other HCM causing mutations such as truncating MyBP-C and missense MYH7 which have been shown to shift myosin to the active disordered relaxed state. If Mant-ATP assays show myosin is maintaining SRX along with an inactive conformation this suggests MyBP-C missense mutations act through a different mechanism.

Effects of grafted myofibrillar protein as a phosphate replacer in brined pork loin

For the development of healthier meat products, the grafted myofibrillar protein was evaluated as an ingredient that can substitute phosphate in brined loin. Individual brine solutions, consisting of salt (negative control, NP), salt + sodium tripolyphosphate (positive control, PC), salt + myofibrillar protein without grafting (MP), salt + myofibrillar protein grafted at high concentration (GMP-H), and salt + myofibrillar protein grafted at low concentration (GMP-L), were added to the pork loin by 40% of their weight. Differential scanning calorimetry demonstrated that MP and GMP-H lowered the thermal energy for the transition of myosin and actin, thereby improving the thermal stability of pork loin and increasing protein solubility. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that thicker protein bands appeared in MP and GMP-H samples while exhibiting increased pH values, moisture content, water holding capacity, and processing yield. Accordingly, the shear force of MP and GMP-H decreased. Lipid oxidation of pork loin was increased in MP, whereas it decreased in GMP-H. Thus, GMP-L is a potential substitute for phosphate since it improves physicochemical properties and prevents the lipid oxidation of pork loin.

Structural OFF/ON transitions of myosin in relaxed porcine myocardium predict calcium-activated force

Contraction in striated muscle is initiated by calcium binding to troponin complexes, but it is now understood that dynamic transition of myosin between resting, ordered OFF states on thick filaments and active, disordered ON states that can bind to thin filaments is critical in regulating muscle contractility. These structural OFF to ON transitions of myosin are widely assumed to correspond to transitions from the biochemically defined, energy-sparing, super-relaxed (SRX) state to the higher ATPase disordered-relaxed (DRX) state. Here we examined the effect of 2'-deoxy-ATP (dATP), a naturally occurring energy substrate for myosin, on the structural OFF to ON transitions of myosin motors in porcine cardiac muscle thick filaments. Small-angle X-ray diffraction revealed that titrating dATP in relaxation solutions progressively moves the myosin heads from ordered OFF states on the thick filament backbone to disordered ON states closer to thin filaments. Importantly, we found that the structural OFF to ON transitions are not equivalent to the biochemically defined SRX to DRX transitions and that the dATP-induced structural OFF to ON transitions of myosin motors in relaxed muscle are strongly correlated with submaximal force augmentation by dATP. These results indicate that structural OFF to ON transitions of myosin in relaxed muscle can predict the level of force attained in calcium-activated cardiac muscle. Computational modeling and stiffness measurements suggest a final step in the OFF to ON transition may involve a subset of DRX myosins that form weakly bound cross-bridges prior to becoming active force-producing cross-bridges.

Abstract 9592: The Super Relaxed State of Myosin Contributes to the Pathogenesis of Right Ventricular Dysfunction in Pulmonary Hypertension

Introduction: Patients with pulmonary hypertension (PH) often succumb to right ventricular (RV) failure yet little is known regarding the underlying pathological mechanisms. Recently, the transition of myosin from the disordered relaxed (DRX) to the super relaxed (SRX) state during diastole has been identified as a pathological feature contributing to myocardial dysfunction in heart failure. Using synchrotron radiation (SR) x-ray diffraction (SAXS), the proximity of myosin heads relative to actin in the in situ beating rat heart can be evaluated in different layers of the myocardial wall. We performed SR SAXS at different timepoints post-PH induction with simultaneous PV-loop acquisition to assess if SRX transition contributes to progressive global RV dysfunction in a rat model of PH. Methods: PH was induced in rats using the Sugen5416/3-week 10% hypoxia method, with rats being returned to normoxia for 3- (SuHx3wk) or 6-weeks (SuHx6wk) after the hypoxic period. SR SAXS was performed at BL40XU, SPring-8 Synchrotron, Japan. Global and site-specific RV myofilament phosphorylation status was also examined. Results: Compared to control rats (n=7), SuHx3wk (n=7) and SuHx6wk (n=7) rats exhibited progressive increases in RV size, RV end systolic pressure (ESP), RV dP/dt max and dP/dt min (all P<0.05 vs. Control). In the deeper subendocardial layer of the RV free wall, there were virtually no myosin heads within the proximity of actin at end diastole in SuHx6wk rats, whereas in the control and SuHx3wk rats, around 30-50% of myosin heads were in proximity to actin (P<0.05, SuHx6wk vs. Control and SuHx3wk). No significant differences in diastolic sarcomere length were found, suggesting a transition of myosin from DRX to SRX in the SuHx6wk rats. Importantly, indices for global RV function (ESP, dP/dt max, dP/dt min ) significantly correlated with diastolic myosin mass transfer values. Analysis of RV myofilament phosphorylation revealed a progressive and significant reduction in MLC-2v phosphorylation over the time course of PH in rats. Conclusion: PH is associated with progressive changes in global RV function, which may be attributed to a transition from DRX to SRX state of myosin in the RV. Reduced MLC-2v phosphorylation may underlie the DRX to SRX transition in PH.

Semi-Implicit Time Integration with Hessian Eigenvalue Corrections for a Larger Time Step in Molecular Dynamics Simulations.

In molecular dynamics simulations, the limited time step size has been a barrier to simulating long-time behaviors. Implicit time integration methods allow markedly larger time steps than the standard explicit time method, although they have major drawbacks such as overheads solving linear systems and instability of Newton iterations. To overcome these issues, we propose a semi-implicit time integration scheme, the semi-implicit Hessian correction (SimHec) scheme, for overdamped Langevin dynamics. The method focuses on the Hessian matrices of bonded and nonbonded interactions, where components with large negative Hessian eigenvalues are cut off in the linear approximation of momentum equations to avoid instability. The narrow band Hessian matrix enables an efficient parallelized linear solution with an overlapping approximation. We tested SimHec for the interdomain fluctuations in adenylate kinase and the powerstroke transition of myosin II using a coarse-grained protein model. SimHec reproduced the same dynamics as the explicit method, although the transition dynamics tended to be accelerated and fluctuations in bonded potentials were slightly reduced. These deviations were corrected using a hybrid method, SimHec-H, which adds explicit time steps after the semi-implicit time step. The proposed scheme allowed us to use time steps 50-200 times larger than those in explicit time integration, which resulted in a speedup factor of 7-30 taking the overhead into account.

Regulated Model of Steady-State Cardiac Length-Dependent Activation

Recent data suggests length-dependent activation derives from titin interacting with super-relaxed myosin crossbridges on the thick filament. This feature was incorporated into an existing model of crossbridge dynamics (Caremani et al, 2015) by replacing the detached post-ATP-hydrolysis state with an equilibrium mixture of rapidly interacting super-relaxed, detached, and weakly bound crossbridges. The model also included: (1) cooperative activation of the thin filament by both Ca++ and strongly bound crossbridges, (2) loosely coupled interaction that allows Ca++ to dissociate from troponin C (TnC) before strong crossbridges detach, (3) Ca++ bound more strongly to TnC when a strong crossbridge is also bound nearby. As suggested by data (Sun & Irving, 2015), the equilibrium between super-relaxed and detached crossbridges equaled 3:1 for a long sarcomere length. We supposed that the reduced forces exerted by titin on the thick filament at low sarcomere length made it more likely to form a super-relaxed crossbridge. As the force exerted by titin increases, the super-relaxed myosin transitions to an unfettered detached state and becomes available to form weak crossbridges. We altered the Gibbs free energy to form a super-relaxed crossbridge by less than 1 kT as titin's force decreased over sarcomere lengths from 2.25 to 1.9 µm. This caused the maximal steady-state force for isometric contraction at saturating Ca++ to reduce by ∼ 25%, as data suggests (Dobesh et al, 2002). The model thus unites existing data and provides a more quantitative understanding of how titin interacts with the thick filament at different sarcomere lengths. We anticipate that this model insight and more extensive data will lead to deeper understanding of cardiac length-dependent activation. (Supported by the Center for Engineering MechanoBiology through a grant from NSF's STC program (CMMI):15-48571).

A programmable DNA origami nanospring that reveals force-induced adjacent binding of myosin VI heads.

Mechanosensitive biological nanomachines such as motor proteins and ion channels regulate diverse cellular behaviour. Combined optical trapping with single-molecule fluorescence imaging provides a powerful methodology to clearly characterize the mechanoresponse, structural dynamics and stability of such nanomachines. However, this system requires complicated experimental geometry, preparation and optics, and is limited by low data-acquisition efficiency. Here we develop a programmable DNA origami nanospring that overcomes these issues. We apply our nanospring to human myosin VI, a mechanosensory motor protein, and demonstrate nanometre-precision single-molecule fluorescence imaging of the individual motor domains (heads) under force. We observe force-induced transitions of myosin VI heads from non-adjacent to adjacent binding, which correspond to adapted roles for low-load and high-load transport, respectively. Our technique extends single-molecule studies under force and clarifies the effect of force on biological processes.

Structural Transitions in Myosin I Detected by Conventional and Pulsed EPR of a Bifunctional Spin Label

We have employed electron paramagnetic resonance (EPR) of a bifunctional spin label (BSL) to develop high-resolution constraints for the myosin catalytic domain in the presence and absence of actin. Two complementary EPR techniques were employed to measure protein orientation (continuous-wave EPR, CW-EPR) and intra-protein distances (double electron-electron resonance, DEER). The use of BSL greatly enhances the resolution of both techniques, by virtue of its strongly immobilized and stereospecific bifunctional attachment to the protein backbone at two engineered Cys residues. Crucially, both techniques utilized here permit the elucidation of myosin structure while in complex with actin, generating relevant constraints for the refinement of actomyosin structural models, and providing insight for structural changes induced by formation of the complex. In the current work, Dictyostelium myosin II was used as our model system. We measured nucleotide-dependent structural transitions of three key helices within the myosin CD. Three double-Cys sites were engineered, with Cys pairs located on the relay helix, helix HK (upper 50kDa domain) and helix HW (lower 50kDa domain), respectively. BSL on a construct with one of these pairs was used to measure myosin orientation relative to oriented actin; BSL on a construct with two pairs was used to measure interprobe distances. The effect of MgADP binding was clearly detected by EPR, and subsequently modeled using the orientation and distance measurements as constraints. This work was funded by grants from NIH (R01 AR32961, T32 AR07612, P30 AR0507220).

Detection of Ultrafast Mechanical Transitions in β-Cardiac Myosin using High-Speed Optical Trapping

The β isoform of myosin II (MYH7) is the primary myosin heavy chain present in both slow skeletal muscle and the cardiac ventricles of large mammals. In cardiac and skeletal muscle, strong binding of myosin to the thin filament plays an important role in the cooperative activation of contraction. Studying the weak-to-strong transition and the initiation of the force-producing power stroke has been difficult using single molecule techniques because standard optical trapping methods require 10-15 ms to detect actin-myosin interactions. This latency is much longer than the expected time from initial actin-myosin interaction to strong binding and force production. In this work we study full-length, tissue-purified, porcine β-cardiac myosin using an ultrafast optical trapping technique that allows the detection of actin-myosin binding events within 1 ms. This improved time resolution reveals three populations of acto-myosin attachments with lifetimes that differ by more than 10-fold, likely representing three different biochemical and/or mechanical states. The slowest of these rates is ATP dependent at low MgATP concentrations and corresponds well with the expected rate of ATP-induced dissociation. The populations with shorter lifetimes likely represent dissociation from actomyosin states that precede ATP-induced dissociation. The force dependence of these lifetimes and the relative amplitude of each phase reveal information about mechano-chemical transitions in cardiac myosin that have not previously been probed in the single molecule regime. In addition, ensemble averages of individual attachment events reveal the amplitude and timing of both sub-steps of β-cardiac myosin's power stroke. Through these ensemble averages, we can separately resolve the pre-powerstroke/actin-bound state and the post-powerstroke state, representing the first single-molecule observation of the initial actin displacement by cardiac myosin under load. Supported by NIH grant P015GM087253.

Physicochemical properties of surimi gels fortified with dietary fiber

Although dietary fiber provides health benefits, most Western populations have insufficient intake. Surimi seafood is not currently fortified with dietary fiber, nor have the effects of fiber fortification on physicochemical properties of surimi been thoroughly studied. In the present study, Alaska pollock surimi was fortified with 0–8g/100g of long-chain powdered cellulose as a source of dietary fiber. The protein/water concentrations in surimi were kept constant by adding an inert filler, silicon dioxide in inverse concentrations to the fiber fortification. Fiber-fortified surimi gels were set at 90°C. The objectives were to determine (1) textural and colour properties; (2) heat-induced gelation (dynamic rheology); and (3) protein endothermic transitions (differential scanning calorimetry) of surimi formulated with constant protein/water, but variable fiber content. Fiber fortification up to 6g/100g improved (P<0.05) texture and colour although some decline occurred with 8g/100g of fiber. Dynamic rheology correlated with texture and showed large increase in gel elasticity, indicating enhanced thermal gelation of surimi. Differential scanning calorimetry showed that fiber fortification did not interfere with thermal transitions of surimi myosin and actin. Long-chain fiber probably traps water physically, which is stabilized by chemical bonding with protein within surimi gel matrix. Based on the present study, it is suggested that the fiber–protein interaction is mediated by water and is physicochemical in nature.

A three‐prong strategy to develop functional food using protein isolates recovered from chicken processing by‐products with isoelectric solubilization/precipitation

Skin-on bone-in chicken drumsticks were processed with isoelectric solubilization/precipitation to recover muscle proteins. The drumsticks were used as a model for dark chicken meat processing by-products. The main objective of this study was conversion of dark chicken meat processing by-products to restructured functional food product. An attempt was made to develop functional food product that would resemble respective product made from boneless skinless chicken breast meat. A three-prong strategy to address diet-driven cardiovascular disease (CVD)with a functional food was used in this study. The strategy included addition of three ingredients with well-documented cardiovascular benefits: (i) ω-3 polyunsaturated fatty acid-rich oil (flaxseed-algae, 9:1); (ii) soluble fiber; and (iii) salt substitute. Titanium dioxide, potato starch, polyphosphate, and transglutaminase were also added. The batters were formulated and cooked resulting in heat-set gels. Color (L*a*b*), texture (torsion test, Kramer shear test, and texture profile analysis), thermal denaturation (differential scanning calorimetry), and gelation (dynamic rheology) of chicken drumstick gels and chicken breast gels were determined and compared. Chicken drumstick gels generally had comparable color and texture properties to the gels made from chicken breast meat. The endothermic transition (thermal denaturation) of myosin was more pronounced and gelation properties were better for the drumstick gels. This study demonstrated a feasibility to develop functional food made of muscle proteins recovered with isoelectric solubilization/precipitation from low-value dark chicken meat processing by-products. The functional food developed in this study was enriched with CVD-beneficial nutrients and had comparable instrumental quality attributes to respective products made of chicken breast meat. Although the results of this study point towards the potential for a novel, marketable functional food product, sensory tests and storage stability study are recommended.

Three Distinct Actin-Attached Structural States of Myosin in Muscle Fibers

We have used thiol cross-linking and electron paramagnetic resonance (EPR) to resolve structural transitions of myosin's light chain domain (LCD) and catalytic domain (CD) that are associated with force generation. Spin labels were incorporated into the LCD of muscle fibers by exchanging spin-labeled regulatory light chain for endogenous regulatory light chain, with full retention of function. To trap myosin in a structural state analogous to the elusive posthydrolysis ternary complex A.M′.D.P, we used pPDM to cross-link SH1 (Cys707) to SH2 (Cys697) on the CD. LCD orientation and dynamics were measured in three biochemical states: relaxation (A.M.T), SH1-SH2 cross-linked (A.M′.D.P analog), and rigor (A.M.D). EPR showed that the LCD of cross-linked fibers has an orientational distribution intermediate between relaxation and rigor, and saturation transfer EPR revealed slow rotational dynamics indistinguishable from that of rigor. Similar results were obtained for the CD using a bifunctional spin label to cross-link SH1-SH2, but the CD was more disordered than the LCD. We conclude that SH1-SH2 cross-linking traps a state in which both the CD and LCD are intermediate between relaxation (highly disordered and microsecond dynamics) and rigor (highly ordered and rigid), supporting the hypothesis that the cross-linked state is an A.M′D.P analog on the force generation pathway.

Physicochemical changes in surimi with salt substitute

Protein endothermic transitions (thermal denaturation), rheological properties (protein gelation), and fundamental texture properties (shear stress and strain at mechanical fracture) of Alaska pollock surimi gels made with 0 (control), 1, 2, and 3g/100g of salt (NaCl) were determined and compared with equal molar concentration of salt substitute. Salt and salt substitute shifted the onset of myosin transition to higher temperature and resulted in larger myosin peaks (i.e., transition enthalpy). Endothermic transitions showed similar trends to rheological properties. The elastic modulus (G′) increased when salt or salt substitute was added to surimi, except at the highest concentration of salt and salt substitute. Salt and salt substitute also induced the onset of protein gelation (i.e., as measured by significant increase of G′) at lower temperature. Surimi gels with salt substitute and salt at equal molar concentrations had similar texture properties (shear stress and strain). Based on the present study, salt substitute can be used in the development of low-sodium surimi seafood products without significant change in gelation and texture.

Dynamic rheology and thermal transitions of surimi seafood enhanced with ω-3-rich oils

Surimi-based seafood products are widely accepted and enjoyed worldwide. The U.S. consumption increased in 1980s; however, it leveled thereafter. Food products nutrified with ω-3 polyunsaturated fatty acids (PUFAs) are in increasing demand due to demonstrated health benefits. Currently, surimi seafood is not nutrified with ω-3 PUFAs. In the present study, surimi seafood was nutritionally-enhanced with ω-3 PUFAs-rich oils (flaxseed, algae, menhaden, krill, and blend). Protein endothermal transitions, heat-induced gelation (elastic modulus, G′), and fundamental texture properties (shear stress) of Alaska pollock surimi nutrified with ω-3 PUFAs-rich oils (flaxseed, algae, menhaden, krill, and blend) were determined and compared to Alaska pollock surimi without oil (control). Differential scanning calorimetry showed that oil addition enhanced thermal transition of actin and did not compromise the transition of myosin. The addition of oil improved heat-induced protein gelation as demonstrated with dynamic rheology. Elastic modulus increased when oil was added. There were no differences ( P > 0.05) in shear stress between surimi gels with and without oil, indicating that nutrification with ω-3 PUFAs-rich oils within the ranges tested did not alter gel strength. This study demonstrates that the nutritional value and gelation of surimi seafood can be enhanced without altering texture properties by addition of ω-3 PUFAs-rich oils.

Dynamic rheology and endothermic transitions of proteins recovered from chicken-meat processing by-products using isoelectric solubilization/precipitation and addition of TiO2

Chicken-meat processing generates large quantities of by-products (backs, necks, etc.). Dark chicken-meat processing by-products present the lowest value and greatest challenge. Therefore, recovery of functional proteins from this source for inclusion in food products resembling those from light chicken-meat presents the greatest value addition and opportunity. Novel isoelectric solubilization/precipitation (ISP) was applied to model, dark chicken-meat processing by-products (skin-on bone-in chicken drumsticks) to recover muscle proteins. Thermal denaturation (endothermic transitions), gelation (elasticity, G′), and fundamental texture properties (shear stress and strain at mechanical fracture) of the ISP-recovered proteins were determined with differential scanning calorimetry (DSC), dynamic rheometer, and torsion test, respectively; and compared to boneless skinless chicken breast. Endothermic transition of myosin was not detected only when TiO2 was not added, while the ISP-recovered proteins with TiO2 showed small myosin peak and large actin peak. However, the level of TiO2 addition did not affect thermal transition/denaturation of the ISP-recovered proteins. The ISP-recovered proteins had a greater transition for actin compared to chicken breast, suggesting that ISP predisposes this protein to thermal denaturation. Similar to endothermic transitions, elasticity (G′) generally increased when TiO2 was added to the ISP-recovered proteins. Gels made of chicken breast had the highest (P < 0.05) shear stress (i.e., gel strength), but gels made of the ISP-recovered chicken proteins had greater (P < 0.05) shear strain (i.e., gel cohesiveness). Addition of TiO2 to the ISP-recovered proteins resulted in increased (P < 0.05) gel strength. Based on the present study, addition of TiO2 is suggested for the development of restructured food products based on proteins recovered from dark chicken-meat processing by-products using ISP. Although the results of this study point towards a novel food product, further studies are recommended.

Myosin‐actin cross‐bridge kinetics explain variation in single skeletal muscle fiber function in humans

Our objective was to determine if myosin-actin cross-bridge kinetics, measured in their intact myofilament lattice, could account for variation in skeletal muscle fiber type specific performance among healthy and diseased humans. We applied small amplitude sinusoidal analysis for the first time to human skeletal muscle fibers obtained from 9 heart failure patients and 6 controls of similar age and activity level. In both groups, myosin heavy chain (MHC) isoform contractile velocity (I<IIA<IIX) was inversely related to myosin-actin attachment time (ton). The rate of myosin transition between weakly- and strongly-bound states (2πb) increased with MHC velocity, suggesting the myosin-actin detachment time (toff) decreases with faster contractions. These results suggest that total myosin-actin cycle time (ton + toff) decreases with contractile velocity due to reductions in both ton and toff in humans. Heart failure patients had an increased ton, which compensated for their reduced cross-bridge number due to MHC content loss, resulting in similar isometric tensions compared to healthy controls. Collectively, these results indicate the usefulness of cross-bridge kinetics and highlight their potential use for examining the effects of disease, muscle activity and aging on single fiber function. Support: NIH HL-077418 and AG-031303

Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Rigor to Post-Rigor Transition in Myosin V: Link between the Dynamics and the Supporting Architecture

The detachment kinetics from actin upon ATP binding is a key step in the reaction cycle of myosin V. We show that a network of residues, constituting the allostery wiring diagram (AWD), that trigger the rigor (R) to post-rigor (PR) transition, span key structural elements from the ATP and actin-binding regions. Several of the residues are in the 33 residue helix (H18), P loop, and switch I. Brownian dynamics simulations show that a hierarchy of kinetically controlled local structural changes leads to the opening of the "cleft" region, resulting in the detachment of the motor domain from actin. Movements in switch I and P loop facilitate changes in the rest of the motor domain, in particular the rotation of H18, whose stiffness within the motor domain is crucial in the R --> PR transition. The finding that residues in the AWD also drive the kinetics of the R --> PR transition shows how the myosin architecture regulates the allosteric movements during the reaction cycle.