Muscle Myosin Research Articles

Virus-free transfection, transient expression, and purification of human cardiac myosin in mammalian muscle cells for biochemical and biophysical assays

Myosin expression and purification is important for mechanistic insights into normal function and mutation induced changes. The latter is particularly important for striated muscle myosin II where mutations cause several debilitating diseases. However, the heavy chain of this myosin is challenging to express and the standard protocol, using C2C12 cells, relies on viral infection. This is time and work intensive and associated with infrastructural demands and biological hazards, limiting widespread use and hampering fast generation of a wide range of mutations. We here develop a virus-free method to overcome these challenges. We use this system to transfect C2C12 cells with the motor domain of the human cardiac myosin heavy chain. After optimizing cell transfection, cultivation and harvesting conditions, we functionally characterized the expressed protein, co-purified with murine essential and regulatory light chains. The gliding velocity (1.5–1.7 µm/s; 25 °C) in the in vitro motility assay as well as maximum actin activated catalytic activity (kcat; 8–9 s−1) and actin concentration for half maximal activity (KATPase; 70–80 µM) were similar to those found previously using virus based infection. The results should allow new types of studies, e.g., screening of a wide range of mutations to be selected for further characterization.

The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy

BackgroundClinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells.MethodA gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation.ResultsFor the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iβ levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion.ConclusionsThe spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

Biliary Atresia Remnants Revisited: Myogenesis, Hepatic Duct-Like Structures, and Fate of Peribiliary Glands.

Proximal levels of excised remnants from youngest infants may reveal early features of biliary atresia (BA). A targeted IHC survey was applied to 34 most proximal 2 levels in 17 BA remnants excised at age 10-74 days including 7 = <30 days old and 6 control hepatic ducts (HD). Severity of inflammation and extent of active fibroplasia do not distinguish proximal remnants in younger (n = 7) and older (n = 10) infants. In 27/34 levels of 14/17 remnants, reactive stroma is focally SM-MHC-2 (+), marking smooth muscle myosin, termed reactive myogenesis (RM), that is absent in controls. RM facilitates identification of 3 novel hepatic duct remnants (HDR): an HD-like collagen collar lined by degenerating cholangiocytes (n = 5); erosion defects in loose reactive stroma (n = 14); solitary foci of hyperplastic squamoid epithelium (n = 4). Peribiliary glands are either hyperplastic or atretic and typically lack RM. Minimally inflammed end-stage lesions in BA remnants occur at youngest ages favoring prenatal onset. Three novel HDR are defined. RM, a useful surrogate for HDR, is a prevalent inappropriate stromal reaction in proximal remnants of uncertain biological significance. RM is the source of mature smooth muscle in BA remnants.

Intraluminal chloride regulates lung branching morphogenesis: involvement of PIEZO1/PIEZO2

BackgroundClinical and experimental evidence shows lung fluid volume as a modulator of fetal lung growth with important value in treating fetal lung hypoplasia. Thus, understanding the mechanisms underlying these morphological dynamics has been the topic of multiple investigations with, however, limited results, partially due to the difficulty of capturing or recapitulating these movements in the lab. In this sense, this study aims to establish an ex vivo model allowing the study of lung fluid function in branching morphogenesis and identify the subsequent molecular/ cellular mechanisms.MethodsEx vivo lung explant culture was selected as a model to study branching morphogenesis, and intraluminal injections were performed to change the composition of lung fluid. Distinct chloride (Cl−) concentrations (5.8, 29, 143, and 715 mM) or Cl− channels inhibitors [antracene-9-carboxylic acid (A9C), cystic fibrosis transmembrane conductance regulator inhibitor172 (CFTRinh), and calcium-dependent Cl− channel inhibitorA01 (CaCCinh)] were injected into lung lumen at two timepoints, day0 (D0) and D2. At D4, morphological and molecular analyses were performed in terms of branching morphogenesis, spatial distribution (immunofluorescence), and protein quantification (western blot) of mechanoreceptors (PIEZO1 and PIEZO2), neuroendocrine (bombesin, ghrelin, and PGP9.5) and smooth muscle [alpha-smooth muscle actin (α-SMA) and myosin light chain 2 (MLC2)] markers.ResultsFor the first time, we described effective intraluminal injections at D0 and D2 and demonstrated intraluminal movements at D4 in ex vivo lung explant cultures. Through immunofluorescence assay in in vivo and ex vivo branching morphogenesis, we show that PGP9.5 colocalizes with PIEZO1 and PIEZO2 receptors. Fetal lung growth is increased at higher [Cl−], 715 mM Cl−, through the overexpression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. In contrast, intraluminal injection of CFTRinh or CaCCinh decreases fetal lung growth and the expression of PIEZO1, PIEZO2, ghrelin, bombesin, MLC2, and α-SMA. Finally, the inhibition of PIEZO1/PIEZO2 by GsMTx4 decreases branching morphogenesis and ghrelin, bombesin, MLC2, and α-SMA expression in an intraluminal injection-independent manner.ConclusionsOur results identify PIEZO1/PIEZO2 expressed in neuroendocrine cells as a regulator of fetal lung growth induced by lung fluid.

Smooth muscle contribution to vaginal viscoelastic response

Smooth muscle cells contribute to the mechanical function of various soft tissues, however, their contribution to the viscoelastic response when subjected to multiaxial loading remains unknown. The vagina is a fibromuscular viscoelastic organ that is exposed to prolonged and increased pressures with daily activities and physiologic processes such as vaginal birth. The vagina changes in geometry over time under prolonged pressure, known as creep. Vaginal smooth muscle cells may contribute to creep. This may be critical for the function of vaginal and other soft tissues that experience fluctuations in their biomechanical environment. Therefore, the objective of this study was to develop methods to evaluate the contribution of smooth muscle to vaginal creep under multiaxial loading using extension – inflation tests. The vaginas from wildtype mice (C57BL/6 × 129SvEv; 3–6 months; n = 10) were stimulated with various concentrations of potassium chloride then subjected to the measured in vivo pressure (7 mmHg) for 100 s. In a different cohort of mice (n = 5), the vagina was stimulated with a single concentration of potassium chloride then subjected to 5 and 15 mmHg. A laser micrometer measured vaginal outer diameter in real-time. Immunofluorescence evaluated the expression of alpha-smooth muscle actin and myosin heavy chain in the vaginal muscularis (n = 6). When smooth muscle contraction was activated, vaginal creep behavior increased compared to the relaxed state. However, increased pressure decreased the active creep response. This study demonstrated that extension – inflation protocols can be used to evaluate smooth muscle contribution to the viscoelastic response of tubular soft tissues.

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex.

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.

Cryo-EM structure of the folded-back state of human β-cardiac myosin.

A large proportion of cardiac myosin heads are sequestered in an off-state during cardiac relaxation. Increasing exertion converts these heads to an on-state that are turned off during relaxation, a process essential for proper heart function. Point mutations that shift the equilibrium towards the on-state can lead to hypercontractility and the development of hypertrophic cardiomyopathy (HCM). The off-state is equated with an inactive, folded-back structural state called the interacted-head motif (IHM). The IHM is a regulatory feature of all muscle and non-muscle class-2 myosins. The off-state is equated with an inactive, folded-back structural state called the interacted-head motif (IHM). The IHM is a regulatory feature of all muscle and non-muscle myosins from class-2. In this work we report the cryo-EM structure of the human β-cardiac myosin IHM to 3.6 Å resolution. The structure reveals that the interface stabilizing the IHM are hot spots of HCM mutations. Importantly, the cardiac myosin IHM structure is significantly different from the previously described smooth muscle myosin IHM, revealing that the IHM structure is tailored to the distinct need of a given muscle type. This work now provides the missing puzzle piece needed to fully understand the molecular basis of inherited cardiomyopathy. It also paves the way for a personalized medicine approach to the design of new molecules that influence the stability of the IHM, to properly regulate cardiac contraction.

Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction.

Mutations in the genes encoding striated muscle myosins lead to a variety of diseases including hypertrophic (HCM) and dilated (DCM) cardiomyopathies (MYH7) and distal arthrogryposis syndromes (MYH3 and MYH8). We have used a muscle myoblast cell line to express and purify recombinant human beta-cardiac myosin and human embryonic skeletal muscle myosins, comparing the WT versions of these proteins with an array of disease-causing variants. HCM-causing mutations in the beta-cardiac motor domain often destabilize a folded back, autoinhibited state of myosin, leading to an increase in the number of heads available within the sarcomere to interact with actin and produce force, which in turn results in the hypercontractility characteristic of HCM. Destabilization is seen for mutations that alter residues involved in the regions of myosin important for forming the intramolecular contacts in the folded back state. However, mutations affecting residues remote from these interfaces can also increase the number of available heads by destabilizing the pre-power stroke state of myosin that enables formation of the folded back state. HCM mutations that affect only myosin motor function without affecting the stability of the folded back state are less common. In contrast, we have studied a variety of DCM-causing mutations and found that most DCM-mutant myosins exhibit decreases in one or more aspects of motor function, including actin-activated ATPase activity and in vitro gliding velocity. Current efforts focus on correlating the effects of distal arthrogryposis syndrome-causing mutations on MYH3 biomechanical function and contrasting the effects of these mutations with paralogous mutations in MYH7.

Small molecules modulating force production: A new perspective against myosin-associated diseases.

Myosins are ATP-dependent molecular motors involved in almost all processes of life. These motors are associated with various human diseases such as various cardiomyopathies, spasticity, deafness or malaria. Nowadays, the most promising approach to treat myosin-associated diseases is the design of small-molecule drugs able to specifically modulate the force produced by these motors. Camzyos (mavacamten) is a specific inhibitor recently approved by the FDA to treat adults with symptomatic class II-III obstructive hypertrophic cardiomyopathy. Omecamtiv mecarbil (OM) also targets β-cardiac myosin but in this case, it is an activator of contraction, currently in late Phase 3 for heart failure. Here we present how a methodology combining X-ray crystallography, molecular dynamics and functional assays allows to study the mechanism of action of some of these compounds. We have determined that despite their antagonistic effects on force production, OM and Mava surprisingly target the same pocket. MPH-220 is a Blebbistatin derivative identified as an inhibitor of skeletal myosin-2 (SkMyo2) and a promising treatment against muscle spasticity. Structures of other inhibitors bound in this pocket with distinct IC50 for three muscle myosins have also provided clues about the parameters that control potency and specificity. KNX-002 is a new inhibitor of Plasmodium falciparum myosin A (PfMyoA). PfMyoA is essential for both red blood cells invasion and parasite motility and is a first-order pharmacological target against malaria. Our structure of PfMyoA complexed to KNX-002 shows how the inhibitor selectively targets an undescribed pocket in the motor, sequestering the myosin in a state detached from actin and preventing hydrolysis. Altogether, these results show how small molecules modulating the force produced by myosins are promising avenues to treat myosin-associated diseases and opens up new approaches to the design of more selective and potent compounds.

Myosin-binding protein C (MyBP-C) stabilizes, but is not the sole determinant of, SRX myosin in cardiac muscle.

The myosin Super Relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with MyBP-C in the C-zone, proximal to the sarcomere center. To define how MyBP-C and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic MyBP C knockout mice (McConnell, 2001) and those expressing MyBP-C with a 271 residue N-terminal truncation (Lynch, 2021).

Conformational and energetic basis of actomyosin force generation.

Interactions between myosin and actin are essential in producing different types of cellular forces. Two key players in muscle contraction, myosin and actin, slide along each other to produce force using the energy from ATP hydrolysis. Targeting the motor domain of cardiac myosin, several small molecules have been proposed to treat cardiomyopathy. A clear mechanistic picture for the allosteric control in the actomyosin complex can facilitate computer-aided drug discovery. Atomistic simulation approaches such as molecular dynamics (MD) are limited for studying the motor functional cycle owing to the slow timescale as well as a lack of various intermediate structures. Here, integrating comparative modeling and enhanced sampling simulations, we reveal how the human cardiac myosin generates force in the cross-bridge cycle at atomic resolution. Initial conformational ensembles for different actomyosin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or transient interactions with actin. We find that the actin-binding cleft closure is allosterically coupled to the myosin core transitions and products release from the active site. Furthermore, a gate between switch I and II is suggested to control phosphate release at the pre-powerstroke state. The predicted metastable states and corresponding energetics are consistent with available experimental data and provide insights into the timing of elementary mechanochemical events.

Effects of blebbistatin on force production, actin motility and the force-velocity relation produced by myosin II filaments and myofibrils.

Blebbistatin is a myosin II-specific inhibitor that is often used to study the relation between force generation and Pi release. The goal of this study was to investigate the effects of blebbistatin on force production and the actin motility while interacting with arrays of myosin motors. We used myosin filaments isolated from mammalian smooth muscles, rabbit psoas and the anterior byssus retractor (ABRM) muscles extracted from fresh Mytilus edulis in different experiments. We also used whole myofibrils isolated from the rabbit psoas. The force during myosin-actin interactions was measured with a system of micro-fabricated cantilevers, and the sliding velocity of actin was measured with a standard in-vitro motility assay. The force-velocity relation was measured in myofibrils with atomic force cantilevers. The force produced by the myosin filaments upon interaction with actin decreased with increasing concentrations of blebbistatin (5 μM-12.5 μM). The force produced by smooth and skeletal muscle myosin filaments were 95.93 ± 12.05 pN/μm before blebbistatin treatment, 71.45 ± 5.94 pN/μm with 5 μm blebbistatin, 62.05 ± 9.44 pN/μm with 7.5 μM blebbistatin, 38.28 ± 8.77 pN/μm with 10 μm blebbistatin and dropped to zero with 12.5 μM blebbistatin. All these differences were significantly different (p<0.0001). Sliding velocity of actin filaments also decreased consistently with increasing concentrations of blebbistatin. Before treatment, filaments slid over HMM at 3.68 ± 0.06 μm/s, decreasing to 2.30 ± 0.056 μm/s with 1 μM, 1.27 ± 0.043 μm/s with 5 μM, and 1.44 ± 0.032 μm/s with 30 μM. The maximal velocity decrease was observed at 5 μM of blebbistatin. These differences were significantly different (P<0.0001). There was a downward shift in the force-velocity relation in myofibrils treated with blebbistatin. These results suggest that blebbistatin regulates the mechanics of myosin motors working collectively in different experimental preparations.

Caldesmon enhances motility inhibition by myosin dephosphorylation.

Some studies have suggested that smooth muscle caldesmon (hCaD) participates in force maintenance at low myosin light chain (LC20) phosphorylation. To elucidate the effects of hCaD on the molecular mechanics of dephosphorylating smooth muscle myosin, we injected myosin light chain phosphatase (MLCP) during in vitro motility measurements performed in the presence of hCaD (400 nM) or its absence. Because hCaD increases the binding force of unphosphorylated smooth muscle myosin to actin, we hypothesized that, upon LC20 dephosphorylation, the fraction (fmot) and the average velocity (vavg) of moving actin filaments would decrease at a greater rate in the presence of hCaD. We fitted sigmoidal curves to fmot and vavg and estimated the rate constant (k) and the midpoint (t0) of the decrease. k was larger in the presence of hCaD (3.6×10−1±6.4×10−2 s−1) than without (1.8×10−1±1.4×10−2 s−1), p = 0.039, n = 5. Additionally, t0 was smaller in the presence of hCaD (32±3 s) than without (44±2 s), p = 0.008, n = 5. These results can be explained either by an increased dephosphorylation rate in the presence of hCaD or by an increase of the reattachment of dephosphorylated myosin to actin. In line with previous experiments that demonstrated that hCaD inhibits the actomyosin ATPase, our sigmoidal fits also show a lower vavg in the presence of hCaD (0.45±0.04 µm/s) than without (0.63±0.06 µm/s), p=0.04, before the addition of MLCP. However, hCaD had no effect on k or t0 of vavg during MLCP injection. The lack of concordance between the fmot and vavg data may be due to the fact that the vavg data are noisier after dephosphorylation. Our results agree with previous data obtained at the whole muscle level that showed that hCaD increases the rate of relaxation of arterial smooth muscle and accelerates the decay in tension of skinned smooth muscle strips.

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.

Dynamic coordination of the lever-arm swing of myosin II in DNA origami-based thick filament.

Muscle myosins work in motor ensembles and must adapt their power stroke in response to mechanical actions by surrounding motors. Understanding the coordination of power strokes is essential for bridging microscopic molecular function and macroscopic muscle contraction, but the details of this phenomenon remain elusive. Here we used high-speed atomic force microscopy and dark-field microscopy to directly visualize the dynamics of the lever-arm swing (power stroke) and weak bindings of individual human skeletal myosins in DNA origami-based synthetic thick filaments. We observed weak bindings with short durations (less than 1 ms) and the two-step lever-arm swing. Also, we observed spatially local power stroke coordination, and our three-dimensional numerical model explained how mechanical communication between myosins achieved coordination. We extended the model to three-dimensional sarcomere structure in which one actin filament was surrounded by three thick filaments and predicted the local power stroke coordination was spatially periodic and propagated toward the contraction direction. We confirmed that a structural mismatch between the myosin head spacing (42.8 nm) and the actin helical pitch (36 nm) caused the coordination while improving contraction speed and energy efficiency. Our findings reveal a key physical basis of efficient muscle contraction.

Mechanisms of cardiac myosin replacement.

A recent mass spectrometry-based proteomic study from our lab demonstrated that newly synthesized myosin molecules are randomly inserted into preexisting thick filaments in adult mouse hearts in vivo. Therefore, we hypothesized that myosin molecules exist in a dynamic equilibrium between a filamentous pool required for contractility, and a monomeric pool required for myosin replacement. To test this hypothesis, we replaced 27 ± 7% of the cardiac myosin regulatory light chain (RLC) in adult mice with fluorescent RLC (GFP-RLC), via adeno-associated viral transfection. We visualized the random distribution of GFP-RLC within thick filaments in intact hearts with 2-photon microscopy, and the exchange of GFP-RLC between these filaments by quantifying fluorescence recovery after photobleaching (FRAP). Rapid FRAP occurred within minutes, when photobleaching small, ∼1-µm3 areas, of partial sarcomeres. These data demonstrate that the GFP-RLC exchanges between the thick filaments and a monomeric pool on a timescale that is orders of magnitude faster than that of protein replacement (t1/2 = 10.8 days). To determine whether the mechanism of this exchange may involve the folding of monomeric myosin, GFP-labeled myosin molecules were extracted from the transfected hearts and their Stokes radii were measured over a range of ionic strengths, using microfluidic diffusional sizing. At low ionic strengths (150 mM KCl), filament formation was favored, but the monomeric fraction of GFP-labeled myosin demonstrated a radius of 12.6±1.1 nm. At higher ionic strengths (400-600 mM KCl), the radius was increased to 18.0 nm. Similar radii have been reported for smooth muscle myosin monomers in folded and extended conformations. Together, these data suggest that myosin molecules exist in rapid equilibrium between a filamentous and monomeric pool within the sarcomere, and individual monomers may adopt a folded, compact conformation to regulate their exchange into, and out of filaments.

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.

Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle.

The myosin super-relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with myosin-binding protein C (MyBP-C) in the sarcomere C-zone. To define how cardiac MyBP-C (cMyBP-C) and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic cMyBP-C null mice and those expressing cMyBP-C with a 271-residue N-terminal truncation. Utilizing super-resolution microscopy, we determined the lifetime and subsarcomeric location of individual fluorescent-ATP turnover events within isolated cardiac myofibrils. The proportion of SRX myosin demonstrated a gradient along the half-thick filament, highest in the P- and C-zones (72 ± 9% and 71 ± 6%, respectively) and lower in the D-zone (45 ± 10%), which lies farther from the sarcomere center and lacks cMyBP-C, suggesting a possible role for cMyBP-C in stabilizing the SRX. However, myofibrils from cMyBP-C null mice demonstrated an ∼40% SRX reduction, not only within the now cMyBP-C-free C-zone (49 ± 9% SRX), but also within the D-zone (22 ± 5% SRX). These data suggest that the influence of cMyBP-C on the SRX state is not limited to the C-zone but extends along the thick filament. Interestingly, myofibrils with N-terminal truncated cMyBP-C had an SRX content and spatial gradient similar to the cMyBP-C null, indicating that the N terminus of cMyBP-C is necessary for cMyBP-C's role in enhancing the SRX gradient along the entire thick filament. Given that SRX myosin exist as a gradient along the thick filament that is highest in the C-zone, even in the absence of cMyBP-C or its N-terminus, an inherent bias must exist in the structure of the thick filament to stabilize the SRX state.

Endothelial Cell Markers Are Inferior to Vascular Smooth Muscle Cells Markers in Staining Vasa Vasorum and Are Non-Specific for Distinct Endothelial Cell Lineages in Clinical Samples.

Current techniques for the detection of vasa vasorum (VV) in vascular pathology include staining for endothelial cell (EC) markers such as CD31 or VE-cadherin. However, this approach does not permit an objective assessment of vascular geometry upon vasospasm and the clinical relevance of endothelial specification markers found in developmental biology studies remains unclear. Here, we performed a combined immunostaining of rat abdominal aorta (rAA) and human saphenous vein (hSV) for various EC or vascular smooth muscle cell (VSMC) markers and found that the latter (e.g., alpha smooth muscle actin (α-SMA) or smooth muscle myosin heavy chain (SM-MHC)) ensure a several-fold higher signal-to-noise ratio irrespective of the primary antibody origin, fluorophore, or VV type (arterioles, venules, or capillaries). Further, α-SMA or SM-MHC staining allowed unbiased evaluation of the VV area under vasospasm. Screening of the molecular markers of endothelial heterogeneity (mechanosensitive transcription factors KLF2 and KLF4, arterial transcription factors HES1, HEY1, and ERG, venous transcription factor NR2F2, and venous/lymphatic markers PROX1, LYVE1, VEGFR3, and NRP2) have not revealed specific markers of any lineage in hSV (although KLF2 and PROX1 were restricted to venous endothelium in rAA), suggesting the need in high-throughput searches for the clinically relevant signatures of arterial, venous, lymphatic, or capillary differentiation.

Millet shell polyphenols ameliorate atherosclerosis development by suppressing foam cell formation

Polyphenols are bioactive compounds that occur naturally in plants, and they are widely used for disease prevention and health maintenance. In present study, the effects of millet shell polyphenols (MSPs) in thwarting atherosclerosis were explored. The results found that MSPs effectively inhibited the ability of macrophages to phagocytose lipids, and reduced the secretion of inflammatory factors IL-1β and TNF-α by obstructing the expression of STAT3 and NF-κB in macrophages. Eventually, MSPs hindered the formation of macrophage-derived foam cells. On the other hand, MSPs promoted the transformation of HASMCs from synthesis to contraction by regulating the gene expression levels of smooth muscle myosin heavy chain (SMMHC), desmin (DES), smoothelin (SMTN) and elastin (ELN). Lipid phagocytosis inhibited along with this process, thereby reducing the formation of smooth muscle cell-derived foam cells. In addition, experiments in ApoE−/− mice also showed that MSPs increased high-density lipoprotein cholesterol (HDL-C). Collectively, MSPs play a role in preventing atherosclerosis by impeding foam cell production. This study offers an integrative strategy for thwarting plaque formation in the early stages of atherosclerosis in cardiovascular disease.