Cardiac Myosin and Thin Filament as Targets for Lead and Cadmium Divalent Cations.

Lead and cadmium are heavy metals widely distributed in the environment and contribute significantly to cardiovascular morbidity and mortality. Using Leadmium Green dye, we have shown that lead and cadmium enter cardiomyocytes, distributing throughout the cell. Using an in vitro motility assay, we have shown that sliding velocity of actin and native thin filaments over myosin decreases with increasing concentrations of Pb2+ and Cd2+. Significantly lower concentrations of Pb2+ and Cd2+ (0.6 mM) were required to stop sliding of thin filaments over myosin compared to the stopping actin sliding over the same myosin (1.1-1.6 mM). Lower concentration of Cd2+ (1.1 mM) needed to stop actin sliding over myosin compared to the Pb2++Cd2+ combination (1.3 mM) and lead alone (1.6 mM). There were no differences found in the effects of lead and cadmium cations on relative force developed by myosin heads or number of actin filaments bound to myosin. Sliding velocity of actin over myosin in the left atrium, right and left ventricles changed equally when exposed to the same dose of the same metal. Thus, we have demonstrated for the first time that Pb2+ and Cd2+ can directly affect myosin and thin filament function, with Cd2+ exerting a more toxic influence on myosin function compared to Pb2+.

Cell blebbing novel therapeutic possibilities to counter metastasis.

Cells constantly reshape there plasma membrane and cytoskeleton during physiological and pathological processes (Hagmann et al. in J Cell Biochem 73:488-499, 1999). Cell blebbing, the formation of bulges or protrusions on the cell membrane, is related to mechanical stress, changes in intracellular pressure, chemical signals, or genetic anomalies. These membrane bulges interfere with the force balance of actin filaments, microtubules, and intermediate filaments, the basic components of the cytoskeleton (Charras in J Microsc 231:466-478, 2008). In the past, these blebs with circular structures were considered apoptotic markers (Blaser et al. in Dev Cell 11:613-627, 2006). Cell blebbing activates phagocytes and promotes the rapid removal of intrinsic compartments. However, recent studies have revealed that blebbing is associated with dynamic cell reorganization and alters the movement of cells in-vivo and in-vitro (Charras and Paluch in Nat Rev Mol Cell Biol 9:730-736, 2008). During tumor progression, blebbing promotes invasion of cancer cells into blood, and lymphatic vessels, facilitating tumor progression and metastasis (Weems et al. in Nature 615:517-525, 2023). Blebbing is a dominant feature of tumor cells generally absent in normal cells. Restricting tumor blebbing reduces anoikis resistance (survival in suspension) (Weems et al. in Nature 615:517-525, 2023). Hence, therapeutic intervention with targeting blebbing could be highly selective for proliferating pro-metastatic tumor cells, providing a novel therapeutic pathway for tumor metastasis with minimal side effects. Here, we review the association between cell blebbing and tumor cells, to uncover new research directions and strategies for metastatic cancer therapy. Finaly, we aim to identify the druggable targets of metastatic cancer in relation to cell blebbing.

Alterations in cardiac contractile and regulatory proteins contribute to age-related cardiac dysfunction in male rats.

Aging is associated with cardiac contractile abnormalities, but the etiology of these contractile deficits is unclear. We hypothesized that cardiac contractile and regulatory protein expression is altered during aging. To investigate this possibility, left ventricular (LV) lysates were prepared from young (6 months) and old (24 months) Fischer344 rats. There are no age-related changes in SERCA2 expression or phospholamban phosphorylation. Additionally, neither titin isoform expression nor phosphorylation differed. However, there is a significant increase in ß-isoform of the myosin heavy chain (MyHC) expression and phosphorylation of TnI and MyBP-C during aging. In permeabilized strips of papillary muscle, force and Ca2+ sensitivity are reduced during aging, consistent with the increase in ß-MyHC expression and TnI phosphorylation. However, the increase in MyBP-C phosphorylation during aging may represent a mechanism to compensate for age-related contractile deficits. In isolated cardiomyocytes loaded with Fura-2, the peak of the Ca2+ transient is reduced, but the kinetics of the Ca2+ transient are not altered. Furthermore, the extent of shortening and the rates of both sarcomere shortening and re-lengthening are reduced. These results demonstrate that aging is associated with changes in contractile and regulatory protein expression and phosphorylation, which affect the mechanical properties of cardiac muscle.

Actomyosin forces in cell migration: Moving beyond cell body retraction.

In textbook illustrations of migrating cells, actomyosin contractility is typically depicted as the contraction force necessary for cell body retraction. This dogma has been transformed by the molecular clutch model, which acknowledges that actomyosin traction forces also generate and transmit biomechanical signals at the leading edge, enabling cells to sense and shape their migratory path in mechanically complex environments. To fulfill these complementary functions, the actomyosin system assembles a gradient of contractile energy along the front-rear axis of migratory cells. Here, we highlight the hierarchic assembly and self-regulatory network structure of the actomyosin system and explain how the kinetics of different nonmuscle myosin II (NM II) paralogs synergize during contractile force generation. Our aim is to emphasize how protrusion formation, cell adhesion, contraction, and retraction are spatiotemporally integrated during different modes of migration, including chemotaxis and durotaxis. Finally, we hypothesize how different NM II paralogs might tune aspects of migration in vivo, highlighting future research directions.

Enhanced expression of the myogenic factor Myocyte enhancer factor-2 in imaginal disc myoblasts activates a partial, but incomplete, muscle development program.

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.

Tight junctions control lumen morphology via hydrostatic pressure and junctional tension.

Formation of fluid-filled lumina by epithelial tissues is essential for organ development. How cells control the hydraulic and cortical forces to control lumen morphology is not well understood. Here, we quantified the mechanical role of tight junctions in lumen formation using MDCK-II cysts. We found that the paracellular ion barrier formed by claudin receptors is not required for the hydraulic inflation of a lumen. However, the depletion of the zonula occludens scaffold resulted in lumen collapse and folding of apical membranes. Combining quantitative measurements of hydrostatic lumen pressure and junctional tension with modeling enabled us to explain lumen morphologies from the pressure-tension force balance. Tight junctions promote lumen inflation by decreasing cortical tension via the inhibition of myosin. In addition, our results suggest that excess apical area contributes to lumen opening. Overall, we provide a mechanical understanding of how epithelial cells use tight junctions to modulate tissue and lumen shape.

In vitro and in vivo preclinical pharmacokinetic characterization of aficamten, a small molecule cardiac myosin inhibitor.

Aficamten, a small molecule selective inhibitor of cardiac myosin, was characterised in preclinical in vitro and in vivo studies.Protein binding in human plasma was 10.4% unbound and ranged from 1.6% to 24.9% unbound across species. Blood-to-plasma ratios ranged from 0.69 to 1.14 across species. Aficamten hepatic clearance in human was predicted to be low from observed high metabolic stability in vitro in human liver microsomes. Aficamten demonstrated high permeability in Caco-2 cell monolayers.Aficamten in vivo clearance was low across species at 8.8, 2.1, 3.3, and 11 mL/min/kg in mouse, rat, dog, and monkey, respectively. The volume of distribution was low-to-high ranging from 0.53 in rat to 11 L/kg in dog. Oral bioavailability ranged from 41% in monkey to 98% in mouse.Aficamten was metabolised in vitro to eight metabolites with hydroxylated metabolites M1a and M1b predominating. CYP phenotyping indicated multiple CYPs (2C8, 2C9, 2D6, and 3A4) contributing to the metabolism of aficamten.Human clearance (1.1 mL/min/kg) and volume of distribution (6.5 L/kg) were predicted using 4-species allometry employing 'rule-of-exponents'. A predicted 69 hour half-life is consistent with observed half-life in human Phase-1.No CYP-based drug-drug interaction liability as a precipitant was predicted for aficamten.

The Aryl Hydrocarbon Receptor Regulates Invasiveness and Motility in Acute Myeloid Leukemia Cells through Expressional Regulation of Non-Muscle Myosin Heavy Chain IIA.

Acute myeloid leukemia (AML) is the most prevalent type of hematopoietic malignancy. Despite recent therapeutic advancements, the high relapse rate associated with extramedullary involvement remains a challenging issue. Moreover, therapeutic targets that regulate the extramedullary infiltration of AML cells are still not fully elucidated. The Aryl Hydrocarbon Receptor (AHR) is known to influence the progression and migration of solid tumors; however, its role in AML is largely unknown. This study explored the roles of AHR in the invasion and migration of AML cells. We found that suppressed expression of AHR target genes correlated with an elevated relapse rate in AML. Treatment with an AHR agonist on patient-derived AML cells significantly decreased genes associated with leukocyte trans-endothelial migration, cell adhesion, and regulation of the actin cytoskeleton. These results were further confirmed in THP-1 and U937 AML cell lines using AHR agonists (TCDD and FICZ) and inhibitors (SR1 and CH-223191). Treatment with AHR agonists significantly reduced Matrigel invasion, while inhibitors enhanced it, regardless of the Matrigel's stiffness. AHR agonists significantly reduced the migration rate and chemokinesis of both cell lines, but AHR inhibitors enhanced them. Finally, we found that the activity of AHR and the expression of NMIIA are negatively correlated. These findings suggest that AHR activity regulates the invasiveness and motility of AML cells, making AHR a potential therapeutic target for preventing extramedullary infiltration in AML.

Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle.

Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. Because the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP overconsumption. Following this, we assessed its short-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently performing global proteomics profiling in dissected soleus myofibres. After a 4 week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. KEY POINTS: No cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. Applying Mavacamten, a small molecule directly targeting the myofilaments, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. Treating a mouse model of nemaline myopathy in vivo with Mavacamten for 4 weeks, remodelled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. Short-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy.

Filamin A regulates platelet shape change and contractile force generation via phosphorylation of the myosin light chain.

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin-crosslinking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner.

Multiscale modeling shows how 2'-deoxy-ATP rescues ventricular function in heart failure.

2'-deoxy-ATP (dATP) improves cardiac function by increasing the rate of crossbridge cycling and Ca[Formula: see text] transient decay. However, the mechanisms of these effects and how therapeutic responses to dATP are achieved when dATP is only a small fraction of the total ATP pool remain poorly understood. Here, we used a multiscale computational modeling approach to analyze the mechanisms by which dATP improves ventricular function. We integrated atomistic simulations of prepowerstroke myosin and actomyosin association, filament-scale Markov state modeling of sarcomere mechanics, cell-scale analysis of myocyte Ca[Formula: see text] dynamics and contraction, organ-scale modeling of biventricular mechanoenergetics, and systems level modeling of circulatory dynamics. Molecular and Brownian dynamics simulations showed that dATP increases the actomyosin association rate by 1.9 fold. Markov state models predicted that dATP increases the pool of myosin heads available for crossbridge cycling, increasing steady-state force development at low dATP fractions by 1.3 fold due to mechanosensing and nearest-neighbor cooperativity. This was found to be the dominant mechanism by which small amounts of dATP can improve contractile function at myofilament to organ scales. Together with faster myocyte Ca[Formula: see text] handling, this led to improved ventricular contractility, especially in a failing heart model in which dATP increased ejection fraction by 16% and the energy efficiency of cardiac contraction by 1%. This work represents a complete multiscale model analysis of a small molecule myosin modulator from single molecule to organ system biophysics and elucidates how the molecular mechanisms of dATP may improve cardiovascular function in heart failure with reduced ejection fraction.

Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition.

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein α-catenin. Using correlative cryo-fluorescence microscopy and cryo-electron tomography, we identify F-actin featuring domains of nanoscale oscillating curvature at cytoskeleton-adhesion interfaces enriched in zyxin, a marker of actin-myosin generated traction forces. We next introduce a reconstitution system for visualizing F-actin in the presence of myosin forces with cryo-electron microscopy, which reveals morphologically similar superhelical F-actin spirals. In simulations, transient forces mimicking tugging and release of filaments by motors produce spirals, supporting a mechanistic link to myosin's ATPase mechanochemical cycle. Three-dimensional reconstruction of spirals uncovers extensive asymmetric remodeling of F-actin's helical lattice. This is recognized by α-catenin, which cooperatively binds along individual strands, preferentially engaging interfaces featuring extended inter-subunit distances while simultaneously suppressing rotational deviations to regularize the lattice. Collectively, we find that myosin forces can deform F-actin, generating a conformational landscape that is detected and reciprocally modulated by a mechanosensitive protein, providing a direct structural glimpse at active force transduction through the cytoskeleton.

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release.

Inherited mutations in human beta-cardiac myosin (M2ß) can lead to severe forms of heart failure. The E525K mutation in M2ß is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2ß subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 µM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2ß S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

Research reviews on myosin head interactions with F-actin.

The sliding filament theory and the cross-bridge model have been fundamental in understanding muscle contraction. While the cross-bridge model explains the interaction between a single myosin head and actin filament, the native myosin molecule consists of two heads. This review explores the possibility and mechanism of two-headed binding in myosin II to the actin. Recent studies using electron tomography and resonance energy transfer have provided evidence in support of the occurrence of two-headed binding. The flexibility of the regulatory light chain (RLC) appears to play a significant role in enabling this binding mode. However, high-resolution structures of the RLCs in the two-headed bound state have not yet been reported. Resolving these structures, possibly through sub-tomogram averaging or single-particle analysis, would provide definitive proof of the conformational flexibility of RLCs and their role in facilitating two-headed binding. Further investigations are also required to address questions such as the predominance of two-headed versus single-headed binding and the influence of the state of each of the heads on the other. An understanding of the mechanism of two-headed binding is crucial for developing a comprehensive model of the cross-bridge cycle of the native myosin molecule.

Talin2 binds to non-muscle myosin IIa and regulates cell attachment and fibronectin secretion.

Talin2 is localized to large focal adhesions and is indispensable for traction force generation, invadopodium formation, cell invasion as well as metastasis. Talin2 has a higher affinity toward ß-integrin tails than talin1. Moreover, disruption of the talin2-ß-integrin interaction inhibits traction force generation, invadopodium formation and cell invasion, indicating that a strong talin2-ß-integrin interaction is required for talin2 to fulfill these functions. Nevertheless, the role of talin2 in mediation of these processes remains unknown. Here we show that talin2 binds to the N-terminus of non-muscle myosin IIA (NMIIA) through its F3 subdomain. Moreover, talin2 co-localizes with NMIIA at cell edges as well as at some cytoplasmic spots. Talin2 also co-localizes with cortactin, an invadopodium marker. Furthermore, overexpression of NMIIA promoted the talin2 head binding to the ß1-integrin tail, whereas knockdown of NMIIA reduced fibronectin and matrix metalloproteinase secretion as well as inhibited cell attachment on fibronectin-coated substrates. These results suggest that talin2 binds to NMIIA to control the secretion of extracellular matrix proteins and this interaction modulates cell adhesion.

eEF1α2 is required for actin cytoskeleton homeostasis in the aging muscle.

The translation elongation factor eEF1α (eukaryotic elongation factor 1α) mediates mRNA translation by delivering aminoacyl-tRNAs to ribosomes. eEF1α also has other reported roles, including the regulation of actin dynamics. However, these distinct roles of eEF1α are often challenging to uncouple and remain poorly understood in aging metazoan tissues. The genomes of mammals and Drosophila encode two eEF1α paralogs, with eEF1α1 expressed ubiquitously and eEF1α2 expression more limited to neurons and muscle cells. Here, we report that eEF1α2 plays a unique role in maintaining myofibril homeostasis during aging in Drosophila. Specifically, we generated an eEF1α2 null allele, which was viable and showed two distinct muscle phenotypes. In young flies, the mutants had thinner myofibrils in indirect flight muscles that could be rescued by expressing eEF1α1. With aging, the muscles of the mutant flies began showing abnormal distribution of actin and myosin in muscles, but without a change in actin and myosin protein levels. This age-related phenotype could not be rescued by eEF1α1 overexpression. These findings support an unconventional role of Drosophila eEF1α2 in age-related homeostasis of muscle myofibers.

Myosin superfamily members during myelin formation and regeneration.

Myelin is an insulator that forms around axons that enhance the conduction velocity of nerve fibers. Oligodendrocytes dramatically change cell morphology to produce myelin throughout the central nervous system (CNS). Cytoskeletal alterations are critical for the morphogenesis of oligodendrocytes, and actin is involved in cell differentiation and myelin wrapping via polymerization and depolymerization, respectively. Various protein members of the myosin superfamily are known to be major binding partners of actin filaments and have been intensively researched because of their involvement in various cellular functions, including differentiation, cell movement, membrane trafficking, organelle transport, signal transduction, and morphogenesis. Some members of the myosin superfamily have been found to play important roles in the differentiation of oligodendrocytes and in CNS myelination. Interestingly, each member of the myosin superfamily expressed in oligodendrocyte lineage cells also shows specific spatial and temporal expression patterns and different distributions. In this review, we summarize previous findings related to the myosin superfamily and discuss how these molecules contribute to myelin formation and regeneration by oligodendrocytes.

Innovative pharmacological approaches to hypertrophic cardiomyopathy: The emerging role of Aficamten.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disorder characterized by left ventricular hypertrophy (LVH), which can lead to left ventricular outflow tract (LVOT) obstruction. Traditional treatments often provide limited symptom relief and may not adequately reduce the LVOT gradient. Myosin inhibitors, such as Aficamten , offer a new therapeutic approach by modulating myocardial contractility and improving symptoms. This paper evaluated the efficacy and safety of Aficamten in patients with symptomatic HCM. We conducted a comprehensive literature review of studies evaluating Aficamten for symptomatic HCM, including clinical trials and observational studies up to July 2024. Data on efficacy, safety, and patient outcomes were extracted and analyzed from a total of 10 studies involving 1,067 patients. Aficamten demonstrated substantial efficacy in reducing the LVOT gradient, with dose-dependent reductions ranging from 3.6 % to 48.6 %. It also improved symptoms, with 82.3 % of patients experiencing reduced left ventricular ejection fraction (LVEF) and notable improvements in New York Heart Association (NYHA) functional class. Exercise capacity was enhanced, as indicated by increased peak oxygen uptake. Safety profiles were generally favorable, though some serious adverse events, such as atrial fibrillation and cardiac dysfunction, were reported. Aficamten was well-tolerated overall, with manageable dose-dependent adverse effects. Aficamten represents a promising advance in the management of symptomatic HCM, offering significant reductions in LVOT gradient and improvement in symptoms and exercise capacity. Its safety profile is generally favorable, although ongoing monitoring is necessary to manage potential adverse effects. Future research should focus on long-term outcomes, comparative effectiveness, and real-world evidence.