Actin Research Articles

Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited in response to inotropic interventions

Mavacamten is a FDA-approved small-molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin toward ordered off states close to the thick filament backbone. It remains elusive whether these myosin heads in the off state(s) can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by 1) Ca2+, 2) increased chronotropy [heart rate (HR)], 3) stretch, and 4) β-adrenergic (β-AR) stimulation, all known physiological inotropic interventions. At the molecular level, we show that Ca2+ increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed state to the active disordered relaxed state. At the myofilament level, both Ca2+ and passive lengthening can shift mavacamten-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with HR in mavacamten-treated animals. Finally, we show that β-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are at least partially activable, thus preserving cardiac reserve mechanisms.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Synthesis and Biophysical Characterization of Fingolimod Derivatives as Cardiac Troponin Antagonists.

Calcium binding to cardiac troponin C (cTnC) in the thin filaments acts as a trigger for cardiac muscle contraction. The N-lobe of cTnC (NcTnC) undergoes a conformational change in the presence of calcium that allows for interaction with the switch region of cardiac troponin I (cTnISP), releasing its inhibitory effect on the thin filament structure. The small molecule fingolimod inhibits cTnC-cTnISP interactions via electrostatic repulsion between its positively charged tail and positively charged residues in cTnISP and acts as a calcium desensitizer of the contractile myofilaments. Here we investigate the structure-activity relationship of the fingolimod hydrophobic headgroup and show that increasing the alkyl chain length increases both its affinity for NcTnC and its inhibitory effect on the NcTnC-cTnISP interaction and that decreasing flexibility completely abolishes these effects. Strikingly, the longer derivatives have no effect on the calcium affinity of cTnC, suggesting that they act as better inhibitors.

Cardiac manifestations of human ACTA2 variants recapitulated in a zebrafish model

The ACTA2 gene encodes actin α2, a major smooth muscle protein in vascular smooth muscle cells. Missense variants in the ACTA2 gene can cause inherited thoracic aortic diseases with characteristic symptoms, such as dysfunction of smooth muscle cells in the lungs, brain vessels, intestines, pupils, bladder, or heart. We identified a heterozygous missense variant of Gly148Arg (G148R) in a patient with a thoracic aortic aneurysm, dissection, and left ventricular non-compaction. We used zebrafish as an in vivo model to investigate whether or not the variants might cause functional or histopathological abnormalities in the heart. Following the fertilization of one-cell stage embryos, we injected in vitro synthesized ACTA2 mRNA of wild-type, novel variant G148R, or the previously known pathogenic variant Arg179His (R179H). The embryos were maintained and raised for 72 h post-fertilization for a heart analysis. Shortening fractions of heart were significantly reduced in both pathogenic variants. A histopathological evaluation showed that the myocardial wall of ACTA2 pathogenic variants was thinner than that of the wild type, and the total cell number within the myocardium was markedly decreased in all zebrafish with pathogenic variants mRNAs. Proliferating cell numbers were also significantly decreased in the endothelial and myocardial regions of zebrafish with ACTA2 variants compared to the wild type. These results demonstrate the effects of ACTA2 G148R and R179H on the development of left ventricle non-compaction and cardiac morphological abnormalities. Our study highlights the previously unknown significance of the ACTA2 gene in several aspects of cardiovascular development.

Mechanical interaction of myosin and native thin filament in the disused rat soleus muscle

The disuse of skeletal limb muscles occurs in a variety of conditions, yet our comprehension of the molecular mechanisms involved in adaptation to disuse remains incomplete. We studied the mechanical characteristics of actin-myosin interaction using an in vitro motility assay and isoform composition of myosin heavy and light chains by dint of SDS-PAGE in soleus muscle of both control and hindlimb-unloaded rats. 14 days of hindlimb unloading led to the increased maximum sliding velocity of actin, reconstituted, and native thin filaments over rat soleus muscle myosin by 24 %, 19 %, and 20 %, respectively. The calcium sensitivity of the “pCa-velocity” relationship decreased. There was a 26 % increase in fast myosin heavy chain IIa (MHC IIa), a 22 % increase in fast myosin light chain 2 (MLC 2f), and a 13 % increase in fast MLC 1f content. The content of MLC 1s/v, typical for slow skeletal muscles and cardiac ventricles did not change. At the same time, MLC 1s, typical only for slow skeletal muscles, disappeared. The maximum velocity of soleus muscle native thin filaments was 24 % higher compared to control ones sliding over the same rabbit myosin. Therefore, both myosin and native thin filament kinetics could influence the mechanical characteristics of the soleus muscle. Additionally, the MLC 1s and MLC 1s/v ratio may contribute to the mechanical characteristics of slow skeletal muscle, along with MHC, MLC 2, and MLC 1 slow/fast isoforms ratio.

Filamin C Deficiency Impairs Sarcomere Stability and Activates Focal Adhesion Kinase through PDGFRA Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Truncating mutations in filamin C (FLNC) are associated with dilated cardiomyopathy and arrhythmogenic cardiomyopathy. FLNC is an actin-binding protein and is known to interact with transmembrane and structural proteins; hence, the ablation of FLNC in cardiomyocytes is expected to dysregulate cell adhesion, cytoskeletal organization, sarcomere structural integrity, and likely nuclear function. Our previous study showed that the transcriptional profiles of FLNC homozygous deletions in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly comparable to the transcriptome profiles of hiPSC-CMs from patients with FLNC truncating mutations. Therefore, in this study, we used CRISPR-Cas-engineered hiPSC-derived FLNC knockout cardiac myocytes as a model of FLNC cardiomyopathy to determine pathogenic mechanisms and to examine structural changes caused by FLNC deficiency. RNA sequencing data indicated the significant upregulation of focal adhesion signaling and the dysregulation of thin filament genes in FLNC-knockout (FLNCKO) hiPSC-CMs compared to isogenic hiPSC-CMs. Furthermore, our findings suggest that the complete loss of FLNC in cardiomyocytes led to cytoskeletal defects and the activation of focal adhesion kinase. Pharmacological inhibition of PDGFRA signaling using crenolanib (an FDA-approved drug) reduced focal adhesion kinase activation and partially normalized the focal adhesion signaling pathway. The findings from this study suggest the opportunity in repurposing FDA-approved drug as a therapeutic strategy to treat FLNC cardiomyopathy.

Roles of chain stretch and concentration gradients in capillary thinning of polymer solutions

Abstract Polymers inhibit the breakup of a liquid filament thinning under surface tension. The coupling of elasticity, capillarity and inertia leads to the well-known beads-on-a-string (BOAS) formation. Additionally, under different conditions, smaller satellite drops can form along the liquid bridge between the main beads. The development of BOAS and satellite drops is controlled by the rheology of the polymer solution. In this study, we consider the roles played by finite extensibility and anisotropic drag on the formation of satellite beads. In particular, we show that the more stretching a polymer chain can undergo, satellite beads are suppressed. The latter stages of capillary thinning has been shown to result in a phase separation resulting in what is referred to as a blistering pattern. We thus also conduct simulations of an inhomogeneous dilute polymer model that considers the competing effects of diffusion and stress gradients. We show that polymer is pulled axially towards the region connecting string and bead. This simple model does not predict a phase separation, but does reveal that pinchoff could be inhibited by the buildup of polymer concentration.

Abstract WP223: Cerebral Visual Impairment in ACTA2 Patients Assessed by Virtual Reality Testing

Introduction: Heterozygous missense mutations disrupting arginine 179 (Arg179) in the ACTA2 gene which codes for the smooth muscle-specific isoform of α-actin, cause Multisystemic Smooth Muscle Dysfunction Syndrome (MSMDS). The clinical manifestations of this genetic condition include congenital mydriasis, patent ductus arteriosus, stroke, and/or aortic aneurysms or dissections among many others. White matter injury, impaired autoregulation, and neurovascular coupling (due to the cerebral vasculopathy affecting cerebral perfusion) are known factors that alter neural network performance and processing speed. The lack of a standardized ophthalmologic examination that detects cerebral visual impairment (CVI), precludes an early diagnosis and creates missed opportunities for intervention. There is a paucity of data regarding CVI in MSMDS patients, and through this novel work, we present data defining their visual processing deficits. Hypothesis: We hypothesize that: 1) individuals with MSMDS will exhibit more pronounced deficits in visual-spatial processing compared to the control group, and 2) their performance will deteriorate as the complexity of visual tasks increases. Methods: Using a virtual reality (VR) based visual search task system, we characterized the impairment of higher-order visual processing in MSMDS patients (n = 4) as compared to healthy controls (n = 13). The VR-based task required searching for targets on a screen among varying levels of visual complexity. Reaction time, gaze error, shifted ellipse area, success rate, and pursuit saccade ratio were statistically analyzed with unpaired group t-tests, with significance being defined as p < 0.05. Results: Compared to controls, MSMDS patients took longer to identify the correct target, were less accurate in finding the target, and exhibited greater impairment in performance with an increasing number of distractors. Both groups showed similar task compliance, measured using an off-screen count metric. Conclusion: This novel work demonstrates that MSMDS patients exhibit impaired higher-order visual processing. This highlights the need for clinical tools that can assess these difficulties with quantitative methods to guide therapies and improve functional outcomes.

Plasma-resonance-assisted filament in a high-pressure microwave discharge

Self-consistent steady-state structure of a thin plasma filament maintained in atmospheric pressure argon by a rotating transverse electric field of ∼1 cm wavelength is simulated numerically under conditions when the local plasma resonance condition is satisfied at some surface inside the filament. The equilibrium radial distributions of the plasma parameters (temperature and density) and the field amplitude inside the filament are calculated based on the solution of the heat equation and the Maxwell equations. It is shown that the occurrence of strong resonant peaks of the radial component of the electric field and the power of Joule losses in the region of critical density at the periphery of the filament provide the energy deposition sufficient to achieve a relatively high gas temperature and a strongly supercritical plasma density in its central region. In relation to the values of these basic parameters of the filament and the lower limit of the external radiation power supporting it, the results obtained are in satisfactory agreement with the data of the earlier experiment.

Proposing a Caputo-Land System for active tension. Capturing variable viscoelasticity

Accurate cell-level active tension modeling for cardiomyocytes is critical to understanding cardiac functionality on a subject-specific basis. However, cell-level models in the literature fail to account for viscoelasticity and inter-subject variations in active tension, which are relevant to disease diagnostics and drug screening, e.g., for cardiotoxicity. Thus, we propose a fractional order system to model cell-level active tension by extending Land's state-of-the-art model of cardiac contraction. Our approach features the (left) Caputo derivative of six state variables that identify the mechanistic origins of viscoelasticity in a myocardial cell in terms of the thin filament, thick filament, and length-dependent interactions. This proposed CLS is the first of its kind for active tension modeling in cells and demonstrates notable subject-specificity, with smaller mean square errors than the reference model relative to cell-level experiments across subjects, promising greater clinical relevance than its counterparts in the literature by highlighting the contribution of different cellular mechanisms to apparent viscoelastic cell behavior, and how it could vary with disease.

Umbrella sampling simulations of cardiac thin filament reveal thermodynamic consequences of troponin I inhibitory peptide mutations

Umbrella sampling simulations of cardiac thin filament reveal thermodynamic consequences of troponin I inhibitory peptide mutations

Leiomodin-2 inhibits actomyosin interactions and perturbs the structure of the thin filament

Leiomodin-2 inhibits actomyosin interactions and perturbs the structure of the thin filament

A missense variant in the Z-disc protein α-actinin 2: Evidence for a sarcomere protein at the nexus of thick and thin filament regulation

A missense variant in the Z-disc protein α-actinin 2: Evidence for a sarcomere protein at the nexus of thick and thin filament regulation

Assessing thin filament lengths in young healthy human skeletal muscle—a role for fiber type?

Assessing thin filament lengths in young healthy human skeletal muscle—a role for fiber type?

Decay of Taylor–Green flow type initial conditions in a two-dimensional domain

Geophysical flows are often two-dimensional (2D) in nature. The decay dynamics of the canonical, symmetric initial conditions defined by the Taylor–Green (TG) flow is simulated in a 2D domain. The standard lattice Boltzmann equation method with the Bhatnagar–Gross–Krook collision model is employed. The vortex dynamics is investigated for bounded and unbounded flows with various sets of Gaussian vortices and varying Reynolds numbers. The formation of thin filaments and the generation of small-scale structures near the no-slip walls are found. To characterize the decay properties, the integral quantities, namely, kinetic energy, E(t), enstrophy, Ω(t) , and palinstrophy, P(t), are tracked as a function of the eddy-turnover-time. The initial vortex population in the domain dictates the decay rate of these integral quantities. The energy spectra, E(k), are computed to investigate the effect of domain boundaries and vortex populations on energy distribution among different length scales for Re=50000 . The evolution of TG flow in a bounded domain shows an exponent close to −3, which may be attributed to the generation of thin filaments and small-scale structures in vortex-wall interactions. Exponent changes with an increase in the vortex populations.

Identification of a hub gene VCL for atherosclerotic plaques and discovery of potential therapeutic targets by molecular docking

BackgroundAtherosclerosis (AS) is a pathology factor for cardiovascular diseases and instability of atherosclerotic plaques contributes to acute coronary events. This study identified a hub gene VCL for atherosclerotic plaques and discovered its potential therapeutic targets for atherosclerotic plaques.MethodsDifferential expressed genes (DEGs) were screened between unstable and stable plaques from GSE120521 dataset and then used for construction of a protein-protein interactions (PPI) network. Through topological analysis, hub genes were identified within this PPI network, followed by construction of a diagnostic model. GSE41571 dataset was utilized to validate the diagnostic model. A key hub gene was identified and its association with immune characteristics and pathways were further investigated. Molecular docking and molecular dynamics (MD) simulation were employed to discover potential therapeutic targets.ResultsAccording to the PPI network, 3 tightly connected protein clusters were found. Topological analysis identified the top 5 hub genes, Vinculin (VCL), Dystrophin (DMD), Actin alpha 2 (ACTA2), Filamin A (FLNA), and transgelin (TAGLN). Among these hub genes, VCL had the highest diagnostic value. VCL was selected for further analysis and we found that VCL was negatively correlated with immune score and AS-related inflammatory pathways. Next, we identified 408 genes that were highly correlated with VCL and determined potential drug candidates. The results from molecular docking and MD simulation showed compound DB07117 combined with VCL protein stably, the binding energy is -7.7 kcal/mol, indicating that compound DB07117 was a potential inhibitor of VCL protein.ConclusionThis study identified VCL as a key gene for atherosclerotic plaques and provides a potential therapeutic target of VCL for the treatment of atherosclerotic plaques.

Unravelling the role of Sildenafil and SB204741 in suppressing fibrotic potential of peritoneal fibroblasts obtained from PD patients.

Introduction: Peritoneal fibrosis (PF) results in technique failure in peritoneal dialysis (PD) patients. Peritoneal fibroblasts are characterized by increase in the ACTA2 gene, responsible for alpha smooth muscle actin (α-SΜΑ), extracellular matrix (ECM) production, and inflammatory cytokines production, which are the are key mediators in the pathogenesis of PF. 5-hydroxytryptamine (5-HT; serotonin) induces ECM synthesis in fibroblasts in a transforming growth factor-beta 1 (TGF-β1) dependent manner. The purpose of our study was to identify the potential mechanism and role of sildenafil and 5HT2B receptor inhibitor (SB204741) combination in attenuating PD-associated peritoneal fibrosis. Methods: Studies were performed to determine the effect of TGF-β1, sildenafil, and SB204741 on human peritoneal fibroblasts (HPFBs) isolated from the parietal peritoneum of patients in long-term PD patients (n = 6) and controls (n = 6). HPFBs were incubated with TGF-β1 (10ng/mL) for 1h and later with TGF-β1 (10ng/mL)/[sildenafil (10µM) or SB204741 (1µM)] and their combination for 24h (post-treatment strategy). In the pre-treatment strategy, HPFBs were pre-treated with sildenafil (10µM) or SB204741 (1µM) and a combination of the two for 1h and later with only TGF-β1 (10ng/mL) for 24h. Results: The anti-fibrotic effects of the combination of sildenafil and SB204741 were greater than that of each drug alone. In TGF-β1-stimulated HPFBs, pro-fibrotic genes (COL1A1, COL1A2, ACTA2, CTGF, FN1, and TGFB1) exhibited higher expression than in controls, which are crucial targets of sildenafil and SB204741 against peritoneal fibrosis. The synergistic approach played an anti-fibrotic role by regulating the pro- and anti-fibrotic gene responses as well as inflammatory cytokine responses. The combination treatment significantly attenuated peritoneal fibrosis, as evident by the almost complete amelioration of ACTA2 expression, restoration of anti-fibrotic genes (MMP2/TIMP1), and, at least, by reducing the expression of pro-inflammatory cytokines (IFN-γ, IL-4, IL-17, IL-1β, IL-6, TNF-α, and TGF-β1) along with an increase in IL-10 levels. Discussion: Taken together, the above research evidences that the combination of sildenafil and SB204741 may have therapeutic potential in suppressing peritoneal fibrosis due to peritoneal dialysis.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Shahulinema minutum gen. & sp. nov. (Oculatellaceae) and Euryhalinema shahuense sp. nov. (Nodosilineaceae), two new Cyanobacteria isolated from Shahu Lake in China

ABSTRACT Two novel cyanobacteria (strains SH01 and SH02) with thin filaments and simple morphology were isolated from Shahu Lake in China. The two strains were morphologically similar to the thin, simple filamentous cyanobacteria. The 16S rRNA gene data phylogeny revealed the distinct lineage of SH01, which was nearest to the clade incorporating the genera Kaiparowitsia, Thermoleptolyngbya and Aerofilum. The strain SH02, together with other Euryhalinema species, grouped in a unique cluster, in which the type species Euryhalinema mangrovii was included. The 16S rRNA gene sequences of SH01 and SH02 showed maximum similarities as 94.2% and 98.5%, respectively, compared to those of established genera and other Euryhalinema species to which they were phylogenetically related. Furthermore, the ITS secondary structures for both strains SH01 and SH02 revealed significant distinction and uniqueness when compared with their respective reference strains (the genera of Oculatellaceae for SH01 and Euryhalinema species for SH02). To sum up, these results verified the establishment of strain SH01 as a novel genus named Shahulinema gen. nov., and strain SH02 as the fourth novel species under the genus Euryhalinema, as E. shahuense sp. nov.

Peripheral thickening of the sarcomeres and pointed end elongation of the thin filaments are both promoted by SALS and its formin interaction partners.

During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.