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The blood-spinal cord barrier (BSCB) is a physiological barrier between the blood and spinal cord parenchyma. This study aims to determine whether Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, can protect the BSCB using in vivo models. The Evans blue fluorescence assay was used to detect leakage of the BSCB. Western blotting was used to define alterations in ROCK-related and tight junction (TJ) protein expression. Immunofluorescence triple-staining was used to evaluate histologic alterations in TJs. Locomotor function was evaluated using the open-field test, the Basso-Beattie-Bresnahan score, and footprint analysis. Two peaks of BSCB leakage after spinal cord injury (SCI) occurred at 24 h and 5 days. The ROCK inhibitor reduced the BSCB leakage at the second peak after SCI. Moreover, the ROCK inhibitor ameliorated the integrity of the BSCB and improved motor function recovery after SCI by regulating the phosphorylation of myosin phosphatase subunit-1 (MYPT1) and cofilin. ROCK inhibitors might protect the BSCB, which provides a new strategy for transitioning SCI treatment from the bench to bedside., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The endothelial barrier regulates interstitial fluid homeostasis by transcellular and paracellular means. Dysregulation of this semipermeable barrier may lead to vascular leakage, edema, and accumulation of pro-inflammatory cytokines, inducing microvascular hyperpermeability. Investigating the molecular pathways involved in those events will most probably provide novel therapeutic possibilities in pathologies related to endothelial barrier dysfunction. Metformin (MET) is an anti-diabetic drug, opposes malignancies, inhibits cellular transformation, and promotes cardiovascular protection. In the current study, we assess the protective effects of MET in LPS-induced lung endothelial barrier dysfunction and evaluate the role of P53 in mediating the beneficial effects of MET in the vasculature. We revealed that this biguanide (MET) opposes the LPS-induced dysregulation of the lung microvasculature, since it suppressed the formation of filamentous actin stress fibers, and deactivated cofilin. To investigate whether P53 is involved in those phenomena, we employed the fluorescein isothiocyanate (FITC) - dextran permeability assay, to measure paracellular permeability. Our observations suggest that P53 inhibition increases paracellular permeability, and MET prevents those effects. Our results contribute towards the understanding of the lung endothelium and reveal the significant role of P53 in the MET-induced barrier enhancement., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Circulating tumor cells (CTCs) are associated with a higher risk of metastasis in tumor patients. The adhesion and arrest of CTCs at a secondary site is an essential prerequisite for the occurrence of tumor metastasis. CTC reattachment has shown to be dependent on microtentacle (McTN) formation in vivo. However, the specific molecular mechanism of McTN formation in suspended cancer cells remains largely unclear. Here, we demonstrated that the activation of Notch-1 signaling triggers McTN formation to facilitate cell reattachment in suspended cell culture conditions. Moreover, molecular mechanistic studies revealed that McTN formation is governed by the balance between microtubule-driven outgrowth and actomyosin-driven cell contractility. The activation of Notch-1 downregulates the acetylation level of microtubules via the Cdc42/HDAC6 pathway, which contributes to microtubule polymerization. Simultaneously, Notch-1 signaling-induced Cdc42 activation also reduced phosphorylation of myosin regulatory light chain, leading to cell contractility attenuation. Altogether, these results defined a novel mechanism by which Notch-1 signaling disturbs the balance between the expansion of microtubules and contraction of the cortical actin, which promotes McTN formation and cell reattachment. Our findings provide a new perspective on the effective therapeutic target to prevent CTC reattachment., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

REF52 fibroblasts have a well-developed contractile machinery, the most prominent elements of which are actomyosin stress fibers with highly ordered organization of actin and myosin IIA filaments. The relationship between contractile activity and turnover dynamics of stress fibers is not sufficiently understood. Here, we simultaneously measured the forces exerted by stress fibers (using traction force microscopy or micropillar array sensors) and the dynamics of actin and myosin (using photoconversion-based monitoring of actin incorporation and high-resolution fluorescence microscopy of myosin II light chain). Our data revealed new features of the crosstalk between myosin II-driven contractility and stress fiber dynamics. During normal stress fiber turnover, actin incorporated all along the stress fibers and not only at focal adhesions. Incorporation of actin into stress fibers/focal adhesions, as well as actin and myosin II filaments flow along stress fibers, strongly depends on myosin II activity. Myosin II-dependent generation of traction forces does not depend on incorporation of actin into stress fibers per se, but still requires formin activity. This previously overlooked function of formins in maintenance of the actin cytoskeleton connectivity could be the main mechanism of formin involvement in traction force generation., (Copyright © 2021. Published by Elsevier B.V.)

Compromised endothelial-cell (EC) barrier function is a hallmark of inflammatory diseases. mTOR inhibitors, widely applied as clinical therapies, cause pneumonitis through mechanisms that are not yet fully understood. This study aimed to elucidate the EC mechanisms underlying the pathogenesis of pneumonitis caused by mTOR inhibition (mTORi). Mice with EC-specific deletion of mTOR complex components ( Mtor , Rptor or Rictor ) were administered LPS to induce pulmonary injury. Cultured ECs were treated with pharmacologic inhibitors, siRNA, or overexpression plasmids. EC barrier function was evaluated in vivo with Evans blue assay and in vitro by measurement of transendothelial electrical resistance and albumin flux. mTORi increased basal and TNFα-induced EC permeability, which was caused by myosin light chain (MLC) phosphorylation-dependent cell contraction. Inactivation of mTOR kinase activity by mTORi triggered PKCδ/p38/NF-κB signaling that significantly upregulated TNFα-induced MLCK (MLC kinase) expression, whereas Raptor promoted the phosphorylation of PKCα/MYPT1 independently of its interaction with mTOR, leading to suppression of MLCP (MLC phosphatase) activity. EC-specific deficiency in mTOR, Raptor or Rictor aggravated lung inflammation in LPS-treated mice. These findings reveal that mTORi induces PKC-dependent endothelial MLC phosphorylation, contraction, and hyperpermeability that promote pneumonitis.

Herein, a β-1,3-d-glucan based microcarrier, yeast cell wall microparticles (YPs), was used to develop a food-source-based nano-in-micro oral delivery system for ulcerative colitis (UC) treatment. Briefly, lactoferrin (Lf), which targets intestinal epithelial cells, was used to encapsulate emodin (EMO) to form nanoparticles (EMO-NPs), and then loaded into YPs with the natural macrophages targeting ability, forming a final formula with two outer-inner targeting layers (EMO-NYPs). These dual-targeting strategy could enhance the dual-effects of EMO in anti-inflammatory and mucosal repair effects respectively. As expected, cell uptake assessment confirmed that EMO-NPs and EMO-NYPs could target on the Lf and dection-1 receptors on the membranes of Caco-2 cells and macrophages, respectively. Importantly, EMO-NYPs showed the best anti-UC effects compared to EMO-NPs and free EMO, by inhibiting NF-κB pathway to anti-inflammation and promoting intestinal mucosa repair via MLCK/pMLC2 pathway. The results show that EMO-NYPs are a promising food-based oral delivery system in anti-UC., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to 'listen' to receptor inputs and respond to directional reprogramming., (© 2021. The Author(s).)

Atrial light chains (ALC1) are naturally present in adult heart atria, while ventricular light chains (VLC1) are predominant in ventricles. Degradation of VLC1 and re‑expression of ALC1 in heart ventricles are associated with heart disorders in response to pressure overload. The aim of the current study was to investigate changes in myosin light chain expression after simulated ischemia and simulated reperfusion (sI/sR). Human cardiomyocytes (HCM) isolated from adult heart ventricles were subjected to chemical ischemia. The control group was maintained under aerobic conditions. Myocyte injury was determined by testing lactate dehydrogenase (LDH) activity. The gene expression of ALC1, VLC1 and MMP‑2 were assessed by reverse transcription‑quatitive PCR. Additionally, protein synthesis was measured using ELISA kits and MMP‑2 activity was measured by zymography. The results revealed that LDH activity was increased in sI/sR cell‑conditioned medium (P=0.02), confirming the ischemic damage of HCM. ALC1 gene expression and content in HCM were also increased in the sI/sR group (P=0.03 and P<0.001, respectively), while VLC1 gene expression after sI/sR was decreased (P=0.008). Furthermore, MMP‑2 gene expression and synthesis were lower in the sI/sR group when compared with the aerobic control group (P<0.001 and P=0.03, respectively). MMP‑2 activity was also increased in sI/sR cell‑conditioned medium (P=0.006). In conclusion, sI/sR treatment led to increased ALC1 and decreased VLC1 expression in ventricular cardiomyocytes, which may constitute an adaptive mechanism to altered conditions and contribute to the improvement of heart function.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca 2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Evidence suggests that sedative dexmedetomidine can prevent intestinal dysfunction. However, the specific mechanisms of its protective effects against burn-induced intestinal barrier injury remain unclear. We aimed to explore the possible positive effects of dexmedetomidine on burn-induced intestinal barrier injury and the effects the myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) signalling pathway in an experimental model of burn injury., Methods: In this study, the plasma concentration of fluorescein isothiocyanate-labelled dextran (FITC-dextran) was measured. Histological changes were evaluated using haematoxylin and eosin (HE) staining. Tight junction proteins were evaluated by western blot and immunofluorescence analyses to assess the structural integrity of intestinal tight junctions. The level of inflammation was detected by enzyme-linked immunosorbent assay (ELISA)., Results: The results shows that the increase in intestinal permeability caused by burn injury is accompanied by histological damage to the intestine, decreases in the expression of the tight junction proteins Zonula Occludens-1 (ZO-1) and Occludin, increases in inflammatory cytokine levels and elevation of both MLCK protein expression and MLC phosphorylation. After dexmedetomidine treatment, the burn-induced changes were ameliorated., Conclusions: In conclusion, dexmedetomidine exerted an anti-inflammatory effect and protected tight junction complexes against burn‑induced intestinal barrier damage by inhibiting the MLCK/p-MLC signalling pathways., (Copyright © 2021 Elsevier Ltd and ISBI. All rights reserved.)

Myosin light chain 9 (MYL9) is a regulatory light chain of myosin, which plays an important role in various biological processes including cell contraction, proliferation and invasion. MYL9 expresses abnormally in several malignancies including lung cancer, breast cancer, prostate cancer, malignant melanoma and others, which is closely related to the poor prognosis, but the clinical significance for its expression varies with different types of cancer tissues. Further elucidating the molecular mechanism of MYL9 in various types of malignant tumor metastasis is of great significance for cancer prevention and treatment. At the same time, as a molecular marker and potential target, MYL9 may have great clinical value in the early diagnosis, prognosis prediction, and targeted treatment of malignant tumors.

In a number of elongated cells, such as fungal hyphae, a vesicle cluster is observed at the growing tip. This cluster, called a Spitzenkörper, has been suggested to act as a vesicle supply center, yet analysis of its function is challenging, as a majority of components identified thus far are essential for growth. Here, we probe the function of the Spitzenkörper in the human fungal pathogen Candida albicans, using genetics and synthetic physical interactions (SPI). We show that the C. albicans Spitzenkörper is comprised principally of secretory vesicles. Mutant strains lacking the Spitzenkörper component myosin light chain 1 (Mlc1) or having a SPI between Mlc1 and either another Spitzenkörper component, the Rab GTPase Sec4, or prenylated green fluorescent protein (GFP), are viable and still exhibit a Spitzenkörper during filamentous growth. Strikingly, all of these mutants formed filaments with increased diameters and extension rates, indicating that Mlc1 negatively regulates myosin V, Myo2, activity. The results of our quantitative studies reveal a strong correlation between filament diameter and extension rate, which is consistent with the vesicle supply center model for fungal tip growth. Together, our results indicate that the Spitzenkörper protein Mlc1 is important for growth robustness and reveal a critical link between filament morphology and extension rate. IMPORTANCE Hyphal tip growth is critical in a range of fungal pathogens, in particular for invasion into animal and plant tissues. In Candida albicans, as in many filamentous fungi, a cluster of vesicles, called a Spitzenkörper, is observed at the tip of growing hyphae that is thought to function as a vesicle supply center. A central prediction of the vesicle supply center model is that the filament diameter is proportional to the extension rate. Here, we show that mutants lacking the Spitzenkörper component myosin light chain 1 (Mlc1) or having synthetic physical interactions between Mlc1 and either another Spitzenkörper component or prenylated GFP, are defective in filamentous growth regulation, exhibiting a range of growth rates and sizes, with a strong correlation between diameter and extension rate. These results suggest that the Spitzenkörper is important for growth robustness and reveal a critical link between filament morphology and extension rate.

Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role., (© 2021. The Author(s).)

Force generation by the molecular motor myosin II (MII) at the actin cortex is a universal feature of animal cells. Despite its central role in driving cell shape changes, the mechanisms underlying MII regulation at the actin cortex remain incompletely understood. Here we show that myosin light chain kinase (MLCK) promotes MII turnover at the mitotic cortex. Inhibition of MLCK resulted in an alteration of the relative levels of phosphorylated regulatory light chain (RLC), with MLCK preferentially creating a short-lived pRLC species and Rho-associated kinase (ROCK) preferentially creating a stable ppRLC species during metaphase. Slower turnover of MII and altered RLC homeostasis on MLCK inhibition correlated with increased cortex tension, driving increased membrane bleb initiation and growth, but reduced bleb retraction during mitosis. Taken together, we show that ROCK and MLCK play distinct roles at the actin cortex during mitosis; ROCK activity is required for recruitment of MII to the cortex, while MLCK activity promotes MII turnover. Our findings support the growing evidence that MII turnover is an essential dynamic process influencing the mechanical output of the actin cortex.

Astrocytes, which can be obtained from neural stem cells (NSCs) by adding serum and/or recombinant proteins in culture media or by passaging NSCs repeatedly, are expected to be applicable in regenerative medicine for the treatment of neurodegenerative diseases. However, astrocytes obtained using existing methods are costly and have poor quality. The stiffness of culture surfaces has been reported to affect astrocytic differentiation of adult NSCs. However, the influence of surface stiffness on astrocytic differentiation of embryonic NSCs has not yet been reported. In this study, we showed that astrocytic differentiation of embryonic NSCs was increased on soft surfaces (1 kPa and 12 kPa) compared with the NSCs on stiff surfaces (2.8 GPa) in serum-free condition. Furthermore, di-phosphorylated myosin regulatory light chain (PP-MRLC) was decreased in embryonic NSCs cultured on the soft surfaces than the cells on the stiff surfaces. Additionally, astrocytic differentiation of embryonic NSCs was induced by a Ras homolog associated kinase (ROCK) inhibitor, which decreased PP-MRLC in NSCs. These results suggest that decreasing the PP-MRLC of embryonic NSCs on soft surfaces or treating NSCs with a ROCK inhibitor is a good method to prepare astrocytes for application in regenerative medicine., (© 2021. The Author(s).)

Phosphorylation of myosin regulatory light chain (MRLC) can regulate muscle contraction and thus affect actomyosin dissociation and meat quality. The objective of this study was to explore the mechanism by how MRLC phosphorylation regulates actomyosin dissociation and thus develop strategies for improving meat quality. Here, the phosphorylation status of MRLC was modulated by myosin light chain kinase and myosin light chain kinase inhibitor. MRLC phosphorylation at Ser17 decreased the kinetic energy and total energy of actomyosin, thus stabilized the structure, facilitating the interaction between myosin and actin; this was one possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation. Moreover, MRLC phosphorylation at Ser17 was beneficial to the formation of ionic bonds, hydrogen bonds, and hydrophobic interaction between myosin and actin, and was the second possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The normal function of heart muscle depends on its ability to contract more strongly at longer length. Increased venous filling stretches relaxed heart muscle cells, triggering a stronger contraction in the next beat- the Frank-Starling relation. Conversely, heart muscle cells are inactivated when they shorten during ejection, accelerating relaxation to facilitate refilling before the next beat. Although both effects are essential for the efficient function of the heart, the underlying mechanisms were unknown. Using bifunctional fluorescent probes on the regulatory light chain of the myosin motor we show that its N-terminal domain may be captured in the folded OFF state of the myosin dimer at the end of the working-stroke of the actin-attached motor, whilst its C-terminal domain joins the OFF state only after motor detachment from actin. We propose that sequential folding of myosin motors onto the filament backbone may be responsible for shortening-induced de-activation in the heart., (© 2021. The Author(s).)

Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott-Aldrich syndrome protein (N-WASP), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Epigenetic gene silencing of the tumor suppressor death-associated protein kinase 1 (DAPK1) is implicated in the progression of malignant gliomas. However, the mechanism underlying the repression of DAPK1 in gliomas remains elusive. In this study, we identified the existence of DAPK1-inositol 1,4,5-trisphosphate receptor (IP 3 R)-interacting protein (ITPRIP) -myosin regulatory light polypeptide 9 (MYL9) complex in malignant glioma cells. Lentivirus co-infection and coimmunoprecipitation showed that ITPRIP bound with the death domain (DD) of DAPK1 in vitro. Further, dissociating ITPRIP-DAPK1 interaction inhibited glioma tumor growth in vitro but not in vivo. Moreover, knockdown of ITPRIP or DAPK1 impaired the ternary complex formation, whereas MYL9 knockdown did not affect ITPRIP-DAPK1 association. We further found that ITPRIP recruited MYL9 to the kinase domain (KD) of DAPK1, and in turn impeded the phosphorylation of MYL9. Accordingly, interference of ITPRIP enhanced the suppressive effects of DAPK1-KD on glioma progression both in vitro and in vivo. Our results demonstrate that ITPRIP plays a crucial role in the inhibition of DAPK1 and enhancement of tumorigenic properties of malignant glioma cells., (Copyright © 2021. Published by Elsevier Inc.)

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca 2+ concentration and decreased Ca 2+ sensitivity of LC20 phosphorylation, named Ca 2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca 2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca 2+ -induced Ca 2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca 2+ /calmodulin (CaM)-dependent protein kinase kinase-β (CaMKKβ) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKβ and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC 20 ) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC 20 . Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of V max and K m for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC 20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC 20 . The in situ rate of MLC 20 phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the V max values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC 20 , and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

Palmitoylethanolamide (PEA) offers a strong protection against BBB disruption and neurological deficits after cerebral ischaemic/reperfusion (I/R) injury. To date, these BBB protective effects of PEA are mainly attributed to PPARα-mediated actions. However, whether PEA protects against BBB disruption through direct regulation of cytoskeletal microfilaments remains unknown. Here, we identified PEA as a Rho-associated protein kinase (ROCK2) inhibitor (IC 50  = 38.4 ± 4.8 μM). In vitro data suggested that PEA reduced the activation of ROCK/MLC signaling and stress fiber formation within microvascular endothelial cells (ECs) after oxygen-glucose deprivation (OGD), and consequently attenuated early (0-4 h) EC barrier disruption. These actions of PEA could not be blocked by the PPARα antagonist GW6471. In summary, the present study described a previously unexplored role of PEA as a ROCK2 inhibitor, and propose a PPARα-independent mechanism for pharmacological effects of PEA., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We investigated the mechanisms associated with E22K mutation in myosin regulatory light chain (RLC), found to cause hypertrophic cardiomyopathy (HCM) in humans and mice. Specifically, we characterized the mechanical profiles of papillary muscle fibers from transgenic mice expressing human ventricular RLC wild-type (Tg-WT) or E22K mutation (Tg-E22K). Because the two mouse models expressed different amounts of transgene, the B6SJL mouse line (NTg) was used as an additional control. Mechanical experiments were carried out on Ca 2+ - and ATP-activated fibers and in rigor. Sinusoidal analysis was performed to elucidate the effect of E22K on tension and stiffness during activation/rigor, tension-pCa, and myosin cross-bridge (CB) kinetics. We found significant reductions in active tension (by 54%) and stiffness (active by 40% and rigor by 54%). A decrease in the Ca 2+ sensitivity of tension (by ∆pCa ~ 0.1) was observed in Tg-E22K compared with Tg-WT fibers. The apparent (=measured) rate constant of exponential process B (2πb: force generation step) was not affected by E22K, but the apparent rate constant of exponential process C (2πc: CB detachment step) was faster in Tg-E22K compared with Tg-WT fibers. Both 2πb and 2πc were smaller in NTg than in Tg-WT fibers, suggesting a kinetic difference between the human and mouse RLC. Our results of E22K-induced reduction in myofilament stiffness and tension suggest that the main effect of this mutation was to disturb the interaction of RLC with the myosin heavy chain and impose structural abnormalities in the lever arm of myosin CB. When placed in vivo, the E22K mutation is expected to result in reduced contractility and decreased cardiac output whereby leading to HCM., Sub-Discipline: Bioenergetics., Database: The data that support the findings of this study are available from the corresponding authors upon reasonable request., Animal Protocol: BK20150353 (Soochow University)., Research Governance: School of Nursing: Hua-Gang Hu: seuboyh@163.com; Soochow University: Chen Ge chge@suda.edu.cn., (© 2021 Federation of European Biochemical Societies.)

Mutations in the cardiac myosin regulatory light chain (RLC, MYL2 gene) are known to cause inherited cardiomyopathies with variable phenotypes. In this study, we investigated the impact of a mutation in the RLC (K104E) that is associated with hypertrophic cardiomyopathy (HCM). Previously in a mouse model of K104E, older animals were found to develop cardiac hypertrophy, fibrosis, and diastolic dysfunction, suggesting a slow development of HCM. However, variable penetrance of the mutation in human populations suggests that the impact of K104E may be subtle. Therefore, we generated human cardiac myosin subfragment-1 (M2β-S1) and exchanged on either the wild type (WT) or K104E human ventricular RLC in order to assess the impact of the mutation on the mechanochemical properties of cardiac myosin. The maximum actin-activated ATPase activity and actin sliding velocities in the in vitro motility assay were similar in M2β-S1 WT and K104E, as were the detachment kinetic parameters, including the rate of ATP-induced dissociation and the ADP release rate constant. We also examined the mechanical performance of α-cardiac myosin extracted from transgenic (Tg) mice expressing human wild type RLC (Tg WT) or mutant RLC (Tg K104E). We found that α-cardiac myosin from Tg K104E animals demonstrated enhanced actin sliding velocities in the motility assay compared with its Tg WT counterpart. Furthermore, the degree of incorporation of the mutant RLC into α-cardiac myosin in the transgenic animals was significantly reduced compared with wild type. Therefore, we conclude that the impact of the K104E mutation depends on either the length or the isoform of the myosin heavy chain backbone and that the mutation may disrupt RLC interactions with the myosin lever arm domain., (© 2021 Rasicci et al.)

In this study, we assessed the super relaxed (SRX) state of myosin and sarcomeric protein phosphorylation in two pathological models of cardiomyopathy and in a near-physiological model of cardiac hypertrophy. The cardiomyopathy models differ in disease progression and severity and express the hypertrophic (HCM-A57G) or restrictive (RCM-E143K) mutations in the human ventricular myosin essential light chain (ELC), which is encoded by the MYL3 gene. Their effects were compared with near-physiological heart remodeling, represented by the N-terminally truncated ELC (Δ43 ELC mice), and with nonmutated human ventricular WT-ELC mice. The HCM-A57G and RCM-E143K mutations had antagonistic effects on the ATP-dependent myosin energetic states, with HCM-A57G cross-bridges fostering the disordered relaxed (DRX) state and the RCM-E143K model favoring the energy-conserving SRX state. The HCM-A57G model promoted the switch from the SRX to DRX state and showed an ∼40% increase in myosin regulatory light chain (RLC) phosphorylation compared with the RLC of normal WT-ELC myocardium. On the contrary, the RCM-E143K-associated stabilization of the SRX state was accompanied by an approximately twofold lower level of myosin RLC phosphorylation compared with the RLC of WT-ELC. Upregulation of RLC phosphorylation was also observed in Δ43 versus WT-ELC hearts, and the Δ43 myosin favored the energy-saving SRX conformation. The two disease variants also differently affected the duration of force transients, with shorter (HCM-A57G) or longer (RCM-E143K) transients measured in electrically stimulated papillary muscles from these pathological models, while no changes were displayed by Δ43 fibers. We propose that the N terminus of ELC (N-ELC), which is missing in the hearts of Δ43 mice, works as an energetic switch promoting the SRX-to-DRX transition and contributing to the regulation of myosin RLC phosphorylation in full-length ELC mice by facilitating or sterically blocking RLC phosphorylation in HCM-A57G and RCM-E143K hearts, respectively., (© 2021 Sitbon et al.)

Cardiovascular diseases have attracted our full attention not only because they are the main cause of mortality and morbidity in many countries but also because the therapy for and cure of these maladies are among the major challenges of the medicine in the 21st century [...].

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great value for studies of human cardiac development, drug discovery, disease modeling, and cell therapy. However, the mixed cardiomyocyte subtypes (ventricular-, atrial-, and nodal-like myocytes) and the maturation heterogeneity of hPSC-CMs restrain their application in vitro and in vivo. Myosin light chain 2 (MYL2, encoding the ventricular/cardiac muscle isoform MLC2v protein) is regarded as a ventricular-specific marker of cardiac myocardium; however, its restricted localization to ventricles during human heart development has been questioned. Consequently, it is currently unclear whether MYL2 definitively marks ventricular hESC-CMs. Here, by using a MYL2-Venus hESC reporter line, we characterized a time-dependent increase of the MYL2-Venus positive (MLC2v-Venus + ) hESC-CMs during differentiation. We also compared the molecular, cellular, and functional properties between the MLC2v-Venus + and MYL2-Venus negative (MLC2v-Venus - ) hESC-CMs. At early differentiation stages of hESC-CMs, we reported that both MLC2v-Venus - and MLC2v-Venus + CMs displayed ventricular-like traits but the ventricular-like cells from MLC2v-Venus + hESC-CMs displayed more developed action potential (AP) properties than that from MLC2v-Venus - hESC-CMs. Meanwhile, about a half MLC2v-Venus - hESC-CM population displayed atrial-like AP properties, and a half showed ventricular-like AP properties, whereas only ~ 20% of the MLC2v-Venus - hESC-CMs expressed the atrial marker nuclear receptor subfamily 2 group F member 2 (NR2F2, also named as COUPTFII). At late time points, almost all MLC2v-Venus + hESC-CMs exhibited ventricular-like AP properties. Further analysis demonstrates that the MLC2v-Venus + hESC-CMs had enhanced Ca 2+ transients upon increase of the MLC2v level during cultivation. Concomitantly, the MLC2v-Venus + hESC-CMs showed more defined sarcomeric structures and better mitochondrial function than those in the MLC2v-Venus - hESC-CMs. Moreover, the MLC2v-Venus + hESC-CMs were more sensitive to hypoxic stimulus than the MLC2v-Venus - hESC-CMs. These results provide new insights into the development of human ventricular myocytes and reveal a direct correlation between the expression profile of MLC2v and ventricular hESC-CM development. Our findings that MLC2v is predominantly a ventricular marker in developmentally immature hESC-CMs have implications for human development, drug screening, and disease modeling, and this marker should prove useful in overcoming issues associated with hESC-CM heterogeneity.

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

Exposure to hydrochloric acid (HCl) leads acutely to asthma-like symptoms, acute respiratory distress syndrome (ARDS), including compromised alveolo-capillary barrier, and respiratory failure. To better understand the direct effects of HCl on pulmonary endothelial function, we studied the characteristics of HCl-induced endothelial barrier dysfunction in primary cultures of human lung microvascular endothelial cells (HLMVEC), defined the involved molecular pathways, and tested the potentially beneficial effects of Heat Shock Protein 90 (HSP90) inhibitors. HCl impaired barrier function in a time- and concentration-dependent manner and was associated with activation of Protein Kinase B (AKT), Ras homolog family member A (RhoA) and myosin light chain 2 (MLC2), as well as loss of plasmalemmal VE-cadherin, rearrangement of cortical actin, and appearance of inter-endothelial gaps. Pre-treatment or post-treatment of HLMVEC with AUY-922, a third-generation HSP90 inhibitor, prevented and restored HCl-induced endothelial barrier dysfunction. AUY-922 increased the expression of HSP70 and inhibited the activation (phosphorylation) of extracellular-signal regulated kinase (ERK) and AKT. AUY-922 also prevented the HCl-induced activation of RhoA and MLC2 and the internalization of plasmalemmal VE-cadherin. We conclude that, by increasing the expression of cytoprotective proteins, interfering with actomyosin contractility, and enhancing the expression of junction proteins, inhibition of HSP90 may represent a useful approach for the management of HCl-induced endothelial dysfunction and acute lung injury.

Coordinated gastric smooth muscle contraction is critical for proper digestion and is adversely affected by a number of gastric motility disorders. In this study we report that the secreted protein Mfge8 (milk fat globule-EGF factor 8) inhibits the contractile responses of human gastric antrum muscles to cholinergic stimuli by reducing the inhibitory phosphorylation of the MYPT1 (myosin phosphatase-targeting subunit (1) subunit of MLCP (myosin light chain phosphatase), resulting in reduced LC20 (smooth muscle myosin regulatory light chain (2) phosphorylation. Mfge8 reduced the agonist-induced increase in the F-actin/G-actin ratios of β-actin and γ-actin1. We show that endogenous Mfge8 is bound to its receptor, α8β1 integrin, in human gastric antrum muscles, suggesting that human gastric antrum muscle mechanical responses are regulated by Mfge8. The regulation of gastric antrum smooth muscles by Mfge8 and α8 integrin functions as a brake on gastric antrum mechanical activities. Further studies of the role of Mfge8 and α8 integrin in regulating gastric antrum function will likely reveal additional novel aspects of gastric smooth muscle motility mechanisms., (© 2021. The Author(s).)

Comprehensive proteomic analyses of human and murine platelets established an extraordinary intracellular repertoire of signaling components, which control crucial functions. The spectrum of platelet serine/threonine protein kinases (more than 100) includes the AGC family (protein kinase A, G, C [PKA, PKG, PKC]), the mitogen-activated protein kinases (MAPKs), and others. PKA and PKG have multiple significantly overlapping substrates in human platelets, which possibly affect functions with clear "signaling nodes" of regulation by multiple protein kinases/phosphatases. Signaling nodes are intracellular Ca 2+ stores, the contractile system (myosin light chains), and other signaling components such as G-proteins, protein kinases, and protein phosphatases. An example for this fine-tuning is the tyrosine kinase Syk, a crucial component of platelet activation, which is controlled by several serine/threonine and tyrosine protein kinases as well as phosphatases. Other protein kinases including PKA/PKG modulate protein phosphatase 2A, which may be a master regulator of MAPK signaling in human platelets. Protein kinases and in particular MAPKs are targeted by an increasing number of clinically used inhibitors. However, the precise regulation and fine-tuning of these protein kinases and their effects on other signaling components in platelets are only superficially understood-just the beginning. However, promising future approaches are in sight., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells., (Copyright © 2021. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear., Aim of the Study: This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease., Materials and Methods: The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D., Results: The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2)., Conclusions: AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Age-associated changes alter calcium-activated potassium channel (BK Ca ) expression of colon. Sphingolipids (SLs) are important cell membrane structural components; altered composition of SLs may affect BK Ca expression. This study investigated the mechanism by which sphingosine-1-phosphate (S1P) contributes to age-associated contractile dysfunction., Methods: Fifty male Sprague Dawley rats of different ages were randomly assigned to five age-groups, namely 3, 6, 12, 18, and 24 months. BK Ca expression, S1P levels, and phosphorylated myosin light chain (p-MLC) levels were tested in colonic tissues. In the absence and presence of S1P treatment, BK Ca expression, p-MLC levels, and intracellular calcium mobilization were tested in vitro. BK Ca small interfering RNA (siRNA) was used to investigate whether p-MLC expression and calcium mobilization were affected by BK Ca in colonic smooth muscle cells (SMCs). The expressions of phosphorylated protein kinase B, c-Jun N-terminal kinases (JNKs), extracellular-regulated protein kinases, nuclear factor kappa-B (NF-κB), and protein kinase C ζ (PKC ζ ) were examined to investigate the correlation between S1P and BK Ca ., Key Results: Sphingosine-1-phosphate levels and sphingosine-1-phosphate receptor 2 (S1PR2) and BK Ca expressions were upregulated and p-MLC expression was downregulated in the colonic tissues, age dependently. In the cultured SMCs, S1P treatment increased BK Ca expression and reduced calcium concentration and p-MLC was observed. BK Ca siRNA increased calcium concentration, and p-MLC levels significantly compared with control. We also showed that S1P upregulated BK Ca through PKC ζ , JNK, and NF-κB pathways., Conclusions and Inferences: In conclusion, S1P and S1PR2 participate in age-associated contractile dysfunction via BK Ca upregulation through PKC ζ , JNK, and NF-κB pathways., (© 2021 John Wiley & Sons Ltd.)

The role of regulatory light chains (RLCs) in cardiac muscle function has been elucidated progressively over the past decade. The RLCs are among the earliest expressed markers during cardiogenesis and persist through adulthood. Failing hearts have shown reduced RLC phosphorylation levels and that restoring baseline levels of RLC phosphorylation is necessary for generating optimal force of muscle contraction. The signalling mechanisms triggering changes in RLC phosphorylation levels during disease progression remain elusive. Uncovering this information may provide insights for better management of heart failure patients. Given the cardiac chamber-specific expression of RLC isoforms, ventricular RLCs have facilitated the identification of mature ventricular cardiomyocytes, opening up possibilities of regenerative medicine. This review consolidates the standing of RLCs in cardiac development and disease and highlights knowledge gaps and potential therapeutic advancements in targeting RLCs.

Myosin-based regulation in the heart muscle modulates the number of myosin motors available for interaction with calcium-regulated thin filaments, but the signaling pathways mediating the stronger contraction triggered by stretch between heartbeats or by phosphorylation of the myosin regulatory light chain (RLC) remain unclear. Here, we used RLC probes in demembranated cardiac trabeculae to investigate the molecular structural basis of these regulatory pathways. We show that in relaxed trabeculae at near-physiological temperature and filament lattice spacing, the RLC-lobe orientations are consistent with a subset of myosin motors being folded onto the filament surface in the interacting-heads motif seen in isolated filaments. The folded conformation of myosin is disrupted by cooling relaxed trabeculae, similar to the effect induced by maximal calcium activation. Stretch or increased RLC phosphorylation in the physiological range have almost no effect on RLC conformation at a calcium concentration corresponding to that between beats. These results indicate that in near-physiological conditions, the folded myosin motors are not directly switched on by RLC phosphorylation or by the titin-based passive tension at longer sarcomere lengths in the absence of thin filament activation. However, at the higher calcium concentrations that activate the thin filaments, stretch produces a delayed activation of folded myosin motors and force increase that is potentiated by RLC phosphorylation. We conclude that the increased contractility of the heart induced by RLC phosphorylation and stretch can be explained by a calcium-dependent interfilament signaling pathway involving both thin filament sensitization and thick filament mechanosensing., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism., Competing Interests: LJ, GN, GW, NR, GC, DS, SB, JM, LM, MM, AR, AD, MO No competing interests declared, (© 2021, Julian et al.)

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet-fed Ldlr-/- mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps - all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Purpose: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR., Methods and Materials: Lewis lung carcinoma (LLC) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days before 15 Gy CONV IR. Tumors were similarly harvested and analyzed., Results: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase, abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR, including reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors., Conclusions: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of the microenvironmental changes differentially regulated between CONV and FLASH IR., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Glucagon-like peptide-2 (GLP-2), an intestinotrophic hormone, has drawn considerable attention worldwide due to its potential to promote intestinal development. We investigated the effects and mechanisms of GLP-2 against lipopolysaccharide (LPS)-induced intestinal inflammation and injury both in vitro and in vivo. Forty healthy piglets weaned at the age of 28 days with similar body weight (BW) were assigned to four in vivo treatments with ten piglets each: (i) nonchallenged control; (ii) LPS-challenged control; (iii) LPS + low dose GLP-2; and (iv) LPS + high dose GLP-2. Piglets were subcutaneously injected with phosphate-buffered saline supplemented with GLP-2 at doses of 0, 0, 2, and 10 nmol/kg BW per day for seven consecutive days. The piglets were challenged with an intraperitoneal injection with 100 μg/kg LPS on day 14 to induce intestinal damage. After that, the gene and protein expression levels of representative tight junction proteins and myosin light-chain kinase (MLCK)/phosphorylated myosin light chain (pMLC), as well as proinflammatory cytokine levels were determined using quantitative reverse transcription polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay methods. A high dose of GLP-2 pretreatment increased intestinal permeability by downregulating and redistributing tight junction proteins (p < .05), for example, zona occluden-1 (ZO-1) and occludin. GLP-2 decreased the transcription of proinflammatory cytokines genes including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α in small intestines (p < .05). GLP-2 prevented the LPS-induced increase in the expression of MLCK dose-dependently and the increase in pMLC levels in the duodenum, jejunum, and ileum. To assess further the protective effect of GLP-2 on LPS-induced intestinal barrier injury after weaning and its possible mechanism, an in vitro intestinal epithelial barrier model was established with IPEC-J2 monolayers and treated with 100 μg/ml LPS with or without 1 × 10 -8  mol/L GLP-2 pretreatment. The in vitro analysis included control, LPS, and GLP-2 + LPS treatments. GLP-2 treatment alleviated the destructive effect of LPS on barrier permeability by restoring the expression and ultrastructure of ZO-1 and occludin (p < .05). In addition, GLP-2 reversed the LPS-induced MLCK hyperexpression and pMLC hyperphosphorylation (p < .05). Taken together, our findings revealed a mechanism by which GLP-2 alleviated LPS-challenged intestinal barrier injury and inflammation in weaned piglets and IPEC-J2 cells via the MLCK/pMLC signaling pathway., (© 2020 Wiley Periodicals LLC.)

Protein interacting with C-kinase 1 (PICK1) is a 415-aa multidomain scaffold protein encoded by the PICK1 gene. Accumulating evidence suggests that PICK1 is involved in the progression of cancer. However, the role of PICK1 in gastric cancer (GC) remains largely unknown. Using integrated analysis of publicly available GC transcriptome data from the Gene Expression Omnibus (GEO) database and immunohistochemistry analysis of samples obtained from clinical GC patients, we found that PICK1 expression was significantly down-regulated in gastric tumor tissues in comparison with adjacent normal tissues. Our analyses also revealed that decreased expression of PICK1 conferred a disadvantage on overall survival time in GC patients. Additionally, PICK1 expression showed a strong association with the epithelial-mesenchymal transition (EMT) pathway, and PICK1 might represent a functional bridge for EMT. Moreover, PICK1 expression was significantly decreased in the EMT subtype of GC and was negatively correlated with the expression of fibronectin 1 (FN1) and myosin light chain 9 (MYL9) mRNAs. Thus, our study provides evidence that PICK1 is a promising biomarker for the molecular etiology of GC.

Smooth muscle is an integral part of hollow organs. Many of them are constantly subjected to mechanical forces that alter organ shape and modify the properties of smooth muscle. To understand the molecular mechanisms underlying smooth muscle function in its dynamic mechanical environment, a new paradigm has emerged that depicts evanescence of myosin filaments as a key mechanism for the muscle's adaptation to external forces in order to maintain optimal contractility. Unlike the bipolar myosin filaments of striated muscle, the side-polar filaments of smooth muscle appear to be less stable, capable of changing their lengths through polymerization and depolymerization (i.e., evanescence). In this review, we summarize accumulated knowledge on the structure and mechanism of filament formation of myosin II and on the influence of ionic strength, pH, ATP, myosin regulatory light chain phosphorylation, and mechanical perturbation on myosin filament stability. We discuss the scenario of intracellular pools of monomeric and filamentous myosin, length distribution of myosin filaments, and the regulatory mechanisms of filament lability in contraction and relaxation of smooth muscle. Based on recent findings, we suggest that filament evanescence is one of the fundamental mechanisms underlying smooth muscle's ability to adapt to the external environment and maintain optimal function. Finally, we briefly discuss how increased ROCK protein expression in asthma may lead to altered myosin filament stability, which may explain the lack of deep-inspiration-induced bronchodilation and bronchoprotection in asthma., (© 2021 Wang et al.)

Background: Metastatic prostate cancer is a fatal disease despite multiple new approvals in recent years. Recent studies revealed that circular RNAs (circRNAs) can be involved in cancer metastasis. Defining the role of circRNAs in prostate cancer metastasis and discovering therapeutic targets that block cancer metastasis is of great significance for the treatment of prostate cancer., Methods: The circSOBP levels in prostate cancer (PCa) were determined by qRT-PCR. We evaluated the function of circSOBP using a transwell assay and nude mice lung metastasis models. Immunofluorescence assay and electron microscopic assay were applied to determine the phenotypes of prostate cancer cells' migration. We used fluorescence in situ hybridization assay to determine the localization of RNAs. Dual luciferase and rescue assays were applied to verify the interactions between circSOBP, miR-141-3p, MYPT1, and phosphomyosin light chain (p-MLC2)., Results: We observed that circSOBP level was significantly lower in PCa specimens compared with adjacent noncancerous prostate specimens, and was correlated with the grade group of PCa. Overexpression of circSOBP suppressed PCa migration and invasion in vitro and metastasis in vivo. CircSOBP depletion increased migration and invasion and induced amoeboid migration of PCa cells. Mechanistically, circSOBP bound miR-141-3p and regulated the MYPT1/p-MLC2 axis. Moreover, the depletion of MYPT1 reversed the inhibitory effect of circSOBP on the migration and invasion of PCa cells. Complementary intronic Alu elements induced but were not necessary for the formation of circSOBP. The nuclear export of circSOBP was mediated by URH49., Conclusion: Our results suggest that circSOBP suppresses amoeboid migration of PCa cells and inhibits migration and invasion through sponging miR-141-3p and regulating the MYPT1/p-MLC2 axis., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway., Methods: Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively., Results: We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α., Conclusions: The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane. Video abstract.

Perturbed Nicotinamide adenine dinucleotide (NAD + ) homeostasis is involved in cancer progression and metastasis. Quinolinate phosphoribosyltransferase (QPRT) is the rate-limiting enzyme in the kynurenine pathway participating in NAD + generation. In this study, we demonstrated that QPRT expression was upregulated in invasive breast cancer and spontaneous mammary tumors from MMTV-PyVT transgenic mice. Knockdown of QPRT expression inhibited breast cancer cell migration and invasion. Consistently, ectopic expression of QPRT promoted cell migration and invasion in breast cancer cells. Treatment with QPRT inhibitor (phthalic acid) or P2Y 11 antagonist (NF340) could reverse the QPRT-induced invasiveness and phosphorylation of myosin light chain. Similar reversibility could be observed following treatment with Rho inhibitor (Y16), ROCK inhibitor (Y27632), PLC inhibitor (U73122), or MLCK inhibitor (ML7). Altogether, these results indicate that QPRT enhanced breast cancer invasiveness probably through purinergic signaling and might be a potential prognostic indicator and therapeutic target in breast cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Cheng, Chen, Lin, Chen, Kuo and Chang.)

Morbidity and mortality associated with heart disease is a growing threat to the global population, and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca 2+ sensitivity of contraction for both genotypes, but this reduction in pCa 50 was nearly twice as large for WT-RLC versus N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed up myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM. NEW & NOTEWORTHY Mavacamten is a pharmaceutical that binds to myosin, and it is under investigation as a therapy for some forms of heart disease. We show that mavacamten reduces isometric tension and Ca 2+ sensitivity of contraction in skinned myocardial strips from a mouse model of hypertrophic cardiomyopathy that expresses the N47K mutation in cardiac myosin regulatory light chain. Mavacamten reduces contractility by decreasing strong cross-bridge binding, partially due to faster cross-bridge nucleotide handling rates that speed up myosin detachment.

The numbers of patients with inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD), have been increasing over time, worldwide; however, the pathogenesis of IBD is multifactorial and has not been fully understood. Myosin light chain 9 and 12a and 12b (Myl9/12) are known as ligands of the CD69 molecule. They create "Myl9 nets" that are often detected in inflamed site, which play a crucial role in regulating the recruitment and retention of CD69-expressing effector cells in inflamed tissues. We demonstrated the strong expression of Myl9/12 in the inflamed gut of IBD patients and mice with DSS-induced colitis. The administration of anti-Myl9/12 Ab to mice with DSS-induced colitis ameliorated the inflammation and prolonged their survival. The plasma Myl9 levels in the patients with active UC and CD were significantly higher than those in patients with disease remission, and may depict the disease severity of IBD patients, especially those with UC. Thus, our results indicate that Myl9/12 are involved in the pathogenesis of IBD, and are likely to be a new therapeutic target for patients suffering from IBD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yokoyama, Kimura, Ito, Hayashizaki, Endo, Wang, Yagi, Nakagawa, Kato, Matsubara and Nakayama.)

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-Cre ERT2 EpCAM fl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)