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The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was used to compare the expression levels of MYL9 in non-small-cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis was used to investigate the pathways associated with MYL9. The BioGRID database was used to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. The protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using Western blot (WB). Epithelial cell adhesion molecule (EpCAM) expression in different cell groups was profiled using flow cytometry analysis. Coimmunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. In addition, the direct protein interaction between MYL9 and MYO19 was explored using a glutathione-S-transferase (GST) pull-down assay. In NSCLC patients, MYL9 was significantly downregulated both in vivo and in cell cultures and had a high enrichment score in the EMT pathway. Scratch assays pointed to its inhibitory effect on cancer cell migration. WB showed that MYL9 could suppress EMT marker protein expression in NSCLC cells. Flow cytometry found that MYL9 greatly reduced the distribution of EpCAM on the cell surface. MYO19 was pinpointed as a potential target of MYL9, as confirmed by coimmunoprecipitation and GST pull-down assays. Rescue experiments confirmed that MYO19 could enhance cell migration, promote the expression of EMT markers, and increase EpCAM levels on the cell surface, but these effects were reserved by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19. NEW & NOTEWORTHY Myosin light chain 9 (MYL9) is downregulated in non-small-cell lung cancer (NSCLC). MYL9 suppresses epithelial-mesenchymal transition (EMT) in NSCLC cells. MYL9 binds to myosin 19 (MYO19). MYL9/MYO19 signaling inhibits EMT in NSCLC.

Desmosomes play a crucial role in maintaining tissue barrier integrity, particularly in mechanically stressed tissues. The assembly of desmosomes is regulated by the cytoskeleton and its regulators, and desmosomes also function as a central hub for regulating F-actin. However, the specific mechanisms underlying the crosstalk between desmosomes and F-actin remain unclear. Here, we identified that ARHGAP32, a Rho GTPase-activating protein, is located in desmosomes through its interaction with desmoplakin (DSP) via its GAB2-interacting domain (GAB2-ID). We confirmed that ARHGAP32 is required for desmosomal organization, maturation and length regulation. Notably, loss of ARHGAP32 increased formation of F-actin stress fibers and phosphorylation of the regulatory myosin light chain Myl9 at T18/S19. Inhibition of ROCK activity in ARHGAP32-knockout (KO) cells effectively restored desmosomal organization and the integrity of epithelial cell sheets. Moreover, loss of DSP impaired desmosomal ARHGAP32 location and led to decreased actomyosin contractility. ARHGAP32 with a deletion of the GAB2-ID domain showed enhanced association with RhoA in the cytosol and failed to rescue the desmosomal organization in ARHGAP32-KO cells. Collectively, our study unveils that ARHGAP32 associates with and regulates desmosomes by interacting with DSP. This interaction potentially facilitates the crosstalk between desmosomes and F-actin., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the β-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gα s ) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gα s gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1 + macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs., (© 2024. The Author(s).)

The physiological importance of cardiac myosin regulatory light chain (RLC) phosphorylation by its dedicated cardiac myosin light chain kinase has been established in both humans and mice. Constitutive RLC-phosphorylation, regulated by the balanced activities of cardiac myosin light chain kinase and myosin light chain phosphatase (MLCP), is fundamental to the biochemical and physiological properties of myofilaments. However, limited information is available on cardiac MLCP. In this study, we hypothesized that the striated muscle-specific MLCP regulatory subunit, MYPT2, targets the phosphatase catalytic subunit to cardiac myosin, contributing to the maintenance of cardiac function in vivo through the regulation of RLC-phosphorylation. To test this hypothesis, we generated a floxed-PPP1R12B mouse model crossed with a cardiac-specific Mer-Cre-Mer to conditionally ablate MYPT2 in adult cardiomyocytes. Immunofluorescence microscopy using the gene-ablated tissue as a control confirmed the localization of MYPT2 to regions where it overlaps with a subset of RLC. Biochemical analysis revealed an increase in RLC-phosphorylation in vivo. The loss of MYPT2 demonstrated significant protection against pressure overload-induced hypertrophy, as evidenced by heart weight, qPCR of hypertrophy-associated genes, measurements of myocyte diameters, and expression of β-MHC protein. Furthermore, mantATP chase assays revealed an increased ratio of myosin heads distributed to the interfilament space in MYPT2-ablated heart muscle fibers, confirming that RLC-phosphorylation regulated by MLCP, enhances cardiac performance in vivo. Our findings establish MYPT2 as the regulatory subunit of cardiac MLCP, distinct from the ubiquitously expressed canonical smooth muscle MLCP. Targeting MYPT2 to increase cardiac RLC-phosphorylation in vivo may improve baseline cardiac performance, thereby attenuating pathological hypertrophy., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Published by Elsevier Inc.)

Background: The tumor microenvironment (TME) is an important factor that regulates the progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) are the main mesenchymal cells in the TME and play a vital role in tumor progression; however, the specific underlying mechanisms require further study., Methods: Multiple single-cell and transcriptome data were analyzed and validated. Primary CAFs isolation, CCK8 assay, co-culture assay, western blotting, multiple immunofluorescence, qRT-PCR, ELISA, immunoprecipitation, ChIP, double luciferase, and animal experiments were used to explore the potential mechanism of MYL9 regulation in CRC., Results: Our findings revealed that MYL9 was predominantly localized and expressed in CAFs rather than in CRC cells, and bioinformatics analysis revealed that high MYL9 expression was strongly associated with poor overall and disease-free survival in various tumors. In addition, high MYL9 expression is closely associated with M2 macrophage infiltration, which can lead to an immunosuppressive microenvironment in CRC, making it insensitive to immunotherapy. Mechanically, MYL9 can regulate the secretion of CAFs on CCL2 and TGF-β1, thus affecting the immune microenvironment and progression of CRC. In addition, MYL9 bounded with IQGAP1 to regulate CCL2 and TGF-β1 secretion through the ERK 1/2 pathway, and CCL2 and TGF-β1 synergistically promoted CRC cells progression through the PI3K-AKT pathway. Furthermore, MYL9 promotes epithelial-mesenchymal transition (EMT) in CRC. During the upstream regulation of MYL9 in CAFs, we found that the EMT transcription factor ZEB1 could bind to the MYL9 promoter in CAFs, enhancing the activity and function of MYL9. Therefore, MYL9 is predominantly expressed in CAFs and can indirectly influence tumor biology and EMT by affecting CAFs protein expression in CRC., Conclusions: MYL9 regulates the secretion of cytokines and chemokines in CAFs, which can affect the immune microenvironment of CRC and promote CRC progression. The relationship between MYL9 expression and CRC clinical staging and immunotherapy is closer in CAFs than in tumor cells; therefore, studies using CAFs as a model deserve more attention when exploring tumor molecular targets in clinical research., (© 2023. The Author(s).)

Objective: This study explored the mechanism of squamous cervical cancer (SCC) progression., Methods: Reverse transcription-quantitative polymerase chain reaction and western blotting were used to evaluate the expression of myosin light chain 9 ( MYL9 ) in SCC tissues and cell lines. Furthermore, Transwell and Boyden assays were used to assess the function of MYL9 in SCC progression. In addition, the levels of lactate and aerobic glycolysis were used to explore the detailed mechanism of MYL9 in SCC., Results: The mRNA and protein levels of MYL9 were elevated in SCC tissues, and MYL9 knockdown inhibited the migration and invasion of SCC cell lines. A mechanistic study demonstrated that MYL9 promotes SCC migration and invasion by enhancing aerobic glycolysis and increasing the activity of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway., Conclusions: MYL9 was upregulated in SCC, and it enhanced JAK2/STAT3 pathway activity and promoted metastasis and glycolysis in SCC., Competing Interests: Declaration of conflicting interestThe authors declare no conflicts of interest.

Background: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3 , regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure., Methods: We generated the knock-in mice ( Mylk3 +/fs and Mylk 3 fs/fs ) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation ( MYLK3 +/fs ) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154)., Results: Both mice ( Mylk3 +/fs and Mylk 3 fs/fs ) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_ MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_ MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the V max for ventricular myosin regulatory light chain phosphorylation without affecting the K m . LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3 +/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3 / PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes., Conclusions: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure., Competing Interests: Disclosures None.

Distant metastasis is one of the leading causes of cancer-related mortality of colorectal cancer (CRC). Dysregulation of E3 ubiquitin ligases has been implicated in acting vital roles in multiple cancers. In this study, we found that the E3 ubiquitin ligase, PRPF19 was positively correlated with liver metastasis, and predicted a worse clinical outcome in CRC. However, the biological effects and the underlying molecular mechanisms of PRPF19 in CRC remain elusive thus far. We illustrated that PRPF19 promoted the migration and invasion capability of CRC cells in both gain- and loss- of function assays. Mechanistically, we uncovered that myosin light chain 9 (MYL9) was the downstream substrate of PRPF19. PRPF19 enhanced the stability of MYL9 via K63-linked ubiquitination, and promoted the migration and invasion capability of CRC cells in an MYL9-mediated manner. Furthermore, the Src-YAP1 cascade was identified as the downstream effector mechanism by which the PRPF19/MYL9 axis promoted metastasis in CRC. Taken together, our findings highlighted that the PRPF19/MYL9 axis served as a novel mechanism in CRC metastasis, which provided an attractive therapeutic strategy for CRC treatment., (© 2023. The Author(s).)

Cardiac contraction is modulated by the phosphorylation state of myosin regulatory light chain 2 (MLC-2v). The level of MLC-2v phosphorylation is dependent on the opposing activities of MLC kinases and phosphatases. The predominant MLC phosphatase found in cardiac myocytes contains Myosin Phosphatase Targeting Subunit 2 (MYPT2). Overexpression of MYPT2 in cardiac myocytes results in a decreased level of MLC phosphorylation, reduced left ventricular contraction, and induction of hypertrophy; however, the effect of knocking out MYPT2 on cardiac function is unknown. We obtained heterozygous mice containing a MYPT2 null allele from the Mutant Mouse Resource Center. These mice were produced in a C57BL/6N background which lack MLCK3, the main regulatory light chain kinase in cardiac myocytes. We found that mice null for MYPT2 were viable and had no obvious phenotypic abnormality when compared to WT mice. Additionally, we determined that WT C57BL/6N mice had a low basal level of MLC-2v phosphorylation, which was significantly increased when MYPT2 was absent. At 12-weeks, MYPT2 KO mice had smaller hearts and showed downregulation of genes involved in cardiac remodeling. Using cardiac echo, we found that 24-week-old male MYPT2 KO mice had decreased heart size with increased fractional shortening compared to their MYPT2 WT littermates. Collectively, these studies highlight the important role that MYPT2 plays in cardiac function in vivo and demonstrate that its deletion can partially compensate for the lack of MLCK3., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Myosin light chain plays a vital regulatory function in a large-scale cellular physiological procedure, however, the role of myosin light chain 5 (MYL5) in breast cancer has not been reported. In this study, we aimed to elucidate the effects of MYL5 on clinical prognosis and immune cell infiltration, and further explore the potential mechanism in breast cancer patients., Methods: In this study, we first explored the expression pattern and prognostic value of MYL5 in breast cancer across multiple databases, including Oncomine, TCGA, GTEx, GEPIA2, PrognoScan, and Kaplan-Meier Plotter. The correlations of MYL5 expression with immune cell infiltration and associational gene markers in breast cancer were analyzed by using the TIMER, TIMER2.0, and TISIDB databases. The enrichment and prognosis analysis of MYL5-related genes were implemented by using LinkOmics datasets., Results: We found that there was a low expression of MYL5 in breast cancer than in corresponding normal tissue by analyzing the data from Oncomine and TCGA datasets. Furthermore, research showed the prognosis of the MYL5 high-expression group was better than the low-expression group in breast cancer patients. Furthermore, MYL5 expression is markedly related to the tumor-infiltrating immune cells (TIICs), including cancer-associated fibroblast, B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, and dendritic cell, and related to immune molecules as well as the associated gene markers of TIICs., Conclusion: MYL5 can serve as a prognostic signature in breast cancer and is associated with immune infiltration. This study first offers a relatively comprehensive understanding of the oncogenic roles of MYL5 for breast cancer., Competing Interests: The author declares that there are no conflicts of interest., (Copyright © 2023 Minghe Lv.)

Our previous transcriptomic study identified MYL4 (myosin light chain 4) to be associated with muscle development and growth in pigs. In this study, we aimed to investigate the genetic variation of MYL4 and determine the effect of these variations in regulating MYL4 expression and muscle fiber size. After screening the regulatory region of MYL4 in Large White pigs, we identified nine completely linked single nucleotide polymorphisms within the MYL4 regulatory region, which showed two haplotypes (H1 and H2). And in MYL4 may affect the activity of the promoter region and regulate the traits of porcine muscle. The results of Western blotting and qRT-PCR showed that haplotype H2 significantly (p < 0.01) increased the relative mRNA and protein expression of MYL4 gene in pig LD tissues, and tissue sections also showed the number of genotype H2H2 Significantly higher than genotype H1H1, in conclusion, our results suggest that MYL4 may promote muscle growth and development and can affect the transcriptional activity of MYL4 through haplotype H1 and haplotype H2., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

In this study, we aimed to investigate whether short-term and low-dose treatment with hydroxychloroquine (HCQ), an antimalarial drug, can modulate heart function in a preclinical model of dilated cardiomyopathy (DCM) expressing the D94A mutation in cardiac myosin regulatory light chain (RLC) compared with healthy non-transgenic (NTg) littermates. Increased interest in HCQ came with the COVID-19 pandemic, but the risk of cardiotoxic side effects of HCQ raised concerns, especially in patients with an underlying heart condition, e.g., cardiomyopathy. Effects of HCQ treatment vs. placebo (H 2 O), administered in Tg-D94A vs. NTg mice over one month, were studied by echocardiography and muscle contractile mechanics. Global longitudinal strain analysis showed the HCQ-mediated improvement in heart performance in DCM mice. At the molecular level, HCQ promoted the switch from myosin's super-relaxed (SRX) to disordered relaxed (DRX) state in DCM-D94A hearts. This result indicated more myosin cross-bridges exiting a hypocontractile SRX-OFF state and assuming the DRX-ON state, thus potentially enhancing myosin motor function in DCM mice. This bottom-up investigation of the pharmacological use of HCQ at the level of myosin molecules, muscle fibers, and whole hearts provides novel insights into mechanisms by which HCQ therapy mitigates some abnormal phenotypes in DCM-D94A mice and causes no harm in healthy NTg hearts.

The effects of cardiomyopathic mutations E56G, M149V, and E177G in the MYL3 gene encoding essential light chain of human ventricular myosin (ELCv), on the functional properties of cardiac myosin and its isolated head (myosin subfragment 1, S1) were investigated. Only the M149V mutation upregulated the actin-activated ATPase activity of S1. All mutations significantly increased the Ca2+-sensitivity of the sliding velocity of thin filaments on the surface with immobilized myosin in the in vitro motility assay, while mutations E56G and M149V (but not E177G) reduced the sliding velocity of regulated thin filaments and F-actin filaments almost twice. Therefore, despite the fact that all studied mutations in ELCv are involved in the development of hypertrophic cardiomyopathy, the mechanisms of their influence on the actin-myosin interaction are different.

L. plantarum 1.0386 repairs intestinal epithelial tight junction injury, and the present study was designed to further explore the role of its postbiotics, including the surface protein (1.0386-Slp), peptidoglycan (1.0386-PG) and exopolysaccharide (1.0386-EPS). The results showed that they all could improve the lipopolysaccharide (LPS)-induced decrease of transepithelial electrical resistance, increase of paracellular permeability, release of inflammatory factors, and disruption of tight junctions in Caco-2 cells, and the repairing effect of 1.0386-Slp was better than those of 1.0386-PG and 1.0386-EPS, and was similar to that of L. plantarum 1.0386. Moreover, either L. plantarum 1.0386 or 1.0386-Slp intervention significantly increased the expression of miR-200c inhibited by LPS, while the miR-200c inhibitor weakened the ability of 1.0386-Slp to promote the expression of tight junction proteins (ZO-1, occludin and claudin-1). Meanwhile, 1.0386-Slp restored the distribution of tight junction proteins and inhibited the increase of NF-κB p65, MLC and pMLC protein expression evoked by LPS. However, the addition of miR-200c inhibitors or mimics weakened or strengthened the down-regulation of MLCK-MLC pathway protein expression by 1.0386-Slp, respectively. In summary, 1.0386-Slp may be the main efficacy component of L. plantarum 1.0386, and miR-200c may be involved in the process of 1.0386-Slp inhibiting the MLCK pathway to repair intestinal epithelial tight junction injury.

Rho-associated coiled-coil containing protein kinase 1 (ROCK1) intracellular cell signaling pathway regulates cell morphology, polarity, and cytoskeletal remodeling. We observed the activation of ROCK1/myosin light chain (MLC2) signaling pathway in buffalopox virus (BPXV) infected Vero cells. ROCK1 depletion by siRNA and specific small molecule chemical inhibitors (Thiazovivin and Y27632) resulted in a reduced BPXV replication, as evidenced by reductions in viral mRNA/protein synthesis, genome copy numbers and progeny virus particles. Further, we demonstrated that ROCK1 inhibition promotes deadenylation of viral mRNA (mRNA decay), mediated via inhibiting interaction with PABP [(poly(A)-binding protein] and enhancing the expression of CCR4-NOT (a multi-protein complex that plays an important role in deadenylation of mRNA). In addition, ROCK1/MLC2 mediated cell contraction, and perinuclear accumulation of p-MLC2 was shown to positively correlate with viral mRNA/protein synthesis. Finally, it was demonstrated that the long-term sequential passage (P = 50) of BPXV in the presence of Thiazovivin does not select for any drug-resistant virus variants. In conclusion, ROCK1/MLC2 cell signaling pathway facilitates BPXV replication by preventing viral mRNA decay and that the inhibitors targeting this pathway may have novel therapeutic effects against buffalopox., (© 2022. The Author(s).)

Background: Myosin family genes, including those encoding myosin heavy chain 6, myosin heavy chain 7, myosin light chain 3, and myosin light chain 2 (MYL2), are important genetic factors in congenital heart disease (CHD). However, how these genes contribute to CHD in the Han Chinese population remains unclear., Methods: We sequenced myosin family genes in a Han Chinese cohort comprising 412 CHD patients and 213 matched controls in the present study. A zebrafish model was used to evaluate the pathogenicity of rare mutations in MYL2., Results: We identified 30 known mutations and 12 novel mutations. Furthermore, the contributions of two novel mutations, MYL2 p.Ile158Thr and p.Val146Met, to CHD were analyzed. The p.Ile158Thr mutation increased MYL2 expression. In zebrafish embryos, injection of myl2b-targeting morpholinos led to aberrant cardiac structures, an effect that was reversed by expression of wild-type MYL2 but not MYL2 p.Ile158Thr and pVal146Met., Conclusions: Overall, our findings suggest that MYL2 p.Ile158Thr and p.Val146Met contribute to the etiology of CHD. The results also indicate the importance of MYL2 in heart formation., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

In this study, we discuss a female patient referred to cardiology with left ventricular hypertrophy at mid-ventricular segments resulting in a mid-cavitary obstruction and a left ventricular apical aneurysm. The patient had normal epicardial coronary arteries, but presented with recurrent cerebrovascular events. The patient had a positive family history for sudden cardiac death. Cardiac MRI detected positive features of left ventricular mid-cavity obstruction, left ventricular apical aneurysm and delayed gadolinium enhancement, with Holter monitoring assessment displaying segments of non-sustained ventricular tachycardia. Genetic analysis detected an myosin light chain 3 ( MYL3 ) gene mutation. The patient will be referred to receive an implantable cardioverter defibrillator.The MYL3 gene mutation is a rare variant in patients with familial hypertrophic cardiomyopathy. To our knowledge, the presence of a left ventricular apical aneurysm has not been previously reported in literature concerning the MYL3 gene mutation. The presence of this abnormality further increases the risk of sudden cardiac death., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I 11 /I 10 ) together with the force-pCa relationship over a full range of [Ca 2+ ] and at a sarcomere length of 2.1 μm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca 2+ sensitivity of force and left shifted the I 11 /I 10 -pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca 2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations., Competing Interests: The authors declare no competing interest.

Introduction: Recent studies have shown that myosin light chain 9 (MYL9) plays a vital role in immune infiltration, tumor invasion, and metastasis; however, the prognostic and immunological role of MYL9 has not been reported. The purpose of this study was to explore the potential prognostic and immunological roles of MYL9 in human cancers by public datasets mainly including the cancer genome atlas (TCGA) and Gene expression omnibus., Methods: The expression pattern and prognostic value of MYL9 were analyzed across multiple public datasets in different cancer. The correlations between MYL9 expression and immune infiltration among multiple cancers were analyzed by using the TIMER2.0. The MYL9-related gene enrichment analysis was implemented by mainly using KEGG and GO datasets., Results: MYL9 was lowly expressed in most cancers, such as breast cancer, lung adenocarcinoma and squamous cell carcinoma, and stomach adenocarcinoma; but it was highly expressed in several cancers, such as cholangiocarcinoma, head and neck squamous cell carcinoma, and liver hepatocellular carcinoma. Furthermore, MYL9 expression was distinctively associated with prognosis in adrenocortical carcinoma, colon adenocarcinoma, brain glioma, lung cancer, ovarian cancer, gastric cancer, breast cancer, blood cancer, and prostate cancer patients. The expressions of MYL9 were significantly associated with the infiltration of cancer-associated fibroblasts, B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, dendritic cell in different tumors as well as immune markers. In addition, we found that the functional mechanisms of MYL9 involved muscle contraction and focal adhesion., Conclusion: MYL9 can serve as a prognostic signature in pan-cancer and is associated with immune infiltration. This pan-cancer study is the first to show a relatively comprehensive understanding of the prognostic and immunological roles of MYL9 across different cancers., (© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Vasorin (VASN) is an important transmembrane protein associated with development and disease. However, it is not clear whether the death of mice with VASN deficiency (VASN -/- ) is related to cardiac dysfunction. The aim of this research was to ascertain whether VASN induces pathological cardiac hypertrophy by targeting myosin light chain 7 (MYL7). VASN -/- mice were produced by CRISPR/Cas9 technology and inbreeding. PCR amplification, electrophoresis, real-time PCR and Western blotting were used to confirm VASN deficiency. Cardiac hypertrophy was examined by blood tests, histological analysis and real-time PCR, and key downstream factors were identified by RNA sequencing and real-time PCR. Western blotting, immunohistochemistry and electron microscopy analysis were used to confirm the downregulation of MYL7 production and cardiac structural changes. Our results showed that sudden death of VASN -/- mice occurred 21-28 days after birth. The obvious increases in cardiovascular risk, heart weight and myocardial volume and the upregulation of hypertrophy marker gene expression indicated that cardiac hypertrophy may be the cause of death in young VASN -/- mice. Transcriptome analysis revealed that VASN deficiency led to MYL7 downregulation, which induced myocardial structure abnormalities and disorders. Our results revealed a pathological phenomenon in which VASN deficiency may lead to cardiac hypertrophy by downregulating MYL7 production. However, more research is necessary to elucidate the underlying mechanism., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Genetics crucially contributes to cardiovascular diseases (CVDs), the global leading cause of death. Since the majority of CVDs can be prevented by early intervention there is a high demand for the identification of predictive causative genes. While genome wide association studies (GWAS) correlate genes and CVDs after diagnosis and provide a valuable resource for such causative candidate genes, often preferentially those with previously known or suspected function are addressed further. To tackle the unaddressed blind spot of understudied genes, we particularly focused on the validation of human heart phenotype-associated GWAS candidates with little or no apparent connection to cardiac function. Building on the conservation of basic heart function and underlying genetics from fish to human we combined CRISPR/Cas9 genome editing of the orthologs of human GWAS candidates in isogenic medaka with automated high-throughput heart rate analysis. Our functional analyses of understudied human candidates uncovered a prominent fraction of heart rate associated genes from adult human patients impacting on the heart rate in embryonic medaka already in the injected generation. Following this pipeline, we identified 16 GWAS candidates with potential diagnostic and predictive power for human CVDs., Competing Interests: The authors have declared that no competing interests exist.

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.)

Background: Idiopathic pulmonary fibrosis (IPF) has high mortality worldwide. The CD247 molecule (CD247, as known as T-cell surface glycoprotein CD3 zeta chain) has been reported as a susceptibility locus in systemic sclerosis, but its correlation with IPF remains unclear., Methods: Datasets were acquired by researching the Gene Expression Omnibus (GEO). CD247 was identified as the hub gene associated with percent predicted diffusion capacity of the lung for carbon monoxide (Dlco% predicted) and prognosis according to Pearson correlation, logistic regression, and survival analysis., Results: CD247 is significantly downregulated in patients with IPF compared with controls in both blood and lung tissue samples. Moreover, CD247 is significantly positively associated with Dlco% predicted in blood and lung tissue samples. Patients with low-expression CD247 had shorter transplant-free survival (TFS) time and more composite end-point events (CEP, death, or decline in FVC >10% over a 6-month period) compared with patients with high-expression CD247 (blood). Moreover, in the follow-up 1st, 3rd, 6th, and 12th months, low expression of CD247 was still the risk factor of CEP in the GSE93606 dataset (blood). Thirteen genes were found to interact with CD247 according to the protein-protein interaction network, and the 14 genes including CD247 were associated with the functions of T cells and natural killer (NK) cells such as PD-L1 expression and PD-1 checkpoint pathway and NK cell-mediated cytotoxicity. Furthermore, we also found that a low expression of CD247 might be associated with a lower activity of TIL (tumor-infiltrating lymphocytes), checkpoint, and cytolytic activity and a higher activity of macrophages and neutrophils., Conclusion: These results imply that CD247 may be a potential T cell-derived disease severity and prognostic biomarker for IPF., 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 Li, Chen, Li, Wang, Li, Ning and Chen.)

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).)

Abstract: Rhabdomyosarcoma (RMS) is a common malignant soft tissue sarcoma, which is the third most common soft tissue sarcoma after malignant fibrohistoma and liposarcoma. The discovery of potential postbiomarkers could lead to early and more effective treatment measures to reduce the mortality of RMS. The discovery of biomarker is expected to be the direction of targeted therapy, providing a new direction for the precise treatment of RMS.Gene Expression Omnibus database was used to download the tow gene profiles, GSE28511 and GSE135517. GEO2R was applied to identify differently expressed genes (DEGs) between RMS and normal group. Database for Annotation, Visualization and Integrated Discovery and Metascape can perform the enrichment analysis for the DEGs. Protein-protein interaction network was constructed, and the hub genes was identified by the Cytoscape. Expression and overall survival analysis of hub genes were performed.A total of 15 common DEGs were screened between RMS and normal tissues. The enrichment analysis here showed that the DEGs mainly enriched in the muscle filament sliding, myofibril, protein complex, sarcomere, myosin complex, nuclear chromosome, and tight junction. The 6 hub genes (DNA Topoisomerase II Alpha, Insulin Like Growth Factor 2, HIST1H4C, Cardiomyopathy Associated 5, Myosin Light Chain 2 [MYL2], Myosin Heavy Chain 2) were identified. Compared with the normal tissues, MYL2 were down-regulated in the RMS tissues. RMS patients with low expression level of MYL2 had poorer overall survival times than those with high expression levels (P < .05).In summary, lower expression of MYL2 was 1 prediction for poor prognosis of RMS. MYL2 is hope to be the target of therapy, which leads to more effective treatment and reduces the mortality rate of RMS., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

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).)

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.

Proper regulation of genome architecture and activity is essential for the development and function of multicellular organisms. Histone modifications, acting in combination, specify these activity states at individual genomic loci. However, the methods used to study these modifications often require either a large number of cells or are limited to targeting one histone mark at a time. Here, we developed a new method called Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE) that uses Expansion Microscopy (ExM) to visualize and quantify multiple histone modifications at non-repetitive genomic regions in single cells at a spatial resolution of ∼75 nm. Using SCEPTRE, we distinguished multiple histone modifications at a single housekeeping gene, quantified histone modification levels at multiple developmentally-regulated genes in individual cells, and evaluated the relationship between histone modifications and RNA polymerase II loading at individual loci. We find extensive variability in epigenetic states between individual gene loci hidden from current population-averaged measurements. These findings establish SCEPTRE as a new technique for multiplexed detection of combinatorial chromatin states at single genomic loci in single cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 [...].

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.)

Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of substrate-dependent gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin motor consisting of a class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. Therefore, the binding of TgMLC1 to TgGAP45 is considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation completely abrogate the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete. IMPORTANCE Gliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention or treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely held "linear motor" model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of, and between cells of the hosts they infect., (Copyright © 2021 Rompikuntal et al.)

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the myocardium characterized by a hypertrophic left ventricle with a preserved or increased ejection fraction. Cardiac hypertrophy is often asymmetrical, which is associated with left ventricular outflow tract obstruction. Myocyte hypertrophy, disarray, and myocardial fibrosis constitute the histological features of HCM. HCM is a relatively benign disease but an important cause of sudden cardiac death in the young and heart failure in the elderly. Pathogenic variants (PVs) in genes encoding protein constituents of the sarcomeres are the main causes of HCM. PVs exhibit a gradient of effect sizes, as reflected in their penetrance and variable phenotypic expression of HCM. MYH7 and MYBPC3 , encoding β-myosin heavy chain and myosin binding protein C, respectively, are the two most common causal genes and responsible for ≈40% of all HCM cases but a higher percentage of HCM in large families. PVs in genes encoding protein components of the thin filaments are responsible for ≈5% of the HCM cases. Whereas pathogenicity of the genetic variants in large families has been firmly established, ascertainment causality of the PVs in small families and sporadic cases is challenging. In the latter category, PVs are best considered as probabilistic determinants of HCM. Deciphering the genetic basis of HCM has enabled routine genetic testing and has partially elucidated the underpinning mechanism of HCM as increased number of the myosin molecules that are strongly bound to actin. The discoveries have led to the development of mavacamten that targets binding of the myosin molecule to actin filaments and imparts beneficial clinical effects. In the coming years, the yield of the genetic testing is expected to be improved and the so-called missing causal gene be identified. The advances are also expected to enable development of additional specific therapies and editing of the mutations in HCM.

Tight junction dysregulation and epithelial damage contribute to intestinal barrier loss in patients with acute liver failure (ALF); however, the regulatory mechanisms of these processes remain poorly understood. The aim of the present study was to investigate the changes of intestinal tight junction and intestinal mucosa in mice with ALF and their mechanisms. In the present study, ALF was induced in mice through an intraperitoneal injection of D‑galactosamine and lipopolysaccharide (D‑GalN/LPS), and the morphological changes of the liver or small intestine were analyzed using hematoxylin and eosin staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The intestinal tissues and isolated serum were analyzed using western blotting, immunofluorescence staining and ELISA. D‑GalN/LPS‑induced mice exhibited signs of hepatocyte necrosis, alongside inflammatory cell infiltration into the liver tissue and partial microvilli detachment in the small intestinal mucosa. TEM demonstrated that the intestinal epithelial tight junctions were impaired, whereas SEM micrographs revealed the presence of abnormal microvilli in D‑GalN/LPS‑induced mice. In addition, the expression levels of phosphorylated (p)‑myosin light chain (MLC), MLC kinase (MLCK) and Rho‑associated kinase (ROCK) were significantly increased in the D‑GalN/LPS‑induced mice compared with those in the control mice, whereas the subsequent inhibition of MLCK or ROCK significantly reduced p‑MLC expression levels. Conversely, the expression levels of occludin and zonula occludens‑1 (ZO‑1) were significantly decreased in the D‑GalN/LPS‑induced mice, and the inhibition of MLCK or ROCK significantly increased occludin and ZO‑1 protein expression levels compared with those in the control group. Changes in the serum levels of tumor necrosis factor‑α (TNF‑α) and interleukin (IL)‑6 were similar to the trend observed in p‑MLC expression levels. In conclusion, the findings of the present study suggested that in a D‑GalN/LPS‑induced ALF model, TNF‑α and IL‑6 signaling may increase MLCK and ROCK expression levels, further mediate phosphorylation of MLC, which may result in tight junction dysregulation and intestinal barrier dysfunction.

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.

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.

The integrity of the intestinal barrier is critical for homeostasis. In this study, we investigated the protective effect of pterostilbene (PTE) on the intestinal epithelium barrier. In vitro results of transepithelial electrical resistance (TEER) in Caco-2 cells indicated that PTE counteracted tumor necrosis factor α (TNFα)-induced barrier damage. In vivo PTE pretreatment markedly ameliorated intestinal barrier dysfunction induced by dextran sulfate sodium (DSS). Notably, intestinal epithelial tight junction (TJ) molecules were restored by PTE in mice exposed to DSS. The mechanism study revealed that PTE prevented myosin light-chain kinase (MLCK) from driving phosphorylation of MLC (p-MLC), which is crucial for maintaining intestinal TJ stability. Furthermore, PTE blunted translocation of NF-κB subunit p65 into the nucleus to downregulate MLCK expression and then to safeguard TJs and barrier integrity. These findings suggest that PTE protected the intestinal epithelial barrier through the NF-κB- MLCK/p-MLC signal pathway.

Purpose: Variants in genes encoding sarcomeric proteins are the most common cause of inherited cardiomyopathies. However, the underlying genetic cause remains unknown in many cases. We used exome sequencing to reveal the genetic etiology in patients with recessive familial cardiomyopathy., Methods: Exome sequencing was carried out in three consanguineous families. Functional assessment of the variants was performed., Results: Affected individuals presented with hypertrophic or dilated cardiomyopathy of variable severity from infantile- to early adulthood-onset and sudden cardiac death. We identified a homozygous missense substitution (c.170C>A, p.[Ala57Asp]), a homozygous translation stop codon variant (c.106G>T, p.[Glu36Ter]), and a presumable homozygous essential splice acceptor variant (c.482-1G>A, predicted to result in skipping of exon 5). Morpholino knockdown of the MYL3 orthologue in zebrafish, cmlc1, resulted in compromised cardiac function, which could not be rescued by reintroduction of MYL3 carrying either the nonsense c.106G>T or the missense c.170C>A variants. Minigene assay of the c.482-1G>A variant indicated a splicing defect likely resulting in disruption of the EF-hand Ca 2+ binding domains., Conclusions: Our data demonstrate that homozygous MYL3 loss-of-function variants can cause of recessive cardiomyopathy and occurrence of sudden cardiac death, most likely due to impaired or loss of myosin essential light chain function.

Pacific oyster (Crassostrea gigas) is one of the most widely cultivated shellfish species in the world. Because of its economic value and complex life cycle involving drastic changes from a free-swimming larva to a sessile juvenile, C. gigas has been used as a model for developmental, environmental, and aquaculture research. However, due to the lack of genetic tools for functional analysis, gene functions associated with biological or economic traits cannot be easily determined. Here, we reported a successful application of CRISPR/Cas9 technology for knockout of myosin essential light chain gene (CgMELC) in C. gigas. C. gigas embryos injected with sgRNAs/Cas9 contained extensive indel mutations at the target sites. The mutant larvae showed defective musculature and reduced motility. In addition, knockout of CgMELC disrupted the expression and patterning of myosin heavy chain positive myofibers in larvae. Together, these data indicate that CgMELC is involved in larval muscle contraction and myogenesis in oyster larvae.

Head and neck squamous cell carcinoma (HNSCC) is one of the most malignant cancers with poor prognosis worldwide. Emerging evidence indicates that competing endogenous RNAs (ceRNAs) are involved in various diseases, however, the regulatory mechanisms of ceRNAs underlying HNSCC remain unclear. In this study, we retrieved differentially expressed long non-coding RNAs (DElncRNAs), messenger RNAs (DEmRNAs) and microRANs (DEmiRNAs) from The Cancer Genome Atlas database and constructed a ceRNA-based risk model in HNSCC by integrated bioinformatics approaches. Functional enrichment analyses showed that DEmRNAs might be involved in extracellular matrix related biological processes, and protein-protein interaction network further selected out prognostic genes, including MYL1 and ACTN2. Importantly, co-expressed RNAs identified by weighted co-expression gene network analysis constructed the ceRNA networks. Moreover, AC114730.3, AC136375.3, LAT and RYR3 were highly correlated to overall survival of HNSCC by Kaplan-Meier method and univariate Cox regression analysis, which were subsequently implemented multivariate Cox regression analysis to build the risk model. Our study provides a deeper understanding of ceRNAs on the regulatory mechanisms, which will facilitate the expansion of the roles on the ceRNAs in the tumorigenesis, development and treatment of HNSCC.

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.

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.)