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Background: Blocking the RhoA/ROCK II/MLC 2 (Ras homolog gene family member A/Rho kinase II/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the RhoA/ROCK II/MLC 2 signaling pathway changes the pathogenic processes of the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH., Methods: Scalp acupuncture (SA) therapy was performed on rats with ICH at the acupuncture point "Baihui"-penetrating "Qubin," and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the RhoA/ROCK II/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging., Results: We found that ROCK II acts as a promoter of the RhoA/ROCK II/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the pre-intervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK II, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at "Baihui"-penetrating "Qubin" and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the RhoA/ROCK II/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats., Conclusion: This study found that these experimental data indicated that SA at "Baihui"-penetrating "Qubin" could preserve BBB integrity and neurological function recovery after ICH by inhibiting RhoA/ROCK II/MLC 2 signaling pathway activation and by regulating endothelial cell-related proteins., (© 2024 The Authors. Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.)

Background: Control of angiogenesis is widely considered a therapeutic strategy, but reliable control methods are still under development. Phosphorylation of myosin light chain 2 (MLC2), which regulates actin-myosin interaction, is critical to the behavior of vascular endothelial cells (ECs) during angiogenesis. MLC2 is phosphorylated by MLC kinase (MLCK) and dephosphorylated by MLC phosphatase (MLCP) containing a catalytic subunit PP1. We investigated the potential role of MLC2 in the pharmacological control of angiogenesis., Methods and Results: We exposed transgenic zebrafish Tg(fli1a:Myr-mCherry) ncv1 embryos to chemical inhibitors and observed vascular development. PP1 inhibition by tautomycetin increased length of intersegmental vessels (ISVs), whereas MLCK inhibition by ML7 decreased it; these effects were not accompanied by structural dysplasia. ROCK inhibition by Y-27632 also decreased vessel length. An in vitro angiogenesis model of human umbilical vein endothelial cells (HUVECs) showed that tautomycetin increased vascular cord formation, whereas ML7 and Y-27632 decreased it. These effects appear to be influenced by regulation of cell morphology rather than cell viability or motility. Actin co-localized with phosphorylated MLC2 (pMLC2) was abundant in vascular-like elongated-shaped ECs, but poor in non-elongated ECs. pMLC2 was associated with tightly arranged actin, but not with loosely arranged actin. Moreover, knockdown of MYL9 gene encoding MLC2 reduced total MLC2 and pMLC2 protein and inhibited angiogenesis in HUVECs., Conclusion: The present study found that MLC2 is a pivotal regulator of angiogenesis. MLC2 phosphorylation may be involved in the regulation of of cell morphogenesis and cell elongation. The functionally opposite inhibitors positively or negatively control angiogenesis, probably through the regulating EC morphology. These findings may provide a unique therapeutic target for angiogenesis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Kiyomi Tsuji-Tamura reports financial support was provided by Japan Society for the Promotion of Science (JSPS). If there are other authors, they 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 © 2023. Published by Elsevier Inc.)

Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact., 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 © 2024 Elsevier B.V. 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.

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.

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.)

Coronary artery spasm (CAS) is an intense vasoconstriction of coronary arteries that causes total or subtotal vessel occlusion. The cardioprotective effect of sirtuin-1 (SIRT1) has been extensively highlighted in coronary artery diseases. The aims within this study include the investigation of the molecular mechanism by which SIRT1 alleviates CAS. SIRT1 expression was first determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis in an endothelin-1 (ET-1)-induced rat CAS model. Interaction among SIRT1, nuclear factor-kappaB (NF-κB), myosin light chain kinase/myosin light chain-2 (MLCK/MLC2), and ET-1 was analyzed using luciferase reporter assay, RT-qPCR, and Western blot analysis. After ectopic expression and depletion experiments in vascular smooth muscle cells (VSMCs), contraction and proliferation of VSMCs and expression of contraction-related proteins (α-SMA, calponin, and SM22α) were measured by collagen gel contraction, 5-ethynyl-2'-deoxyuridine (EdU) assay, RT-qPCR, and Western blot analysis. The obtained results showed that SIRT1 expression was reduced in rat CAS models. However, overexpression of SIRT1 inhibited the contraction and proliferation of VSMCs in vitro. Mechanistic investigation indicated that SIRT1 inhibited NF-κB expression through deacetylation. Moreover, NF-κB could activate the MLCK/MLC2 pathway and upregulate ET-1 expression by binding to their promoter regions, thus inducing VSMC contraction and proliferation in vitro. In vivo experimental results also revealed that SIRT1 alleviated CAS through regulation of the NF-κB/MLCK/MLC2/ET-1 signaling axis. Collectively, our data suggested that SIRT1 could mediate the deacetylation of NF-κB, disrupt the MLCK/MLC2 pathway, and inhibit the expression of ET-1 to relieve CAS, providing a theoretical basis for the prospect of CAS treatment and prevention. NEW & NOTEWORTHY Rat coronary artery spasm models exhibit reduced expression of SIRT1. Overexpression of SIRT1 inhibits contraction and proliferation of VSMCs. SIRT1 inhibits NF-κB through deacetylation to modulate VSMC contraction and proliferation. NF-κB activates the MLCK/MLC2 pathway. NF-κB upregulates ET-1 to modulate VSMC contraction and proliferation.

Mutations in CAPN3 cause limb girdle muscular dystrophy R1 (LGMDR1, formerly LGMD2A) and lead to progressive and debilitating muscle wasting. Calpain 3 deficiency is associated with impaired CaMKIIβ signaling and blunted transcriptional programs that encode the slow-oxidative muscle phenotype. We conducted a high-throughput screen on a target of CaMKII ( Myl2 ) to identify compounds to override this signaling defect; 4 were tested in vivo in the Capn3 knockout (C3KO) model of LGMDR1. The leading compound, AMBMP, showed good exposure and was able to reverse the LGMDR1 phenotype in vivo , including improved oxidative properties, increased slow fiber size, and enhanced exercise performance. AMBMP also activated CaMKIIβ signaling, but it did not alter other pathways known to be associated with muscle growth. Thus, AMBMP treatment activates CaMKII and metabolically reprograms skeletal muscle toward a slow muscle phenotype. These proof-of-concept studies lend support for an approach to the development of therapeutics for LGMDR1., Competing Interests: M.J.S. and A.D.P. are co-founders of MyoGene Bio and are members of its scientific advisory board. M.J.S., I.K., J.C., and V.J. are inventors on a patent pending pertaining to new chemical entities of AMBMP., (© 2020 The Authors.)

Myosin is an essential motor protein, which in muscle is comprised of two molecules each of myosin heavy-chain (MHC), the essential or alkali myosin light-chain 1 (MLC1), and the regulatory myosin light-chain 2 (MLC2). It has been shown previously that MLC2 phosphorylation at two canonical serine residues is essential for proper flight muscle function in Drosophila ; however, MLC2 is also phosphorylated at additional residues for which the mechanism and functional significance is not known. We found that a hypomorphic allele of Pkcδ causes a flightless phenotype; therefore, we hypothesized that PKCδ phosphorylates MLC2. We rescued flight disability by duplication of the wild-type Pkcδ gene. Moreover, MLC2 is hypophosphorylated in Pkcδ mutant flies, but it is phosphorylated in rescued animals. Myosin isolated from Pkcδ mutant flies shows a reduced actin-activated ATPase activity, and MLC2 in these myosin preparations can be phosphorylated directly by recombinant human PKCδ. The flightless phenotype is characterized by a shortened and disorganized sarcomere phenotype that becomes apparent following eclosion. We conclude that MLC2 is a direct target of phosphorylation by PKCδ, and that this modification is necessary for flight muscle maturation and function., (Copyright © 2020 by the Genetics Society of America.)

Background: Intestinal epithelial barrier dysfunction, which involves myosin light chain kinase (MLCK) activation, contributes to the occurrence and progression of inflammation in inflammatory bowel disease (IBD). Wogonoside helps maintain intestinal homeostasis in mice with dextran sulfate sodium (DSS)-induced colitis, but it is unclear whether it modulates intestinal barrier function., Purpose: Here, we demonstrate that wogonoside protects against intestinal barrier dysfunction in colitis via the MLCK/pMLC2 pathway both in vivo and in vitro., Methods: Caco-2 cell monolayers treated with the proinflammatory cytokine TNF-α showed barrier dysfunction and were assessed in the absence and presence of wogonoside for various physiological, morphological, and biochemical parameters. Colitis was induced by 3% DSS in mice, which were used as an animal model to explore the pharmacodynamics of wogonoside. We detected MLCK/pMLC2 pathway proteins via western blot analysis, assessed the cytokines IL-13 and IFN-γ via ELISA, tested bacterial translocation via fluorescence in situ hybridization (FISH) and a proper sampling of secondary lymphoid organs for bacterial culture. In addition, the docking affinity of wogonoside and MLCK was observed with DS2.5 software., Results: Wogonoside alleviated the disruption of transepithelial electrical resistance (TER) in TNF-α exposured Caco-2 cell; FITC-dextran hyperpermeability; loss of the tight junction (TJ) proteins occludin, ZO-1 and claudin-1 in Caco-2 cell monolayers; and bacterial translocation in colitic mice. Moreover, wogonoside reduced the levels of the proinflammatory cytokines IL-13 and IFN-γ to maintain intestinal immune homeostasis. Transmission electron microscopy (TEM) confirmed that wogonoside ameliorated the destruction of intestinal epithelial TJs. Wogonoside not only inhibited the cytoskeletal F-actin rearrangement induced by TNF-α, stabilized the cytoskeletal structure, suppressed MLCK protein expression, and reduced MLC2 phosphorylation. In addition, the results of molecular docking analysis showed that wogonoside had a high affinity for MLCK and formed hydrogen bonds with the amino acid residue LYS261 and π bonds with LYS229., Conclusion: Collectively, our study indicates that wogonoside alleviates colitis by protecting against intestinal barrier dysfunction, and the potential mechanism may involve regulation of TJs via the MLCK/pMLC2 signaling pathway. Meanwhile, our study also explains the success of S. baicalensis in the treatment of ulcerative colitis (UC)., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Tumor necrosis factor-α (TNF-α) is an important inflammatory cytokine that plays a key role in neuronal damage. Elevated expression of TNF-α is associated with numerous neurodegenerative diseases including Alzheimer's Disease and Parkinson's Disease. However, the specific mechanism of the signaling events that trigger TNF-α-mediated neurotoxicity remain unknown. In this study, we report that intracerebroventricular injection of TNF-α in rat hippocampal neurons down-regulates MLC2 and up-regulates MARCH8, an essential light chain and regulatory myosin light chain of NM Myosin II, respectively. MARCH8 overexpression attenuates the degradation of MLC2 by promoting its ubiquitination and degradation. Inhibition of MARCH8 by siRNA blocks caspase-3 activation and apoptosis signaling, suggesting that TNF-α-induced apoptosis of neurons is partially dependent on the accumulation of MARCH8 and the ubiquitination of MLC2. Taken together, our data not only clarify the function of MARCH8 in TNF-α-induced neurotoxicity, but also demonstrates that TNF-α promotes the MARCH8-MLC2 mediated apoptosis of hippocampal neurons. Anat Rec, 302:2271-2278, 2019. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

Cardiac contractility is enhanced by phosphorylation of myosin light chain 2 (MLC2) by cardiac-specific MLC kinase (cMLCK), located at the neck region of myosin heavy chain. In normal mouse and human hearts, the level of phosphorylation is maintained relatively constant, at around 30-40% of total MLC2, likely by well-balanced phosphorylation and phosphatase-dependent dephosphorylation. Overexpression of cMLCK promotes sarcomere organization, while the loss of cMLCK leads to cardiac atrophy in vitro and in vivo. In this study, we showed that cMLCK is predominantly expressed at the Z-disc with additional diffuse cytosolic expression in normal adult mouse and human hearts. cMLCK interacts with the Z-disc protein, α-actinin2, with a high-affinity kinetic value of 13.4 ± 0.1 nM through the N-terminus region of cMLCK unique to cardiac-isoform. cMLCK mutant deficient for interacting with α-actinin2 did not promote sarcomeric organization and reduced cardiomyocyte cell size. In contrast, a cMLCK kinase-deficient mutant showed effects similar to wild-type cMLCK on sarcomeric organization and cardiomyocyte cell size. Our results suggest that cMLCK plays a role in sarcomere organization, likely distinct from its role in phosphorylating MLC2, both of which will contribute to the enhancement of cardiac contractility.

Fasudil is a potent Rho-kinase (ROCK) inhibitor and can relax smooth muscle or cardiac muscle contraction through decreasing the phosphorylation level of myosin regulatory light chain (p-MLC 20 or p-MLC2v), while p-MLC2v can function as a transcription factor to promote the NADPH oxidase 2 (NOX2) expression in rat hearts subjected to ischemia/reperfusion (I/R). This study aims to explore whether fasudil can protect the rat hearts against I/R oxidative injury through suppressing NOX2 expression via reduction of p-MLC2v level. The SD rat hearts were subjected to 1h-ischemia plus 3h-reperfusion, which showed myocardial injuries (myocardial fiber loss and disarray, increase of creatine kinase release and myocardial infarction/apoptosis), increase in ROCK activity and nuclear p-MLC 2v level concomitant with up-regulation of NOX2 and H 2 O 2 production; these phenomena were attenuated by fasudil in a dose-dependent manner. Next, we verified the cardioprotective effect of fasudil and the underlying mechanisms in hypoxia-reoxygenation (H/R) -treated H9c2 cells. Consistent with the results in vivo, the H/R-treated H9c2 cells showed cellular injury (increase in apoptotic ratio), elevation in ROCK activity and nuclear p-MLC 2v level, accompanied by up-regulation of NOX2 and H 2 O 2 production; these effects were blocked in the presence of fasudil in a dose-dependent way. Based on these observations, we conclude that beneficial effect of fasudil against myocardial I/R or H/R oxidative injury is related to the suppression of NOX2 expression through decrease of the p-MLC2v level. Our findings also highlight that intervention of MLC2v phosphorylation by drugs may provide a novel strategy to protect heart from I/R oxidative injury., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5 eGFP/w and MLC2v mCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5 eGFP/w and MLC2v mCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The vascular smooth muscle cells (VSMCs) are exposed to interstitial flow induced shear stress that may be sensed by the surface glycocalyx, a surface layer composed primarily of proteoglycans and glycoproteins, to mediate cell contraction during the myogenic response. We, therefore, attempted to elucidate the signal pathway of the glycocalyx mechanotransduction in shear stress regulated SMC contraction. Human umbilical vein SMCs (HUVSMCs) deprived of serum for 3-4 days were exposed to a step increase (0 to 20 dyn/cm 2 ) in shear stress in a parallel plate flow chamber, and reduction in the cell area was quantified as contraction. The expressions of Rho kinase (ROCK) and its downstream signal molecules, the myosin-binding subunit of myosin phosphatase (MYPT) and the myosin light chain 2 (MLC2), were evaluated. Results showed that the exposure of HUVSMCs to shear stress for 30 min induced cell contraction significantly, which was accompanied by ROCK1 up-regulation, re-distribution, as well as MYPT1 and MLC activation. However, these shear induced phenomenon could be completely abolished by heparinase III or Y-27632 pre-treatment. These results indicate shear stress induced VSMC contraction was mediated by cell surface glycocalyx via a ROCK-MLC phosphatase (MLCP) pathway, providing evidence of the glycocalyx mechanotransduction in myogenic response., Competing Interests: The authors declare no competing financial interests.

As a physiological small molecular product from the microbial fermentation of dietary fibers, butyrate plays an important role in maintaining intestinal health. Our previous works have proved that the effect of sodium butyrate (NaB) on the intestinal barrier function is mediated by activation of AMP-activated protein kinase (AMPK). However, the detailed pathway involved remains unknown. Using the calcium switch assay in the Caco-2 cell monolayer model, we found here that NaB activated AMPK mainly by increasing the calcium level, but not the ATP concentration, via promoting store-operated calcium entry (SOCE). Upon the activation of AMPK, NaB promoted the reassembly of tight junctions (TJs) based on reducing the phosphorylation of myosin II regulatory light chain (MLC2) at Ser19 and increasing phosphorylation of protein kinase C β2 (PKCβ2) at Ser660. Inhibiting (protein kinase C β) PKCβ blocked the reassembly of TJs induced by NaB in the barrier monolayer model. These results indicated that NaB could activate the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) pathway to mediate AMPK phosphorylating, which then inhibited the phosphorylation of MLC2 and promoted the phosphorylation of PKCβ2, respectively, so that the downstream molecules of AMPK coordinately contributed to the reassembly of TJs in the Caco-2 barrier model. These results suggested a potential mechanism of butyrate for intestine homeostasis and protection., Competing Interests: The authors declare no conflict of interest.

BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2.

Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injury; however, the mechanisms underlying such injuries are poorly understood. In this study of human HepaRG and primary hepatocytes, we found that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid (BA) efflux and require alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. Short exposure to 6 cholestatic compounds revealed that BC constriction and dilation were associated with disruptions in the ROCK/MLCK/myosin pathway. At the studied concentrations, cyclosporine A and chlorpromazine induced early ROCK activity, resulting in permanent MLC2 phosphorylation and BC constriction. However, fasudil reduced ROCK activity and caused rapid, substantial and permanent MLC2 dephosphorylation, leading to BC dilation. The remaining compounds (1-naphthyl isothiocyanate, deoxycholic acid and bosentan) caused BC dilation without modulating ROCK activity, although they were associated with a steady decrease in MLC2 phosphorylation via MLCK. These changes were associated with a common loss of BC contractions and failure of BA clearance. These results provide the first demonstration that cholestatic drugs alter BC dynamics by targeting the ROCK/MLCK pathway; in addition, they highlight new insights into the mechanisms underlying bile flow failure and can be used to identify new predictive biomarkers of drug-induced cholestasis.

There is growing evidence that microRNAs are implicated in pulmonary arterial hypertension (PAH), but underlying mechanisms remain elusive. Here, we identified that miR-223 was significantly downregulated in chronically hypoxic mouse and rat lungs, as well as in pulmonary artery and pulmonary artery smooth muscle cells (PASMC) exposed to hypoxia. Knockdown of miR-223 increased PASMC proliferation. In contrast, miR-223 overexpression abrogated cell proliferation, migration and stress fiber formation. Administering miR-223 agomir in vivo antagonized hypoxia-induced increase in pulmonary artery pressure and distal arteriole muscularization. RhoB, which was increased by hypoxia, was identified as one of the targets of miR-223. Overexpressed miR-223 suppressed RhoB and inhibited the consequent phosphorylation of myosin phosphatase target subunit (MYPT1) and the expression of myosin light chain of myosin II (MLC2), which was identified as another target of miR-223. Furthermore, serum miR-223 levels were decreased in female patients with PAH associated with congenital heart disease. Our study provides the first evidence that miR-223 can regulate PASMC proliferation, migration, and actomyosin reorganization through its novel targets, RhoB and MLC2, resulting in vascular remodeling and the development of PAH. It also highlights miR-223 as a potential circulating biomarker and a small molecule drug for diagnosis and treatment of PAH.

Hyperplasia and hypertrophy are the two mechanisms by which muscle develops and grows. We study these two mechanisms, during the early development of white muscle in Sparus aurata, by means of histology and the expression of structural and regulatory genes. A clear stage of stratified hyperplasia was identified early in the development of gilthead sea bream but ceased by 35 dph when hypertrophy took over. Mosaic recruitment of new white fibers began as soon as 60 dph. The genes mlc2a and mlc2b were expressed at various levels during the main phases of hyperplasia and hypertrophy. The genes myog and mlc2a were significantly up-regulated during the intensive stratified formation of new fibers and their expression was significantly correlated. Expression of mstn1 and igf1 increased at 35 dph, appeared to regulate the hyperplasia-to-hypertrophy transition, and may have stimulated the expression of mlc2a, mlc2b and col1a1 at the onset of mosaic hyperplasia. The up-regulation of mstn1 at transitional phases in muscle development indicates a dual regulatory role of myostatin in fish larval muscle growth.

Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ∼19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method.

Objective: CC-chemokine receptor 5 (CCR5) plays a pivotal role in reperfusion after stroke. This study assessed and confirmed the effects of CCR5 in experimental stroke via regulation of ROCK/P-MLC pathway., Methods: Male Sprague Dawley (SD) rats were randomly divided into sham group, ischaemia-reperfusion group (I/R group) and DAPTA group (I/R + CCR5 antagonist group). The rats of the I/R group were subjected to transient middle cerebral artery occlusion (tMCAO) for 2 hours, followed by 24 hours of reperfusion. Animals were measured for neurologic deficit, cerebral infarct volume, TUNEL and hematoxylin-eosin (HE) staining. The protein expressions of ROCK2 and P-MLC2(Ser19) were determined by western blot., Results: Pre-treatment with DAPTA displayed significantly improved neurological functional outcome and reduced cerebral lesion compared with the I/R group animals (p < 0.05); HE staining showed that the I/R group had severe neuronal damage in the ischaemia core and penumbral; Compared with the I/R group, ROCK2 and P-MLC2(Ser19) protein expression in the DAPTA group was reduced (p < 0.05)., Conclusions: The data demonstrate that CCR5 is correlated with up-regulation of the expression of ROCK2 and P-MLC2(Ser19) in the ischaemia cortex. Treated with CCR5 antagonist protects the brain against focal cerebral ischaemia-reperfusion injury in rats.

Linezolid (LZD) is an antimicrobial that is commonly used for treatment of vancomycin-resistant Enterococci and methicillin-resistant Staphylococcus aureus infections. However, the development of thrombocytopenia, one of the most frequent adverse side effects of this antimicrobial, can lead to discontinuation of LZD treatment. While clinical studies indicate that risk factors for the development of LZD-induced thrombocytopenia include treatment for >14 consecutive days, renal dysfunction, and chronic liver disease, the fundamental mechanism governing the pathogenesis of this disorder remains unclear. In this study, we aimed to elucidate the mechanism of LZD-induced thrombocytopenia by investigating the impact of LZD treatment on platelet destruction and production using rat platelet-rich plasma (PRP) and human immortalized cell lines, respectively. Compared to the control population, an increase in lactate dehydrogenase release was not detected upon exposure of rat PRP to varying concentrations of LZD, indicating that this compound is not cytotoxic towards platelets. Meanwhile, LZD treatment resulted in a significant dose-dependent increase in the proliferation of HEL human erythroleukemia and MEG-01 human megakaryoblast cells in vitro, but did not influence the differentiation of these cell lines. Lastly, LZD treatment yielded elevated levels of phosphorylation of myosin light chain 2 (MLC2), which regulates platelet release, in MEG-01 cells. Based on these results, we speculate that LZD induces thrombocytopenia by promoting MLC2 phosphorylation and thereby suppressing the release of platelets from mature megakaryocytes. These findings provide the first insight into the mechanism of LZD-mediated thrombocytopenia and may facilitate the development of strategies to treat and/or prevent this disease.

The heart is characterized by a remarkable degree of heterogeneity, the basis of which is a subject of active investigation. Myofilament protein post-translational modifications (PTMs) represent a critical mechanism regulating cardiac contractility, and emerging evidence shows that pathological cardiac conditions induce contractile heterogeneity that correlates with transmural variations in the modification status of myofilament proteins. Nevertheless, whether there exists basal heterogeneity in myofilament protein PTMs in the heart remains unclear. Here we have systematically assessed chamber-specific and transmural variations in myofilament protein PTMs, specifically, the phosphorylation of cardiac troponin I (cTnI), cardiac troponin T (cTnT), tropomyosin (Tpm), and myosin light chain 2 (MLC2). We show that the phosphorylation of cTnI and αTm vary in the different chambers of the heart, whereas the phosphorylation of MLC2 and cTnT does not. In contrast, no significant transmural differences were observed in the phosphorylation of any of the myofilament proteins analyzed. These results highlight the importance of appropriate tissue sampling-particularly for studies aimed at elucidating disease mechanisms and biomarker discovery-in order to minimize potential variations arising from basal heterogeneity in myofilament PTMs in the heart., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Myosin light chain-2 (MYL2, also called MLC-2) is an ~19kDa sarcomeric protein that belongs to the EF-hand calcium binding protein superfamily and exists as three major isoforms encoded by three distinct genes in mammalian striated muscle. Each of the three different MLC-2 genes (MLC-2f; fast twitch skeletal isoform, MLC-2v; cardiac ventricular and slow twitch skeletal isoform, MLC-2a; cardiac atrial isoform) has a distinct developmental expression pattern in mammals. Genetic loss-of-function studies in mice demonstrated an essential role for cardiac isoforms of MLC-2, MLC-2v and MLC-2a, in cardiac contractile function during early embryogenesis. In the adult heart, MLC-2v function is regulated by phosphorylation, which displays a specific 1`expression pattern (high in epicardium and low in endocardium) across the heart. These data along with new data from computational models, genetic mouse models, and human studies have revealed a direct role for MLC-2v phosphorylation in cross-bridge cycling kinetics, calcium-dependent cardiac muscle contraction, cardiac torsion, cardiac function and various cardiac diseases. This review focuses on the regulatory functions of MLC-2 in the embryonic and adult heart, with an emphasis on phosphorylation-driven actions of MLC-2v in adult cardiac muscle, which provide new insights into mechanisms regulating myosin cycling kinetics and human cardiac diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aims: Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca(2+)-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca(2+) transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca(2+) response., Methods and Results: We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure-volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation., Conclusion: Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Dilated cardiomyopathy (DCM) is a disease of the myocardium characterized by left ventricular dilatation and diminished contractile function. Here we describe a novel DCM mutation in the myosin regulatory light chain (RLC), in which aspartic acid at position 94 is replaced by alanine (D94A). The mutation was identified by exome sequencing of three adult first-degree relatives who met formal criteria for idiopathic DCM. To obtain insight into the functional significance of this pathogenic MYL2 variant, we cloned and purified the human ventricular RLC wild-type (WT) and D94A mutant proteins, and performed in vitro experiments using RLC-mutant or WT-reconstituted porcine cardiac preparations. The mutation induced a reduction in the α-helical content of the RLC, and imposed intra-molecular rearrangements. The phosphorylation of RLC by Ca²⁺/calmodulin-activated myosin light chain kinase was not affected by D94A. The mutation was seen to impair binding of RLC to the myosin heavy chain, and its incorporation into RLC-depleted porcine myosin. The actin-activated ATPase activity of mutant-reconstituted porcine cardiac myosin was significantly higher compared with ATPase of wild-type. No changes in the myofibrillar ATPase-pCa relationship were observed in wild-type- or D94A-reconstituted preparations. Measurements of contractile force showed a slightly reduced maximal tension per cross-section of muscle, with no change in the calcium sensitivity of force in D94A-reconstituted skinned porcine papillary muscle strips compared with wild-type. Our data indicate that subtle structural rearrangements in the RLC molecule, followed by its impaired interaction with the myosin heavy chain, may trigger functional abnormalities contributing to the DCM phenotype., (© 2015 FEBS.)

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

Familial hypertrophic cardiomyopathy (HCM) is associated with mutations in sarcomeric proteins, including the myosin regulatory light chain (RLC). Here we studied the impact of three HCM mutations located in the NH2 terminus of the RLC on the molecular mechanism of β-myosin heavy chain (MHC) cross-bridge mechanics using the in vitro motility assay. To generate mutant β-myosin, native RLC was depleted from porcine cardiac MHC and reconstituted with mutant (A13T, F18L, and E22K) or wild-type (WT) human cardiac RLC. We characterized the mutant myosin force and motion generation capability in the presence of a frictional load. Compared with WT, all three mutants exhibited reductions in maximal actin filament velocity when tested under low or no frictional load. The actin-activated ATPase showed no significant difference between WT and HCM-mutant-reconstituted myosins. The decrease in velocity has been attributed to a significantly increased duty cycle, as was measured by the dependence of actin sliding velocity on myosin surface density, for all three mutant myosins. These results demonstrate a mutation-induced alteration in acto-myosin interactions that may contribute to the pathogenesis of HCM., (Copyright © 2014 the American Physiological Society.)

Pyruvate kinase M2 (PKM2) is expressed at high levels during embryonic development and tumour progression and is important for cell growth. However, it is not known whether it directly controls cell division. Here, we found that Aurora B phosphorylates PKM2, but not PKM1, at T45; this phosphorylation is required for PKM2's localization and interaction with myosin light chain 2 (MLC2) in the contractile ring region of mitotic cells during cytokinesis. PKM2 phosphorylates MLC2 at Y118, which primes the binding of ROCK2 to MLC2 and subsequent ROCK2-dependent MLC2 S15 phosphorylation. PKM2-regulated MLC2 phosphorylation, which is greatly enhanced by EGF stimulation or EGFRvIII, K-Ras G12V and B-Raf V600E mutant expression, plays a pivotal role in cytokinesis, cell proliferation and brain tumour development. These findings underscore the instrumental function of PKM2 in oncogenic EGFR-, K-Ras- and B-Raf-regulated cytokinesis and tumorigenesis.

Although calcium is the major regulator of excitation-contraction coupling, myofilament function can also be modulated through post-translational modifications. In particular, phosphorylation and O-GlcNAcylation are key modulators of calcium activation parameters. Among the regulatory proteins of skeletal muscle contraction, the myosin light chain 2 (MLC2) can undergo both types of post-translational modification. During aging or physical inactivity, the phosphorylation status of the slow isoform of MLC2 (sMLC2) does not correlate with calcium sensitivity, suggesting that the O-GlcNAcylation might modulate sMLC2 activity. To increase understanding of the contractile dysfunction associated with muscle atrophy, we studied the phosphorylation/O-GlcNAcylation interplay on the sMLC2. We demonstrate a two-fold decrease of O-GlcNAcylation level on sMLC2 in a rat model of skeletal muscle atrophy (hindlimb unloading), while phosphorylation increased. Both post-translational modifications were mutually exclusive. Their interplay reversed during reloading. The expression of enzymes involved in the phosphorylation and O-GlcNAcylation interplay on sMLC2 was modified on whole protein pattern as well as on myofilament, and was load-dependent. All enzymes were colocalized on the contractile apparatus. Finally, we describe a multienzymatic complex which might finely modulate the phosphorylation/dephosphorylation and O-GlcNAcylation/de-O-GlcNAcylation of sMLC2 that could be involved in the contractile dysfunction of atrophied muscle. Importantly, this complex was localized at the Z-disk, a nodal point of signalling in skeletal muscle.

4-Amino-2-trifluoromethyl-phenyl retinate (ATPR) is a novel all-trans retinoic acid (ATRA) derivative which was reported to have a superior antitumor effect in breast cancer cells. However, little is known about its antitumor effects on human gastric cancer cells and the mechanisms have not been fully elucidated. The results of the present study suggest that in the human gastric carcinoma cell line BGC-823, ATPR plays a more effective role than ATRA at the same dose in inhibiting proliferation, migration and inducing differentiation after the same treatment time. Furthermore, we investigated the preliminary mechanism of ATPR's anti‑migration effect. Immunofluorescence assay demonstrated that claudin-18 positioned from cytoplasm to cell surface following ATPR stimuli. Real-time quantitative RT-PCR and western blot analyses showed that ATPR had significant effects on downregulation of the phosphorylation level of myosin light chain II (MLC II) by suppressing myosin light chain kinase (MLCK) and Rho-associated coiled-coil containing kinase (ROCK), as well as its regulation in the protein expression of RARα and RARβ. Moreover, ATPR increased the activity of myosin phosphatase by inhibiting ROCK. Consequently, ATPR showed more promising antitumor effects than ATRA in BGC-823 in vitro, and it may conduct its anti-migration effects by decreasing the phosphorylation level of MLC II, as well as by regulating MLCK and ROCK as downstream target genes.

Thin (actin) filament accessory proteins are thought to be the regulatory force for muscle contraction in cardiac muscle; however, compelling new evidence suggests that thick (myosin) filament regulatory proteins are emerging as having independent and important roles in regulating cardiac muscle contraction. Key to these new findings is a growing body of evidence that point to an influential and, more recently, direct role for ventricular myosin light chain-2 (MLC2v) phosphorylation in regulating cardiac muscle contraction, function, and disease. This includes the discovery and characterization of a cardiac-specific myosin light chain kinase capable of phosphorylating MLC2v as well as a myosin phosphatase that dephosphorylates MLC2v in the heart, which provides added mechanistic insights on MLC2v regulation within cardiac muscle. Here, we review evidence for an emerging and critical role for MLC2v phosphorylation in regulating cardiac myosin cycling kinetics, function, and disease, based on recent studies performed in genetic mouse models and humans. We further provide new perspectives on future avenues for targeting these pathways as therapies in alleviating cardiac disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood-brain barrier (BBB). This study explored whether inductions of protein kinase C-β (PKC-β) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-β (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-β activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-β.

This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR+Ex subjects were submitted to combined aerobic and resistive exercises, and BR+Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS-PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR+Nut while BR+Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosylation observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atrophy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers., (© 2013.)

Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and carbohydrate metabolism and can be activated either by natural ligands as fatty acids or by synthetic ligands including several environmental chemicals. In this study, two PPARα isoforms (α1 and α2) were analyzed in turbot (Scophthalmus maximus) for a different tissue distribution. PPARα1 was ubiquitously expressed, while the PPARα2 was predominantly expressed in the heart. Following this result, turbot juveniles were exposed by injection to a synthetic selective PPARα agonist, WY-14643, for 14 days. Suppression subtractive hybridization (SSH) was performed with pools of heart samples of control and exposed fish to get insights into PPARα-regulated genes in the heart of juvenile turbot. Four genes were positively identified in the forward-subtracted and 12 genes in the reverse-subtracted cDNA SSH library, corresponding to the down-regulated and up-regulated genes in response to the WY-14643 treatment, respectively. The confirmation of these results in individual samples of juvenile turbot exposed to WY-14643 revealed a statistically significant mRNA induction of two cardiac muscle proteins (myosin light chain 2 and tropomyosin 4), which were shown to be involved in heart contraction and heartbeat regulation in other teleost species. Herewith, we showed for the first time that PPARα2 is predominantly expressed in the heart and that a PPARα agonist can induce the mRNA expression of cardiac muscle proteins in teleosts.

The human DEK proto-oncogene is a nuclear protein with suspected roles in human carcinogenesis. DEK appears to function in several nuclear processes, including transcriptional regulation and modulation of chromatin structure. To investigate the clinicopathological significance of DEK in patients with non-small cell lung cancer (NSCLC), we analyzed DEK immunohistochemistry in 112 NSCLC cases. The results showed that DEK was overexpressed mainly in the nuclear compartment of tumor cells. In squamous cell carcinoma, DEK-positive expression occurred in 47.9% (23/48) of cases, and in lung adenocarcinoma, DEK-positive expression occurred in 67.2% (43/64) of cases and correlated with differentiation, p-TNM stage, and nodal status. Moreover, in lung adenocarcinoma, DEK expression was significantly higher compared with DEK expression in squamous cell carcinoma. Kaplan-Meier analysis showed that patients with low DEK expression had higher overall survival compared with patients with high DEK expression. Depleting DEK expression inhibited cellular proliferation and migration. Furthermore, in DEK-depleted NSCLC cells, we found that RhoA expression was markedly reduced; in conjunction, active RhoA-GTP levels and the downstream effector phosphorylated MLC2 were also reduced. Taken together, DEK depletion inhibited cellular migration in lung cancer cell lines possibly through inactivation of the RhoA/ROCK/MLC signal transduction pathway.

Human heart failure has been associated with a low level of thin-filament protein phosphorylation and an increase in calcium sensitivity of contraction relative to both "control" human heart tissue and tissue from small animal models. However, diverse strategies of human tissue procurement and the reliance on tissue obtained from subjects with end-stage heart failure suggest this may be an incomplete characterization. Therefore, we evaluated cardiac left ventricular (LV) biopsy samples from patients with aortic stenosis undergoing valve replacement who presented either with LV hypertrophy and preserved systolic function (Hyp) or with LV dilation and reduced ejection fraction (Dil). In Hyp, total troponin I (TnI) phosphorylation was markedly increased and myosin light chain 2 (MLC2) phosphorylation was unchanged relative to a control group of patients with normal LV function. Conversely, in Dil, total TnI phosphorylation was significantly reduced compared with control subjects and MLC2 phosphorylation was increased. Site-specific analysis of TnI phosphorylation revealed phenotype-specific differences such that Hyp samples demonstrated significant increases in phosphorylation at serine 22/23 and Dil samples had significant decreases at serine 43. The ratio of phosphorylation at the two sites was biased toward serine 22/23 in Hyp and toward serine 43/45 in Dil. Western blot analysis showed that protein phosphatase-1 was reduced in Hyp and protein phosphatase-2 was reduced in Dil. These data suggest that posttranslational modifications of sarcomeric proteins, both singly and in combination, are stage specific. Defining these changes in progressive heart disease may provide important diagnostic and treatment information.

Myosin is the chemomechanical energy transducer in striated heart muscle. The myosin cross-bridge applies impulsive force to actin while consuming ATP chemical energy to propel myosin thick filaments relative to actin thin filaments in the fiber. Transduction begins with ATP hydrolysis in the cross-bridge driving rotary movement of a lever arm converting torque into linear displacement. Myosin regulatory light chain (RLC) binds to the lever arm and modifies its ability to translate actin. Gene sequencing implicated several RLC mutations in heart disease, and three of them are investigated here using photoactivatable GFP-tagged RLC (RLC-PAGFP) exchanged into permeabilized papillary muscle fibers. A single-lever arm probe orientation is detected in the crowded environment of the muscle fiber by using RLC-PAGFP with dipole orientation deduced from the three-spatial dimension fluorescence emission pattern of the single molecule. Symmetry and selection rules locate dipoles in their half-sarcomere, identify those at the minimal free energy, and specify active dipole contraction intermediates. Experiments were performed in a microfluidic chamber designed for isometric contraction, total internal reflection fluorescence detection, and two-photon excitation second harmonic generation to evaluate sarcomere length. The RLC-PAGFP reports apparently discretized lever arm orientation intermediates in active isometric fibers that on average produce the stall force. Disease-linked mutants introduced into RLC move intermediate occupancy further down the free energy gradient, implying lever arms rotate more to reach stall force because mutant RLC increases lever arm shear strain. A lower free energy intermediate occupancy involves a lower energy conversion efficiency in the fiber relating a specific myosin function modification to the disease-implicated mutant.

The iron-regulated metastasis suppressor, N-myc downstream-regulated gene 1 (NDRG1), is up-regulated by cellular iron depletion mediated by iron chelators and can inhibit cancer cell migration. However, the mechanism of how NDRG1 achieves this effect remains unclear. In this study, we implemented established and newly constructed NDRG1 overexpression and knockdown models using the DU145, HT29, and HCT116 cancer cell lines to investigate the molecular basis by which NDRG1 exerts its inhibitory effect on cell migration. Using these models, we demonstrated that NDRG1 overexpression inhibits cell migration by preventing actin-filament polymerization, stress fiber assembly and formation. In contrast, NDRG1 knockdown had the opposite effect. Moreover, we identified that NDRG1 inhibited an important regulatory pathway mediated by the Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated myosin light chain 2 (pMLC2) pathway that modulates stress fiber assembly. The phosphorylation of MLC2 is a key process in inducing stress fiber contraction, and this was shown to be markedly decreased or increased by NDRG1 overexpression or knockdown, respectively. The mechanism involved in the inhibition of MLC2 phosphorylation by NDRG1 was mediated by a significant (P < 0.001) decrease in ROCK1 expression that is a key kinase involved in MLC2 phosphorylation. Considering that NDRG1 is up-regulated after cellular iron depletion, novel thiosemicarbazone iron chelators (e.g., di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) were demonstrated to inhibit ROCK1/pMLC2-modulated actin-filament polymerization, stress fiber assembly, and formation via a mechanism involving NDRG1. These results highlight the role of the ROCK1/pMLC2 pathway in the NDRG1-mediated antimetastatic signaling network and the therapeutic potential of iron chelators at inhibiting metastasis.

Background: Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined., Methods and Results: We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems., Conclusions: Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Myosin II is a hexameric protein complex consisting of two myosin heavy chains, two myosin essential light chains and two myosin regulatory light chains. Multiple subunit isoforms exist, allowing great diversity in myosin II composition which likely impacts on its contractile properties. Little is known about the evolutionary origin, expression pattern and function of myosin regulatory light chain (MLC2) isoforms. We analysed the evolutionary relationship between smooth muscle (sm), nonmuscle (nm) and nonmuscle-like (nml) MLC2 genes, which encode three homologous proteins expressed in nonmuscle cells. The three genes arose by successive gene duplication events. The high sequence similarity between the tandemly arranged nm- and nml-MLC2 genes is best explained by gene conversion. Urea/glycerol-polyacrylamide gel electrophoresis and RNA analysis were employed to monitor expression of sm-, nm- and nml-MLC2 in human and mouse cell lines. Conspicuous differences between transformed and non-transformed cells were observed, with sm-MLC2 being suppressed in Ras-transformed cells. Our findings shed light on the evolutionary history of three homologous MLC2 proteins and point to isoform-specific cell growth-related roles in nonmuscle cell myosin II contractility., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

Aims: In this paper, we tested the hypothesis that different binding affinities of the C-terminus of human cardiac alkali (essential) myosin light chain (A1) isoforms to the IQ1 motif of the myosin lever arm provide a molecular basis for distinct sarcomeric sorting and inotropic activity., Methods and Results: We employed circular dichroism and surface plasmon resonance spectroscopy to investigate structural properties, secondary structures, and protein-protein interactions of a recombinant head-rod fragments of rat cardiac β-myosin heavy chain aa664-915 with alanine-mutated IQ2 domain (rβ-MYH(664-915)IQ(ala4)) and A1 isoforms [human atrial (hALC1) and human ventricular (hVLC-1) light chains]. Double epitope-tagging competition was used to monitor the intracellular localization of exogenously introduced hALC-1 and hVLC-1 constructs in neonatal rat cardiomyocytes. Contractile functions of A1 isoforms were investigated by monitoring shortening and intracellular-free Ca(2+) (Fura-2) of adult rat cardiomyocytes infected with adenoviral (Ad) vectors using hALC-1 or β-galactosidase as expression cassettes. hALC-1 bound more strongly (greater than three-fold lower K(D)) to rβ-MYH(664-915) than did hVLC-1. Sorting specificity of A1 isoforms to sarcomeres of cardiomyocytes rose in the order hVLC-1 to hALC-1. Replacement of endogenous VLC-1 by hALC-1 in adult rat cardiomyocytes increased contractility while the systolic Ca(2+) signal remained unchanged., Conclusion: Intense myosin binding of hALC-1 provides a mechanism for preferential sarcomeric sorting and Ca(2+)-independent positive inotropic activity.

Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated with myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin-like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated with myosin D via its calmodulin (CaM)-like domain and anchored to the plasma membrane of T. gondii via its N-terminal extension. Molecular modeling suggests that the MyoD-MLC2 complex is more compact than the reported structure of Plasmodium MyoA-myosin A tail-interacting protein (MTIP) complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation., (© 2011 John Wiley & Sons A/S.)

Nanos genes were required for differentiation of the anterior-posterior body axis in the Drosophila embryo. In addition to this somatic function, the nanos protein plays critical roles in the migration and survival of PGCs in vertebrates and invertebrates, but the regulating mechanisms of Nanos is largely undefined during the embyro development. In this study, we report a novel interaction between zebrafish Nanos and myosin light chain II (Mylz2). This stable complex, which is formed through the RNA-binding zinc finger domain and fourteen amino acids at the C-terminus of Nanos, was confirmed by GST pull-down and co-immunoprecipitation. Both of these two parts are required for the interaction with Mylz2, but ninety-one amio acids at N-terminus of Nanos are not required. Using a phospho-myosin light chain 2 (Ser19) antibody, we demonstrate that Nanos downregulates the phosphorylation of the Mylz2 by cotransfecting PRK-FLAG-Na and PRK-HA-My Mylz2 in 93T cell. We discuss the biological roles of the interaction between Nanos and Mylz2 and the Nanos regulating phosphorylation of the Mylz2 in PGCs., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)