Mathematical model of excitation-contraction in a uterine smooth muscle cell

The Bursztyn et al. (2007) paper proposes a mathematical model of excitation-contraction in a myometrial smooth muscle cell (SMC). The model incorporates processes of intracellular Ca^2+ concentration control, myosin light chain (MLC) phosphorylation and stress production. We create a modularized CellML implementation of the model, which is able to simulate these processes against the original data.

Mathematical model of excitation-contraction in a uterine smooth muscle cell

The Bursztyn et al. (2007) paper proposes a mathematical model of excitation-contraction in a myometrial smooth muscle cell (SMC). The model incorporates processes of intracellular Ca^2+ concentration control, myosin light chain (MLC) phosphorylation and stress production. We create a modularized CellML implementation of the model, which is able to simulate these processes against the original data.

Activation of mTOR mediates hyperglycemia-induced renal glomerular endothelial hyperpermeability via the RhoA/ROCK/pMLC signaling pathway

Objective Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro. Materials and methods HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed. Results HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure. Conclusions Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

Effects of selective EP2 receptor agonist, omidenepag, on trabecular meshwork cells, Schlemm’s canal endothelial cells and ciliary muscle contraction

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm’s canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation

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

Dissecting the molecular basis of human interneuron migration in forebrain assembloids from Timothy syndrome

Defects in interneuron migration during forebrain development can disrupt the assembly of cortical circuits and have been associated with neuropsychiatric disease. The molecular and cellular bases of such deficits have been particularly difficult to study in humans due to limited access to functional forebrain tissue from patients. We previously developed a human forebrain assembloid model of Timothy Syndrome (TS), caused by a gain-of-function mutation in CACNA1C which encodes the L-type calcium channel (LTCC) Cav1.2. By functionally integrating human induced pluripotent stem cell (hiPSC)-derived organoids resembling the dorsal and ventral forebrain from patients and control individuals, we uncovered that migration is disrupted in TS cortical interneurons. Here, we dissect the molecular underpinnings of this phenotype and report that acute pharmacological modulation of Cav1.2 can rescue the saltation length but not the saltation frequency of TS migrating interneurons. Furthermore, we find that the defect in saltation length in TS interneurons is associated with aberrant actomyosin function and is rescued by pharmacological modulation of MLC phosphorylation, whereas the saltation frequency phenotype in TS interneurons is driven by enhanced GABA sensitivity and can be restored by GABA receptor antagonism. Overall, these findings uncover multi-faceted roles of LTCC function in human cortical interneuron migration in the context of disease and suggest new strategies to restore interneuron migration deficits.

Activation of mtor Mediates Hyperglycemia-Induced Renal Glomerular Endothelial Hyperpermeability via the RhoA/ROCK/pmlc Signaling Pathway

OBJECTIVE: Hyperglycemia is associated with albuminuria and renal glomerular endothelial dysfunction in patients with diabetic nephropathy. The mTOR and RhoA/ROCK signaling pathways are involved in glomerular filtration barrier (GFB) regulation, but their role in high glucose (HG)-induced GFB dysfunction in human renal glomerular endothelial cells (HRGECs) has not been investigated. This study aimed to investigate the mechanisms of HG-induced GFB dysfunction in vitro.MATERIALS AND METHODS: HRGECs were cultured in vitro and exposed to HG. The horseradish peroxidase–albumin leakage and transendothelial electrical resistance of the endothelial monolayer were measured after HG treatment with or without rapamycin preincubation. A fluorescence probe was used to study the distribution of F-actin reorganization. The phosphorylation levels of myosin light chain (MLC) and mTOR were measured via western blotting. RhoA activity was evaluated via GTPase activation assay. The effects of blocking mTOR or the RhoA/ROCK pathway on endothelial permeability and MLC phosphorylation under HG conditions were observed.RESULTS: HG exposure induced F-actin reorganization and increased MLC phosphorylation, leading to EC barrier disruption. This effect was attenuated by treatment with rapamycin or Y-27632. Phospho-MLC (pMLC) activation in HRGECs was mediated by RhoA/ROCK signaling. mTOR and RhoA/ROCK inhibition or knockdown attenuated pMLC activation, F-actin reorganization and barrier disruption that occurred in response to HG exposure.CONCLUSIONS: Our results revealed that HG stimulation upregulated RhoA expression and activity through an mTOR-dependent pathway, leading to MLC-mediated endothelial cell cytoskeleton rearrangement and glomerular endothelial barrier dysfunction.

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 > 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

Loss of PI3-kinase activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration and intestinal homeostasis

Inositol polyphosphate multikinase (IPMK) is a rate-limiting enzyme in the inositol phosphate (IP) pathway which converts IP3 to IP4 and IP5. In mammalian cells, IPMK can also act as a phosphoinositol-3-kinase (PI3-kinase). We previously found that IPMK is a critical PI3-kinase activator of AKT. Here, we show that IPMK mediates AKT activation by promoting membrane localization and activation of PDK1. The PI3-kinase activity of IPMK is dispensable for membrane localization of AKT, which is entirely controlled by classical PI3-kinase (p110α,ß, γ, δ). By contrast, we found that PDK1 membrane localization was largely independent of classical PI3-kinase. Membrane localization of PDK1 stimulates cell migration by dissociating ROCK1 from inhibitory binding to RhoE and promoting ROCK1-mediated myosin light chain (MLC) phosphorylation. Deletion of IPMK impairs cell migration associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent MLC phosphorylation. To investigate the physiological relevance of IPMK-mediated AKT activation, we generated mice selectively lacking IPMK in epithelial cells of the intestine, where IPMK is highly expressed. Deletion of IPMK in intestinal epithelial cells markedly reduced AKT phosphorylation and diminished numbers of Paneth cells – a crypt-resident epithelial cell type that generates the physiological niche for intestinal stem cells. Ablation of IPMK impaired intestinal epithelial cell regeneration basally and after; chemotherapy-induced damage, suggesting a broad role for IPMK in the activation of AKT and intestinal tissue regeneration. In summary, the PI3-kinase activity of IPMK promotes membrane localization of PDK1, a critical kinase whereby AKT maintains intestinal homeostasis.One Sentence SummaryPI3-kinase activity of IPMK is essential for activation of AKT.

The Calcilytic Drug Calhex-231 Ameliorates Vascular Hyporesponsiveness in Traumatic Hemorrhagic Shock by Inhibiting Oxidative Stress and miR-208a-Mediated Mitochondrial Fission

Background. The calcium-sensing receptor (CaSR) plays a fundamental role in extracellular calcium homeostasis in humans. Surprisingly, CaSR is also expressed in nonhomeostatic tissues and is involved in regulating diverse cellular functions. The objective of this study was to determine if Calhex-231 (Cal), a negative modulator of CaSR, may be beneficial in the treatment of traumatic hemorrhagic shock (THS) by improving cardiovascular function and investigated the mechanisms. Methods. Rats that had been subjected to THS and hypoxia-treated vascular smooth muscle cells (VSMCs) were used in this study. The effects of Cal on cardiovascular function, animal survival, hemodynamics, and vital organ function in THS rats and the relationship to oxidative stress, mitochondrial fusion-fission, and microRNA (miR-208a) were investigated. Results. Cal significantly improved hemodynamics, elevated blood pressure, increased vital organ blood perfusion and local oxygen supply, and markedly improved the survival outcomes of THS rats. Furthermore, Cal significantly improved vascular reactivity after THS in vivo and in vitro. Cal also restored the THS-induced decrease in myosin light chain (MLC) phosphorylation (the key element for VSMC contraction). Inhibition of MLC phosphorylation antagonized the Cal-induced restoration of vascular reactivity following THS. Cal suppressed oxidative stress in THS rats and hypoxic-VSMCs. Meanwhile, THS induced expression of mitochondrial fission proteins Drp1 and Fis1 and decreased expression of mitochondrial fusion protein Mfn1 in vascular tissues. Cal reduced expression of Drp1 and Fis1. In hypoxic-VSMCs, Cal inhibited mitochondrial fragmentation and preserved mitochondrial morphology. In addition, miR-208a mimic decreased Fis1 expression, and miR-208a inhibitor prevented Cal-induced Fis1 downregulation in hypoxic-VSMCs. Conclusion. Calhex-231 exhibits outstanding potential for effective therapy of traumatic hemorrhagic shock, and the beneficial effects result from its protection of vascular function via inhibition of oxidative stress and miR-208a-mediated mitochondrial fission.