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/cm2) 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.

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.

Platelets are highly sensitive blood cells, which play central role in hemostasis and thrombosis. Platelet dense granules carry considerable amount of neurotransmitter glutamate that is exocytosed upon cell activation. As platelets also express glutamate receptors on their surface, it is pertinent to ask whether exposure to glutamate would affect their signalling within a growing thrombus. In this study we demonstrate that, glutamate per se induced synthesis of thrombogenic peptides, plasminogen activator inhibitor-1 and hypoxia-inducible factor-2α, from pre-existing mRNAs in enucleate platelets, stimulated cytosolic calcium entry, upregulated RhoA-ROCK-myosin light chain/myosin light chain phosphatase axis, and elicited extensive shedding of extracellular vesicles from platelets. Glutamate, too, incited platelet spreading and adhesion on to immobilized matrix under arterial shear, raised mitochondrial transmembrane potential associated with generation of reactive oxygen species and induced activation of AMP-activated protein kinase in platelets. Taken together, glutamate switches human platelets to pro-activation phenotype mediated mostly through AMPA receptors and thus targeting glutamate receptors may be a promising anti-platelet strategy.

Tumor cell invasion requires the molecular and physical adaptation of both the cell and its microenvironment. Here we show that tumor cells are able to switch between the use of microvesicles and invadopodia to facilitate invasion through the extracellular matrix. Invadopodia formation accompanies the mesenchymal mode of migration on firm matrices and is facilitated by Rac1 activation. On the other hand, during invasion through compliant and deformable environments, tumor cells adopt an amoeboid phenotype and release microvesicles. Notably, firm matrices do not support microvesicle release, whereas compliant matrices are not conducive to invadopodia biogenesis. Furthermore, Rac1 activation is required for invadopodia function, while its inactivation promotes RhoA activation and actomyosin contractility required for microvesicle shedding. Suppression of RhoA signaling blocks microvesicle formation but enhances the formation of invadopodia. Finally, we describe Rho-mediated pathways involved in microvesicle biogenesis through the regulation of myosin light chain phosphatase. Our findings suggest that the ability of tumor cells to switch between the aforementioned qualitatively distinct modes of invasion may allow for dissemination across different microenvironments.

We investigated effects of age, sex and frailty on contractions, calcium transients and myofilament proteins to determine if maladaptive changes associated with aging were sex-specific and modified by frailty. Ventricular myocytes and myofilaments were isolated from middle-aged (~12 mos) and older (~24 mos) mice. Frailty was assessed with a non-invasive frailty index. Calcium transients declined and slowed with age in both sexes, but contractions were largely unaffected. Actomyosin Mg-ATPase activity increased with age in females but not males; this could maintain contractions with smaller calcium transients in females. Phosphorylation of myosin-binding protein C (MyBP-C), desmin, tropomyosin and myosin light chain-1 (MLC-1) increased with age in males, but only MyBP-C and troponin-T increased in females. Enhanced phosphorylation of MyBP-C and MLC-1 could preserve contractions in aging. Interestingly, the age-related decline in Hill coefficients (r = -0.816; p = 0.002) and increase in phosphorylation of desmin (r = 0.735; p = 0.010), tropomyosin (r = 0.779; p = 0.005) and MLC-1 (r = 0.817; p = 0.022) were graded by the level of frailty in males but not females. In these ways, cardiac remodeling at cellular and subcellular levels is graded by overall health in aging males. Such changes may contribute to heart diseases in frail older males, whereas females may be resistant to these effects of frailty.

Consequences of primary dsysmenorrhea (PD) can be severe. Increased prostaglandin production leads to uterine contraction and insufficient blood flow to the endometrium causing ischemia and pain symptoms. Protein tyrosine kinase/phosphatase activities contribute to the modulation of uterine contraction. In our previous study, we found the synthetic β-methoxyacrylates compound Fluacrypyrim (FAPM), significantly increased protein tyrosine phosphatases (PTPs) activity, resulting in dephosphorylation of tyrosine kinases. In the present study, we found that FAPM near completely inhibited prostaglandin F2α (PGF2α)-, oxytocin-, acetylcholine-, and high K+-induced uterine contractions in rats in vitro, and decreased rat myometrial myosin light chain (MLC20) phosphorylation induced by PGF2α. A structure-activity relationship assay indicated that the β-methoxyacrylates structure of FAPM is crucial for the inhibition of PGF2α-induced uterine contractions. FAPM caused a concentration-dependent parallel rightward shift of the concentration-response curve induced by oxytocin, dose-dependently reduced the number of abdominal constrictions and increased the latency time in PGF2α- and acetic acid-induced writhing test in mice in vivo. Furthermore, FAPM considerably inhibited the development of Carr-induced rat paw edemas and thexylene-induced mouse ear edemas. Taken together, our results indicate that FAPM exerts antinociceptive and anti-inflammatory effects in vivo with considerable potential as a novel uterine relaxant.

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.

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.

Generating atrial-like cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) is crucial for modeling and treating atrial-related diseases, such as atrial arrythmias including atrial fibrillations. However, it is essential to obtain a comprehensive understanding of the electrophysiological properties of these cells. The objective of the present study was to investigate the molecular, electrical, and biophysical properties of several ion channels, especially NaV1.5 channels, in atrial hiPSC cardiomyocytes. Atrial cardiomyocytes were obtained by the differentiation of hiPSCs treated with retinoic acid (RA). The quality of the atrial specification was assessed by qPCR, immunocytofluorescence, and western blotting. The electrophysiological properties of action potentials (APs), Ca2+ dynamics, K+ and Na+ currents were investigated using patch-clamp and optical mapping approaches. We evaluated mRNA transcript and protein expressions to show that atrial cardiomyocytes expressed higher atrial- and sinoatrial-specific markers (MYL7, CACNA1D) and lower ventricular-specific markers (MYL2, CACNA1C, GJA1) than ventricular cardiomyocytes. The amplitude, duration, and steady-state phase of APs in atrial cardiomyocytes decreased, and had a shape similar to that of mature atrial cardiomyocytes. Interestingly, NaV1.5 channels in atrial cardiomyocytes exhibited lower mRNA transcripts and protein expression, which could explain the lower current densities recorded by patch-clamp. Moreover, Na+ currents exhibited differences in activation and inactivation parameters. These differences could be explained by an increase in SCN2B regulatory subunit expression and a decrease in SCN1B and SCN4B regulatory subunit expressions. Our results show that a RA treatment made it possible to obtain atrial cardiomyocytes and investigate differences in NaV1.5 channel properties between ventricular- and atrial-like cells.