PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Ion Channel and Structural Remodeling in Obesity-Mediated Atrial Fibrillation.

BACKGROUND: Epidemiological studies have established obesity as an independent risk factor for atrial fibrillation (AF), but the underlying pathophysiological mechanisms remain unclear. Reduced cardiac sodium channel expression is a known causal mechanism in AF. We hypothesized that obesity decreases Nav1.5 expression via enhanced oxidative stress, thus reducing INa, and enhancing susceptibility to AF. METHODS: To elucidate the underlying electrophysiological mechanisms a diet-induced obese mouse model was used. Weight, blood pressure, glucose, F2-isoprostanes, NOX2 (NADPH oxidase 2), and PKC (protein kinase C) were measured in obese mice and compared with lean controls. Invasive electrophysiological, immunohistochemistry, Western blotting, and patch clamping of membrane potentials was performed to evaluate the molecular and electrophysiological phenotype of atrial myocytes. RESULTS: Pacing-induced AF in 100% of diet-induced obese mice versus 25% in controls (P<0.01) with increased AF burden. Cardiac sodium channel expression, INa and atrial action potential duration were reduced and potassium channel expression (Kv1.5) and current (IKur) and F2-isoprostanes, NOX2, and PKC-α/δ expression and atrial fibrosis were significantly increased in diet-induced obese mice as compared with controls. A mitochondrial antioxidant reduced AF burden, restored INa, ICa,L, IKur, action potential duration, and reversed atrial fibrosis in diet-induced obese mice as compared with controls. CONCLUSIONS: Inducible AF in obese mice is mediated, in part, by a combined effect of sodium, potassium, and calcium channel remodeling and atrial fibrosis. Mitochondrial antioxidant therapy abrogated the ion channel and structural remodeling and reversed the obesity-induced AF burden. Our findings have important implications for the management of obesity-mediated AF in patients. Graphic Abstract: A graphic abstract is available for this article.

MEIS-mediated suppression of human prostate cancer growth and metastasis through HOXB13-dependent regulation of proteoglycans.

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced.

Decisions regarding the treatment of patients with early-stage prostate cancer are often based on the risk that the cancer could grow and spread quickly. However, it is not always straightforward to predict how the cancer will behave. Studies from 2017 and 2018 found that samples of less aggressive prostate cancer have higher levels of a group of proteins called MEIS proteins. MEIS proteins help control the production of numerous other proteins, which could affect the behavior of prostate cancer cells in many ways. VanOpstall et al. ­ including some of the researchers that performed the 2017 and 2018 studies ­ have investigated how MEIS proteins affect prostate cancer. When prostate cancer cells are implanted into mice, they result in tumors. VanOpstall et al. found that tumors that produced MEIS proteins grew more slowly. Next, MEIS proteins were extracted from the prostate cancer cells and were found to interact with another protein called HOXB13, which regulates the activity of numerous genes. When the cells were genetically modified to prevent HOXB13 being produced, the protective effect of MEIS proteins was lost. MEIS proteins work with HOXB13 to regulate the production of several other proteins, in particular a protein called Decorin that can suppress tumors. When MEIS proteins and HOXB13 are present, the cell produces more Decorin and the tumors grow more slowly and are less likely to spread. VanOpstall et al. found that blocking Decorin production rendered MEIS proteins less able to slow the spread of prostate cancer. These results suggest that MEIS proteins and HOXB13 are needed to stop tumors from growing and spreading, and some of this ability is by prompting production of Decorin. This study explains how MEIS proteins can reduce prostate cancer growth, providing greater confidence in using them to determine whether aggressive treatment is needed. A greater understanding of this pathway for tumor suppression could also provide an opportunity for developing anti-cancer drugs.

In Vivo Restoration of Myocardial Conduction With Carbon Nanotube Fibers.

BACKGROUND: Impaired myocardial conduction is the underlying mechanism for re-entrant arrhythmias. Carbon nanotube fibers (CNTfs) combine the mechanical properties of suture materials with the conductive properties of metals and may form a restorative solution to impaired myocardial conduction. METHODS: Acute open chest electrophysiology studies were performed in sheep (n=3). Radiofrequency ablation was used to create epicardial conduction delay after which CNTf and then silk suture controls were applied. CNTfs were surgically sewn across the right atrioventricular junction in rodents, and acute (n=3) and chronic (4-week, n=6) electrophysiology studies were performed. Rodent toxicity studies (n=10) were performed. Electrical analysis of the CNTf-myocardial interface was performed. RESULTS: In all cases, the large animal studies demonstrated improvement in conduction velocity using CNTf. The acute rodent model demonstrated ventricular preexcitation during sinus rhythm. All chronic cases demonstrated resumption of atrioventricular conduction, but these required atrial pacing. There was no gross or histopathologic evidence of toxicity. Ex vivo studies demonstrated contact impedance significantly lower than platinum iridium. CONCLUSIONS: Here, we show that in sheep, CNTfs sewn across epicardial scar acutely improve conduction. In addition, CNTf maintain conduction for 1 month after atrioventricular nodal ablation in the absence of inflammatory or toxic responses in rats but only in the paced condition. The CNTf/myocardial interface has such low impedance that CNTf can facilitate local, downstream myocardial activation. CNTf are conductive, biocompatible materials that restore electrical conduction in diseased myocardium, offering potential long-term restorative solutions in pathologies interrupting efficient electrical transduction in electrically excitable tissues.

Molecular Insights into the Short QT Syndrome.

Short QT syndrome (SQTS) is a myocardial conduction disorder characterized by a short QT interval on electrocardiogram and predisposition to familial atrial fibrillation and/or sudden cardiac death. Genetic SQTS is primarily caused by one or more cardiac ion channelopathies, in which either impaired depolarization currents or enhanced repolarization currents shorten cardiac action potential duration. Given that QT interval duration is not always predictive of arrhythmia burden and risk of death in SQTS, there is a need to understand the molecular mechanisms of the condition to improve risk prognostication and potential pharmacologic treatment. In the last decade, several computational advances and in vitro preclinical studies have provided insight into the molecular mechanisms underlying congenital SQTS. In this review, we discuss recent findings in SQTS molecular mechanisms and correlate these advances with clinical guidelines for SQTS diagnosis and treatment.

The posterior neural plate in axolotl gives rise to neural tube or turns anteriorly to form somites of the tail and posterior trunk.

Classical grafting experiments in the Mexican axolotl had shown that the posterior neural plate of the neurula is no specified neuroectoderm but gives rise to somites of the tail and posterior trunk. The bipotentiality of this region with neuromesodermal progenitor cell populations was revealed more recently also in zebrafish, chick, and mouse. We reinvestigated the potency of the posterior plate in axolotl using grafts from transgenic embryos, immunohistochemistry, and in situ hybridization. The posterior plate is brachyury-positive except for its more anterior parts which express sox2. Between anterior and posterior regions of the posterior plate a small domain with sox2+ and bra+ cells exists. Lineage analysis of grafted GFP-labeled posterior plate tissue revealed that posterior GFP+ cells move from dorsal to ventral, form the posterior wall, turn anterior bilaterally, and join the gastrulated paraxial presomitic mesoderm. More anterior sox2+/GFP+ cells, however, are integrated into the developing spinal cord. Tail notochord is formed from axial mesoderm involuted already during gastrulation. Thus the posterior neural plate is a postgastrula source of paraxial mesoderm, which performs an anterior turn, a novel morphogenetic movement. More anterior plate cells, in contrast, do not turn anteriorly but become specified to form tail spinal cord.

Recent Advances in BLV Research.

Different animal models have been proposed to investigate the mechanisms of Human T-lymphotropic Virus (HTLV)-induced pathogenesis: rats, transgenic and NOD-SCID/γcnull (NOG) mice, rabbits, squirrel monkeys, baboons and macaques. These systems indeed provide useful information but have intrinsic limitations such as lack of disease relevance, species specificity or inadequate immune response. Another strategy based on a comparative virology approach is to characterize a related pathogen and to speculate on possible shared mechanisms. In this perspective, bovine leukemia virus (BLV), another member of the deltaretrovirus genus, is evolutionary related to HTLV-1. BLV induces lymphoproliferative disorders in ruminants providing useful information on the mechanisms of viral persistence, genetic determinants of pathogenesis and potential novel therapies.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy-tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans.

Phospho-NHE3 forms membrane patches and interacts with beta-actin to sense and maintain constant direction during cell migration.

The Na(+)/H(+) exchanger NHE3 colocalizes with beta-actin at the leading edge of directionally migrating cells. Using human osteosarcoma cells (SaOS-2), rat osteoblasts (calvaria), and human embryonic kidney (HEK) cells, we identified a novel role for NHE3 via beta-actin in anode and cathode directed motility, during electrotaxis. NHE3 knockdown by RNAi revealed that NHE3 expression is required to achieve constant directionality and polarity in migrating cells. Phosphorylated NHE3 (pNHE3) and beta-actin complex formation was impaired by the NHE3 inhibitor S3226 (IC50 0.02µM). Fluorescence cross-correlation spectroscopy (FCCS) revealed that the molecular interactions between NHE3 and beta-actin in membrane protrusions increased 1.7-fold in the presence of a directional cue and decreased 3.3-fold in the presence of cytochalasin D. Data from flow cytometric analysis showed that membrane potential of cells (Vmem) decreases in directionally migrating, NHE3-deficient osteoblasts and osteosarcoma cells whereas only Vmem of wild type osteoblasts is affected during directional migration. These findings suggest that pNHE3 has a mechanical function via beta-actin that is dependent on its physiological activity and Vmem. Furthermore, phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels increase while PIP2 remains stable when cells have persistent directionality. Both PI3 kinase (PI3K) and Akt expression levels change proportionally to NHE3 levels. Interestingly, however, the content of pNHE3 level does not change when PI3K/Akt is inhibited. Therefore, we conclude that NHE3 can act as a direction sensor for cells and that NHE3 phosphorylation in persistent directional cell migration does not involve PI3K/Akt during electrotaxis.

Activation of P2X7R and downstream effects in bleomycin treated lung epithelial cells.

Changes in intracellular calcium concentration [Ca(2+)](i) are believed to influence the proliferation and differentiation of airway epithelial cells both in vivo and in vitro. In the present study, using mouse alveolar epithelial E10 cells, we demonstrated that the treatment of lung epithelial cells with BLM resulted in elevated intracellular Ca(2+) levels. BLM further increased P2rx7 mRNA expression and P2X7R protein levels, paralleled by increased PKC-ß1 levels. BLM treatment or stimulation of the P2X7R with the P2X7R agonist BzATP induced translocation of PKC-ß1 from the cytoplasm to the membrane. The expression of PKC-ß1 was repressed by the P2X7R inhibitor oxATP, suggesting that PKC-ß1 is downstream of P2X7R activation. Furthermore, cells exposed to BLM contained increased amounts of P2X7R and PKC-ß1 in Cav-1 containing lipid raft fractions. The comparison of lung tissues from wild-type and P2rx7(-/-) mice revealed decreased protein and mRNA levels of PKC-ß1 and CaM as well as decreased immunoreactivity for PKC-ß1. The knockdown of P2X7R in alveolar epithelial cells resulted also in a loss of PKC-ß1. These data suggest that the effect of P2X7R on expression of PKC-ß1 detected in alveolar epithelial cells is also functioning in the animal model. Immunohistochemical evaluation of fibrotic lungs derived from a BLM-induced mouse model revealed a strong increase in PKC-ß1 immunoreactivity. The present experiments demonstrated that the increased expression of P2X7R influences PKC-ß1. We predict that increased Ca(2+) concentration stimulates PKC-ß1, whereas the prerequisite for activating PKC-ß1 after P2X7R increase remained to be determined. Our findings suggest that PKC-ß1 is important in the pathogenesis of pulmonary fibrosis.

Persistent directional cell migration requires ion transport proteins as direction sensors and membrane potential differences in order to maintain directedness.

BACKGROUND: Ion transport proteins generate small electric fields that can induce directional cell motility; however, little is known about their mechanisms that lead to directedness. We investigated Na, K-ATPase (NaKA) and Na+/H+ exchanger isoforms (NHE1 and 3) in SaOS-2 and Calvarial osteoblasts, which present anode- and cathode- directed motility, during electrotaxis. RESULTS: Significant colocalizations of NaKA with vinculin and pNHE3 with ß-actin were observed to occur at the leading edges of cells. The directedness were attenuated when NaKA or NHE3 was inhibited, confirming their implication in directional sensing. Depending on the perceived direction, a divergent regulation in PIP2 levels as a function of NHE3 and NaKA levels was observed, suggesting that PIP2 may act as a spatiotemporal regulator of the cell membrane during electrotaxis. Moreover, at the same places where pNHE3 accumulates, bubble-shaped H+ clouds were observed, suggesting a physio-mechanical role for NHE3. The cell membrane becomes hyperpolarized at the front and depolarized at the back, which confirms NaKA activity at the leading edge. CONCLUSION: We suggest a novel role for both NaKA and NHE3 that extends beyond ion translocation and conclude that they can act as directional sensors and Vmem as a regulatory cue which maintain the persistent direction in electrotaxis.

Local calcium elevation and cell elongation initiate guided motility in electrically stimulated osteoblast-like cells.

BACKGROUND: Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10-15 V/cm) and weak (< or = 5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation. CONCLUSION/SIGNIFICANCE: Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions.