Lack of authentic atrial fibrillation in commonly used murine atrial fibrillation models.

The mouse is a useful preclinical species for evaluating disease etiology due to the availability of a wide variety of genetically modified strains and the ability to perform disease-modifying manipulations. In order to establish an atrial filtration (AF) model in our laboratory, we profiled several commonly used murine AF models. We initially evaluated a pharmacological model of acute carbachol (CCh) treatment plus atrial burst pacing in C57BL/6 mice. In an effort to observe micro-reentrant circuits indicative of authentic AF, we employed optical mapping imaging in isolated mouse hearts. While CCh reduced atrial refractoriness and increased atrial tachyarrhythmia vulnerability, the left atrial (LA) excitation patterns were rather regular without reentrant circuits or wavelets. Therefore, the atrial tachyarrhythmia resembled high frequency atrial flutter, not typical AF per se. We next examined both a chronic angiotensin II (Ang II) infusion model and the surgical model of transverse aortic constriction (TAC), which have both been reported to induce atrial and ventricular structural changes that serve as a substrates for micro-reentrant AF. Although we observed some extent of atrial remodeling such as fibrosis or enlarged LA diameter, burst pacing-induced atrial tachyarrhythmia vulnerability did not differ from control mice in either model. This again suggested that an AF-like pathophysiology is difficult to demonstrate in the mouse. To continue searching for a valid murine AF model, we studied mice with a cardiac-specific deficiency (KO) in liver kinase B1 (Cardiac-LKB1), which has been reported to exhibit spontaneous AF. Indeed, the electrocardiograms (ECG) of conscious Cardiac-LKB1 KO mice exhibited no P waves and had irregular RR intervals, which are characteristics of AF. Histological evaluation of Cardiac-LKB1 KO mice revealed dilated and fibrotic atria, again consistent with AF. However, atrial electrograms and optical mapping revealed that electrical activity was limited to the sino-atrial node area with no electrical conduction into the atrial myocardium beyond. Thus, Cardiac-LKB1 KO mice have severe atrial myopathy or atrial standstill, but not AF. In summary, the atrial tachyarrhythmias we observed in the four murine models were distinct from typical human AF, which often exhibits micro- or macro-reentrant atrial circuits. Our results suggest that the four murine AF models we examined may not reflect human AF well, and raise a cautionary note for use of those murine models to study AF.

Thoracic Surgeon Impressions of the Impact of the COVID-19 Pandemic on Lung Cancer Care-Lessons from the First Wave in Canada.

BACKGROUND: COVID-19 has invariably changed the way lung cancer surgical care is provided in Canada. Despite relevant management guidelines, the way in which cancer care has been affected has yet to be described for thoracic surgical populations. Routine lung cancer physiologic and staging assessments are unique in that they are droplet producing and aerosolizing procedures. Our objective was to quantify the effect of the COVID-19 pandemic on surgical lung cancer care as perceived by practicing thoracic surgeons during the first wave of the pandemic in Canada. METHODS: An electronic survey was distributed to members of the Canadian Association of Thoracic Surgeons. The survey was designed to determine surgeon perception of lung cancer preoperative care during the Canadian pandemic-instilled period of resource reallocation compared to standard care. Planned analyses were exploratory in nature; with count and frequency distributions of responses quantified. RESULTS: Fifty-three thoracic surgeons completed the survey. Responses were collected from all Canadian provinces. Little change in access to preoperative imaging was noted. However, a significant decrease in access to lung function and bronchoscopy testing occurred. Pulmonary surgery was perceived to be lengthier with reduced operating theater availability. Despite decreased OR access, only 40% of surgeons were aware of respective institutional mitigation strategies. SUMMARY: The COVID-19 pandemic has had an impact on standard lung cancer care preoperative workup. Further inquiry using institutional data is warranted to quantify its impact on cancer patient outcomes. Assessing the extent and effects of newly present barriers to standard lung cancer care is essential in forming appropriate mitigation strategies and planning for future pandemic waves.

Microglial Gi-dependent dynamics regulate brain network hyperexcitability.

Microglial surveillance is a key feature of brain physiology and disease. Here, we found that Gi-dependent microglial dynamics prevent neuronal network hyperexcitability. By generating MgPTX mice to genetically inhibit Gi in microglia, we show that sustained reduction of microglia brain surveillance and directed process motility induced spontaneous seizures and increased hypersynchrony after physiologically evoked neuronal activity in awake adult mice. Thus, Gi-dependent microglia dynamics may prevent hyperexcitability in neurological diseases.

Protease-Activated Receptor-2 Regulates Neuro-Epidermal Communication in Atopic Dermatitis.

Background: Activation of protease-activated receptor-2 (PAR2) has been implicated in inflammation, pruritus, and skin barrier regulation, all characteristics of atopic dermatitis (AD), as well as Netherton syndrome which has similar characteristics. However, understanding the precise role of PAR2 on neuro-immune communication in AD has been hampered by the lack of appropriate animal models. Methods: We used a recently established mouse model with epidermal overexpression of PAR2 (PAR2OE) and littermate WT mice to study the impact of increased PAR2 expression in epidermal cells on spontaneous and house dust mite (HDM)-induced skin inflammation, itch, and barrier dysfunction in AD, in vivo and ex vivo. Results: PAR2OE newborns displayed no overt abnormalities, but spontaneously developed dry skin, severe pruritus, and eczema. Dermatological, neurophysiological, and immunological analyses revealed the hallmarks of AD-like skin disease. Skin barrier defects were observed before onset of skin lesions. Application of HDM onto PAR2OE mice triggered pruritus and the skin phenotype. PAR2OE mice displayed an increased density of nerve fibers, increased nerve growth factor and endothelin-1 expression levels, alloknesis, enhanced scratching (hyperknesis), and responses of dorsal root ganglion cells to non-histaminergic pruritogens. Conclusion: PAR2 in keratinocytes, activated by exogenous and endogenous proteases, is sufficient to drive barrier dysfunction, inflammation, and pruritus and sensitize skin to the effects of HDM in a mouse model that mimics human AD. PAR2 signaling in keratinocytes appears to be sufficient to drive several levels of neuro-epidermal communication, another feature of human AD.

Designing a Green Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis in Animals.

A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.

Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish.

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.

A rapid fluorogenic GPCR-ß-arrestin interaction assay.

Detection of protein-protein interactions involved in signal transduction in live cells and organisms has a variety of important applications. We report a fluorogenic assay for G protein-coupled receptor (GPCR)-ß-arrestin interaction that is genetically encoded, generalizes to multiple GPCRs, and features high signal-to-noise because fluorescence is absent until its components interact upon GPCR activation. Fluorescence after protease-activated receptor-1 activation developed in minutes and required specific serine-threonine residues in the receptor carboxyl tail, consistent with a classical G protein-coupled receptor kinase dependent ß-arrestin recruitment mechanism. This assay provides a useful complement to other in vivo assays of GPCR activation.

Visualizing Dynamics of Cell Signaling In Vivo with a Phase Separation-Based Kinase Reporter.

Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.

Inactivating mutations in Drosha mediate vascular abnormalities similar to hereditary hemorrhagic telangiectasia.

The transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-ß or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-ß and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an overrepresentation of rare nonsynonymous variants of DROSHA Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino-directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.

Structural Basis for Activity and Specificity of an Anticoagulant Anti-FXIa Monoclonal Antibody and a Reversal Agent.

Coagulation factor XIa is a candidate target for anticoagulants that better separate antithrombotic efficacy from bleeding risk. We report a co-crystal structure of the FXIa protease domain with DEF, a human monoclonal antibody that blocks FXIa function and prevents thrombosis in animal models without detectable increased bleeding. The light chain of DEF occludes the FXIa S1 subsite and active site, while the heavy chain provides electrostatic interactions with the surface of FXIa. The structure accounts for the specificity of DEF for FXIa over its zymogen and related proteases, its active-site-dependent binding, and its ability to inhibit substrate cleavage. The inactive FXIa protease domain used to obtain the DEF-FXIa crystal structure reversed anticoagulant activity of DEF in plasma and in vivo and the activity of a small-molecule FXIa active-site inhibitor in vitro. DEF and this reversal agent for FXIa active-site inhibitors may help support clinical development of FXIa-targeting anticoagulants.

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis.

Systemic Metronidazole May Not Reduce Posthemorrhoidectomy Pain: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Hemorrhoidectomy is associated with significant postoperative pain. Oral metronidazole has been recommended as an adjunct to improve posthemorrhoidectomy analgesia. OBJECTIVE: This study aimed to evaluate the impact of oral metronidazole on patient-reported pain following hemorrhoidectomy. DATA SOURCES: We conducted a systematic search in the MEDLINE, EMBASE, ISI Web of Science, and Cochrane Central Register of Controlled Trials databases. STUDY SELECTION: Randomized controlled trials examining adults who underwent surgical hemorrhoidectomy were included. Participants in an active intervention group received oral metronidazole postoperatively, and those in a control group received placebo or usual care. Postoperative pain was assessed for at least 3 days postoperatively. INTERVENTION: A random-effects model was used. MAIN OUTCOMES MEASURES: The primary outcome was pain during the first 2 postoperative weeks, measured on a visual analogue scale. The secondary outcome was time to return to normal activities. RESULTS: Patients who received oral metronidazole had significantly lower reported pain scores on postoperative day 1 (standardized mean difference, -0.87 ± 0.44; 95% CI, -1.73 to -0.015; p = 0.046; n = 4) and day 4 (standardized mean difference, -1.43 ± 0.71; 95% CI, -2.83 to -0.037; p = 0.044; n = 3). Metronidazole use was associated with a significantly shorter time to return to normal activities (standardized mean difference, -0.76 ± 0.34; 95% CI, -1.43 to -0.088, p = 0.027). The improvements disappeared in a sensitivity analysis excluding the largest trial with a high risk of bias, and no significance was observed during the remaining postoperative days. LIMITATIONS: The meta-analysis was limited by lack of double blinding, absence of a placebo, and unclear or high risk of bias in a proportion of the included trials. CONCLUSIONS: Although a favorable adverse effect profile supports consideration of oral metronidazole to reduce posthemorrhoidectomy pain, pooled analysis reveals inconsistent results with no pain reduction on most postoperative days. The current recommendation for routine prescription of oral metronidazole should be reevaluated in the absence of additional well-designed trials.

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Platelet and Erythrocyte Sources of S1P Are Redundant for Vascular Development and Homeostasis, but Both Rendered Essential After Plasma S1P Depletion in Anaphylactic Shock.

RATIONALE: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. OBJECTIVE: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. METHODS AND RESULTS: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus-response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P2 receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P1 receptor was dispensable for survival despite its importance for maintaining vascular integrity. CONCLUSIONS: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.

Factor XIa-specific IgG and a reversal agent to probe factor XI function in thrombosis and hemostasis.

Thrombosis is a major cause of morbidity and mortality. Current antithrombotic drugs are not ideal in that they must balance prevention of thrombosis against bleeding risk. Inhibition of coagulation factor XI (FXI) may offer an improvement over existing antithrombotic strategies by preventing some forms of thrombosis with lower bleeding risk. To permit exploration of this hypothesis in humans, we generated and characterized a series of human immunoglobulin Gs (IgGs) that blocked FXIa active-site function but did not bind FXI zymogen or other coagulation proteases. The most potent of these IgGs, C24 and DEF, inhibited clotting in whole human blood and prevented FeCl3-induced carotid artery occlusion in FXI-deficient mice reconstituted with human FXI and in thread-induced venous thrombosis in rabbits at clinically relevant doses. At doses substantially higher than those required for inhibition of intravascular thrombus formation in these models, DEF did not increase cuticle bleeding in rabbits or cause spontaneous bleeding in macaques over a 2-week study. Anticipating the desirability of a reversal agent, we also generated a human IgG that rapidly reversed DEF activity ex vivo in human plasma and in vivo in rabbits. Thus, an active site-directed FXIa-specific antibody can block thrombosis in animal models and, together with the reversal agent, may facilitate exploration of the roles of FXIa in human disease.

Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo.

Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful for imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here, we report the design and characterization of a GFP-based fluorogenic protease reporter, dubbed ZipGFP. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.

Sphingosine 1-phosphate receptor-1 in cardiomyocytes is required for normal cardiac development.

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts via G protein-coupled receptors. The S1P receptor S1P1, encoded by S1pr1, is expressed in developing heart but its roles there remain largely unexplored. Analysis of S1pr1 LacZ knockin embryos revealed ß-galactosidase staining in cardiomyocytes in the septum and in the trabecular layer of hearts collected at 12.5 days post coitus (dpc) and weak staining in the inner aspect of the compact layer at 15.5 dpc and later. Nkx2-5-Cre- and Mlc2a-Cre-mediated conditional knockout of S1pr1 led to ventricular noncompaction and ventricular septal defects at 18.5 dpc and to perinatal lethality in the majority of mutants. Further analysis of Mlc2a-Cre conditional mutants revealed no gross phenotype at 12.5 dpc but absence of the normal increase in the number of cardiomyocytes and the thickness of the compact layer at 13.5 dpc and after. Consistent with relative lack of a compact layer, in situ hybridization at 13.5 dpc revealed expression of trabecular markers extending almost to the epicardium in mutants. Mutant hearts also showed decreased myofibril organization in the compact but not trabecular myocardium at 12.5 dpc. These results suggest that S1P signaling via S1P1 in cardiomyocytes plays a previously unknown and necessary role in heart development in mice.

A mutation in the atrial-specific myosin light chain gene (MYL4) causes familial atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia, is a growing epidemic with substantial morbidity and economic burden. Mechanisms underlying vulnerability to AF remain poorly understood, which contributes to the current lack of highly effective therapies. Recognizing mechanistic subtypes of AF may guide an individualized approach to patient management. Here, we describe a family with a previously unreported syndrome characterized by early-onset AF (age <35 years), conduction disease and signs of a primary atrial myopathy. Phenotypic penetrance was complete in all mutation carriers, although complete disease expressivity appears to be age-dependent. We show that this syndrome is caused by a novel, heterozygous p.Glu11Lys mutation in the atrial-specific myosin light chain gene MYL4. In zebrafish, mutant MYL4 leads to disruption of sarcomeric structure, atrial enlargement and electrical abnormalities associated with human AF. These findings describe the cause of a rare subtype of AF due to a primary, atrial-specific sarcomeric defect.