Development of an atrial transseptal puncture procedure in horses to access the left heart: An ultrasound-guided jugular vein and transhepatic approach.

BACKGROUND: Radiofrequency ablation has been successfully applied to treat right atrial arrhythmias in horses. Ablation of left-sided arrhythmias requires a retrograde transarterial approach which is complicated. In human medicine, the left atrium is accessed through transseptal puncture (TSP) of the fossa ovalis (FO) using a caudal approach via the femoral vein. OBJECTIVES: To develop a zero fluoroscopy TSP technique for horses using a jugular vein (cranial) and transhepatic (caudal) approach. STUDY DESIGN: In vivo experimental study. METHODS: Transseptal puncture was performed in 18 horses admitted for euthanasia and donated for scientific research under general anaesthesia: using a jugular vein approach (10 horses), a transhepatic approach (2 horses) or both (6 horses). Radiofrequency energy was applied on a guidewire to perforate the FO and allow sheath advancement under intracardiac and transthoracic echocardiographic guidance. Puncture lesions were inspected post-mortem. RESULTS: Transseptal puncture was successful in 17/18 horses, of which 15/16 jugular vein approaches and 5/8 transhepatic approaches. Failure was due to technical malfunction, inability to advance the guidewire toward the heart and inability to advance the sheath through the FO. Intracardiac echocardiography was essential to safely guide the puncture process. Atrial arrhythmias caused by the TSP occurred in 13/18 horses. Puncture lesions were found in the right atrium in the FO region, and left atrium ventral to pulmonary vein ostium III. MAIN LIMITATIONS: Because in several horses two approaches were tested consecutively, it cannot be excluded that the second TSP was performed at the previous puncture site. Due to the developmental nature of the study the approaches were not randomised and did not allow comparison. CONCLUSION: Transseptal puncture is feasible in horses using ultrasound guidance and allows for electrophysiological exploration of the left heart. Further studies are needed to evaluate post-operative follow-up.

Morphological evidence of a potential arrhythmogenic substrate in the caudal and cranial vena cava in horses.

BACKGROUND: Three-dimensional electro-anatomical mapping, previously performed in horses with atrial arrhythmias, has demonstrated the medial region of the caudal vena cava (CaVC), 1-8 cm caudal to the fossa ovalis, as an anatomical predilection site for atrial tachycardia associated with areas of slow conduction and conduction block. Slow conduction has also been recorded in the cranial vena cava (CrVC). OBJECTIVES: To investigate the morphological characteristics of the myocardial sleeves (MS) in the CaVC and CrVC, in order to identify a potential substrate of right sided atrial arrhythmias. STUDY DESIGN: Cross sectional. METHODS: Post-mortem dissection of 37 hearts from adult warmblood horses without known cardiovascular disease. Macroscopic examination of the myocardial distribution, evaluated the MS area, length, width, and shape in the CaVC and the CrVC. At least 2 samples from each vena cava MS were histologically examined using Masson's trichrome staining. RESULTS: Myocardial sleeves into the medial CaVC and into the CrVC were observed in all horses and showed variations in distribution, shape, and size between horses. Their mean ± standard deviation length from the limbus into the CaVC reached 5.7 ± 1.0 cm (maximum 8.3 cm), and from the azygos vein into the CrVC 5.3 ± 1.6 cm (maximum 8.6 cm). Myocardium-free islands were observed in the CaVC and CrVC in 30% and 6% of horses, respectively. Histologically, MS showed a non-uniform myocardial fibre arrangement, with presence of fibroadipose tissue, features known to result in slow conduction and pro-arrhythmia. MAIN LIMITATIONS: Study only included horses without history of atrial arrhythmia. CONCLUSIONS: Myocardial sleeves are present in both CaVC and CrVC, showing anatomical variations between horses. Tissue characteristics known to favour re-entry were identified indicating that these venae cavae MS are a potential substrate for atrial tachyarrhythmias and a target for treatment by ablation.

CONTEXTE: La modélisation électro-anatomique tridimensionnelle, réalisée auparavant chez des chevaux souffrant d'arythmies atriales, a démontré que la région médiale de la veine cave caudale (CaVC), 1-8 cm caudalement à la fossae ovalis, représente un site anatomique de prédilection pour la tachycardie auriculaire associée à des zones de conduction ralentie et des blocs de conduction. Une conduction ralentie a aussi été enregistrée dans la veine cave crâniale (CrVC). OBJECTIFS: Investiguer les caractéristiques morphologiques des manchons myocardiques (Myocardial sleeve;MS) dans les CAVC et CrVC, afin d'identifier un substrat potentiel d'arythmies atriales du côté droit du cœur. TYPE D'ÉTUDE: Étude transversale. MÉTHODES: Dissection post-mortem de 37 cœurs de chevaux à sang chaud adultes sans historique de maladie cardiovasculaire. Lors de l'examen macroscopique de la distribution myocardique, la surface des MS, leur longueur, largeur et forme ont été évaluées dans les CAVC et CrVC. Les MS ont été examinées microscopiquement dans au moins 2 échantillons de chaque veine cave, en utilisant la coloration de Masson Trichrome. RÉSULTATS: Les MS à l'aspect médial de la CaVC et à l'intérieur de la CrVC ont été inspectées chez tous les chevaux et ont montré des variations de distribution, forme et grandeur entre chevaux. Leur moyenne de longueur ± déviation standard du limbe de la fossae ovalis dans la CaVC était de 5.7 ± 1.0 cm (maximum 8.3 cm) et de la veine azygos dans la CrVC, de 5.3 ± 1.6 cm (maximum 8.6 cm). Des Îlots dénudés de myocarde ont été observé dans la CaVC et CrVC dans 30% et 6% des chevaux respectivement. À l'histologie, les MS ont montré des fibres myocardiques organisées de façon non-uniforme, avec présence de tissue fibroadipeux, caractéristiques connues pour entraîner une conduction lente et favoriser l'arythmie. LIMITES PRINCIPALES: Étude incluant seulement des chevaux sans historique d'arythmie atriale. CONCLUSION: Les MS sont présentes dans les CaVC et CrVC, démontrant les variations anatomiques entre chevaux. Des caractéristiques tissulaires connues pour favoriser une conduction ralentie ont été identifiés indiquant que les MS représentent un substrat potentiel des tachyarythmies atriales et une cible pour un traitement par ablation.

Successful caudal vena cava and pulmonary vein isolation in healthy horses using 3D electro-anatomical mapping and a contact force-guided ablation system.

BACKGROUND: Recently, treatment of equine atrial tachycardia by three-dimensional electro-anatomical mapping (3D EAM) and radiofrequency catheter ablation (RFCA) has been described. Myocardial sleeves in the caudal vena cava and pulmonary veins are a potential trigger for initiation and perpetuation of atrial tachycardia and atrial fibrillation in the horse. Isolation of these myocardial sleeves by RFCA may be an effective treatment for these arrhythmias. OBJECTIVES: To describe the feasibility of 3D EAM and RFCA to isolate caudal vena cava and pulmonary veins in adult horses using 3D mapping and a contact force (CF)-guided ablation system. STUDY DESIGN: In vivo experiments. METHODS: 3D EAM and RFCA was performed in five horses without cardiovascular disease under general anaesthesia, using the CF-guided system CARTO®3. Point-by-point RFCA aimed for isolation of caudal vena cava and pulmonary veins. Radiofrequency energy was delivered in power-controlled mode with a target power of 45 W, CF between 10 and 15 g and 30 mL/min irrigation rate, until an ablation-index of 450-500 was reached. RESULTS: In the right atrium, myocardial sleeves of the caudal vena cava were isolated (n = 5). In the left atrium, isolation of ostium II (n = 3), ostium III (n = 1) and ostium I, II and III en bloc (n = 1) was performed. Successful isolation was confirmed by entrance and exit block. MAIN LIMITATIONS: Horses were euthanised at the end of the procedure, so long term effects such as potential reconnection of isolated veins could not be studied. CONCLUSIONS: This is the first description of 3D EAM and RFCA with CARTO®3 in horses, thereby showing the technical feasibility and successful caudal vena cava and pulmonary vein isolation. CF measurement allowed monitoring of catheter-tissue contact, resulting in efficient acute lesion creation as confirmed by entrance and exit block. This is a promising treatment for cardiac arrhythmias in horses.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation.

Crosstalk between metabolism and stress-responsive signaling is essential to maintaining cellular homeostasis. One way this crosstalk is achieved is through the covalent modification of proteins by endogenous, reactive metabolites that regulate the activity of key stress-responsive transcription factors such as NRF2. Several metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 regulatory protein KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolic pathways to NRF2 activation. We found that succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 transcriptional signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Optimization of 3-aminotetrahydrothiophene 1,1-dioxides with improved potency and efficacy as non-electrophilic antioxidant response element (ARE) activators.

Activating NRF2-driven transcription with non-electrophilic small molecules represents an attractive strategy to therapeutically target disease states associated with oxidative stress and inflammation. In this study, we describe a campaign to optimize the potency and efficacy of a previously identified bis-sulfone based non-electrophilic ARE activator 2. This work identifies the efficacious analog 17, a compound with a non-cytotoxic profile in IMR32 cells, as well as ARE activators 18 and 22, analogs with improved cellular potency. In silico drug-likeness prediction suggested the optimized bis-sulfones 17, 18, and 22 will likely be of pharmacological utility.

Circadian disruption enhances HSF1 signaling and tumorigenesis in Kras-driven lung cancer.

Disrupted circadian rhythmicity is a prominent feature of modern society and has been designated as a probable carcinogen by the World Health Organization. However, the biological mechanisms that connect circadian disruption and cancer risk remain largely undefined. We demonstrate that exposure to chronic circadian disruption [chronic jetlag (CJL)] increases tumor burden in a mouse model of KRAS-driven lung cancer. Molecular characterization of tumors and tumor-bearing lung tissues revealed that CJL enhances the expression of heat shock factor 1 (HSF1) target genes. Consistently, exposure to CJL disrupted the highly rhythmic nuclear trafficking of HSF1 in the lung, resulting in an enhanced accumulation of HSF1 in the nucleus. HSF1 has been shown to promote tumorigenesis in other systems, and we find that pharmacological or genetic inhibition of HSF1 reduces the growth of KRAS-mutant human lung cancer cells. These findings implicate HSF1 as a molecular link between circadian disruption and enhanced tumorigenesis.

CRY2 missense mutations suppress P53 and enhance cell growth.

Disruption of circadian rhythms increases the risk of several types of cancer. Mammalian cryptochromes (CRY1 and CRY2) are circadian transcriptional repressors that are related to DNA-repair enzymes. While CRYs lack DNA-repair activity, they modulate the transcriptional response to DNA damage, and CRY2 can promote SKP1 cullin 1-F-box (SCF)FBXL3-mediated ubiquitination of c-MYC and other targets. Here, we characterize five mutations in CRY2 observed in human cancers in The Cancer Genome Atlas. We demonstrate that two orthologous mutations of mouse CRY2 (D325H and S510L) accelerate the growth of primary mouse fibroblasts expressing high levels of c-MYC. Neither mutant affects steady-state levels of overexpressed c-MYC, and they have divergent impacts on circadian rhythms and on the ability of CRY2 to interact with SCFFBXL3 Unexpectedly, stable expression of either CRY2 D325H or of CRY2 S510L robustly suppresses P53 target-gene expression, suggesting that this may be a primary mechanism by which they influence cell growth.

YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.

Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxides as non-electrophilic antioxidant response element (ARE) activators.

The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.

Defining the Functional Targets of Cap'n'collar Transcription Factors NRF1, NRF2, and NRF3.

The NRF transcription factors NRF1, NRF2, and NRF3, are a subset of Cap'n'collar transcriptional regulators which modulate the expression of genes harboring antioxidant-response element (ARE) sequences within their genomic loci. Despite the emerging physiological importance of NRF family members, the repertoire of their genetic targets remains incompletely defined. Here we use RNA-sequencing-based transcriptional profiling and quantitative proteomics to delineate the overlapping and differential genetic programs effected by the three NRF transcription factors. We then create consensus target gene sets regulated by NRF1, NRF2, and NRF3 and define the integrity of these gene sets for probing NRF activity in mammalian cell culture and human tissues. Together, our data provide a quantitative assessment of how NRF family members sculpt proteomes and transcriptomes, providing a framework to understand the critical physiological importance of NRF transcription factors and to establish pharmacologic approaches for therapeutically activating these transcriptional programs in disease.

Small-Molecule Stimulators of NRF1 Transcriptional Activity.

The transcription factor nuclear factor erythroid 2-related factor 1 (NRF1) maintains proteostasis and promotes cellular resilience by stimulating the transcription of proteasomal subunits and a host of protective enzymes. Although NRF1 activation would likely be beneficial in a number of disease states, information regarding its ligandability and upstream regulation are lacking. Herein we report a high-throughput chemical screen that identified selective stimulators of NRF1-driven transcription, including unannotated inhibitors of the ubiquitin proteasome system (UPS) as well as two non-UPS-targeted compounds that synergistically activate NRF1 in the context of submaximal UPS inhibition. This work introduces a suite of tool molecules to study the NRF1 transcriptional response and to uncover the druggable components governing NRF1 activity in cells.

A list of fish species that are potentially exposed to pesticides in edge-of-field water bodies in the European Union--a first step towards identifying vulnerable representatives for risk assessment.

Surrogate species are used in standard toxicity tests for the environmental risk assessment of chemicals. Test results are then extrapolated to the situation in the field, which is often associated with a large degree of uncertainty. Since a vulnerable species in the field is not only characterised by its intrinsic sensitivity to a stressor but also by its potential for exposure and its population resilience, the identification of focal species based on these three components of vulnerability is needed for a more ecologically relevant risk assessment. This study listed European fish species that are susceptible to pesticide exposure in the field and thus achieved the first step towards identifying focal species for the risk assessment of pesticides for fish in Europe. A step-wise filtering approach was applied to list freshwater fish species that are native to Europe and widespread in the European Union, which inhabit streams, ditches or ponds in agricultural landscapes and therefore, are at an elevated risk of being exposed to pesticides. Out of the 579 fish species occurring in European freshwater, 27 species met the filtering criteria. The resulting list was verified based on monitoring studies that were conducted in agricultural landscapes over the past 20 years. Focal fish species that can be used for a more ecologically relevant environmental risk assessment of pesticides in Europe can be identified from the produced list of species by further assessing their ecological (life history and dispersal characteristics) and intrinsic sensitivities.