Prevalence and ablation of different anatomic locations of atrial tachicardias in the electrophysiology lab of a single large center

Funding Acknowledgements Type of funding sources: None. Introduction Focal atrial tachycardias (ATs) can arise from several different anatomic regions both in the right atrium (RA) and left atrium (LA). The prevalence of focal atrial tachycardia is not well known. A European study of young males applying for pilot licenses demonstrated that 0.34% had asymptomatic atrial tachycardia and 0.46% had symptomatic atrial tachycardia1. It is well-recognized that these foci do not occur randomly throughout the atria but tend to cluster at characteristic anatomic locations. However, the distribution of these sites in the total amount of ATs is not well recognized. Purpose The objective of this study was to determine the prevalence of different anatomic locations of ATs in the electrophysiology lab of a single large center (300 ablations per year) and to verify the site of ablation. Methods We collected 150 consecutive patients submitted to catheter ablation of ATs between January 2010 to December 2020. Anatomic localization of the atrial focus was performed during tachicardia by analysis of endocardial activation sequence. Results The distribution of the different anatomic locations is rappresented in the figure. In 134 patients (89%) ATs were localized in the RA. In the RA, these foci mainly occured along the crista terminalis (32%), the perinodal region (22,6%), the anterior right sided septum, near the foramen ovale (9,3%), the posterior right-sided septum (8%), the ostium of the coronary sinus (5,3%), the tricuspid annulus (5,3%), the superior vena cava (4,6%), the infero-lateral wall (2,5%), the right atrial appendage (0,6%) and the cavotricuspid histhmus (0,6%). In the LA (11%), foci occur predominantly at the pulmonary vein ostia (5,2%) and less commonly at the mitral annulus (2,5%), the left sided septum (0,6%), the appendage ridge (0,6%), the roof (0,6%) and the anterior wall (0,6%). For each location of AT, the ablation was performed at the earliest activation site, but about the perinodal ATs, the ablation was performed at the non coronary sinus of Valsalva of the aortic root, regardless the earliest activation site, for avoiding to create damages to the atrio-ventricular (AV) conduction system. Conclusions ATs mainly arise from the RA. Crista terminalis is the most common site but the perinodal region is the second more frequent site. The ablation of ATs from the perinodal region is challenging for the risk of damages to the AV conduction system. The relatively low prevalence of ATs arising from the pulmonary veins could be explained for the likely degeneration of these ATs in atrial fibrillation. Abstract Figure.

The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability

Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.

“It’s not even the leaders out here who have any say at all in how long they’re gonna have to wait”: A study of waiting time, power, and acceptance

Waiting time is an exercise of power. When citizens such as welfare clients, asylum seekers, or inmates encounter the state, they experience lengthy waiting time. Their cases are often delayed; their appointments are postponed or canceled. This creates uncertainty, and studies on the lived experiences of waiting time show that this makes citizens accept that they have to wait. In this study, I argue that citizens accept that they have to wait because they believe that frontline workers have no power to reduce waiting time. I explore this through an ethnographic study of citizens on social assistance at an activation site in Denmark where they perform labor to receive their benefits. The study makes three contributions to the existing scholarship on waiting time. First, it combines observations of both frontline workers’ decisions about waiting time and how citizens experience these decisions. I explore how the frontline workers make citizens wait to perform labor and leave at the end of the day. The length of waiting changes daily, and citizens are rarely informed about how long they have to wait. I observe that citizens accept the waiting time even though they associate it with frustration, demotivation, and uncertainty. Second, what engenders this acceptance is the frontline workers’ tendency to deflect “blame away” from their power to impose waiting time. When they justify their decisions, they deflect blame either “upwards” (toward the rules or their management) or “downwards” (toward troublesome citizens). Third, the study shows that waiting time do not only reflect structural asymmetries between the citizens and the frontline workers. Citizens accept that they have to wait, because they build trust with the frontline workers and believe that the reduction of waiting time is beyond their control. This shows that the power of waiting time is also relationally produced.

P955Non-invasive mapping: what is the minimal number of electrodes needed to obtain a good spatial resolution of the activation map?

Introduction and aims The 12-lead ECG is highly inaccurate for localization of the site of origin of supraventricular and ventricular arrhythmias. Non-invasive mapping systems (ECGI) based on a high number of electrodes recording the electrical activity on the surface of the torso have already proven good accuracy for mapping different arrhythmic substrates. The aim of this study was to assess what is the minimal number of leads needed to obtain a precise mapping with the ECGI. Methods This study enrolled 14 patients (9 male, median age 50 (44-58) years) referred to our center for catheter ablation of premature ventricular contractions (PVC). Patients underwent pre-procedural ECGI using the epicardial and endocardial mapping system . This system uses the DICOM images from contrast computed tomography of the heart and up to 28 adhesive electrodes with 8 leads each, adding up to 224 body-surface leads. All patients underwent invasive electroanatomical mapping and ablation with the magnetic navigation system. We analysed the number of recording leads used to construct the non-invasive activation map of the PVCs and the accuracy and the spatial resolution of the map when comparing to the invasive map. We then reprocessed the exam, using progressively less leads until we only left the leads placed in the standard 12 lead ECG positions and evaluated the concordance with the invasive map as well as the spatial resolution. We considered an earliest activation site (EAS) area of 1 cm2 a good spatial resolution and using a ROC curve we calculated the minimal number of leads necessary to obtain a good spatial resolution. Results The median number of electrodes used for the initial map was 170 (138-177). Concordance between non-invasive and invasive site of origin occurred in 11 out of 14 patients. The results are presented in the Table. The minimal number of electrodes to have a good spatial resolution was 100. The area of EAS was significantly lower when using more than 100 leads, respectively 0.65 (0.5-1) cm2 versus 3 (1.6-5) cm2, p < 0.001. Conclusions The minimal number of leads to achieve a good spatial resolution was high. Reducing the number of leads resulted in a significant decrease in spatial resolution and a lower concordance rate. ECGI data Number of adhesive electrodes Median number of leads Amycard/Carto concordance Median area of EAS in the ECGI (cm2) Maximal nº electrodes 170 (138-177) 11/14 0.64 (0.5-0.9) 12 electrodes 76 (61-80) 11/14 1.6 (1.4-2.6) 6 electrodes (2 Ant, 2 Lat ,2 Post) 38 (32-44) 9/14 4.3 (3.2-5.4) 12 leads 12 0/14 - Ant anterior; Lat: lateral; Post: posterior; EAS: early activation site. Abstract Figure. Area of EAS according to the N of leads

A48 COLITIS FAVORS THE EXPANSION OF BACTERIA THAT ACTIVATE PAR2 AMPLIFYING INFLAMMATORY RESPONSE

Background Proteolytic imbalance has been described in patients with inflammatory bowel disease (IBD) and in different models of experimental colitis. Although the proteases reported to be increased are mainly from the host, the role of bacterial proteases has recently emerged, as they can promote inflammation, in part, through activation of Protease-activated receptors (PARs). PAR2 deficient mice are resistant to inflammation and PAR2 activation affects multiple aspects of the tissue response to injury. However, PAR2 communicates with other receptors such as toll-like and other PARs, which are important in multiple immune signaling pathways. Thus, the direct implication of PAR2 in colitis, and specifically the activation of the external domain by microbial proteases, remains unclear. Aims To study the role of PAR2 and bacterial proteases in experimental colitis. Methods C57BL/6 and protease-resistant PAR2 (R38E-PAR2) mice, in which activation site of PAR2 is missing, were given 3.5% dextran sodium sulfate (DSS) in drinking water for 5 days followed by 2 days of water. Control C57BL/6 (wild-type) and R38E-PAR2 mice received only water. Percent weight change was evaluated along the study. Fecal microbiota (16S Ilumina), expression of proinflammatory genes (Nanostring), gut permeability (Ussing chamber), proteolytic activities (colorimetric assay) and bacterial translocation (plating) were measured in all mice at sacrifice. Inflammation was determined by analyzing stool consistency, fecal blood and by microscopic scores (Cooper score). Bacteria with proteolytic activity were isolated using agar-media with protein as main nutrient and bacterial capacity to release the external domain of PAR2 was tested in cells harbouring luciferase at the PAR2 N terminus. Results Wild-type, but not R38E-PAR2 mice, had increased expression of several pro-inflammatory genes, such as tnf, map3k and tlr, gut dysfunction and increased intestinal permeability, increased bacterial translocation into spleen and altered microbiota profiles following DSS treatment. DSS induced colitis in both mouse strains, but clinical and microscopic scores were significantly lower in R38E-PAR2 compared with wild-type mice. DSS increased the abundance of opportunistic pathogens such as Enterococcus and Staphylococcus only in wild-type mice. Proteolytic phenotype of these bacteria, and their capacity to cleave the external domain of PAR2, was confirmed. Conclusions Mice lacking the activation site of PAR2 are protected from DSS-induced colitis. DSS treatment leads to expansion of bacteria releasing active proteases, which may mediate injury through PAR2. These results suggest that intestinal injury promotes microbial proteolytic imbalance which in turn, exacerbates inflammation. Funding Agencies CAG, CCC, CIHR

Clinical Study Start-Up: Overview of the Process and Expected Challenges

Start-up phase is the base of any project at any type of businesses, and CROs are not an exception.Initially, the emphasis was concentrated on conducting the study, with little attention paid to study start-ups (SSUs), but as stakeholders become more and more aware that the effectiveness of SSU processes is directly related to shorter clinical periods, interest in this opportunity is growing. SSU covers a lot of activities starting from sites identification and completing the enrollment of the first patient - in between this includes site selection and pre-study visits, trial documents submission to competent authorities, contract and budget execution,vendors approval and setup,site activation,site initiation visits and, at last, enrolling the first patient. Each step has multiple components and requires utmost care and attention to minimize the likelihood of study delivery delays from the beginning.

Electrostatically-directed Pd-catalysis in combination with C–H activation: site-selective coupling of remote chlorides with fluoroarenes and fluoroheteroarenes

Electrostatically-directed oxidative addition is compatible with a subsequent C–H activation step, enabling site-selective coupling of remote chlorides with fluoroarenes and fluoroheteroarenes.

Identification of a Heme Activation Site on the MD-2/TLR4 Complex

Lipopolysaccharide (LPS), the first-identified TLR4 agonist, binds myeloid differentiation factor-2 (MD-2) in association with TLR4 to initiate TLR4 signaling. LPS binds to a large hydrophobic pocket in MD-2 and directly bridges the MD-2/TLR4 heterodimer. The MD-2/TLR4 complex also recognizes a diverse number of endogenous molecules released from injured cells called damage-associated molecular patterns or DAMPs. One such DAMP is heme. Large amounts of heme can be released intravascularly by trauma, sepsis, malaria and red blood cell disorders such as sickle cell disease (SCD). Recent studies underscore the importance of heme-mediated MD-2/TLR4 activation in inflammation, vessel occlusion, lethality and pulmonary injury in SCD. Therefore, we examined human MD-2 for potential heme activation sites. Recombinant MD-2 (rMD-2) was produced by transfecting Chinese hamster ovary (CHO) cells with human MD-2 plasmids. After 72 hours, Western blots of the CHO-conditioned media demonstrated soluble rMD-2 was present. Heme was shown to bind rMD-2 using pull-down assays utilizing heme-agarose or biotin-heme with streptavidin-agarose coupled with MD-2 Western blots of the pellet. These pull-down assays of rMD2 were inhibited by excess heme, indicating specific binding of heme to rMD-2. UV/visible scanning spectroscopy (250 - 550 nm) of purified rMD-2 in the presence or absence of heme, confirmed specific rMD-2-heme binding. In silico analyses combining both structure and sequence-based methods, identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44 (Figure 1). To determine whether MD-2 mutations at these two sites affect heme-MD-2/TLR4 signaling, HEK293 cells were transfected with plasmids encoding human MD-2, TLR4, CD14 and an NF-κB luciferase reporter. After 24 hours, transfected cells were stimulated with heme (10 μM) or LPS (10 ng/ml) for 6 hours and NF-κB luciferase reporter activity was measured. Heme or LPS treatment elicited robust luciferase activity. The addition of both heme and LPS had an additive effect on NF-κB luciferase activity. Absence of an MD-2, TLR4 or CD14 plasmid abolished NF-κB luciferase reporter responses to heme and/or LPS. When plasmids encoding MD-2 point mutants W23A or Y34A were introduced into MD-2, heme-induced NF-κB luciferase activity was inhibited 91-92% compared to WT-MD-2. The S33A MD-2 mutant stimulated NF-κB luciferase activity by 40%. NF-κB activation by LPS was marginally affected by the same mutants. Biotin-heme/streptavidin-agarose pulled down 68% less W23A mutant MD-2 and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the other potential heme binding site, heme-induced NF-κB luciferase activity was increased in mutants Y36A (120%), C37A (121%) and I44A (230%) compared to WT-MD-2. These data suggest that amino acids W23 and Y34 on MD-2 are specific for heme binding and TLR4 signaling. This heme activation site was targeted for potential inhibitors using virtual screening. The virtual screen identified 60 potential inhibitors for screening in heme-stimulated primary human umbilical vein endothelial cells (HUVEC) and a human U-937 monocyte cell line. Four of these molecules inhibited Weibel-Palade body P-selectin and von Willebrand factor expression in HUVEC and IL-8 secretion by U-937 cells stimulated with heme. We conclude that heme activates MD-2/TLR4 signaling at residues W23 and Y34 on MD-2, which might be a drugable target in SCD and other hemolytic diseases. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.