Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

Comparison of electrogram characteristics in persistent atrial fibrillation.

INTRODUCTION: Multiple analysis techniques evaluate electrograms during atrial fibrillation (AF), but none have been established to guide catheter ablation. This study compares electrogram properties recorded from multiple right (RA) and left atrial (LA) sites. METHODS: Multisite LA/RA mapping (281 ± 176/239 ± 166 sites/patient) was performed in 42 patients (30 males, age 63 ± 9 years) undergoing first (n = 32) or redo-AF ablation (n = 10). All electrogram recordings were visually reviewed and artifactual signals were excluded leaving a total of 21 846 for analysis. Electrogram characteristics evaluated were cycle length (CL), amplitude, Shannon's entropy (ShEn), fractionation interval, dominant frequency, organizational index, and cycle length of most recurrent morphology (CLR ) from morphology recurrence plot analysis. RESULTS: Electrogram characteristics were correlated to each other. All pairwise comparisons were significant (p < .001) except for dominant frequency and CLR (p = .59), and amplitude and dominant frequency (p = .38). Only ShEn and fractionation interval demonstrated a strong negative correlation (r = -.94). All other pairwise comparisons were poor to moderately correlated. The relationships are highly conserved among patients, in the RA versus LA, and in those undergoing initial versus redo ablations. Antiarrhythmic drug therapy did not have a significant effect on electrogram characteristics, except minimum ShEn. Electrogram characteristics associated with ablation outcome were shorter minimum CLR , lower minimum ShEn, and longer mimimum CL. There was minimal overlap between the top 10 sites identified by one electrogram characteristic and the top 10 sites identified by the other 10 characteristics. CONCLUSION: Multiple techniques can be employed for electrogram analysis in AF. In this analysis of eight different electrogram characteristics, seven were poorly to moderately correlated and do not identify similar locations. Only some characteristics were predictive of ablation outcome. Further studies to consider electrogram properties, perhaps in combination, for categorizing and/or mapping AF are warranted.

Association between high-density mapping of atypical atrial flutter, clinical outcomes and healthcare utilization.

BACKGROUND: Success of atypical atrial flutter (AAFL) ablation has historically been limited by difficulty mapping the complex re-entrant circuits involved. While high-density (HD) mapping has become commonplace in clinical practice, there are limited data on outcomes of HD versus non-HD mapping for AAFL ablation. OBJECTIVE: To compare clinical outcomes and healthcare utilization using HD mapping versus non-HD mapping for AAFL ablation. METHODS: Retrospective analysis of all AAFL procedures between 2005 and 2022 at an academic medical center was conducted. Procedures utilizing a 16-electrode HD Grid catheter and Precision mapping system were compared to procedures using prior generation 10-20 electrode spiral catheters and the Velocity system (Abbott, IL). Cox regression models and Poisson regression models were utilized to examine procedural and healthcare utilization outcomes. Models were adjusted for left ventricular ejection fraction, CHA2DS2-VASc, and history of prior ablation. RESULTS: There were 108 patients (62% HD mapping) included in the analysis. Baseline clinical characteristics were similar between groups. Use of HD mapping was associated with a higher rate of AAFL circuit delineation (92.5% vs. 76%; p = .014) and a greater adjusted procedure success rate, defined as non-inducibility at procedure end, (aRR (95% CI) 1.26 (1.02-1.55) p = .035) than non-HD mapping. HD mapping was also associated with a lower rate of ED visits (aIRR (95% CI) 0.32 (0.14-0.71); p = .007) and hospitalizations (aIRR (95% CI) 0.32 (0.14-0.68); p = .004) for AF/AFL/HF through 1 year. While there was a lower rate of recurrent AFL through 1 year among HD mapping cases (aHR (95% CI) 0.60 (0.31-1.16) p = .13), statistical significance was not met likely due to the low sample size and higher rate of ambulatory rhythm monitoring in the HD group (61% vs. 39%, p = .025). CONCLUSION: Compared to non-HD mapping, AAFL ablation with HD mapping is associated with improvements in the ability to define the AAFL circuit, greater procedural success, and a reduction in the number of ED visits and hospitalization for AF/AFL/HF.

Implementation of an intravenous sotalol initiation protocol: Implications for feasibility, safety, and length of stay.

INTRODUCTION: Oral sotalol initiation requires a multiple-day, inpatient admission to monitor for QT prolongation during loading. A 1-day intravenous (IV) sotalol loading protocol was approved by the United States Food and Drug Administration in March 2020, but limited data on clinical use and administration currently exists. This study describes implementation of an IV sotalol protocol within an integrated health system, provides initial efficacy and safety outcomes, and examines length of stay (LOS) compared with oral sotalol initiation. METHODS: IV sotalol was administered according to a prespecified initiation protocol to adult patients with refractory atrial or ventricular arrhythmias. Baseline characteristics, safety and feasibility outcomes, and LOS were compared with patients receiving oral sotalol over a similar time period. RESULTS: From January 2021 to June 2022, a total of 29 patients (average age 66.0 ± 8.6 years, 27.6% women) underwent IV sotalol load and 20 patients (average age 60.4 ± 13.9 years, 65.0% women) underwent oral sotalol load. The load was successfully completed in 22/29 (75.9%) patients receiving IV sotalol and 20/20 (100%) of patients receiving oral sotalol, although 7/20 of the oral sotalol patients (35.0%) required dose reduction. Adverse events interrupting IV sotalol infusion included bradycardia (seven patients, 24.1%) and QT prolongation (three patients, 10.3%). No patients receiving IV or oral sotalol developed sustained ventricular arrhythmias before discharge. LOS for patients completing IV load was 2.6 days shorter (mean 1.0 vs. 3.6, p < .001) compared with LOS with oral load. CONCLUSION: IV sotalol loading has a safety profile that is similar to oral sotalol. It significantly shortens hospital LOS, potentially leading to large cost savings.

Esophageal temperature management during cryoballoon ablation for atrial fibrillation.

INTRODUCTION: Esophageal thermal injury (ETI) is a well-recognized complication of atrial fibrillation (AF) ablation. Previous studies have demonstrated that direct esophageal cooling reduces ETI during radiofrequency AF ablation. The purpose of this study was to evaluate the use of an esophageal warming device to prevent ETI during cryoballoon ablation (CBA) for AF. METHODS: This prospective, double-blinded study enrolled 42 patients with symptomatic AF undergoing CBA. Patients were randomized to the treatment group with esophageal warming (42°C) using recirculated water through a multilumen, silicone tube inserted into the esophagus (EnsoETM®; Attune Medical) (WRM) or the control group with a luminal single-electrode esophageal temperature monitoring probe (LET). Patients underwent upper endoscopy esophagogastroduodenoscopy (EGD) the following day. ETI was classified into four grades. RESULTS: Baseline patient characteristics were similar between groups. Procedural characteristics including number of freezes, total freeze time, early freeze terminations, coldest balloon temperature, procedure duration, posterior wall ablation, and proton pump inhibitor and transesophageal echocardiogram use before procedure were not different between groups. The EGD was completed in 40/42 patients. There was significantly more ETI in the WRM group compared to the LET group (n = 8 [38%] vs. n = 1 [5%], p = 0.02). All ETI lesions were grade 1 (erythema) or 2 (superficial ulceration). Total freeze time in the left inferior pulmonary vein was predictive of ETI (360 vs. 300 s, p = 0.03). CONCLUSION: Use of a luminal heat exchange tube for esophageal warming during CBA for AF was paradoxically associated with a higher risk of ETI.

Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Attenuation of Oxidative Injury With Targeted Expression of NADPH Oxidase 2 Short Hairpin RNA Prevents Onset and Maintenance of Electrical Remodeling in the Canine Atrium: A Novel Gene Therapy Approach to Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K+ current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. METHODS: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. RESULTS: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKACh by a mechanism involving frequency-dependent activation of PKCε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. CONCLUSIONS: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.

Role of t-tubule remodeling on mechanisms of abnormal calcium release during heart failure development in canine ventricle.

The goal of this work was to investigate the role of t-tubule (TT) remodeling in abnormal Ca2+ cycling in ventricular myocytes of failing dog hearts. Heart failure (HF) was induced using rapid right ventricular pacing. Extensive changes in echocardiographic parameters, including left and right ventricular dilation and systolic dysfunction, diastolic dysfunction, elevated left ventricular filling pressures, and abnormal cardiac mechanics, indicated that severe HF developed. TT loss was extensive when measured as the density of total cell volume, derived from three-dimensional confocal image analysis, and significantly increased the distances in the cell interior to closest cell membrane. Changes in Ca2+ transients indicated increases in heterogeneity of Ca2+ release along the cell length. When critical properties of Ca2+ release variability were plotted as a function of TT organization, there was a complex, nonlinear relationship between impaired calcium release and decreasing TT organization below a certain threshold of TT organization leading to increased sensitivity in Ca2+ release below a TT density threshold of 1.5%. The loss of TTs was also associated with a greater incidence of triggered Ca2+ waves during rapid pacing. Finally, virtually all of these observations were replicated by acute detubulation by formamide treatment, indicating an important role of TT remodeling in impaired Ca2+ cycling. We conclude that TT remodeling itself is a major contributor to abnormal Ca2+ cycling in HF, reducing myocardial performance. The loss of TTs is also responsible for a greater incidence of triggered Ca2+ waves that may play a role in ventricular arrhythmias arising in HF.NEW & NOTEWORTHY Three-dimensional analysis of t-tubule density showed t-tubule disruption throughout the whole myocyte in failing dog ventricle. A double-linear relationship between Ca2+ release and t-tubule density displays a steeper slope at t-tubule densities below a threshold value (∼1.5%) above which there is little effect on Ca2+ release (T-tubule reserve). T-tubule loss increases incidence of triggered Ca2+ waves. Chemically induced t-tubule disruption suggests that t-tubule loss alone is a critical component of abnormal Ca2+ cycling in heart failure.

Two RNA Tunnel Inhibitors Bind in Highly Conserved Sites in Dengue Virus NS5 Polymerase: Structural and Functional Studies.

Dengue virus (DENV) NS5 RNA-dependent RNA polymerase (RdRp), an important drug target, synthesizes viral RNA and is essential for viral replication. While a number of allosteric inhibitors have been reported for hepatitis C virus RdRp, few have been described for DENV RdRp. Following a diverse compound screening campaign and a rigorous hit-to-lead flowchart combining biochemical and biophysical approaches, two DENV RdRp nonnucleoside inhibitors were identified and characterized. These inhibitors show low- to high-micromolar inhibition in DENV RNA polymerization and cell-based assays. X-ray crystallography reveals that they bind in the enzyme RNA template tunnel. One compound (NITD-434) induced an allosteric pocket at the junction of the fingers and palm subdomains by displacing residue V603 in motif B. Binding of another compound (NITD-640) ordered the fingers loop preceding the F motif, close to the RNA template entrance. Most of the amino acid residues that interacted with these compounds are highly conserved in flaviviruses. Both sites are important for polymerase de novo initiation and elongation activities and essential for viral replication. This work provides evidence that the RNA tunnel in DENV RdRp offers interesting target sites for inhibition.IMPORTANCE Dengue virus (DENV), an important arthropod-transmitted human pathogen that causes a spectrum of diseases, has spread dramatically worldwide in recent years. Despite extensive efforts, the only commercial vaccine does not provide adequate protection to naive individuals. DENV NS5 polymerase is a promising drug target, as exemplified by the development of successful commercial drugs against hepatitis C virus (HCV) polymerase and HIV-1 reverse transcriptase. High-throughput screening of compound libraries against this enzyme enabled the discovery of inhibitors that induced binding sites in the RNA template channel. Characterizations by biochemical, biophysical, and reverse genetics approaches provide a better understanding of the biological relevance of these allosteric sites and the way forward to design more-potent inhibitors.

Recent advances in gene therapy for atrial fibrillation.

Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments for AF are suboptimal as they are not targeting the molecular mechanisms underlying AF. In this regard, gene therapy is emerging as a promising approach for mechanism-based treatment of AF. In this review, we summarize recent advances and challenges in gene therapy for this important cardiovascular disease.

Use of a novel 4D intracardiac echocardiography catheter to guide interventional electrophysiology procedures.

INTRODUCTION: Standard two-dimensional (2D), phased-array intracardiac echocardiography (ICE) is routinely used to guide interventional electrophysiology (EP) procedures. A novel four-dimensional (4D) ICE catheter (VeriSight Pro, Philips) can obtain 2D and three-dimensional (3D) volumetric images and cine-videos in real-time (4D). The purpose of this study was to determine the early feasibility and safety of this 4D ICE catheter during EP procedures. METHODS: The 4D ICE catheter was placed from the femoral vein in ten patients into various cardiac chambers to guide EP procedures requiring transseptal catheterization, including ablation for atrial fibrillation and left atrial appendage closure. 2D- and 3D-ICE images were acquired in real-time by the electrophysiologist. A dedicated imaging expert performed digital steering to optimize and postprocess 4D images. RESULTS: Eight patients underwent pulmonary vein isolation (cryoballoon in seven patients, pulsed field ablation in one, additional radiofrequency left atrial ablation in one). Two patients underwent left atrial appendage closure. High quality images of cardiac structures, transseptal catheterization equipment, guide sheaths, ablation tools, and closure devices were acquired with the ICE catheter tip positioned in the right atrium, left atrium, pulmonary vein, coronary sinus, right ventricle, and pulmonary artery. There were no complications. CONCLUSION: This is the first experience of a novel deflectable 4D ICE catheter used to guide EP procedures. 4D ICE imaging is safe and allows for acquisition of high-quality 2D and 3D images in real-time. Further use of 4D ICE will be needed to determine its added value for each EP procedure type.

Impact of pre-ablation weight loss on the success of catheter ablation for atrial fibrillation.

INTRODUCTION: Obesity is an established risk factor for recurrent atrial fibrillation (AF) after ablation. The impact of pre-procedure weight changes on freedom from AF (FFAF) after ablation in obese and nonobese patients is unknown. METHODS: A single-center retrospective cohort study of patients undergoing pulmonary vein isolation was performed. Before ablation, all candidates were encouraged to adopt healthy lifestyle habits according to American Heart Association guidelines, including weight loss, by their physician. The primary endpoint was FFAF through 1-year after completion of the 3-month blanking period. RESULTS: Of the 601 patients (68% male; average age 62.1 ± 10.3 years) included in analysis, 234 patients (38.9%) were obese (body mass index ≥ 30) and 315 (52.4%) had paroxysmal AF. FFAF was observed in 420 patients (69.9%) at 15 months. Percent change in weight that occurred during the year before ablation independently predicted FFAF through 15-months in all patients (adjusted odds ratio = 1.17, 95% confidence interval: 1.11-1.23). Subgroup analyses based on paroxysmal vs persistent AF, presence of obesity, and history of prior ablation were performed. Percent change in weight over the year before ablation was independently associated with FFAF in all subgroups except nonobese patients with persistent AF. CONCLUSION: Pre-ablation weight loss was associated with FFAF in both obese and nonobese patients. Further studies are needed to define the optimal approach to weight loss before AF ablation.

Patient-reported outcomes after cryoballoon ablation are equivalent between moderate sedation and general anesthesia.

INTRODUCTION: Moderate sedation (MS) during cryoballoon ablation (CBA) avoids risks of general anesthesia (GA) and improves electrophysiology (EP) lab throughput. However, one barrier to the use of MS is the potential for patient discomfort. The objective of this study was to compare patient-reported outcome measures following CBA for paroxysmal atrial fibrillation (pAF) under MS and GA. METHODS AND RESULTS: Consecutive patients undergoing a first CBA for pAF under GA or MS were prospectively enrolled. The sedation method was assigned based on patient and provider preference, and perceived airway risk. The primary outcomes were quality of recovery (measured using a validated 40 question survey; QoR-40) and likelihood to recommend (LTR) the procedure and sedation method (measured by Likert scale). Secondary outcomes were acute pulmonary vein (PV) isolation rate, procedure, fluoroscopy and ablation times, and complication rates. Forty-seven GA and 53 MS patients were included. The mean age was 64.9 ± 9.4 years and mean CHA2 DS2 -VASc score was 2.0 ± 1.4. QoR-40 scores were 184.6 ± 16.4 for GA and 187.6 ± 10.2 for MS (P = .28). LTR responses were similar between groups. Mean procedure times were 148.2 ± 56.0 minutes for GA and 129.4 ± 31.4 minutes for MS (P = .038). Fluoroscopy and ablation times were similar between groups. A total of 100% (409/409) of PVs were acutely isolated. One hemopericardium occurred in the MS group requiring pericardiocentesis. CONCLUSION: MS for CBA offers an alternative to GA that is safe and well-tolerated by patients with comparable success rates and improved EP lab throughput.

Repeat pulmonary vein isolation with or without FIRM-guided ablation for recurrent atrial fibrillation with pulmonary vein reconnection.

INTRODUCTION: Focal impulse and rotor modulation (FIRM) ablation can be used to target nonpulmonary vein (PV) sources of atrial fibrillation (AF). No published studies have compared freedom from atrial fibrillation (FFAF) after pulmonary vein reisolation (PVRI) plus FIRM to PVRI alone in patients with reconnected PVs undergoing repeat ablation. METHODS: A 3:1 matched retrospective cohort study was performed on 21 patients with recurrent AF and PV reconnection who underwent PVRI plus FIRM-guided ablation and 63 patients with recurrent AF treated with PVRI alone at a single institution. All patients in the PVRI-alone cohort had cryoballoon PVRI at the time of repeat ablation without additional lesion sets for AF. Cases were matched based on the type of AF (paroxysmal vs nonparoxysmal), left atrial diameter (±4 mm), left ventricular ejection fraction (±10%), duration of AF (±18 months), and age (±5 years). The primary endpoint was FFAF after a 3-month blanking period. RESULTS: Out of 53 total FIRM cases performed at Northwestern Memorial Hospital between 2015 and 2017, 21 patients had PVRI plus FIRM for recurrent AF with PV reconnection. These patients had an average of 3.3 ± 2.1 rotors (60% left atrial) ablated. Over a median follow-up time of 24.7 months (interquartile range, 13-36 months), patients in the PVRI-alone cohort demonstrated a higher rate of FFAF (n = 35; 55.6%) than patients in the PVRI plus FIRM-guided ablation cohort (n = 7; 33.3%) (logrank P = .049). CONCLUSION: In patients undergoing repeat ablation for AF with PV reconnection, PVRI plus FIRM did not increase FFAF compared to PVRI alone.

Patient characteristics as predictors of recurrence of atrial fibrillation following cryoballoon ablation.

BACKGROUND: While several studies have evaluated predictors for atrial fibrillation (AF) recurrence following catheter ablation, there are limited data specific to cryoballoon ablation (CBA). METHODS: We analyzed a prospective registry of patients at a single institution who underwent CBA. Recurrence of AF (RAF) was defined as recurrence of AF by 12-month follow-up, excluding the 3-month blanking period. Univariate analysis was performed to evaluate predictors of RAF. Receiver operating characteristic analysis was used to compare and evaluate the performance of various risk scores for discriminating risk of RAF. RESULTS: There were 542 patients included in the analysis with mean age 61.3 ± 10.6 years, 67.9% male, and 51.6% paroxysmal AF (PAF). Overall, only left atrial diameter (LAD) > 40 mm and ERAF (early recurrence of AF within 0-3 month blanking period) were significant predictors of RAF. In the PAF specific subgroup, LAD > 40 mm, AF duration > 12 months, prior stroke or transient ischemic attack, ERAF, and having previously failed an antiarrhythmic drug were significant predictors of RAF. In persistent AF (PeAF) subgroup, obstructive sleep apnea (OSA) and ERAF were significant predictors of RAF. Out of clinical risk scores tested, BASEAF2 had the highest performance with area under the curve of 0.646 (95% confidence interval [0.548, 0.708]; P < .01). CONCLUSIONS: In this single-center retrospective study of CBA, we found only LAD > 40 mm and ERAF to be predictors of RAF. We identified OSA as a potential targetable risk factor in PeAF patients undergoing CBA. Out of risk scores tested for discriminating risk of RAF, BASEAF2 had the best performance.

Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 µM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Constitutive Expression of a Dominant-Negative TGF-ß Type II Receptor in the Posterior Left Atrium Leads to Beneficial Remodeling of Atrial Fibrillation Substrate.

RATIONALE: Fibrosis is an important structural contributor to formation of atrial fibrillation (AF) substrate in heart failure. Transforming growth factor-ß (TGF-ß) signaling is thought to be intricately involved in creation of atrial fibrosis. OBJECTIVE: We hypothesized that gene-based expression of dominant-negative type II TGF-ß receptor (TGF-ß-RII-DN) in the posterior left atrium in a canine heart failure model will sufficiently attenuate fibrosis-induced changes in atrial conduction and restitution to decrease AF. Because AF electrograms are thought to reflect AF substrate, we further hypothesized that TGF-ß-RII-DN would lead to increased fractionation and decreased organization of AF electrograms. METHODS AND RESULTS: Twenty-one dogs underwent injection+electroporation in the posterior left atrium of plasmid expressing a dominant-negative TGF-ß type II receptor (pUBc-TGFß-DN-RII; n=9) or control vector (pUBc-LacZ; n=12), followed by 3 to 4 weeks of right ventricular tachypacing (240 bpm). Compared with controls, dogs treated with pUBC-TGFß-DN-RII demonstrated an attenuated increase in conduction inhomogeneity, flattening of restitution slope and decreased duration of induced AF, with AF electrograms being more fractionated and less organized in pUBc-TGFß-DN-RII versus pUBc-LacZ dogs. Tissue analysis revealed a significant decrease in replacement/interstitial fibrosis, p-SMAD2/3 and p-ERK1/2. CONCLUSIONS: Targeted gene-based reduction of TGF-ß signaling in the posterior left atrium-with resulting decrease in replacement fibrosis-led to beneficial remodeling of both conduction and restitution characteristics of the posterior left atrium, translating into a decrease in AF and increased complexity of AF electrograms. In addition to providing mechanistic insights, this data may have important diagnostic and therapeutic implications for AF.

Left atrial volume predicts atrial fibrillation recurrence after radiofrequency ablation: a meta-analysis.

Aims: Left atrial (LA) diameter is a predictor of atrial fibrillation (AF) recurrence following radiofrequency catheter ablation (RFA). However, LA volume (LAV) is more accurate in assessing LA size. Studies evaluating LAV as a predictor of AF recurrence are contradictory; therefore, we performed a meta-analysis to assess whether LAV is an independent predictor of AF recurrence following RFA. Methods and results: All studies reporting LAV/LAV index (LAVi) as a predictor of AF recurrence following RFA were included. For studies reporting mean LAV/ LAVi in patients with and without AF recurrence, standard difference in means (SDM) and standard errors were calculated, and combined using meta-analytical techniques. For studies reporting adjusted odds ratio (OR) for AF recurrence based on LAV/LAVi, log ORs were combined using generic inverse variance. Twenty one studies (3822 subjects) were included. Meta-analysis of 11 studies (1559 subjects) reporting LAV, showed that patients with AF recurrence had a higher mean LA volume compared to patients with no recurrence (SDM 0.801; CI 0.387-1.216). Data from 9 studies (1425 subjects) comparing LAVi showed that, patients with AF recurrence had a higher mean LAVi compared to patients with no recurrence (SDM-0.596; CI 0.305-0.888). Thirteen studies (2886 patients) reporting ORs for AF recurrence based on LAV/ LAVi, showed that LAV/LAVi was independently predictive of AF recurrence post-RFA (OR-1.032, CI- 1.012-1.052). Conclusions: Patients with AF recurrence following RFA have a higher mean LAV/LAVi compared to patients with no recurrence. Large LAV/LAVi increases the odds of AF recurrence post RFA.

A Conserved Pocket in the Dengue Virus Polymerase Identified through Fragment-based Screening.

We performed a fragment screen on the dengue virus serotype 3 RNA-dependent RNA polymerase using x-ray crystallography. A screen of 1,400 fragments in pools of eight identified a single hit that bound in a novel pocket in the protein. This pocket is located in the polymerase palm subdomain and conserved across the four serotypes of dengue virus. The compound binds to the polymerase in solution as evidenced by surface plasmon resonance and isothermal titration calorimetry analyses. Related compounds where a phenyl is replaced by a thiophene show higher affinity binding, indicating the potential for rational design. Importantly, inhibition of enzyme activity correlated with the binding affinity, showing that the pocket is functionally important for polymerase activity. This fragment is an excellent starting point for optimization through rational structure-based design.