Contribution of circulating Mfge8 to human T2DM and cardiovascular disease.

MFGE8 is a major exosome (EV) protein known to mediate inflammation and atherosclerosis in type 2 diabetes mellitus (T2DM) in animal studies. The pathophysiological role of this protein in obesity, T2DM, and cardiovascular disease is less investigated in humans. Earlier we reported a rare Asian Indian population-specific missense variant (rs371227978; Arg148His) in the MFGE8 gene associated with increased circulating Mfge8 and T2DM. We have further investigated the role of Mfge8 with T2DM risk in additional Asian Indians (n = 4897) and Europeans and other multiethnic cohorts from UK Biobank (UKBB) (n = 455,808) and the US (n = 1150). We also evaluated the exposure of Mfge8-enriched human EVs in zebrafish (ZF) for their impact on cardiometabolic organ system. Most individual carriers of Arg148His variant not only had high circulating Mfge8 but also revealed a positive significant correlation with glucose (r = 0.42; p = 4.9 × 10-04), while the non-carriers showed a negative correlation of Mfge8 with glucose (r = -0.38; p = 0.001) in Asian Indians. The same variant was monomorphic in non-South Asian ethnicities. Even without the variant, serum Mfge8 correlated significantly with blood glucose in other non-South Asian ethnicities (r = 0.47; p = 2.2 × 10-13). Since Mfge8 is an EV marker, we tested the exposure of Mfge8-enriched human EVs to ZF larvae as an exploratory study. The ZF larvae showed rapid effects on insulin-sensitive organs, developing fatty liver disease, heart hypertrophy and exhibiting redundant growth with poor muscular architecture with and without the high-fat diet (HFD). In contrast, the control group fishes developed fatty liver disease and heart hypertrophy only after the HFD feeding. Backed with strong support from animal studies on the role of Mfge8 in obesity, insulin resistance, and atherosclerosis, the current research suggests that circulating Mfge8 may become a potential marker for predicting the risk of T2DM and cardiovascular disease in humans.

Cardioneuroablation for Drug Refractory Vasovagal Syncope After COVID-19 Infection.

Cardioneuroablation is a novel approach to treat patients with recurrent vasovagal syncope (VVS), targeting the ganglionated plexi around the atria and thus reducing the vagal input to the heart. This study reports a case of drug-refractory VVS after COVID-19 infection, successfully managed with cardioneuroablation.

In Situ Complexation of sgRNA and Cas12a Improves the Performance of a One-Pot RPA-CRISPR-Cas12 Assay.

Due to their ability to selectively target pathogen-specific nucleic acids, CRISPR-Cas systems are increasingly being employed as diagnostic tools. "One-pot" assays that combine nucleic acid amplification and CRISPR-Cas systems (NAAT-CRISPR-Cas) in a single step have emerged as one of the most popular CRISPR-Cas biosensing formats. However, operational simplicity comes at a cost, with one-pot assays typically being less sensitive than corresponding two-step NAAT-CRISPR-Cas assays and often failing to detect targets at low concentrations. It is thought that these performance reductions result from the competition between the two enzymatic processes driving the assay, namely, Cas-mediated cis-cleavage and polymerase-mediated amplification of the target DNA. Herein, we describe a novel one-pot RPA-Cas12a assay that circumvents this issue by leveraging in situ complexation of the target-specific sgRNA and Cas12a to purposefully limit the concentration of active Cas12a during the early stages of the assay. Using a clinically relevant assay against a DNA target for HPV-16, we show how this in situ format reduces competition between target cleavage and amplification and engenders significant improvements in detection limit when compared to the traditional one-pot assay format, even in patient-derived samples. Finally, to gain further insight into the assay, we use experimental data to formulate a mechanistic model describing the competition between the Cas enzyme and nucleic acid amplification. These findings suggest that purposefully limiting cis-cleavage rates of Cas proteins is a viable strategy for improving the performance of one-pot NAAT-CRISPR-Cas assays.

The impact of atrial fibrillation on clinical outcomes in heart failure with mid-range and preserved ejection fraction patients.

BACKGROUND: The combined effect of left ventricular ejection fraction (LVEF) and atrial fibrillation (AF) on clinical outcomes in heart failure (HF) remains complex. OBJECTIVE: In this post hoc analysis of the TOPCAT trial, we aimed to evaluate the impact of AF on clinical outcomes in patients with HF stratified by LVEF range. METHODS: A total of 3442 patients were included, stratified into 3 groups according to LVEF range-HF with mid-range ejection fraction (HFmrEF), LVEF of 45%-50% (n = 823); HF with preserved ejection fraction (HFpEF), LVEF of 51%-60% (n = 1682); and HF with normal ejection fraction (HFnEF), LVEF >60% (n = 937)-and subdivided according to the presence of AF at enrollment. Cox regression analysis was used to define independent associations between AF and clinical outcomes. RESULTS: AF was prevalent in 38.6% in HFmrEF, 34.6% in HFpEF, and 33.7% in HFnEF (P = .07). AF was associated with worse primary outcome in each subgroup and with HF hospitalizations and worse cardiovascular mortality in HFpEF and HFnEF. The hazard ratio for the primary outcome in those with AF compared with sinus rhythm (SR) was 1.11 (1.01-1.22; P = .03) in HFmrEF, 1.20 (1.11-1.29; P < .001) in HFpEF, and 1.16 (1.05-1.28; P = .004) in HFnEF. When LVEF was treated as a continuous variable, there was a linear negative association between LVEF and the effect of AF vs SR for the primary end point and HF hospitalizations and a linear positive association for cardiovascular mortality. CONCLUSION: Compared with SR, AF was independently associated with worse outcomes across all LVEF ranges.

Synergistic Effects of Weight Loss and Catheter Ablation: Can microRNAs Serve as Predictive Biomarkers for the Prevention of Atrial Fibrillation Recurrence?

In atrial fibrillation (AF), multifactorial pathologic atrial alterations are manifested by structural and electrophysiological changes known as atrial remodeling. AF frequently develops in the context of underlying cardiac abnormalities. A critical mechanistic role played by atrial stretch is played by abnormal substrates in a number of conditions that predispose to AF, including obesity, heart failure, hypertension, and sleep apnea. The significant role of overweight and obesity in the development of AF is known; however, the differential effect of overweight, obesity, cardiovascular comorbidities, lifestyle, and other modifiable risk factors on the occurrence and recurrence of AF remains to be determined. Reverse remodeling of the atrial substrate and subsequent reduction in the AF burden by conversion into a typical sinus rhythm has been associated with weight loss through lifestyle changes or surgery. This makes it an essential pillar in the management of AF in obese patients. According to recently published research, microRNAs (miRs) may function as post-transcriptional regulators of genes involved in atrial remodeling, potentially contributing to the pathophysiology of AF. The focus of this review is on their modulation by both weight loss and catheter ablation interventions to counteract atrial remodeling in AF. Our analysis outlines the experimental and clinical evidence supporting the synergistic effects of weight loss and catheter ablation (CA) in reversing atrial electrical and structural remodeling in AF onset and in recurrent post-ablation AF by attenuating pro-thrombotic, pro-inflammatory, pro-fibrotic, arrhythmogenic, and male-sex-associated hypertrophic remodeling pathways. Furthermore, we discuss the promising role of miRs with prognostic potential as predictive biomarkers in guiding approaches to AF recurrence prevention.

Sex differences in atrial fibrillation ablation outcomes in patients with heart failure.

BACKGROUND: There is a lack of data on the impact of sex on the outcomes of patients with heart failure (HF) undergoing atrial fibrillation (AF) ablation. We aimed to analyze the association of sex with outcomes of atrial fibrillation ablation in patients with heart failure. METHODS: The National Readmissions Database (NRD) was analyzed from 2016 to 2019 to identify patients ≥ 18 years old with heart failure (HF) undergoing AF ablation. The outcomes of interest included peri-procedural complications, in-hospital mortality, resource utilization, and unplanned 1-year readmissions. The final cohort was divided into patients with HFrEF and HFpEF and outcomes were compared between males and females in both cohorts. RESULTS: A total of 23,277 patients with HF underwent AF ablation between 2016 and 2019, of which 14,480 had HFrEF and 8,797 had HFpEF. Among patients with HFrEF, 61.6% were males and 38.4% were females whereas, among patients with HFpEF, 35.4% were males and 64.6% were females. On a multivariable-adjusted analysis, in patients with HFrEF, there was no difference in the odds of in-hospital mortality, peri-procedural complications, or 1-year HF-related/AF-related/all-cause readmissions between males and females. In patients with HFpEF, females had a higher risk 1-year HF-related readmissions (adjusted hazards ratio: 1.46; 95% CI: 1.13-1.87; p = 0.01), without any difference in the 1-year AF-related/all-cause readmissions, in-hospital mortality, or peri-procedural complications. CONCLUSION: Our results show that females with HFrEF undergoing AF ablation have similar outcomes whereas females with HFpEF have higher 1-year HF readmissions with no difference in the other outcomes, compared to males.

Rhythm vs Rate Control Strategy for Atrial Fibrillation: A Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Rhythm control, either with antiarrhythmic drugs or catheter ablation, and rate control strategies are the cornerstones of atrial fibrillation (AF) management. Despite the increasing role of rhythm control over the past few years, it remains inconclusive which strategy is superior in improving clinical outcomes. OBJECTIVES: This study summarizes the total and time-varying evidence regarding the efficacy of rhythm- vs rate-control strategies in the management of AF. METHODS: We systematically perused the MEDLINE, CENTRAL (Cochrane Central Register of Controlled Trials), and Web of Science databases for randomized controlled trials from inception to November 2023. We included studies that compared the efficacy of rhythm control (ie, antiarrhythmic drugs classes Ia, Ic, or III, AF catheter ablation, and electrical cardioversion) and rate control (ie, beta-blocker, digitalis, or calcium antagonist) strategies among patients with nonvalvular AF. The primary outcome was cardiovascular (CV) death, whereas secondary outcomes included all-cause death, stroke, hospitalization for heart failure (HF), sinus rhythm at the end of the follow-up, and rhythm control-related adverse events. A cumulative meta-analysis to assess temporal trends and a meta-regression analysis using the percentage of ablation use was performed. RESULTS: We identified 18 studies with a total of 17,536 patients (mean age: 68.6 ± 9.7 years, 37.9% females) and a mean follow-up of 28.5 months. Of those, 31.9% had paroxysmal AF. A rhythm control strategy reduced CV death (HR: 0.78; 95% CI: 0.62-0.96), stroke (HR: 0.801; 95% CI: 0.643-0.998), and hospitalization for HF (HR: 0.80; 95% CI: 0.69-0.94) but not all-cause death (HR: 0.86; 95% CI: 0.73-1.02) compared with a rate control strategy. This benefit was driven by contemporary studies, whereas more ablation use within the rhythm control arm was associated with improved outcomes, except stroke. CONCLUSIONS: In patients with AF, a contemporary rhythm control strategy leads to reduced CV mortality, HF events, and stroke compared with a rate control strategy.

Atrial fibrillation in the setting of cardiac amyloidosis - A review of the literature.

Cardiac amyloidosis (CA) is related to the aggregation of insoluble fibrous deposits of misfolded proteins within the myocardium. Transthyretin amyloidosis (ATTR) and immunoglobulin light-chain amyloidosis are the main forms of CA. Atrial fibrillation (AF) is a common arrhythmia in CA patients, especially in those with ATTR amyloidosis. Increased atrial preload and afterload, atrial enlargement, enhanced atrial wall stress, and autonomic dysfunction are the main mechanisms of AF in CA patients. CA is associated with the formation of endocardial thrombi and systemic embolism. The promoters of thrombogenesis include endomyocardial damage, blood stasis, and hypercoagulability. The prevalence of thrombi in patients with AF remains elevated despite long-term anticoagulation. Consequently, transesophageal ultrasound examinations before cardioversion should be performed to exclude endocardiac thrombi despite anticoagulation. Furthermore, the CHA2DS2-VASc score should not be used to assess the thromboembolic risk in CA patients with AF. Rate control is challenging in patients with CA, while rhythm control is the preferred treatment option, especially in the early stages of the disease process. Although catheter ablation is an effective treatment option, more data are needed to explore the role of the procedure in CA patients.

Automated identification of atrial fibrillation from single-lead ECGs using multi-branching ResNet.

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia, which is clinically identified with irregular and rapid heartbeat rhythm. AF puts a patient at risk of forming blood clots, which can eventually lead to heart failure, stroke, or even sudden death. Electrocardiography (ECG), which involves acquiring bioelectrical signals from the body surface to reflect heart activity, is a standard procedure for detecting AF. However, the occurrence of AF is often intermittent, costing a significant amount of time and effort from medical doctors to identify AF episodes. Moreover, human error is inevitable, as even experienced medical professionals can overlook or misinterpret subtle signs of AF. As such, it is of critical importance to develop an advanced analytical model that can automatically interpret ECG signals and provide decision support for AF diagnostics. Methods: In this paper, we propose an innovative deep-learning method for automated AF identification using single-lead ECGs. We first extract time-frequency features from ECG signals using continuous wavelet transform (CWT). Second, the convolutional neural networks enhanced with residual learning (ReNet) are employed as the functional approximator to interpret the time-frequency features extracted by CWT. Third, we propose to incorporate a multi-branching structure into the ResNet to address the issue of class imbalance, where normal ECGs significantly outnumber instances of AF in ECG datasets. Results and Discussion: We evaluate the proposed Multi-branching Resnet with CWT (CWT-MB-Resnet) with two ECG datasets, i.e., PhysioNet/CinC challenge 2017 and ECGs obtained from the University of Oklahoma Health Sciences Center (OUHSC). The proposed CWT-MB-Resnet demonstrates robust prediction performance, achieving an F1 score of 0.8865 for the PhysioNet dataset and 0.7369 for the OUHSC dataset. The experimental results signify the model's superior capability in balancing precision and recall, which is a desired attribute for ensuring reliable medical diagnoses.

Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: -4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = -2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1ß, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

Association between atrial fibrillation and heart failure patient reported outcomes across the ejection fraction spectrum.

BACKGROUND: Atrial fibrillation (AF) is common in patients with heart failure (HF) and is associated with worse clinical outcomes. We evaluated the relationship between AF and longitudinal changes in health-related quality of life (HRQoL) measured by Kansas City Cardiomyopathy Questionnaire (KCCQ) in both HF with preserved (HFpEF) and reduced ejection fraction (HFrEF). METHODS: This is a post-hoc analysis of the TOPCAT and HF-ACTION trials. The effect of AF on KCCQ overall summary scores (OSS), in both trials, was examined using a mixed effects regression model. Patients were divided into 3 groups according to AF status at baseline: patients with a history of AF but no AF detected on ECG at enrollment (Hx AF group), patients with history of AF and AF detected on ECG at enrollment (ECG AF group) and patients with post-randomization new-onset AF (New AF group). RESULTS: In TOPCAT, among 1,710 patients with KCCQ data available, AF was associated with a significantly lower KCCQ-OSS (-3.98; 95% CI -7.21: -0.74) at 48 months, with a significant AF status by time interaction (P = .03). In HF-ACTION, among 1,814 patients with available KCCQ data, AF was associated with a significantly lower KCCQ-OSS (-3.67; 95% CI -6.21: -1.41) at 24 months but there was no significant AF status by time interaction. In both trials, the type of AF was not associated with significant changes in KCCQ-OSS score. CONCLUSION: Ιn patients with both HFpEF and HFrEF, AF was independently associated with worse HRQoL measured by KCCQ.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

Droplet-based microfluidics and enzyme evolution.

Enzymes are widely used as catalysts in the chemical and pharmaceutical industries. While successful in many situations, they must usually be adapted to operate efficiently under nonnatural conditions. Enzyme engineering allows the creation of novel enzymes that are stable at elevated temperatures or have higher activities and selectivities. Current enzyme engineering techniques require the production and testing of enzyme variant libraries to identify members with desired attributes. Unfortunately, traditional screening methods cannot screen such large mutagenesis libraries in a robust and timely manner. Droplet-based microfluidic systems can produce, process, and sort picoliter droplets at kilohertz rates and have emerged as powerful tools for library screening and thus enzyme engineering. We describe how droplet-based microfluidics has been used to advance directed evolution.

Ultrahigh-Throughput, Real-Time Flow Cytometry for Rare Cell Quantification from Whole Blood.

We present an ultrahigh-throughput, real-time fluorescence cytometer comprising a viscoelastic microfluidic system and a complementary metal-oxide-semiconductor (CMOS) linear image sensor-based detection system. The flow cytometer allows for real-time quantification of a variety of fluorescence species, including micrometer-sized particles and cells, at analytical throughputs in excess of 400,000 species per second. The platform integrates a custom C++ control program and graphical user interface (GUI) to allow for the processing of raw signals, adjustment of processing parameters, and display of fluorescence intensity histograms in real time. To demonstrate the efficacy of the platform for rare event detection and its utility as a basic clinical tool, we measure and quantify patient-derived circulating tumor cells (CTCs) in peripheral blood, realizing that detection has a sensitivity of 6 CTCs per million blood cells (0.000006%) with a volumetric throughput of over 3 mL/min.

Noninvasive Vagus Nerve Stimulation in Postural Tachycardia Syndrome: A Randomized Clinical Trial.

BACKGROUND: Low-level transcutaneous stimulation of the auricular branch of the vagus nerve at the tragus is antiarrhythmic and anti-inflammatory in animals and humans. Preliminary studies show that transcutaneous vagus nerve stimulation (tVNS) is beneficial in animal models of postural tachycardia syndrome (POTS). OBJECTIVES: In this study the authors conducted a sham-controlled, double-blind, randomized clinical trial to examine the effect of tVNS on POTS over a 2-month period relative to sham stimulation. METHODS: tVNS (20 Hz, 1 mA below discomfort threshold) was delivered using an ear clip attached to either the tragus (active; n = 12) or the ear lobe (sham; n = 14) for 1 hour daily over a 2-month period. Postural tachycardia was assessed during the baseline and 2-month visit. Heart rate variability based on 5-minute electrocardiogram, serum cytokines, and antiautonomic autoantibodies were measured at the respective time points. RESULTS: Mean age was 34 ± 11 years (100% female; 81% Caucasian). Adherence to daily stimulation was 83% in the active arm and 86% in the sham arm (P > 0.05). Postural tachycardia was significantly less in the active arm compared with the sham arm at 2 months (mean postural increase in heart rate 17.6 ± 9.9 beats/min vs 31.7 ± 14.4 beats/min; P = 0.01). Antiadrenergic autoantibodies and inflammatory cytokines were lower in the active arm compared with the sham arm at 2 months (P < 0.05). Heart rate variability was better in the active arm. No device-related side effects were observed. CONCLUSIONS: Our results support the emerging paradigm of noninvasive neuromodulation to treat POTS. Mechanistically, this effect appears to be related to reduction of antiautonomic autoantibodies and inflammatory cytokines, and improvement in autonomic tone. Further studies are warranted. (Autoimmune Basis for Postural Tachycardia Syndrome; NCT05043051).

Pharmacological Treatments in Heart Failure With Mildly Reduced and Preserved Ejection Fraction: Systematic Review and Network Meta-Analysis.

BACKGROUND: Medical treatment for heart failure with preserved ejection (HFpEF) and heart failure with mildly reduced ejection fraction (HFmrEF) has weaker evidence compared with reduced ejection fraction, despite recent trials with an angiotensin receptor neprilysin inhibitor (ARNI) and sodium glucose co-transporter 2 inhibitors (SGLT2is). OBJECTIVES: The authors aimed to estimate the aggregate therapeutic benefit of drugs for HFmrEF and HFpEF. METHODS: The authors performed a systematic review of MEDLINE, CENTRAL, and Web of Science for randomized trials including patients with heart failure (HF) and left ventricular ejection fraction (LVEF) >40%, treated with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (analyzed together as renin-angiotensin system inhibitors [RASi]), beta-blockers (BBs), mineralocorticoid receptor antagonists (MRAs), digoxin, ARNI, and SGLT2i. An additive component network meta-analysis was performed. The primary outcome was a composite of cardiovascular (CV) death and first hospitalization for heart failure (HHF); secondary outcomes were CV death, total HHF, and all-cause mortality. RESULTS: The authors identified 13 studies with a total of 29,875 patients and a mean LVEF of 56.3% ± 8.7%. ARNI, MRA, and SGLT2i separately, but not RASi, BB, or digoxin, reduced the primary composite outcome compared with placebo. The combination of ARNI, BB, MRA, and SGLT2i was the most effective (HR: 0.47 [95% CI: 0.31-0.70]); this was largely explained by the triple combination of ARNI, MRA, and SGLT2i (HR: 0.56 [95% CI 0.43-0.71]). Results were similar for CV death (HR: 0.63 [95% CI 0.43-0.91] for ARNI, MRA, and SGLT2i) or total HHF (HR: 0.49 [95% CI 0.33-0.71] for ARNI, MRA, and SGLT2i) alone. In a subgroup analysis, only SGLT2i had a consistent benefit among all LVEF subgroups, whereas the triple combination had the greatest benefit in HFmrEF, robust benefit in patients with LVEF 50% to 59%, and a statistically marginal benefit in patients with LVEF ≥60%. CONCLUSIONS: In patients with HF and LVEF>40%, the quadruple combination of ARNI, BB, MRA, and SGLT2i provides the largest reduction in the risk of CV death and HHF; driven by the robust effect of the triple combination of ARNI, MRA, and SGLT2i. The benefit was more pronounced in HFmrEF patients.