The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF

Visual Abstract

H eart failure (HF) is a major global public health problem.Almost 50% of patients with HF suffer from heart failure with a preserved ejection fraction (HFpEF). 1 Patients with HFpEF are more often female, elderly, and have multiple comorbidities such as hypertension, type 2 diabetes mellitus (T2DM), and obesity. 2 To date, w80% of all patients with HFpEF have obesity and 20%-45% have T2DM. 3,47][8][9] EMPEROR-Preserved (Empagliflozin Outcome Trial in Patients With Chronic Heart Failure With Preserved Ejection Fraction), evaluating the effects of the sodium glucose co-transporter 2 inhibitor empagliflozin, was the first study to show a significant reduction on a combined "hard" end point of HF hospitalizations and cardiovascular mortality in patients with HFpEF. 10 Sodium glucose co-transporter 2 inhibitors have metabolic effects, including lowering of plasma glucose levels, and (modest) effects on body weight and blood pressure. 11So, these findings validated the concept that effects on the cardiometabolic profile may be effective in HFpEF. 12,13is is in line with data on other interventions that affect body weight.[16] Clearly, bariatric surgery results in very substantial weight loss, but ancillary effects also have been described, such as incretin response and changes of the gut microbiome, and some refer to bariatric surgery as metabolic surgery. 17Regardless, targeting the unfavorable cardiometabolic profile through weight loss in HFpEF may resemble a powerful treatment strategy to improve outcomes in HFpEF.
A group of drugs that is of particular interest in this respect are the glucagon-like peptide-1 receptor agonists (GLP-1RAs).Long-acting GLP-1RAs exert substantial and sustained body weight losses, 18 and 2 GLP-1RAs are registered for the indication weight management. 19In addition, several GLP-1RAs are approved for effective glucose lowering in patients with T2DM and improve cardiovascular outcomes. 20,21The clinical trials-STEP-HFpEF (Research  Possible mechanisms of action on the cardiovascular system of GLP-1RAs include anti-inflammatory, antiatherosclerotic, vasodilatory, and other hemodynamic effects, and life-long exposure of GLP-1 is associated with prevention of HF. [22][23][24] Semaglutide treatment results in significant reductions in levels of the inflammatory marker C-reactive protein. 25However, it remains elusive what proportion of these effects result from improved glycemic control and significant weight loss and to what extent additional metabolic and cardiovascular effects contribute, transcending reduction in weight. 26erefore, we investigated the cardiometabolic effects of semaglutide in a representative mouse model of HFpEF 27,28 and zoomed in on potential underlying actions and molecular mechanisms as compared to pair feeding (PF)-induced weight loss.

METHODS
A detailed methods section is provided in the Supplemental Appendix.EXERCISE CAPACITY.Exercise capacity was determined using voluntary wheel running.After 2 days of acclimatization to the running wheel, we measured running distance during 5 constitutive days in the week prior to sacrifice and the average running distance (km/d) was calculated afterward.

FASTING GLUCOSE AND GLUCOSE TOLERANCE.
After 12 weeks of intervention, mice were subjected to an oral glucose tolerance test.Fasting glucose was measured after 12 hours of fasting followed by administration of an oral bolus of glucose (2 g/kg) and tail vein blood samples were measured using an Accu-Chek Aviva glucose analyzer (Roche Diagnostics) at multiple time points (0, 15, 30, 60, 90, and 120 minutes).Area under the curve was calculated to determine the rate of glucose clearance, as described previously. 28ASUREMENT OF BODY MASS COMPOSITION.Body mass composition (fat mass, fluid, and lean mass) was determined using minispec LF90II body composition Withaar et al analyzer (Bruker Optics) in week 12 according to manufacturer's protocol.
ECHOCARDIOGRAPHY.Three days prior to sacrifice, echocardiographic measurements were performed using a Vevo 3100 system equipped with a 40-MHz MXX550D linear array transducer (FUJIFILM Visual-Sonics) to assess cardiac dimensional and functional parameters.Offline speckle tracking (VEVO strain, Vevo LAB software version 5.5.1) was used to determine global longitudinal strain and reverse peak longitudinal strain rate. 30SSUE PROCUREMENT AND ORGAN MORPHOMETRY.At week 12, mice were anesthetized with isoflurane and sacrificed.Blood was drawn using apical puncture, collected in EDTA tubes, and spun down, and then the plasma was collected and stored.The heart with all its compartments was rapidly excised, rinsed in ice cold 1 mol/L potassium chloride (Merck Millipore), weighed, and stored for further analyses.
Lungs were excised and their wet weight was measured.Visceral adipose tissue (VAT) was collected and stored.
HISTOLOGICAL ANALYSIS.Left ventricular (LV) mid-transverse sections were fixed in 4% paraformaldehyde before paraffin embedding and cut into 4-mm thick sections.Masson trichrome stain was performed to detect collagen deposition as a measurement of fibrosis, as previously described. 31Cardiomyocyte cross-sectional area (mm 2 ) was visualized with immunofluorescent staining with germ agglutinin fluorescein isothiocyanate (Sigma-Aldrich) as previously reported. 28ANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION.Total RNA from LV tissue was extracted using TRI Reagent (Sigma-Aldrich), and complementary DNA synthesis was performed using the Quanti-Tect RT kit (Qiagen).Cell clustering and uniform manifold approximation and projection construction was performed in Seurat, whereas cell type populations were identified using known marker genes (Supplemental Figure 4) and supplemented with automatic cell type annotation SCINA 36 and scClassify Bioconductor) (Supplemental Figure 5).

Differential expression analyses between conditions
for every cell population were calculated in Seurat 37 using the Wilcoxon rank sum test.The R package Gprofiler (g:Profiler version e106_eg53_p16_65fcd97) was used for gene ontology analyses.Full Semaglutide Protects From HFpEF   For detailed transcriptomic and proteomic statistics, we refer to the extended methods in the Supplemental Appendix.

SEMAGLUTIDE AND PF BOTH RESULTED IN WEIGHT LOSS CAUSED BY A REDUCTION IN FAT MASS.
Treatment with semaglutide was associated with reduced food intake during the first 14 days and normalized afterwards (Figure 1B).Semaglutide resulted in sustained weight loss from first day of treatment onward (Figure 1C).PF resulted in similar weight loss (Figure 1C).In both groups, weight loss was caused by a significant reduction in fat mass in 4 weeks of treatment (19.4-6.2 g for semaglutide; 19.8-8.3 g for PF), as compared to vehicle-treated mice (Figure 1D).measured by voluntary wheel running, improved in mice treated with semaglutide and not in mice on PF (Figure 1G).To better understand the underlying mechanism of semaglutide treatment, we performed proteome analysis of the LV.For this analysis, we compared the effect of semaglutide vs vehicle and the effect of PF vs vehicle.Principal component analysis showed distinct clustering that was more prominent in semaglutide than in mice on PF (Figure 4A).Both semaglutide and PF resulted in a considerable number of uniquely up-or down-regulated proteins (Figure 4B).
Using gene set enrichment analyses on up-regulated proteins in semaglutide-treated mice, we identified pathways related to cardiac cytoskeleton processes and actin filament organization (Figure 4C).Of note, these proteomic pathways were not enriched in PF mice (Supplemental Figure 2).We then explored the systemic effects of semaglutide or PF and performed plasma proteomics.A total of 240 proteins were identified with semaglutide treatment and 116 proteins with PF (Figure 5A).Treatment with semaglutide or PF resulted in a clustering of proteins that were significantly altered related to either intervention (Figure 5B).Subsequent functional enrichment analysis in plasma of semaglutide mice identified uniquely up-regulated pathways involved in antioxidant activity and oxidative-stress regulation with related genes-GPX1, SOD1, PRDX1, PRDX2, GSTP1, GPX3, G6PD-and cytoskeleton regulation with related genes-ACTR2, GMFG, ARPC4, ARPC5, RAC1 (Figure 5C).Similar pathways were not observed in mice that were on PF (Figure 5D).

IN VAT, SEMAGLUTIDE ACTIVATES INTERFERON-g PRODUCTION, REGULATES T-CELL REGULATION AND T-CELL DIFFERENTIATION, AND INHIBITS
EXTRACELLULAR MATRIX ORGANIZATION.Because semaglutide and PF both resulted in fat loss, we performed transcriptome analysis on VAT to investigate the effect of weight loss on VAT.We observed that semaglutide had a clear effect on gene expression levels, but this was not the case for mice on PF (Figure 6A).In VAT of mice treated with semaglutide, we identified several uniquely up-regulated pathways involved in T-cell regulation and T-cell differentiation, as well as pathways involved in interferong production (Figure 6B).In addition, in VAT of semaglutide-treated mice, we also uniquely identified down-regulated pathways related to extracellular matrix organization (Figure 6C).GLP-1RAs, such as semaglutide, are GLP-1 analogs that improve glucose-dependent insulin release by pancreatic bcells, reduce glucagon secretion by acells, and anti-inflammatory effects. 40For this reason GLP-1RAs have traditionally been used for treatment of patients with T2DM to improve glycemic control, for the reduction of cardiovascular events, and slower progression of kidney disease. 23,41GLP-1 also has a direct effect on receptors in the central nervous system and thereby induces weight loss by increased satiety, decreasing hunger and affecting the reward system in various ways leading to better control of food intake.GLP-1RAs are known to induce meaningful weight loss in patients with obesity with or without T2DM. 42,43They attracted strong interest from the cardiovascular field ever since it became clear that GLP-1RAs improve major adverse cardiovascular outcomes including cardiovascular death, myocardial infarction, and stroke in patients with T2DM. 44,45In mechanistic studies, GLP-1RAs were shown to improve cardiac function in patients with   expressed genes that map to the GO term.Color-coding reflects P value after Gprofiler Threshold (P < 0.05).Abbreviations as in Figures 1 and 4. with the results of our experimental study, in which we also observed a reduction in fat mass (>15%) but not in lean mass.

DISCUSSION
In our study, we used a PF protocol to match the semaglutide-induced reduction in food intake, with subsequent weight loss, to find out whether semaglutide exerts additional drug-induced effects beyond weight loss alone.Semaglutide resulted in an w60% food reduction in week 1, w30% in week 2, and w10% in weeks 3 and 4. Matching this food intake, however, was not enough to improve glucose homeostasis in our PF group, as observed with other (true) caloric restriction protocols used in mice.Pak et al 60 showed that prolonged fasting of 10 months resulted in caloric restriction-mediated increase in insulin sensitivity.Furthermore, long term >40% caloric restriction recovered insulin sensitivity, fasting glucose, and insulin levels, 61,62 leading us to believe that our protocol was not long enough to observe these effects in the PF group.
A distinct feature of HFpEF is the presence of multiple comorbidities and together with hypertension and aging, obesity is a main risk factor for HFpEF.Obesity indirectly promotes insulin resistance and hyperglycemia and has additional direct deleterious effect, given that adipose tissue is highly metabolically active and involved in promoting a systemic proinflammatory state. 635][66] Several animal studies and studies with patients with T2DM have already shown that GLP-1RAs have anti-inflammatory and antioxidant properties, and this may reflect a pathway by which semaglutide exerts (a part of) its effects. 26,67,68In this study we validated these antiinflammatory and antioxidant effects: in plasma, semaglutide-but not PF-increases antioxidant enzymes that protect against oxidative stress damage; such as glutathione peroxidase, peroxiredoxin, and catalase.In VAT, semaglutide significantly enriched pathways related to protective inflammatory responses, whereas PF-which was able to reduce fat content in a similar manner-did not have these additional positive effects.Therefore, this study clearly demonstrates the favorable cardiometabolic effects of GLP-1RAs are not solely the result of altered glucose metabolism or reduced VAT volume, but they also result from reduced inflammatory and oxidative stress state. 66,69hibition of systemic metabolic inflammation with glucose-lowering drugs is thought to enhance endothelial and cardiomyocyte function, [70][71][72] because continuous myocardial exposure to high glucose levels induces advanced glycation of contractile proteins and causes alterations in cardiac actin and myosin cytoskeleton that are associated with decreased LV function. 73In our HFpEF animal model, we show that the GLP-1 receptor is mainly present in endothelial cells and not in cardiomyocytes, suggesting that GLP-1RAs do not directly target car-  74,76,77 The effects of GLP-1RAs are not organ-specific and a reduction in fibrosis and improved outcome have also been seen in patients with diabetic kidney disease. 78This suggests that GLP-1RAs may have specific antifibrogenic properties in several organs, including the heart.
Cardiomyocyte stiffness refers to changes in relaxation and filling properties and includes alterations in myofilament deactivation by titin (a giant sarcomeric protein) 79 or in acto-myosin kinetics. 80 our study, treatment with semaglutide resulted in improved cardiac function with less myocardial diastolic stiffness.We observed that semaglutide had no effect on sarcomere stiffness (Fpassive) but resulted in enrichment of actin-myosin and muscle contraction pathways.This suggests that the functional improvement is rather a result of improved actomyosin kinetics than altered sarcomere stiffness.
STUDY LIMITATIONS.In this study we used a multifactorial preclinical HFpEF model that resembles the cardiometabolic human HFpEF phenotype to a large Our results provide mechanistic evidence that GLP-1RAs, particularly semaglutide, could be a potential novel therapeutic option for patients with obesityrelated HFpEF.
Study to Investigate How Well Semaglutide Works in People Living With Heart Failure and Obesity; NCT04788511), STEP-HFpEF-DM (Research Study to Look at How Well Semaglutide Works in People Living With Heart Failure, Obesity, and Type 2 Diabetes; NCT04916470), and a dual acting GLP-1/GIP in Participants With Heart Failure With Preserved A B B R E V I A T I O N S A N D A C R O N Y M S ANGII = angiotensin II Ctrl = control Fpassive = passive stiffness GLP-1RAs = glucagon-like peptide 1 receptor agonists HF = heart failure HFD = high fat diet HFpEF = heart failure with preserved ejection fraction LV = left ventricle PF = pair feeding T2DM = type 2 diabetes mellitus VAT = visceral adipose tissue The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate.For more information, visit the Author Center.Manuscript received November 21, 2022; revised manuscript received May 2, 2023, accepted May 2, 2023.

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 3 Withaar et al O C T O B E R 2 0 2 3 : 1 2 9 8 -1 3 1 4Semaglutide Protects From HFpEF Ejection Fraction and Obesity; NCT04847557)-are currently recruiting participants with obesity-related HFpEF for treatment with semaglutide or tirzepatide.
DATA AVAILABILITY.All supporting data are available within this paper and the Supplemental Appendix.Single nucleus RNA-sequencing data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus and are accessible through GEO series on request.Mass spectrometry data have been deposited to the Pro-teomeXchange Consortium via the PRIDE (Proteomics Identification Database) partner repository with data set identifier PXD034625.ANIMALS.All animal studies were approved by the Centrale Commissie Dierproeven (CCD) license number AVD105002016487 and the Animal Care and User Committee of the Groningen University (permit number 16487-07-04) and conducted in accordance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines. 29Female C57BL6/J mice, 18-22 months old, were purchased from Jackson Laboratory.Mice were housed on a 12-hour light/12hour dark cycle with ad libitum access to chow and water.Echocardiography, mini pump placement, and sacrifice were performed using continuous oxygen and 2%-3% isoflurane anesthesia (Teva Pharmaceuticals). 28EXPERIMENTAL DESIGN.We have shown previously that a murine model of HFpEF has several similarities with human HFpEF. 27,28Briefly, aged female mice were fed a high fat diet (HFD) (60% kcal fat, 20% kcal protein, 20% kcal carbohydrates; Research Diets D17041409) or a low-fat equivalent control (Ctrl) chow (n ¼ 10; 20% kcal fat, 20% kcal protein, 60% kcal carbohydrates; Research Diets D17041407) for 12 consecutive weeks.After 8 weeks of HFD, mice underwent surgery and an ALZET osmotic mini pump (Model 2004) with angiotensin-II (ANGII) (1.25 mg/kg/d) was implanted in a subcutaneous pocket on the back.For Ctrl mice, the subcutaneous pocket was closed without placement of a pump.After pump implantation, mice on HFD were assigned to a group with subcutaneous semaglutide treatment (n ¼ 12) or vehicle treatment, phosphatebuffered saline (n ¼ 16) during last 4 weeks of this study.Semaglutide was uptitrated over 5 days to a final dose of 9 nmol/kg/d, and this final dose was continued during 23 days.To determine the extent to which the cardiometabolic effects of semaglutide occurred by changes in body weight or body composition caused by reduced food intake, we designated a separate group of mice, after 8 weeks and pump implantation, to a pair feeding (PF) (n ¼ 8) protocol.With this technique, the amount of food consumed by the semaglutide groups (grams of chow per day) is exactly matched to that consumed by the pair-fed group on a daily basis.A schematic overview of the experimental design is displayed in Figure 1A.

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1

FIGURE 1
FIGURE 1 Effects of Sema Treatment or PF on Metabolic Parameters

FIGURE 2
FIGURE 2 Effects of Sema Treatment or PF on Cardiac Function and Structure

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 3 Withaar et al O C T O B E R 2 0 2 3 : 1 2 9 8 -1 3 1 4 methodologic details for reagents used, nuclear isolation, library construction, quality control, and single nucleus RNA analysis can be found in the extended methods in the Supplemental Appendix.LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY.Total proteins were isolated from LV and plasma followed by denaturation, reduction, alkylation, and tryptic digestion.Peptides were fractionated off-line with an Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific), and subsequently separated with Easy-nLC 1200 coupled online to an Orbitrap QExactive HF mass spectrometer (Thermo Fisher Scientific).Mass spectrometry data were acquired using a data-independent method as detailed in the extended methods in the Supplemental Appendix.Raw mass spectrometry data were analyzed using the Pulsar search engine (Spectronaut Biognosys).The data were further processed using Perseus software (Maxquant).Enriched protein sets were identified using Gprofiler with significant Gprofiler threshold (P < 0.05).Mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with data set identifier PXD034625.STATISTICAL ANALYSIS.All values are presented as mean AE SEM.Normality of data was tested with Shapiro-Wilk test.Kruskal-Wallis test was used for comparisons among >2 groups followed by Dunn multiple comparisons test with an adjusted P value using GraphPad Prism (version 8.42, GraphPad Software) or SPSS (version 23, IBM Corp).The area under the curve was computed using the trapezoid rule.For proteomics, the false discovery rate was set to 0.01, and data are presented as difference of average (>0.5) and the Àlog 10 Student's t-test (P < 0.05) were used to compare between groups.P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

FIGURE 3
FIGURE 3 Effects of Sema Treatment or PF on Fibrosis and Cardiomyocyte Stiffness

FIGURE 4
FIGURE 4 Effects of Sema Treatment or PF on Differently Expressed Proteins, Cell-type-Specific Gene Expression and Pathways in the LV FIGURE 4 Continued (A) Principal component (PC) analysis of LV identified proteins that uniquely cluster for Ctrl (n ¼ 6), Veh treatment (n ¼ 6), Sema treatment (n ¼ 6), or PF (n ¼ 6).(B) Venn diagram of differently expressed proteins for Sema treatment or PF and their direction vs Veh treatment.(C) Curated Gene Ontology (GO) Biological Processes list of protein-coded genes identified in the LV demonstrating distinctive pathways engaged by Sema treatment vs Veh treatment.Circle size reflects gene ratio, which is the proportion of differentially expressed genes in a pathway divided by all differentially expressed genes that map to GO term.Color-coding reflects P value after Gprofiler Threshold (P < 0.05).(D) Uniform manifold approximation and projection (UMAP) plot of processed single nucleus RNA-sequencing data from LV tissue of combined Ctrl, Veh, Sema, and PF mice.Coloring indicates manual annotation of celltype clusters generated by unsupervised clustering.(E) Overlay of glucagon-like peptide -1 receptor (GLP1r) expression onto UMAP.(F, G) Dot plot showing top 10 most-regulated genes per cell type comparing (F) Sema treatment against Veh treatment and (G) PF against Veh.Dot size represents the absolute log 2 -fold change of a gene, whereas the color indicates positive (POS) (orange) or negative (NEG) (blue) regulation of a gene.(H, I) GO enrichment analysis of up-regulated genes in endothelial (Endo) cells comparing (H) Sema against Veh and (I) PF against Veh treatment.(J) GO enrichment analysis of down-regulated genes in Endo cells comparing PF against Veh treatment.(K, L) GO enrichment analysis of up-regulated genes in cardiomyocytes comparing (K) Sema against Veh and (L) PF against Veh treatment.ATP ¼ adenosine triphosphate; Fibro ¼ fibroblasts; Lymph ¼ lymphatic cells; Meso ¼ mesothelial cells; NADH ¼ nicotinamide adenine dinucleotide; Peri ¼ pericytes; SMC ¼ smooth muscle cells; other abbreviations as in Figures 1 and 2.
In this study we investigated the effects of the GLP-1RA semaglutide in our established multihit mouse model of HFpEF 28 and compared the effects to weight reduction in response to PF.We observed that, by design, semaglutide and PF were associated with the exact degree of weight loss mainly caused by fat mass reduction.However, whereas PF only tended to influence some parameters of HFpEF, semaglutide improved several hallmarks of HFpEF, including reduced LV hypertrophy and fibrosis, improved diastolic dysfunction, reduced lung congestion, and improved exercise capacity.Our study demonstrates that the cardioprotective effects of GLP-1RAs in a model of obesity and HFpEF supersede the effects of weight loss by PF alone and provide mechanistic support for the ongoing STEP-HF study of semaglutide in obesity-related HFpEF.

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 3 Withaar et al O C T O B E R 2 0 2 3 : 1 2 9 8 -1 3 1 4 Semaglutide Protects From HFpEF T2DM. 21,46-52It had already been demonstrated that treatment with GLP-1RAs activates several cardioprotective pathways, prevents HFD-induced insulin resistance and inflammation, improves endothelial function, and ameliorates cardiac function in obese mice. 53In this study, therefore, we conducted detailed studies using single-cell transcriptomics and proteomics of the LV, adipose tissue, and plasma to identify potential underlying mechanisms through which the GLP-1RA semaglutide may improve cardiac function and cardiovascular outcomes in patients with HFpEF.Furthermore, we compared the effects and pathways to the most common intervention to combat obesity-induced HFpEF-dietary measures such as PF.

FIGURE 5
FIGURE 5 Effects of Sema Treatment or PF on Differentially Expressed Plasma Proteins and Pathways

FIGURE 6
FIGURE 6 Effects of Sema Treatment or PF on Differentially Expressed VAT Genes and Pathways

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A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 3 diomyocytes.Recent work by Helmstadter et al74 showed that the endothelium-localized GLP-1 receptor indeed appears to be responsible for the positive effects of GLP-1RAs.In our study we demonstrated that consistent in both -omics data sets, treatment with semaglutide results in enriched pathways related to cytoskeleton organization and mitochondrial function in endothelial cells.We therefore hypothesize that the effects of semaglutide on cardiac function and cardiac structure are primarily the result of improved endothelial function and secondarily to that a possibly improved muscle contraction caused by intimate cell-cell interactions within the heart.75Myocardial diastolic stiffness is among the hallmarks of HFpEF and results from both myocardial fibrosis and increased cardiomyocyte stiffness.Targeting these 2 components of myocardial diastolic stiffness may therefore be an attractive therapeutic intervention for HFpEF.In this study we observed that semaglutide improved cardiac structure and reduced LV hypertrophy through reduced fibrosis and fibrosis mediators.Other vitro studies observed a beneficial effect of GLP-1RAs on ANGII-induced cardiac fibrosis as well.53, Withaar et alJ A C C : B A S I C T O T R A N S L A T I O N A L S C I E N C E V O L .8 , N O . 1 0 , 2 0 2 3 Semaglutide Protects From HFpEF O C T O B E R 2 0 2 3 : 1 2 9 8 -1 3 1 4 extent.Although this model includes aging, obesity, impaired glucose handling, and female sex, the model is still not representative of the entire spectrum of HFpEF.The number of comorbidities in humans with HFpEF often is even larger, and direct translation of our findings into clinical practice must be done cautiously.Also, we only used aged female mice with HFpEF.In humans, the HFpEF phenotype is more prevalent in aging women and less common in aging men, 81 but future studies are needed to investigate whether semaglutide has potential sexspecific effects.Semaglutide was administered in a phase where the HFpEF in part developed, namely after 8 weeks of HFD and at the start of ANGII infusion.Therefore, our timing does not allow us to fully dissect whether semaglutide would prevent incident HFpEF or rather would attenuate prevalent HFpEF or both.In addition, unraveling the role of GLP-1 receptor located on endothelial cells in the context of aging and comorbidities are needed but require lengthy experiments with old animals that require follow-up studies.CLINICAL IMPLICATIONS AND TRANSLATIONAL OUTLOOK.Given their promising cardiovascular effects in patients with T2DM, GLP-1RAs, including semaglutide, are currently being evaluated in patients with HFpEF.However, it remains unclear whether the potential effects of semaglutide are pathway-and drug-specific or whether they are indirectly caused by weight loss.Here, we evaluated the cardiometabolic effects of semaglutide in a validated HFpEF mouse model.We show that the cardiometabolic effects of semaglutide transcends those of weight loss alone, and we demonstrate that semaglutide induces structural and functional changes in the heart and adipose tissue that are associated with improved cardiac structure and cardiac function.Therefore, this study provides mechanistic evidence that semaglutide may represent an effective novel therapeutic option for patients with obesity-related HFpEF.CONCLUSIONS HFpEF is a major and growing public health problem. 82To date, treatment options for HFpEF are limited and novel treatment options are eagerly awaited.Targeting the unfavorable cardiometabolic phenotype may represent an attractive treatment strategy, especially for those patients with obesityrelated HFpEF.Our study shows that the GLP-1RA semaglutide has numerous cardiometabolic effects that extend beyond the effects of weight loss per se.Clinical studies-STEP-HFpEF (NCT04788511) and STEP-HFpEF-DM (NCT04916470) 83 -are already ongoing to address whether semaglutide will improve function in HFpEF, and SELECT (Semaglutide Effects on Heart Disease and Stroke in Patients With Overweight or Obesity; NCT03574597) addresses whether semaglutide affects cardiovascular outcomes in patients with obesity.Preclinical studies such as this study help in understanding the mechanistic basis for observed clinical results.In conclusion, we have shown that in a multifactorial experimental animal model of HFpEF, the cardiometabolic effects of semaglutide extend beyond the effects of weight reduction caused by PF alone.