Hydrogen Sulfide Deficiency and Therapeutic Targeting in Cardiometabolic HFpEF: Evidence for Synergistic Benefit with GLP-1/Glucagon Agonism

Hydrogen Sulfide Deficiency and Therapeutic Targeting in Cardiometabolic HFpEF: Evidence for Synergistic Benefit with GLP-1/Glucagon Agonism | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Hydrogen Sulfide Deficiency and Therapeutic Targeting in Cardiometabolic HFpEF: Evidence for Synergistic Benefit with GLP-1/Glucagon Agonism Jake E. Doiron , View ORCID Profile Mahmoud H. Elbatreek , Huijing Xia , Xiaoman Yu , Natalie D. Gehred , Tatiana Gromova , Jingshu Chen , Ian H. Driver , Naoto Muraoka , Martin Jensen , Smitha Shambhu , W.H. Wilson Tang , Kyle B. LaPenna , Thomas E. Sharp III , Traci T. Goodchild , Ming Xian , Shi Xu , Heather Quiriarte , Timothy D. Allerton , Alexia Zagouras , Jennifer Wilcox , Sanjiv J. Shah , Josef Pfeilschifter , Karl-Friedrich Beck , Thomas M. Vondriska , Zhen Li , David J. Lefer doi: https://doi.org/10.1101/2024.09.16.613349 Jake E. Doiron 2 Department of Pharmacology and Cardiovascular Center, Louisiana State University Health Sciences Center , New Orleans, LA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mahmoud H. Elbatreek 1 Department of Cardiac Surgery, Smidt Heart Institute, Cedars-Sinai Medical Center , Los Angeles, CA 12 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zagazig University , Zagazig, Egypt Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mahmoud H. Elbatreek Huijing Xia 2 Department of Pharmacology and Cardiovascular Center, Louisiana State University Health Sciences Center , New Orleans, LA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xiaoman Yu 1 Department of Cardiac Surgery, Smidt Heart Institute, Cedars-Sinai Medical Center , Los Angeles, CA M.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Natalie D. Gehred 3 Department of Anesthesiology, Medicine & Physiology, David Geffen School of Medicine at University of California , Los Angeles, CA B.A. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tatiana Gromova 3 Department of Anesthesiology, Medicine & Physiology, David Geffen School of Medicine at University of California , Los Angeles, CA B.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jingshu Chen 4 Gordian Biotechnology , South San Francisco, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ian H. Driver 4 Gordian Biotechnology , South San Francisco, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Naoto Muraoka 4 Gordian Biotechnology , South San Francisco, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Martin Jensen 4 Gordian Biotechnology , South San Francisco, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Smitha Shambhu 4 Gordian Biotechnology , South San Francisco, CA M.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site W.H. Wilson Tang 5 Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic , Cleveland, OH 6 Center of Microbiome and Human Health, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic , Cleveland, Cleveland, OH M.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kyle B. LaPenna 2 Department of Pharmacology and Cardiovascular Center, Louisiana State University Health Sciences Center , New Orleans, LA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas E. Sharp III 7 Molecular Pharmacology and Physiology, University of South Florida , Tampa, FL Ph.D Find this author on Google Scholar Find this author on PubMed Search for this author on this site Traci T. Goodchild 1 Department of Cardiac Surgery, Smidt Heart Institute, Cedars-Sinai Medical Center , Los Angeles, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ming Xian 8 Department of Chemistry, Brown University , Providence, RI Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shi Xu 8 Department of Chemistry, Brown University , Providence, RI Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Heather Quiriarte 9 Vascular Metabolism Laboratory, Pennington Biomedical Research Center , Baton Rouge, LA M.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timothy D. Allerton 9 Vascular Metabolism Laboratory, Pennington Biomedical Research Center , Baton Rouge, LA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alexia Zagouras 5 Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic , Cleveland, OH M.D. M.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer Wilcox 6 Center of Microbiome and Human Health, Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic , Cleveland, Cleveland, OH B.S. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sanjiv J. Shah 10 Northwestern University Medicine, Feinberg School of Medicine , Chicago, IL M.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Josef Pfeilschifter 11 Institute of Pharmacology and Toxicology, Goethe University , Frankfurt am Main, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karl-Friedrich Beck 11 Institute of Pharmacology and Toxicology, Goethe University , Frankfurt am Main, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas M. Vondriska 3 Department of Anesthesiology, Medicine & Physiology, David Geffen School of Medicine at University of California , Los Angeles, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Zhen Li 1 Department of Cardiac Surgery, Smidt Heart Institute, Cedars-Sinai Medical Center , Los Angeles, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site David J. Lefer 1 Department of Cardiac Surgery, Smidt Heart Institute, Cedars-Sinai Medical Center , Los Angeles, CA Ph.D. Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: David.Lefer{at}cshs.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Background Heart failure with preserved ejection fraction (HFpEF) is a significant public health concern with limited treatment options. Dysregulated nitric oxide-mediated signaling has been implicated in HFpEF pathophysiology, however, little is known about the role of endogenous hydrogen sulfide (H 2 S) in HFpEF. Objectives This study evaluated H 2 S bioavailability in patients and two animal models of cardiometabolic HFpEF and assessed the impact of H 2 S on HFpEF severity through alterations in endogenous H 2 S production and pharmacological supplementation. We also evaluated the effects of the H 2 S donor, diallyl trisulfide (DATS) in combination with the GLP-1/glucagon receptor agonist, survodutide, in HFpEF. Methods HFpEF patients and two rodent models of HFpEF (“two-hit” L-NAME + HFD mouse and ZSF1 obese rat) were evaluated for H 2 S bioavailability. Two cohorts of two-hit mice were investigated for changes in HFpEF pathophysiology: (1) endothelial cell cystathionine-γ-lyase (EC-CSE) knockout; (2) H 2 S donor, JK-1, supplementation. DATS and survodutide combination therapy was tested in ZSF1 obese rats. Results H 2 S levels were significantly reduced (i.e., 81%) in human HFpEF patients and in both preclinical HFpEF models. This depletion was associated with reduced CSE expression and activity, and increased SQR expression. Genetic knockout of H 2 S -generating enzyme, CSE, worsened HFpEF characteristics, including elevated E/e’ ratio and LVEDP, impaired aortic vasorelaxation and increased mortality. Pharmacologic H 2 S supplementation restored H 2 S bioavailability, improved diastolic function and attenuated cardiac fibrosis corroborating an improved HFpEF phenotype. DATS synergized with survodutide to attenuate obesity, improve diastolic function, exercise capacity, and reduce oxidative stress and cardiac fibrosis. Conclusions H 2 S deficiency is evident in HFpEF patients and conserved across multiple preclinical HFpEF models. Increasing H 2 S bioavailability improved cardiovascular function, while knockout of endogenous H 2 S production exacerbated HFpEF pathology and mortality. These results suggest H 2 S dysregulation contributes to HFpEF and increasing H 2 S bioavailability may represent a novel therapeutic strategy for HFpEF. Furthermore, our data demonstrate that combining H 2 S supplementation with GLP-1/glucagon receptor agonist may provide synergistic benefits in improving HFpEF outcomes. Highlights H 2 S deficiency is evident in both human HFpEF patients and two clinically relevant models. Reduced H 2 S production by CSE and increased metabolism by SQR impair H 2 S bioavailability in HFpEF. Pharmacological H 2 S supplementation improves diastolic function and reduces cardiac fibrosis in HFpEF models. Targeting H 2 S dysregulation presents a novel therapeutic strategy for managing HFpEF. H 2 S synergizes with GLP-1/glucagon agonist and ameliorates HFpEF INTRODUCTION Heart failure with preserved ejection (HFpEF) fraction is a significant public health concern. The increasing prevalence of HFpEF risk factors has resulted in HFpEF diagnoses accounting for 50% of all new heart failure diagnoses, while the subsequent pathophysiology leaves HFpEF patients subject to a 23% 30-day hospital readmission rate and 50-75% 5-year mortality rate 1 - 4 . In order to effectively treat HFpEF it is crucial that we further our understanding of the cardiovascular, and systemic drivers of this devastating CV disease. The influence of the physiologic gaseous mediator, nitric oxide (NO), has been extensively investigated in both preclinical and clinical studies 5 , 6 . While the precise role of NO signaling and nitrosative stress in HFpEF is still being elucidated, little is known about other physiological gaseous molecules, particularly hydrogen sulfide (H 2 S). Since its discovery as an endogenously produced and biologically essential molecule in 1996, H 2 S has garnered significant attention for its role in maintaining whole-body and cardiovascular homeostasis 7 , 8 . H 2 S biosynthesis is regulated through three key enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) 9 - 11 . The major catabolic pathway for H 2 S breakdown occurs in the mitochondria under the control of sulfide quinone oxidoreductase (SQR) 12 . Perturbations in H 2 S bioavailability have been implicated in numerous cardiovascular pathologies, including atherosclerosis, hypertension, myocardial infarction, and heart failure with reduced ejection fraction (HFrEF) 13 - 16 . Investigation of H 2 S in these pathologies has revealed that H 2 S exerts potent antioxidant, anti-inflammatory, metabolic regulatory, and mitochondrial preservation actions 17 - 21 . Additionally, H 2 S has significant interplay with NO, whereby H 2 S augments cardioprotective endothelial nitric oxide synthase (eNOS) signaling 22 . Considering that HFpEF is characterized by derangements in metabolism, inflammation, oxidative stress and NO signaling, there is strong rationale for elucidation of the potential role of alterations in H 2 S signaling in HFpEF. Of the HFpEF phenotypes, cardiometabolic HFpEF is of increasing importance given its reputation as the most prevalent HFpEF phenotype in a syndrome growing in incidence and diagnoses 1 , 23 . Cardiometabolic HFpEF comprises an estimated 33% of the HFpEF patient population and is most frequently comorbid with obesity, diabetes, hypertension, chronic inflammation, and hepatic injury 24 , 25 . Interestingly, H 2 S deficiency has been associated with these independent comorbidities and replenishment of H 2 S has been shown to improve the respective pathologies 10 , 18 , 26 - 30 . Given the complexity of HFpEF, a multi-pronged approach targeting various underlying mechanisms is likely more effective than single-drug treatments. Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are already approved for obesity and diabetes and are recommended by professional and medical societies for mitigation of the cardiovascular risk in patients with type 2 diabetes 31 . These drugs are categorized by the receptors they activate. Single GLP-1RAs activate only the GLP-1 receptor (e.g., liraglutide, dulaglutide, semaglutide), while dual GLP-1 and GIP RAs activate both GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) receptors (e.g., tirzepatide). Both have shown promise in recent HFpEF clinical trials 32 , 33 . Emerging classes include dual GLP-1 and glucagon RAs, which activate both GLP-1 and glucagon receptors (e.g., mazdutide, survodutide) and are currently under development for obesity, diabetes and have shown very powerful effects against metabolic liver disease 34 . Finally, triple GLP-1, GIP and glucagon RAs activate all three receptors (e.g., retatrutide), demonstrating strong anti-obesity effects compared to single and dual agonists 35 , 36 . While the potential of the later two classes in HFpEF is intriguing, their impact remains to be determined. In the present study, we sought to evaluate alterations in H 2 S bioavailability and the role of H 2 S in HFpEF using multiple clinically relevant models of cardiometabolic HFpEF. We also investigated the potential beneficial actions of H 2 S therapy in the form of H 2 S donors in the setting of HFpEF. This study also aimed to explore Survodutide’s therapeutic benefits in HFpEF and investigate if its effects are enhanced when combined with an H 2 S donor. METHODS Human Plasma Samples Plasma samples were collected from either ambulatory patients with HFpEF prospectively enrolled in a heart failure outpatient clinic (with signs and symptoms or heart failure and echocardiogram showing left ventricular ejection fraction ≥50%) or age- and sex-matched healthy participants recruited from the community without existing cardiovascular diseases (confirmed by echocardiography, pulmonary function testing, and cardiac biomarkers) under protocols approved by the Institutional Review Board (IRB# 06-805 and #10-727, respectively) at the Cleveland Clinic. All participants provided written informed consent to participate. Baseline characteristics of all participants are shown in Table 1 . View this table: View inline View popup Download powerpoint Table 1. Baseline Characteristics of participants with HFpEF vs. Healthy Controls. Measurement of Plasma hs-CRP The levels of hsCRP (#EA101010, ORIGENE, Rockville, MD) in plasma samples from control of HFpEF patients were measured with commercially available ELISA assay kits according to manufacturer’s instructions. Experimental Animal Models of HFpEF “Two-hit” Mouse Model of HFpEF Male C57BL/6N (Charles Rivers Laboratories, Wilmington, MA, USA) mice were purchased at 8 weeks of age and allowed to acclimatize for one week prior to study enrollment. Starting at 9 weeks of age, male C57BL/6N were treated with either L-N G -nitro arginine methyl ester (L-NAME, Enzo Biochem, Farmingdale, NY, USA) in the drinking water (0.5 gram/L) and 60% kcal high fat diet (HFD, D12492, Research Diets, New Brunswick, NJ, USA) to induce HFpEF (n = 7-10 per group), or normal drinking water and normal diet (Teklad 2019s, Inotiv, Chicago, IL, USA) (n = 7-10 per group). C57BL/6N mice were maintained on HFpEF treatment for 0, 5 or 10 weeks prior to physiological characterization. Similarly, male endothelial cell-cystathionine-γ-lyase (EC-CSE) knockout (KO) with constitutive Cre expression restricted to the endothelium, EC-CSE transgenic (Tg) and age-matched littermate controls were generated as described previously 37 , 38 . At 9 weeks of age, animals were enrolled to L-NAME and HFD treatment for 18 weeks. Lastly, for the H 2 S treatment intervention with H 2 S donor, JK-1, male C57BL/6N were enrolled at 9 weeks of age and began L-NAME and HFD treatment. At 5 weeks of L-NAME and HFD, mice were further randomized to either receive JK-1 (100µg/kg, b.i.d, i.p.) or vehicle (saline, b.i.d, i.p.). Group sizes of n = 7-10 were determined using a power and sample analysis with the significance level at 5% and power at 80%. ZSF1 Obese Rat Model of Cardiometabolic HFpEF Wistar Kyoto and ZSF1 obese rats (Charles River Laboratories, Wilmington, MA, USA) were purchased at 8 weeks of age to acclimatize for at least 2 weeks prior to study enrollment. Group sizes of n = 6 - 8 were determined using a power and sample analysis with the significance level at 5% and power at 80%. Mice and rats were housed at LSUHSC and CSMC in a temperature controlled and 12-hour light/dark cycle. All studies were LSUHSC and CSMC IACUC (Institutional Animal Care and Use Committee) approved and received care in LSUHSC animal care according to AALAC guidelines. A cohort of male ZSF1 obese rats were subjected to an 8-week treatment regimen starting at 18 weeks of age. Animals were randomly assigned to three groups: vehicle (saline, n=6), Survodutide (MedChemExpress LLC, USA, 30 nmol/kg, subcutaneously, biweekly, n=8), or a combination of Survodutide and DATS (Cayman Chemical, USA, 150 µM/kg, intraperitoneally, daily, n=8). Non-Fasting Blood Glucose A blood drop from the rat tail was used to measure non-fasting blood glucose with an Accu-Check Guide glucometer (Roche, Indianapolis, IN, USA). Hydrogen Sulfide Donors JK-1 was synthesized by Dr. Ming Xian et al. as previously reported 39 . The dosage of JK-1 (100 µg/kg, b.i.d) was determined based on our previous studies of JK-1 in murine models of CV disease 40 - 42 . DATS was prepared as described previously 43 . Exercise Capacity Testing Treadmill exercise capacity of either mice or rats was performed using an IITC Life Science 800 Series rodent specific treadmill (Woodland Hills, CA). For mice, animals were first allowed to acclimate to the treadmill environment for a period of 5 minutes. Following acclimation, the mice were subjected to a warm-up protocol on a flat plane starting at 1 meter per minute that began increasing by 1.1m/min until a final speed of 12 meters/min was achieved after 10 minutes and then subsequently maintained for an additional 5 minutes. After the 15-minute warm-up, the ramp was increased to a 30° incline and the exercise capacity of the mice were recorded during an experimental protocol starting at 12 meters/min that increased by 2 meters/min until a final speed of 18 meters/min was achieved and maintained until the mouse reached a state of exhaustion. Rats were allowed to acclimate to the treadmill environment for a period of 5 minutes. After acclimation, a warm-up protocol on a flat plane was initiated consisting of a starting speed of 6m/min with a gradual increase of 1.5 meters/min until a final speed of 12 meters/min was achieved and maintained for an additional minute. After the 5-minute warm-up, the animal’s exercise capacity was recorded during an experimental run on a flat plane at a consistent speed of 12 meters/min until the animal reached a state of exhaustion. Exhaustion was determined as an animal refusing to run for greater than 5 seconds or an inability of the animal to reach the front of the treadmill for 20 seconds. Data are represented as both exercise distance and work (kg*m) utilizing the animal’s body weight immediately prior to exercise testing. Transthoracic Echocardiography Echocardiography was performed utilizing a Vevo-2100 ultrasound system (Visual Sonics, Toronto, Canada) for mice and rats. Animals were shaved 18 hours prior to experimentation. Animals were induced at 3% isoflurane and maintained at 1-3% isoflurane for echocardiographic evaluation. For mice, recordings pertaining to diastolic function occurred at a target heart rate of >400 beats per minute (bpm) and >450 bpm for systolic measures. For rats, a target heart rate of >300 bpm was used for systolic and diastolic measures. Left ventricular ejection fraction (LVEF) was quantified using an M-mode image across the parasternal short-axis view. Early filling velocity (E), atrial filling velocity (A), and early diastolic tissue velocity (E’) were measured in the four-chamber apical view. Data are represented as averages of three consecutive measurements for each parameter. Systemic and Left Ventricular Hemodynamic Measurements Left ventricular end-diastolic and systemic pressures were measured as previously described. 40 In brief, at the study endpoint, animals were anesthetized at 3% isoflurane until unresponsive to stimuli. The right common carotid artery was further surgically isolated and exposed. Once exposed, the isoflurane was reduced to 1% while a 1.4Fr (for mice) or 1.6Fr (for rats) high-fidelity pressure catheter (Transonic, NY, USA) was inserted and measurements of systemic blood pressures were recorded. The catheter was then advanced into the left ventricular lumen for measurement of left ventricular end-diastolic pressure (LVEDP). Ex Vivo Aortic Ring Vascular Reactivity Thoracic aortas of mice or rats were removed for vascular reactivity testing at sacrifice as previously described. 44 In brief, thoracic aortic rings were first equilibrated in Krebs-Henseleit solution and provided a tension of 0.5 grams for 60 minutes to reach equilibration. The rings were then pretreated with phenylephrine for maximal constriction and then followed with challenges of titrated acetylcholine (10 -9 to 10 -5 M) and subsequently sodium nitroprusside (10 -9 to 10 -5 M) and measured relaxation as compared to phenylephrine maximal contraction. Data are reported as percent relaxation from the maximum contraction to phenylephrine. Hydrogen Sulfide and Sulfane Sulfur Measurements Hydrogen sulfide and sulfane sulfur were measured in plasma and tissue samples utilizing a gas chromatography-sulfur chemiluminescence system (Agilent Technologies, Santa Clara, CA, USA) as previously described 19 . Real-Time PCR Total RNA from tissues was extracted with TRIzol (Life Technologies Corporation, USA). First strand cDNAs were obtained with iScrip Reverse Transcription Supermix (Bio-Rad, USA). Quantitative real-time PCR was performed with SYBR Green qPCR Master Mix (Selleck Chemicals, USA) on CFX Duet (Bio-Rad, USA). 2 -ΔΔCt method was utilized to calculate relative expression to 18s/Tubb5. Primer sequences used in the current study are listed in Supplemental Table 1 . Western blot Immunoblot analysis was performed on samples lysed in RIPA lysis buffer (MedChemExpress, USA) with the addition of Phosphatase Inhibitor (Selleck Chemicals, USA) and Protease Inhibitor Cocktail Mini-Tablet (MedChemExpress, USA) using standard procedures. The protein concentration was determined with BCA Protein Assay Kits (Thermo, USA). Total protein was separated with SDS-PAGE and transferred onto PVDF membranes (Gen Hunter Corporation, USA). Western blot analysis was performed using commercially available antibodies: CSE Polyclonal Antibody (1:2,000, PA5-29725; Thermo, USA), Anti-SQRDL Antibody (1:1,000, 144-09256-20; Ray Biotech, USA). Total protein staining used as loading control was obtained utilizing No-Stain™ Protein Labeling Reagent (Invitrogen, USA). Where mentioned, proteins were assessed using ImageJ software to analyze the optical density of western blots normalized to loading control. Total protein quantification was performed using signals from the entire lane as loading control. Enzyme Activity Assay Enzyme reactions to determine the production of H 2 S from CSE were performed as described previously 45 with slight modification. Briefly, tissues were homogenized in buffer containing 100 mM potassium phosphate, pH 7.4. For enzyme reactions, 0.172 mL of homogenate was incubated with 0.028 mL of substrate mix (10 mM L-cysteine, 2 mM pyridoxal 5 ′ -phosphate) in a sealed vial at 37°C for 30 minutes. After adding 0.400 ml of 1 M sodium citrate buffer (pH 6.0), the mixtures were incubated at 37°C for 15 min with shaking on a rotary shaker to facilitate a release of H 2 S gas from the aqueous phase. After shaking, 0.1 mL of head-space gas was applied to a gas chromatograph as described above. For both reactions, the H 2 S concentration of each sample was calculated against a calibration curve of Na 2 S. Single Nuclei RNA-seq & Analysis The left ventricle of the heart tissue was collected from WKY and ZSF1 Obese Rats at both 14 and 26 weeks of age. The collected tissue was snap-frozen in liquid nitrogen. Heart nuclei were isolated using a lysis buffer consisting 0.25M sucrose, 10mM Tris-Hcl pH 7.5, 25mM KCl, 5mM MgCl2, 45uM Actinomycin D, supplemented with 1X protease inhibitor (G6521,Promega), 0.4U/uL RNasin Ribonuclease Inhibitor (N2515, Promega), 0.2U/ul SuperaseIn (AM2694, ThermoFisher). Briefly, heart tissue samples were minced into smaller pieces with scissors in a 1ml lysis buffer. The minced tissue was homogenized in a dounce homogenizer on ice with 10 strokes of pestle A, followed by 10 strokes of pestle B. The homogenized heart tissue was filtered through a 40 μm cell strainer and centrifuged at 400 x g for 5 min at 4°C. The nuclei pellet was resuspended in 2% BSA in PBS supplemented with Protector RNase inhibitor (03335402001, Sigma-Aldrich) at 0.2 U/ul. Heart nuclei were stained with Sytox red (S34859, Thermo Fisher, Waltham, MA) and sorted and purified through fluorescence-activated cell sorting (FACS). 8000-1000 nuclei from each rat heart were processed using a 10X Genomics microfluidics chip to generate barcoded Gel Bead-In Emulsions according to manufacturer protocols. Indexed single-cell libraries were then created according to 10X Genomics specifications (Chromium Next GEM Single Cell 5ʹ v2.1-Dual Index Libraries). Samples were multiplexed and sequenced in pairs on an Illumina Novaseq X (Illumina, San Diego, CA). The sequenced data were processed into expression matrices with the Cell Ranger Single-cell software 9.0.0 ( https://www.10xgenomics.com/support/software/cell-ranger/latest/release-notes/cr-release-notes ). FASTQ files were obtained from the base-call files from Novaseq X sequencer and subsequently aligned to the rat genome NCBI Rnor6.0, with a read length of 26 bp for cell barcode and unique molecule identifier (UMI) (read 1), 8 bp i7 index read (sample barcode), and 98 bp for actual RNA read (read 2). Each rat sample yielded approximately 300 M reads. Anndata and Scanpy were used to load and preprocess h5ad files for each sample for import into Seurat. Scrublet was used to identify and remove putative doublets before AnnData objects were concatenated by week. Barcodes, features, matrix files, and metadata were extracted into a folder for import into R with Seurat’s Read10X function. 14-week and 26-week Seurat objects were created with CreateSeuratObject. Mitochondrial gene percentage was calculated for each cell in each Seurat object before removing cells containing greater than 5% mitochondrial reads. Mitochondrial genes and genes with fewer than 10 reads across all cells in each object were also removed. Lastly, any cells with fewer than 500 reads were removed. The 14- and 26-week Seurat objects were merged by timepoint and layers joined. Samples were split by week and log-normalized with NormalizeData. FindVariableFeatures was used to identify the top 2000 variable features per week and the features were centered and scaled with ScaleData. Principle Component Analysis (RunPCA function) was run on the split objects with default parameters before layers were integrated together (IntegrateLayers function) with method=RPCAIntegration. A shared nearest-neighbor graph was created with FindNeighbors (15 RPCA dimensions) and clustered with FindClusters (resolution=0.5). Clusters were assigned cell types based on known marker genes. To create the heatmap in Figure 5M , the integrated Seurat object was subset into separate objects by cell type and week. Seurat’s FindMarkers function was used to identify differentially expressed genes in ZSF1 Obese cardiomoyoctes at 14 and 26 weeks, with a Bonferroni-corrected p-value cutoff of 0.05. Results were intersected with a list of known H2S-related enzymes, and the average Log2Fold-Change (FC) was plotted with ggplot2’s geom_tile function. To create the boxpot in Figure 5N , Seurat’s AverageExpression was used with feature=Sqor in cardiomyocytes grouped by rat, week, and condition. Average expression per cell per sample was plotted with GraphPad Prism 10.4. Histology and Fibrosis Cardiac, renal cortex, and hepatic samples were collected and preserved in a 10% zinc formalin buffered solution (CUNZF-5-G, Azer Scientific, Morgantown, PA, USA) for 48 hours, after which they were transferred to a storage solution of 0.01% sodium azide in PBS. The samples were then embedded in paraffin and sectioned into 5 μm thick cross-sections. These sections were stained using either hematoxylin and eosin or picrosirius red with fast green counterstaining. Frozen liver sections from the rats were fixed and stained with Oil Red O as previously described 46 . Images were processed with QuPath software and analyzed using ImageJ to quantify cardiac interstitial and perivascular fibrosis, as well as renal interstitial, perivascular, and periglomerular fibrosis, along with hepatic lipid content. For each animal, a minimum of 10 areas, vessels, or glomeruli within the tissue section were analyzed. Initially, images were converted to 8-bit grayscale with an RGB stack, and the threshold was adjusted to quantify lipid droplet features and the percentage of fibrosis. Glucose tolerance test An oral glucose tolerance test was performed on rats. Fasting blood glucose was measured after a 6-hour fast. Subsequently, an oral glucose bolus (2 g/kg) was administered, and blood glucose levels were monitored at 0, 15, 30, 60, and 120 minutes using an Accu-Chek Guide glucose meter (Roche Diagnostics). Plasma and hepatic lipid assays Plasma and hepatic triglycerides (# MAK266) and cholesterol (# MAK043) were measured by commercially available kits (Millipore Sigma, USA) according to manufacturer’s protocols. ELISA Plasma 8-isoprostane (# 516351 Cayman Chemical, USA) and 3-nitrotyrosine (# NBP2-66363, Novus Biologicals, USA) levels were measured by ELISA kits according to manufacturer’s instructions. Statistical Analysis Data are presented using both mean ± SEM and box plots (median, minimum, and maximum). All statistical analysis were performed in a blinded manner using the Prism 6 and Prism 10 software (GraphPad, San Diego, California). Differences in data among 2 groups were compared using an unpaired student t-test. For comparisons amongst multiple, separate groups of animals enrolled within the same experimental study, multiple unpaired student t tests were utilized. For data involving animal cohorts that were continuously monitored and evaluated according to factors such as timepoints or concentrations, 2-way ANOVA analysis followed by a Bonferroni multiple comparison test was utilized. Survival data was acquired using a Kaplan-Meier survival curve analysis through Prism 6. A p value of <0.05 was considered statistically significant. The presented data may have different numbers of animals per group as only a subset of the mice or rats from each group were used for certain experiments. Additionally, exclusion of animals was carried out due to complications such as procedural failure in invasive hemodynamics measurement, limited amount of sample collected (i.e., plasma volume), and lack of participation in treadmill running. Prior to conducting statistical analysis, an outlier test was performed using a Grubbs test (α = 0.05) through GraphPad to identify and remove any outliers in the data set. RESULTS H 2 S is Depleted in Human HFpEF To investigate the role of H 2 S in HFpEF, we collected plasma samples from patients with confirmed HFpEF (n=46) and a control group without HFpEF (n=45). The characteristics of the patient population are detailed in Table 1 . Our analysis revealed an 81% reduction in circulating H 2 S levels in the HFpEF patients, accompanied by significant decreases in sulfane sulfur levels, a stable metabolite of gaseous H 2 S ( Figure 1A and 1B ). Given that cardiometabolic HFpEF is characterized by low-grade chronic inflammation and that H 2 S possesses anti-inflammatory effects 47 , 48 , we measured circulating hs-CRP levels. We observed elevated levels of the inflammatory marker hs-CRP in the HFpEF group ( Figure 1C ). We also investigated potential correlations between circulating H 2 S and sulfane sulfur levels with the clinical variables presented. However, no statistically significant correlations were found. This may be due to the relatively small sample size of our study, which limits our power to detect such correlations. Download figure Open in new tab Figure 1. Reduced Circulating Hydrogen Sulfide in HFpEF Patients. ( A ) Circulating H 2 S (µM), ( B ) Circulating sulfane sulfur (µM), ( C ) Circulating hs-CRP (µM). Circled number inside bars indicate sample size. Data were analyzed with Student unpaired 2-tail t test. Data are presented as box plots (median, minimum, and maximum). hs-CRP, high-sensitivity C-reactive protein. H 2 S is Reduced in Mouse Two-Hit HFpEF Model Due to Decreased Production by CSE We next employed a well-established “two-hit” cardiometabolic HFpEF mouse model (L-NAME + HFD) 5 , to assess H 2 S bioavailability and HFpEF phenotype after 0, 5, or 10 weeks of treatment as outlined in Supplemental Figure 1A . We first characterized the HFpEF phenotype in this model. Mice with HFpEF exhibited increased body weights and elevated systolic and diastolic blood pressures ( Supplemental Figure 1B-1D ). While left ventricular ejection fraction (LVEF) remained within a preserved range, the E/e’ ratio increased throughout the treatment ( Supplemental Figure 1E and 1F ). Correspondingly, left ventricular catheterization revealed elevated left ventricular end-diastolic pressure (LVEDP), indicating increased myocardial stiffness and reduced compliance ( Supplemental Figure 1G ). Additionally, HFpEF mice displayed substantially diminished exercise capacity, as evidenced by reduced exercise distance ( Supplemental Figure 1H ). Endothelial dysfunction was also noted, with impaired aortic vasoreactivity to acetylcholine. Importantly, these changes in vascular reactivity were independent of altered responses to sodium nitroprusside, suggesting that the observed effects stem from compromised endothelial function ( Supplemental Figure 1I and 1J ). While our study demonstrates that both vascular dysfunction and cardiac abnormalities are present in the HFpEF model, establishing a definitive temporal relationship between these two physiological parameters requires further investigation. It is possible that endothelial dysfunction and impaired vascular reactivity may contribute to the development of cardiac abnormalities in HFpEF. For example, sustained increases in afterload due to impaired vascular compliance could contribute to the development of diastolic dysfunction and increased LVEDP. Further studies, such as longitudinal assessments of vascular function and cardiac parameters in HFpEF models, are necessary to fully elucidate the temporal and causal relationships between these two critical aspects of HFpEF pathophysiology. At baseline, no differences were observed in circulating H 2 S; however, exposure to L-NAME and HFD resulted in a progressive decline in both circulating H 2 S and sulfane sulfur levels ( Figure 2A and 2B ). Similarly, cardiac H 2 S and sulfane sulfur levels decreased progressively throughout the two-hit regimen ( Figure 2C and 2D ). Although H 2 S levels was also reduced in control mice at later timepoints compared to baseline, we did not observe any changes in cardiac physiology as LV E/e’, remained stable over time. Potential contributing factors to this decline could include small sample size, age-related changes in H 2 S metabolism or subtle variations in experimental conditions across timepoints. Download figure Open in new tab Figure 2. Circulating and Myocardial Hydrogen Sulfide Bioavailability in “Two-hit” Murine HFpEF Model. ( A ) Circulating H 2 S (µM), ( B ) Circulating sulfane sulfur (µM), ( C ) Myocardial H 2 S (nmol/g tissue), ( D ) Myocardial sulfane sulfur (nmol/g tissue), ( E ) Myocardial CSE gene expression, ( F ) Myocardial CSE protein expression expressed as fold change, ( G ) Myocardial CSE enzyme activity (nmol H 2 S/g tissue), ( H ) Myocardial SQR gene expression expressed as fold change, ( I ) Myocardial SQR protein expression expressed as fold change. Data in panels A - D were analyzed with multiple Student unpaired 2-tail t tests. Data in panels E - I were from tissues isolated after 10 weeks and analyzed with Student unpaired 2-tail t tests. Data are presented as box plots (median, minimum, and maximum). CSE, cystathionine γ-lyase; SQR, sulfide quinone oxidoreductase. To investigate the underlying causes of H 2 S reduction in HFpEF, we examined the primary vascular H 2 S-producing enzyme, CSE, and the primary H 2 S-metabolizing enzyme, SQR 9 , 10 . In the hearts of HFpEF mice, both gene and protein expression, as well as enzyme activity of CSE, were significantly reduced ( Figure 2E - 2G ), while SQR expression remained comparable to that in control hearts ( Figure 2H and 2I ) Given the systemic nature of HFpEF, we also assessed H 2 S bioavailability in the liver and kidney which are the primary sources of systemic H 2 S production and regulate overall H 2 S bioavailability and metabolism. Indeed, liver is the highest CSE-expressing organ 12 , 49 . We observed significant reductions in hepatic H 2 S, along with decreased CSE gene and protein expression at the 10-week endpoint, although CSE activity did not differ from controls ( Supplemental Figure 2A-2D ). Notably, hepatic SQR gene expression was elevated in HFpEF, while protein levels remained unchanged ( Supplemental Figure 2E and 2F ). In the kidneys, no significant differences in H 2 S levels were noted between control and HFpEF models at the 10-week endpoint ( Supplemental Figure 2G ). However, renal CSE gene and protein expression, as well as enzyme activity, were significantly reduced in HFpEF, while SQR expression levels were similar to controls ( Supplemental Figure 2H-2L ). These changes in systemic H 2 S bioavailability correlate with the worsening HFpEF phenotype. Our data demonstrate that the “two-hit” L-NAME + HFD murine model develops a progressively obese and hypertensive phenotype, accompanied by a systemic depletion of bioavailable H 2 S, primarily due to dysfunctional CSE in multiple organs. CSE Genetic Deficiency Exacerbates While Overexpression Attenuates HFpEF Phenotype To further explore the potential causal relationship between CSE dysfunction and global H 2 S reduction, as well as to assess the impact of decreased H 2 S bioavailability on HFpEF severity, we investigated mice with genetic deletion or overexpression of endothelial cell cystathionine-γ-lyase (EC-CSE). EC-CSE knockout (KO) and transgenic (Tg) mice and their wild-type littermates underwent the same L-NAME and HFD HFpEF protocol. EC-CSE KO mice showed significantly reduced cardiac H 2 S levels and slightly reduced sulfane sulfur levels ( Figure 3A and 3B ). The lack of endothelial CSE resulted in elevated E/e’ ratios and LVEDP ( Figure 3C and 3D ). We also observed a significant impairment in exercise capacity in EC-CSE KO mice ( Figure 3E ). To assess vascular dysfunction in the context of CSE deficiency and HFpEF, we evaluated isolated thoracic aortic rings exposed to increasing concentrations of acetylcholine. Knockout animals demonstrated reduced vasorelaxation in response to acetylcholine, though responsiveness to sodium nitroprusside remained unchanged ( Figure 3F and 3G ). Notably, the genetic deficiency of EC-CSE was associated with increased mortality in our study, underscoring the critical role of H 2 S homeostasis in HFpEF ( Figure 3H ). Diastolic function and mortality from wild type control and EC-CSE KO treated with standard chow are presented in Supplemental Figure 3 . Download figure Open in new tab Figure 3. HFpEF in Endothelial Cell (EC) Cystathionine Gamma Lyase (CSE) Knockout and Transgenic Mice. ( A ) Myocardial H 2 S (nmol/g tissue), ( B ) Myocardial sulfane sulfur (nmol/g tissue), ( C ) Ratio of early mitral diastolic inflow velocity (E) and mitral annular early diastolic velocity (e’), ( D ) Left ventricular end diastolic pressure (LVEDP) in mmHg after 10 weeks, ( E ) Treadmill exercise distance in meters (m), ( F ) Aortic vascular reactivity to acetylcholine (ACh) after 10 weeks expressed as a percent of pre-contraction to norepinephrine, (G) Aortic vascular reactivity to sodium nitroprusside (SNP) after 10 weeks expressed as a percent of pre-contraction to norepinephrine, (H) Survival analysis. ( I ) Myocardial H 2 S (nmol/g tissue), ( J ) Myocardial sulfane sulfur (nmol/g tissue), ( K ) Ratio of early mitral diastolic inflow velocity (E) and mitral annular early diastolic velocity (e’), ( L ) Left ventricular end diastolic pressure (LVEDP) in mmHg after 10 weeks, ( M ) Treadmill exercise distance in meters (m), ( N ) Aortic vascular reactivity to acetylcholine (ACh) after 10 weeks expressed as a percent of pre-contraction to norepinephrine, (O) Aortic vascular reactivity to sodium nitroprusside (SNP) after 10 weeks expressed as a percent of pre-contraction to norepinephrine, (P) Survival analysis. Data in panels A, B, D, I, J and L were analyzed with Student unpaired 2-tail t test. Data in panels C, E, K and M were analyzed with multiple Student unpaired 2-tail t tests. Data in panels F, G, N and O were analyzed with ordinary 2-way ANOVA. Survival data in panels H and P was analyzed with Kaplan-Meier survival analysis. Data in panels A-D and I-L are presented as box plots (median, minimum, and maximum). Data in panels E-G and M-O are presented as mean ± SEM. * Echocardiography, # Treadmill exercise, ⚲ Aortic vascular reactivity, Invasive hemodynamics, H 2 S and sulfane sulfur measurements. These findings are further supported by the studies in EC-CSE overexpressing transgenic mice, which demonstrate significantly higher H 2 S levels and provide protection against the development of HFpEF. Indeed, EC-CSE Tg mice exhibited higher levels of cardiac H 2 S and sulfane sulfur, albeit the latter was not statistically significant ( Figure 3I and 3J ). Transgenic mice had improved cardiac function as indicated by reduced E/e’ ratio and LVEDP ( Figure 3K and 3L ) . We did not observe any significant differences in exercise capacity between transgenic mice and controls ( Figure 3M ) , however the former showed a significant improvement in endothelium-dependent, but not endothelium-independent relaxation ( Figure 3N and 3O ). Remarkably, there was no mortality in both control and transgenic mice throughout the study ( Figure 3P ) . Exogenous H 2 S Administration Ameliorates Systemic HFpEF Dysfunction From a translational perspective, we aimed to determine whether the H 2 S deficiency observed in HFpEF mice could be addressed with an exogenous pharmacological source of H 2 S. We enrolled a cohort of wild-type mice in the two-hit HFpEF protocol, where they received L-NAME and HFD for 5 weeks to induce HFpEF. After this period, mice were randomized to receive either a saline vehicle or the H 2 S donor JK-1 for an additional 5 weeks until the study endpoint ( Supplemental Figure 4A ). Mice treated with JK-1 exhibited significantly higher levels of circulating and cardiac sulfane sulfur, without notable increases in free H 2 S ( Supplemental Figure 4B-4E ). The short half-life of free H 2 S, combined with the pharmacokinetic profile of JK-1, likely accounts for the lack of elevated H 2 S levels; however, the increased sulfane sulfur indicates a significant enhancement in the physiological sulfur pool. Download figure Open in new tab Figure 4. Reduced Circulating Hydrogen Sulfide Bioavailability and HFpEF Severity in the ZSF1 Obese Rat ( A ) Study Timeline, ( B ) Body weight in grams (g), ( C ) Non-fasting blood glucose (mg/dl), ( D ) Systolic blood pressure (mmHg) at 26 weeks, ( E ) Diastolic blood pressure (mmHg) at 26 weeks, ( F ) Left ventricular ejection fraction (LVEF), ( G ) Ratio of early mitral diastolic inflow velocity (E) and mitral annular early diastolic velocity (e’), ( H ) Left ventricular end diastolic pressure (LVEDP) (mmHg) at 26 weeks, ( I ) Treadmill exercise distance expressed in meters (m), ( J ) Circulating H 2 S (µM), ( K ) Circulating sulfane sulfur (µM). Data in panels B, C, F, G, I-K were analyzed with multiple Student unpaired 2-tail t tests. Data in panels D, E and H were analyzed with Student unpaired 2-tail t test. Data are presented as mean ± SEM. Exogenous H 2 S supplementation did not affect body weight, systolic blood pressure but resulted in a significant reduction in diastolic blood pressure ( Supplemental Figure 4F-4H ). Moreover, animals treated with JK-1 showed statistically significant decreases in both E/e’ and LVEDP ( Supplemental Figure 4I and 4J ). JK-1 treatment also significantly improved exercise capacity ( Supplemental Figure 4K ). These results are noteworthy, as HFpEF is characterized by severe exercise intolerance, and our findings suggest that H 2 S therapy enhanced exercise capacity even in the presence of an eNOS inhibitor, indicating that the benefits of H 2 S in HFpEF are likely independent of NO signaling 50 . H 2 S has been shown to exert anti-fibrotic effects in models of myocardial infarction and HFrEF, prompting us to evaluate myocardial interstitial and perivascular fibrosis 40 . We observed significant reductions in both perivascular and interstitial fibrosis following JK-1 treatment ( Supplemental Figure 4L and 4M ). Additionally, our initial characterization of the L-NAME + HFD two-hit model indicated compromised hepatic H 2 S bioavailability. Hepatic H 2 S is crucial for liver health, and diminished hepatic H 2 S signaling has been linked to the development of non-alcoholic fatty liver disease (NAFLD), a condition recently proposed as a predictor of HFpEF diagnosis 26 , 51 . Notably, we found substantial reductions in hepatic lipid content in HFpEF mice treated with JK-1 for 5 weeks, further supporting the potential benefits of H 2 S in this systemic condition ( Supplemental Figures 4N ). Altered H 2 S Production by CSE and Metabolism by SQR Underpins Cardiometabolic HFpEF in ZSF1 Obese Rats To further validate our earlier findings in humans and mice, we investigated H 2 S bioavailability in a clinically relevant model of cardiometabolic HFpEF using ZSF1 obese (Ob) rats 52 , 53 . For this study, we enrolled ZSF1 Ob rats and normotensive, non-obese, non-diabetic control Wistar Kyoto (WKY) rats. We comprehensively characterized the cardiometabolic HFpEF phenotype in ZSF1 Ob rats, monitoring HFpEF progression from week 10 to week 26 of age ( Figure 4A ). Key cardiometabolic parameters, including body weight, blood glucose levels, systolic and diastolic blood pressures, E/e’ ratio, and LVEDP, were significantly elevated in ZSF1 Ob rats compared to WKY controls, while LVEF was preserved, and exercise capacity was impaired ( Figure 4B - 4I ). Notably, circulating H 2 S and sulfane sulfur levels were dramatically reduced as early as 10 weeks of age in the ZSF1 obese rats ( Figure 4J and 4K ). The reduction in circulating H 2 S and sulfane sulfur in the ZSF1 obese rat is very similar to that observed in HFpEF patients (i.e., ∼80-90%). We also examined CSE and SQR expression in the heart, liver, and kidneys. While cardiac CSE gene expression did not differ between WKY and ZSF1 Ob rats, CSE protein levels were lower in the ZSF1 Ob group. Conversely, both SQR gene and protein expressions were elevated in these rats ( Figure 5A - 5D ). In the liver, we observed significant reductions in both CSE gene and protein expression, with unchanged SQR gene expression but increased SQR protein levels in ZSF1 Ob rats ( Figure 5E - 5H ). In the kidneys, CSE expression was increased, while SQR expression was decreased at both the gene and protein levels ( Figure 5I - 5L ). Interestingly, single cell RNA sequencing revealed increased expression of SQR in the cardiomyocytes of ZSF1 Ob rats in both early HFpEF (14 weeks old) and late HFpEF (26 weeks old) ( Figure 5M and 5N ). This analysis also showed that both the H 2 S generator, CBS, and the H 2 S metabolizer, thiosulfate sulfurtransferase (TST), are elevated in the cardiomyocytes only in early HFpEF ( Figure 5M ) . Download figure Open in new tab Figure 5. Reduced Expression of CSE and Increased Expression of SQR in the 26-Week-Old ZSF1 Obese Rat Tissues. ( A ) Myocardial CSE gene expression, ( B ) Myocardial CSE protein expression, ( C ) Myocardial SQR gene expression, ( D ) Myocardial SQR protein expression, ( E ) Hepatic CSE gene expression, ( F ) Hepatic CSE protein expression, ( G ) Hepatic SQR gene expression, (H) Hepatic SQR protein expression, ( I ) Renal CSE gene expression, ( J ) Renal CSE protein expression, ( K ) Renal SQR gene expression, (L) Renal SQR protein expression, ( M ) Heat map showing the expression of H 2 S related genes in rat cardiomyocytes, ( N ) Average SQR expression per cell. Data in A-L were analyzed with Student unpaired 2-tail t test. Data in N was analyzed with Two-Way ANOVA. Data are presented as box plots (median, minimum, and maximum). CSE, cystathionine γ-lyase; SQR, sulfide quinone oxidoreductase. In the heart, interstitial and perivascular fibrosis were pronounced in ZSF1 Ob rats ( Figure 6A and Supplemental Figure 5A ), and H 2 S levels were reduced despite unchanged CSE enzyme activity ( Figure 6B and 6C ). In the liver, lipid content was markedly increased, accompanied by substantial reductions in both H 2 S levels and CSE enzyme activity ( Figure 6D - 6F ). The kidneys of ZSF1 Ob rats also exhibited significant perivascular, interstitial, and periglomerular fibrosis ( Figure 6G and Supplemental Figure 5B and 5C ), with reduced H 2 S levels and CSE enzyme activity observed by week 26 ( Figure 6H and 6I ). Download figure Open in new tab Figure 6. Histopathological Changes and Reduced Hydrogen Sulfide and CSE Enzyme Activity in the ZSF1 Obese Rats. (A) Representative images of cardiac perivascular fibrosis expressed as % collagen fraction at 10 and 26 weeks of age. Tissue stained with Masson’s Trichrome and fast green counterstaining and respective quantification, ( B ) Myocardial H 2 S (nmol/g tissue), ( C ) Myocardial CSE enzyme activity (µmol H 2 S/g tissue/min), ( D ) Representative images of hepatic lipid accumulation (% area) stained with hematoxylin and eosin and respective quantification, ( E ) Hepatic H 2 S (nmol/g tissue), ( F ) Hepatic CSE enzyme activity (µmol/g tissue/min), ( G ) Representative images of renal perivascular fibrosis (% collagen fraction) stained with Masson’s Trichrome and fast green counterstaining and respective quantification , ( H ) Renal H 2 S (nmol/g tissue), ( I ) Renal CSE enzyme activity (µmol/g tissue/min). Data in panels A, B, D, E, G and H were analyzed with multiple Student unpaired 2-tail t tests. Data in panels C, F and I were analyzed with Student unpaired 2-tail t test. Data are presented as mean ± SEM. CSE, cystathionine γ-lyase. These findings further confirm global H 2 S deficiency as a consistent feature across multiple HFpEF models. H 2 S Synergizes with the GLP-1/Glucagon Receptor Survodutide in Ameliorating Cardiometabolic HFpEF in ZSF1 Obese Rats To investigate a clinically relevant approach to treat HFpEF, we examined the therapeutic efficacy of Survodutide, a GLP-1/glucagon receptor agonist in combination with the well-characterized polysulfide H 2 S donor, DATS, in ZSF1 obese rats ( Figure 7A ). We confirmed that DATS treatment significantly increased circulating H 2 S and sulfane sulfur levels ( Supplemental Figure 6A and 6B ). While Survodutide effectively improved metabolic parameters, including reduced body weight, plasma and hepatic triglycerides, and plasma total cholesterol, it unexpectedly impaired glucose tolerance. This likely stems from the glucagon receptor agonism, which may not be completely counteracted by GLP-1 receptor stimulation. Importantly, the addition of DATS synergistically associated with improvements in metabolic parameters ( Figure 7B - 7G ). Download figure Open in new tab Figure 7. H 2 S Donor Synergizes with a dual GLP-1/glucagon agonist and Ameliorates HFpEF in the ZSF1 Obese Rat (A) Study Timeline, ( B ) Body weight in grams, ( C ) Glucose tolerance test, GTT, ( D ) GTT area under the curve, ( E ) Plasma triglycerides, ( F ) Hepatic triglycerides, ( G ) Hepatic cholesterol, ( H ) Systolic blood pressure at 26 th week, ( I ) Diastolic blood pressure, ( J ) Left ventricular ejection fraction (LVEF), ( K ) Ratio of early mitral diastolic inflow velocity (E) and mitral annular early diastolic velocity (e’), ( L ) Left ventricular end diastolic pressure (LVEDP), ( M ) Treadmill exercise distance, ( N ) Plasma 3-nitrotrosine, ( O ) Plasma 8-isoprostane, ( P ) Aortic vascular reactivity to acetylcholine (ACh), (Q) Aortic vascular reactivity to sodium nitroprusside (SNP). Data in panels C-I, L and N-Q were collected at the last timepoint (week 8). Data in panels B, C, J, K, P and Q were analyzed with 2-way ANOVA followed by Sidak’s test. Data in panels D-I, L, N and O were analyzed with one-way ANOVA followed by Tukey test. Data are presented as mean ± SEM. * p < 0.05 vs. Control. * Echocardiography and Treadmill exercise, ⚲ Aortic vascular reactivity and Invasive hemodynamics. Both Survodutide and combination therapy with DATS failed to significantly reduce systemic blood pressure or LVEF ( Figure 7H - 7J ). Notably, Survodutide significantly improved diastolic function, as evidenced by reduced E/e’ and LVEDP, and enhanced exercise capacity. These beneficial effects were further augmented by DATS, resulting in a synergistic improvement in these parameters ( Figure 7K - 7O ). Furthermore, both treatments significantly improved endothelium-dependent and -independent aortic vascular relaxation ( Figure 7P and 7Q ). Remarkably, the combination therapy significantly reduced hepatic lipid content and attenuated cardiac interstitial and perivascular fibrosis and was superior to Survodutide treatment alone ( Figure 8 and Supplemental Figure 6C ). These findings highlight the therapeutic potential of H 2 S supplementation in HFpEF and demonstrate its ability to potentiate the effects of clinically relevant therapies like Survodutide. Download figure Open in new tab Figure 8. Survodutide and DATS Attenuate Hepatic Lipid Content and Cardiac Fibrosis in ZSF1 Obese Rat HFpEF Model. Representative images of hepatic lipid accumulation stained with Oil Red O, and representative images of cardiac fibrosis stained with Masson’s Trichrome and fast green counterstaining and respective quantification. Hepatic lipid content expressed as % area in bar graph. Cardiac interstitial and perivascular fibrosis expressed as % collagen fraction in bar graphs. Data were analyzed with one-way ANOVA followed by Tukey test. Data are presented as mean ± SEM. DISCUSSION In this study, we identified systemic reductions in H 2 S as a potential contributor to the development of cardiometabolic HFpEF, evident in both human patients and two distinct preclinical models. Our data indicate that H 2 S deficiency and HFpEF pathology was remediated following the administration of pharmacological agents that release H 2 S. Our data show that H 2 S deficiency in HFpEF emerges from reduced tissue CSE expression and activity, and increased tissue SQR expression. In addition, we show that H 2 S synergizes with the dual GLP-1/glucagon RA, survodutide, in ameliorating HFpEF. Our findings reveal a remarkable ∼80% reduction in circulating H 2 S levels in plasma from HFpEF patients compared to non-failing controls. This dramatic decrease in H 2 S bioavailability far exceeds the ∼40-50% reduction reported in HFrEF cases 15 , suggesting that H 2 S deficiency may play an equal or more significant role in HFpEF pathophysiology. Correspondingly, our preclinical models demonstrated a 60% reduction in circulating H 2 S in the two-hit mouse model and up to 90% in ZSF1 Ob rats. Notably, the ZSF1 Ob rat exhibits a more pronounced H 2 S deficiency that closely mirrors the clinical observations in HFpEF patients. We further examined H 2 S levels across various organs including the heart, liver, and kidney given the systemic nature of HFpEF. In both the two-hit mouse model and ZSF1 Ob rats, we observed a decline in H 2 S levels in the plasma and peripheral orans. Utilizing the two-hit mouse model, we confirmed the presence of pronounced diastolic dysfunction, exercise intolerance, and metabolic dysregulation, consistent with previous studies 5 , 44 . The observed H 2 S deficiency was primarily attributable to decreased expression and activity of CSE in peripheral organs. While CSE is constitutively expressed in the liver, kidneys, vasculature, and heart, vascular endothelial cells as well as the liver and kidney 10 , 54 are believed to be the primary regulators of circulating H 2 S levels. Our experiments with endothelial CSE knockout mice revealed that the depletion of this physiological sulfur pool exacerbated cardiac and vascular dysfunction. Conversely, exogenous H 2 S therapy with the H 2 S donor, JK-1, resulted in cardiovascular beneficial effects and marked improvements in exercise capacity. Indeed, previous studies show that CSE-derived H 2 S has multiple cardiovascular benefits including vasorelaxation 55 - 57 , angiogenesis regulation 56 , 58 , 59 , atheroprotection 60 - 63 and reduced myocardial remodeling 22 , 43 , 64 , 65 . This study focused on CSE as an H 2 S producing enzyme as CBS is primarily localized to the central nervous system and has limited evidence of involvement in cardiovascular diseases 12 . The role of the mitochondrial 3-MST in HFpEF is currently under investigation by our group. Our observations in ZSF1 Ob rats further corroborate the results from the two-hit model, as this model is characterized by a comprehensive phenotype that includes hypertension, obesity, diabetes, diastolic dysfunction, exercise intolerance, non-alcoholic fatty liver disease (NAFLD), and renal impairment 53 , 66 , 67 . Notably, the ZSF1 Ob rats exhibited even greater reductions in circulating, hepatic, and renal H 2 S levels, potentially elucidating the observed hepatic and renal pathologies in this model 66 , 67 . While both models share reductions in CSE expression, ZSF1 Ob rats demonstrated increased SQR expression. The later enzyme catalyzes the first irreversible step in the metabolism of H 2 S, and thus plays a pivotal role in the regulation of H 2 S-mediated signaling 12 . Previous data show that SQR inhibition attenuated hypertrophy, lung congestion, LV dilatation and myocardial fibrosis in HFrEF 68 . Future studies could explore the potential of SQR inhibitors as a therapeutic strategy for HFpEF. While previous investigations have primarily focused on H 2 S effects within single organ systems, our findings underscore the importance of considering systemic dysregulated H 2 S signaling in HFpEF. The pleiotropic nature of H 2 S suggests that multiple mechanisms may contribute to the observed phenotype. Dysfunction in the liver and kidney, as observed in many HFpEF patients, can significantly impact overall H 2 S bioavailability. For example, hepatic dysfunction, often observed in patients with HFpEF, can lead to impaired H 2 S production and contribute to the systemic H 2 S deficiency observed in our study. This emphasizes the systemic nature of H 2 S dysregulation in HFpEF and highlights the importance of considering the contributions of multiple organs, beyond the heart, to the overall pathophysiology of this complex disease. A key clinically relevant finding from our study is the synergistic benefits of H 2 S when combined with the dual GLP-1/glucagon RA, survodutide, given the complexity of cardiometabolic HFpEF that necessitates a multifaceted treatment approach. While current therapies such as SGLT2 inhibitors and mineralocorticoid receptor antagonists offer some benefit, they have limited impact on mortality in HFpEF patients 69 , 70 . This unmet need drives the search for novel therapies, including GLP-1RAs. Notably, this drug class is clinically used to treat the major comorbidities of HFpEF i.e., obesity and diabetes and recently have gained a considerable attention due to their potential efficacy in a plethora of many diseases. Emerging clinical investigations include cardiovascular diseases, metabolic liver disease, chronic kidney disease, sleep apnea, neurodegenerative disorders, addiction and obesity-associated cancers 71 - 73 . The single GLP-1RA, semaglutide, significantly improved heart failure symptoms and physical limitations, and lowered the risk of major adverse outcomes of heart failure in obese HFpEF patients without (STEP-HFpEF trial) or with (STEP-HFpEF DM trial) type 2 diabetes 33 , 74 . Interestingly, semaglutide attenuated CRP levels and was associated with 10.7% and 6.4% reductions in body weight in both trials, respectively. Mechanistically, the beneficial effects of semaglutide in HFpEF was attributed to the extent of weight loss 75 , reducing inflammation 76 and improving adverse cardiac remodeling 77 . Remarkably, a pooled analysis of four major semaglutide trials (SELECT, FLOW, STEP-HFpEF, and STEP-HFpEF DM) confirmed the substantial efficacy of this drug in heart failure 78 . The remarkable findings of semaglutide in HFpEF encouraged the investigation of the dual GLP-1/GIP RA, tirzepatide, in this condition (SUMMIT trial) 32 . Similar to semaglutide, tirzepatide reduced CRP and body weight (11.6%), and was associated with reduced worsening heart-failure events and improved exercise tolerance. In addition to body weight reduction, other mechanisms for tirzepatide in HFpEF have been suggested, including reduced circulatory volume–pressure overload and systemic inflammation and mitigated cardiovascular–kidney end-organ injury 79 , and reducing paracardiac adipose tissue 80 . The dual GLP-1/glucagon RA including survodutide, is a new emerging class under development and has not been explored in HFpEF. This class was discovered based on oxyntomodulin, an endogenous peptide hormone with a weak dual agonism on GLP-1 and glucagon receptors 81 , to simultaneously target both energy expenditure and food intake and induce weight loss. Survodutide has been examined in a phase 2 randomized, double-blind trial in persons with pre-obesity and obesity without diabetes and led to a dose-dependent reduction in body weight 82 , 83 . A post hoc analysis revealed that survodutide also improved both systolic and diastolic blood pressure 84 . In a 16-week trial, survodutide demonstrated superior efficacy to semaglutide in reducing HbA1c levels and body weight in patients with type 2 diabetes 85 . Recently, survodutide has been revealed to improve metabolic dysfunction-associated steatohepatitis (MASH) with significant improvements in fibrosis in a phase 2 randomized trial 34 . This drug is currently investigated in a phase 3, randomized trial (SYNCHRONIZE-CVOT) to determine its cardiovascular safety and efficacy 86 . Our study represents the first investigation into the beneficial effects of this class in HFpEF. Furthermore, while these drugs offer substantial potential, we hypothesize that combining them with H 2 S donors could synergistically enhance their therapeutic impact and provide a more comprehensive approach to combating this complex disease. Our study indicates that H 2 S may further enhance the weight loss and metabolic benefits of GLP-1RAs. This is supported by observed improvements in lipid profiles and reduced hepatic lipid content. While previous research demonstrated that survodutide reduces both circulating and hepatic lipids in diet-induced obese mice, those experiments showed maintained glycemic control, a finding that differs from our results in ZSF1 Ob rats 87 . This discrepancy may be attributed to species-specific differences. ZSF1 Ob rats have higher baseline glucose levels compared to mice and could be more susceptible to the hyperglycemic effects of glucagon. Additionally, the previous study utilized a shorter subchronic design, unlike our study. Although clinical studies on survodutide have not specifically addressed glucose tolerance, they have shown a reduction in HbA1c levels 85 . The synergy of H 2 S with survodutide on improving metabolic parameters emerges from H 2 S pleiotropic actions in multiple organs and its effect on hepatic and lipid metabolism 88 , 89 . This study reveals a striking synergy between survodutide and H 2 S in enhancing cardiac function, exercise tolerance, and reducing cardiac fibrosis in a ZSF1 Ob model of HFpEF. This synergistic effect is particularly noteworthy when compared to our previous investigation, which demonstrated a potentiation effect with the combination of the SGLT2 inhibitor empagliflozin and H 2 S in the same model. Several key findings emerge from this study. Firstly, survodutide alone demonstrated superior efficacy compared to empagliflozin, which had no significant impact on multiple parameters. Secondly, the combination of H 2 S and survodutide markedly improved exercise capacity, a clinically relevant marker. This combination led to progressive improvement in exercise capacity over time, whereas H 2 S plus empagliflozin only prevented deterioration without further enhancement. Crucially, this study administered survodutide and H 2 S during the advanced, severe stage of HFpEF (between 18 and 26 weeks of age). This contrasts with our previous study, where empagliflozin and H 2 S treatment was initiated earlier (10 to 18 weeks of age) before significant disease progression. STUDY LIMITATIONS This study utilized male ZSF1 Ob rats and murine models of hypertension and obesity-induced HFpEF. While these rodent models are well-established and characterized, they primarily reflect metabolic dysfunction and blood pressure dysregulation, representing only a subset of HFpEF patients. Additionally, the exclusive use of male rats and mice limits the generalizability of our findings. Future research should focus on investigating changes in cardiac and systemic H 2 S bioavailability, as well as the effects of H 2 S therapy, in female animals across various HFpEF subtypes. Another limitation is the use of only endothelial-specific CSE KO and Tg mice, while CSE is highly expressed in other organs like the liver and the kidney. Future studies should focus on global or organ specific CSE knockout. A further limitation is that H 2 S donor therapy was administered early in the development and progression of HFpEF. Specifically, in the murine model, the H 2 S donor JK-1 was given five weeks after the initiation of L-NAME and a high-fat diet, a period during which H 2 S levels remain relatively normal. To rigorously evaluate H 2 S therapy’s efficacy in HFpEF, future experiments should involve administering H 2 S donors at later stages of HFpEF progression. CONCLUSIONS This study offers novel insights into the dysregulation of H 2 S in cardiometabolic HFpEF. We observed significant reductions in H 2 S and sulfane sulfur levels in circulation, as well as in myocardial, hepatic, and renal tissues across multiple clinically relevant HFpEF models. These decreases are primarily attributed to diminished CSE expression and activity and increased SQR expression. Furthermore, enhancing H 2 S bioavailability through pharmacological administration markedly improved the HFpEF phenotype. Future research should focus on the organ-specific effects of H 2 S modulation through genetic models targeting CSE, 3-MST, and SQR, as well as the precise mechanisms underlying the global and local benefits of H 2 S donation in HFpEF. Additionally, the efficacy of SQR inhibitors in HFpEF should be explored. Our findings demonstrate that H 2 S donor therapy synergistically enhances the beneficial effects of GLP-1/glucagon RAs, such as Survodutide, in improving metabolic parameters, diastolic function, exercise capacity, and reducing oxidative stress and fibrosis in a preclinical model of HFpEF. This synergistic interaction suggests that combining H 2 S donors with GLP-1/glucagon RAa may represent a promising therapeutic strategy for the treatment of cardiometabolic HFpEF. Given that cardiometabolic HFpEF is characterized by multi-organ dysfunction, there is an urgent need for innovative therapeutic strategies and a deeper understanding of its pathophysiological drivers. Our findings strongly support a central role for H 2 S deficiency in the pathology of cardiometabolic HFpEF, highlighting the necessity for further investigation into the role of H 2 S in this condition. Clinical Perspectives Clinical competencies This study highlights the critical role of hydrogen sulfide (H 2 S) in heart failure with preserved ejection fraction (HFpEF), underscoring several key competencies for clinicians. First, healthcare providers should recognize the potential for H 2 S deficiency in HFpEF patients, as this deficiency may contribute to disease progression and poor outcomes. Understanding the underlying pathophysiology—specifically the decreased production of H 2 S by cystathionine-γ-lyase (CSE) and its increased metabolism by sulfide quinone oxidoreductase (SQR)—can inform treatment strategies. Additionally, the findings suggest that pharmacological H 2 S supplementation may improve diastolic function and reduce cardiac fibrosis, encouraging clinicians to consider emerging therapies that target H 2 S bioavailability. Importantly, the study demonstrates a synergistic interaction between H 2 S supplementation and GLP-1/glucagon RAs in improving key HFpEF parameters. This finding has significant clinical implications, as it suggests that combining these two therapeutic approaches may offer a more effective treatment strategy than either agent alone for patients with HFpEF. This study ultimately emphasizes the importance of a holistic approach to managing HFpEF, integrating metabolic and inflammatory factors alongside traditional cardiac care. Translational Outlook While the study offers promising insights, several barriers must be addressed for successful clinical translation. Further research is needed to establish optimal dosing, delivery methods, and long-term safety of H 2 S donors in HFpEF patients. It is also crucial to explore how individual patient characteristics, including comorbidities and genetic factors, influence H 2 S metabolism and response to therapy. Robust clinical trials are essential to validate the efficacy of H 2 S supplementation across diverse populations with HFpEF, ensuring that findings are generalizable. Lastly, the integration of H 2 S-related interventions into existing clinical guidelines for HFpEF management will require collaboration among researchers, clinicians, and guideline committees. By addressing these barriers, future research can enhance clinical practice and improve outcomes for patients with HFpEF. Data Sharing Statement The data underlying this article will be shared on reasonable request to the corresponding author. Acknowledgements We thank Alexandra Nevins, Silpa Arkat, and Cell Biology and Bioimaging Core at Pennington Biomedical Research Center for technical support. Footnotes Author/Funding disclosures: These studies were supported by grants from the National Institutes of Health HL146098, HL146514, and HL151398 to D.J.L., HL159428 to T.T.G., AA029984 to T.E.S., AHA Postdoctoral Fellowship to Z.L. (20POST35200075), P20GM135002 and U54GM104940 to T.D.A., and NIH CCTS-Training Grant TL1TR003106 to J.D. D.J.L. serves as an unpaid consultant for Sulfagenix, Inc. The other authors declare no conflicts of interest. Tweet: H 2 S deficiency is linked to HFpEF in humans and animal models. Reduced production by CSE and increased metabolism by SQR impair H 2 S bioavailability. H 2 S donor synergizes with GLP-1/glucagon agonist to ameliorate HFpEF. These findings suggest a promising therapeutic approach for HFpEF. # LeferLab # HeartFailure # H 2 SResearch . New results including a combination therapy of an H2S donor with the GLP-1/glucagon receptor agonist, survodutide, in HFpEF. ABBREVIATIONS AND ACRONYMS CSE cystathionine-γ-lyase GLP-1 glucagon-like peptide 1 GLP-1RA GLP-1 receptor agonist HFpEF heart failure with preserved ejection fraction HFrEF heart failure with reduced ejection fraction L-NAME N(ω)-nitro-L-arginine methyl ester LV left ventricle SNP sodium nitroprusside SQR sulfide quinone oxidoreductase WKY Wistar Kyoto ZSF1 Zucker fatty and spontaneously hypertensive REFERENCES 1. ↵ Savarese G , Becher PM , Lund LH , Seferovic P , Rosano GMC , Coats AJS . Global burden of heart failure: a comprehensive and updated review of epidemiology . Cardiovasc Res . 2023 ; 118 : 3272 – 3287 . doi: 10.1093/cvr/cvac013 OpenUrl CrossRef PubMed 2. Loop MS , Van Dyke MK , Chen L , Brown TM , Durant RW , Safford MM , Levitan EB . Comparison of Length of Stay, 30-Day Mortality, and 30-Day Readmission Rates in Medicare Patients With Heart Failure and With Reduced Versus Preserved Ejection Fraction . 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