AIMS:Overactivity of the sympathetic nervous system is a common underlying mechanism in development and progression of several heart failure with preserved ejection fraction (HFpEF) comorbidities. Decreasing renal sympathetic nerve activity using catheter-based renal denervation (RDN) systems have shown efficacy in treating resistant hypertension and cardiac dysfunction in heart failure with reduced ejection fraction. The purpose of this study was to determine if modulation of renal sympathetic nerve activity by RDN improves cardiac function and exercise tolerance in a clinically-relevant minipig model of cardiometabolic HFpEF. METHODS AND RESULTS:Multiple HFpEF comorbidities were induced in adult female Göttingen minipigs by mineralocorticoid excess and a diet high in cholesterol, fat, fructose, and salt. HFpEF minipigs were randomized to bilateral catheter-RDN (n = 5) treatment or sham-RDN (n = 4). RDN therapy reduced renal sympathetic activity in HFpEF minipigs and increased treadmill exercise duration. Following RDN treatment, sustained improvements in diastolic function including E/e' and left atrial fractional area change were observed. Elevations in resting left ventricular filling and pulmonary pressures in HFpEF minipigs, while indicative of HFpEF severity, were unaffected by RDN treatment. Following RDN treatment, there was a transient reduction in arterial blood pressure and no change in heart rate. CONCLUSIONS:Device-based RDN may be a potential therapeutic strategy to halt the progression of HFpEF by improving cardiac diastolic function and exercise tolerance.

ABSTRACT Acute myocardial infarction (AMI) patients typically present with a constellation of one or more risk factors including hypertension, dyslipidemia, obesity, or diabetes. Neurohormonal modulation has been a mainstay pharmacotherapy in patients; however, patient compliance is a major obstacle towards clinical efficacy due to side effects and lifelong drug regimens. FDA approval of ultrasound renal denervation (uRDN) for the treatment of resistant hypertension raises the possibility of uRDN therapy for additional disease states involving sympathetic overactivity. In the current pilot study, we sought to explore if prior uRDN has cardioprotective effects against AMI in a comorbid‐laden minipig model. Göttingen minipigs (female) were subject to mineralocorticoid excess and a Western high‐fat, high‐salt diet to induce hypertension, obesity, and hyperlipidemia. Minipigs were randomized to bilateral uRDN (n = 5) treatment or sham‐RDN (n = 5) after 4 weeks. After 6 weeks of hypertension and Western high‐fat, high‐salt diet, animals were subjected to a 75‐min left anterior descending coronary artery occlusion followed by 2 weeks of reperfusion. Markers of renal nerve viability, ischemic injury, and cardiac structure and function were assessed. In the uRDN treatment group, there was reduced renal norepinephrine content, improved survival, and reduced myocardial infarct area and calcification compared to sham‐RDN treatment. Preservation of the myocardial performance Tei index indicated preserved systolic and diastolic function 2 weeks post AMI. The beneficial effects of uRDN were independent of any reductions in blood pressure. Our pilot study provides new preliminary evidence regarding the efficacy of uRDN in a preclinical large animal model of AMI that features clinically relevant comorbidities.

Heart failure with preserved ejection fraction (HFpEF) presents significant treatment challenges. We assessed hydrogen sulfide (H2S) bioavailability in HFpEF patients and 2 animal models: the "2-hit" L-NAME + high-fat diet mouse model and ZSF1 obese rats. H2S levels were significantly reduced in patients and both models, linked to decreased cystathionine-γ-lyase expression and increased sulfide quinone oxidoreductase. Cystathionine-γ-lyase knockout worsened HFpEF, whereas pharmacological supplementation with an H2S donor improved diastolic function and reduced cardiac fibrosis. H2S supplement synergized with GLP-1/glucagon agonist and ameliorated HFpEF. These findings suggest that enhancing H2S bioavailability may provide a novel therapeutic strategy for HFpEF.

BACKGROUND:Recent studies suggest aberrant elevation of iNOS (inducible NO synthase) expression and excessive protein s-nitrosylation promote the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, the interplay between NO bioavailability, enzymatic regulation of protein s-nitrosylation by transnitrosylase and denitrosylase, and HFpEF progression remains poorly defined. We investigated the molecular basis of nitrosative stress in HFpEF, focusing on alterations in NO signaling and regulation of protein s-nitrosylation. METHODS:Circulating nitrite (NO bioavailability) and nitrosothiols were quantified in patients with HFpEF. Parallel studies using rodent models of cardiometabolic HFpEF were performed to evaluate cardiac function, NO signaling, and total nitroso species during disease progression. Single-nucleus RNA sequencing and proteomic analysis were conducted to identify regulatory genes and cellular targets of pathological s-nitrosylation. RESULTS:In patients with HFpEF, circulating nitrosothiols were significantly elevated, indicating heightened nitrosative stress, whereas nitrite levels remained unchanged. In ZSF1 Obese (ob) rats, NO bioavailability declined with age, whereas total nitroso species progressively increased as HFpEF worsened. Transcriptomic analysis revealed marked upregulation of a transnitrosylase HBb (hemoglobin-β subunit), validated in both rat and human HFpEF hearts. Enzymatic assays demonstrated aberrant functions of Trx2 (thioredoxin 2) and GSNOR (S-nitrosoglutathione reductase) in ZSF1 Ob hearts. Cell-based experiments confirmed that altered expression or function of HBb, Trx2, and GSNOR resulted in elevated cellular RxNO. Additionally, similar dysregulation of s-nitrosylation dynamics was observed in the peripheral organs, such as the kidneys and liver, in HFpEF. CONCLUSIONS:These data demonstrate that nitrosative stress, evidenced by dysregulated protein s-nitrosylation occurs in the heart and peripheral organs in cardiometabolic HFpEF. Pathological alterations in NO bioavailability resulting from alterations in NOS expression or function alone do not account for this phenotype. Instead, pathological protein s-nitrosylation results in part from the imbalance between transnitrosylase and denitrosylase function. Restoration of physiological levels of protein s-nitrosylation and NO signaling may represent an effective therapeutic target for HFpEF.

Heart failure with preserved ejection fraction (HFpEF) accounts for ∼50% of HF cases. The ZSF1-obese rat model recapitulates clinical features of HFpEF including hypertension, obesity, metabolic syndrome, exercise intolerance, and diastolic dysfunction. We utilized a systems-biology approach to define the metabolic and transcriptional signatures to gain mechanistic insight into pathways contributing to HFpEF development. Male ZSF1-obese, ZSF1-lean hypertensive controls, and WKY (wild-type) controls were compared at 14 weeks of age for extensive physiological phenotyping and left ventricle (LV) tissue harvesting for unbiased-metabolomics, RNA-sequencing, and mitochondrial morphology and function. Utilizing ZSF1-lean and WKY controls enabled a distinction between hypertension-driven molecular changes driving HFpEF pathology, versus hypertension + metabolic syndrome. Comparison of ZSF1-lean vs WKY (ie, hypertension-exclusive effects) revealed metabolic remodeling suggesting increased aerobic glycolysis, decreased β-oxidation, and dysregulated purine and pyrimidine metabolism with few transcriptional changes. ZSF1-obese rats displayed worsened metabolic remodeling and robust transcriptional remodeling highlighted by upregulation of inflammatory genes and downregulation of the mitochondrial structure/function and metabolic processes. Integrated network analysis of metabolomic and RNAseq datasets revealed downregulation of most catabolic energy producing pathways, manifesting in a marked decrease in the energetic state (ie, reduced ATP/ADP, PCr/ATP). Cardiomyocyte ultrastructure analysis revealed decreased mitochondrial area, size, and cristae density, as well as increased lipid droplet content in HFpEF hearts. Impaired mitochondrial function was demonstrated by decreased substrate-mediated respiration and dysregulated calcium handling. Collectively, the integrated omics approach applied here provides a framework to uncover novel genes, metabolites, and pathways underlying HFpEF, with an emphasis on mitochondrial energy metabolism as a potential interventional target.

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.

Malonyl-CoA decarboxylase (MCD), encoded by Mlycd, plays a critical role in regulating malonyl-CoA levels, fatty acid oxidation, and glucose metabolism. To investigate the systemic effects of Mlycd deficiency, we generated a genetically modified rat model with partial loss of Mlycd. Due to high preweaning mortality in homozygous mutants, we focused on heterozygous and wild-type animals for cross-tissue targeted proteomic and metabolomic analyses. Alterations in metabolic and stress-related pathways were observed across the heart, kidney, liver, plasma, and urine. Enrichment of oxidative phosphorylation, fatty acid metabolism, immune responses, and autophagy-related signaling was noted. Elevated levels of oxidative stress-related proteins and disrupted autophagy regulation were particularly evident in the kidney and liver. Additionally, several lysine conjugates, including trimethyllysine, N-methyl-pipecolic acid, and Nε-acetyl-lysine, were decreased in the kidney. These findings demonstrate the widespread physiological impact of Mlycd deficiency and highlight its role in coordinating energy metabolism and cellular stress responses across multiple organ systems.

Summary: Heart failure with preserved ejection fraction (HFpEF) presents significant treatment challenges. We assessed hydrogen sulfide (H2S) bioavailability in HFpEF patients and 2 animal models: the ''2-hit'' L-NAME + high-fat diet mouse model and ZSF1 obese rats. H2S levels were significantly reduced in patients and both models, linked to decreased cystathionine-γ-lyase expression and increased sulfide quinone oxidoreductase. Cystathionine-γ-lyase knockout worsened HFpEF, whereas pharmacological supplementation with an H2S donor improved diastolic function and reduced cardiac fibrosis. H2S supplement synergized with GLP-1/glucagon agonist and ameliorated HFpEF. These findings suggest that enhancing H2S bioavailability may provide a novel therapeutic strategy for HFpEF.

Swine are increasingly used in cardiovascular research due to their anatomical and physiological similarities to humans, particularly for studying diastolic dysfunction. Although MRI offers excellent structural imaging, echocardiography provides superior real-time assessment of diastolic parameters. To address the lack of standardized methods and reduce variability across studies, we present a comprehensive guide for performing echocardiography in Yorkshire pigs, detailing anatomical considerations, equipment requirements, and technical approaches. We describe systematic approaches for obtaining and optimizing right parasternal long- and short-axis views, apical four-chamber, and subcostal imaging windows, with specific attention to anatomical variations from human cardiac orientation and standard clinical transducer positioning. These tomographic views enable a comprehensive assessment of systolic and diastolic function, including ventricular volumes, wall thicknesses, chamber dimensions, ejection fraction, and Doppler measurements of blood flow and tissue velocities. This standardized methodology for echocardiographic images acquisition enhances data reliability in cardiovascular pig models, improving the interpretation of preclinical study results and strengthening translational research outcomes. The protocol also provides consistency for veterinary applications, making echocardiography a preferred modality for longitudinal studies in this valuable translational model.NEW & NOTEWORTHY Anatomical positioning: porcine heart is more central and caudal than human heart; apical views are obtained at sixth to seventh intercostal space; right parasternal views are most reliable in pigs. Technical requirements are as follows: standardized sedation protocol is essential; specialized veterinary procedure table; and integrated electrocardiogram (ECG) monitoring for timing. Clinical value: excellent translational model for cardiovascular research; regular protocol adjustment based on animal size.

BACKGROUND AND PURPOSE:Sodium glucose cotransporter 2 inhibitors (SGLT2i) have emerged as a potent therapy for heart failure with preserved ejection fraction (HFpEF). Hydrogen sulphide (H2S), a well-studied cardioprotective agent, could be beneficial in HFpEF. SGLT2i monotherapy and combination therapy involving an SGLT2i and H2S donor in two preclinical models of cardiometabolic HFpEF was investigated. EXPERIMENTAL APPROACH:Nine-week-old C57BL/6N mice received L-NAME and a 60% high fat diet for five weeks. Mice were then randomized to either control, SGLT2i monotherapy or SGLT2i and H2S donor, SG1002, for five additional weeks. Ten-week-old ZSF1 obese rats were randomized to control, SGLT2i or SGLT2i and SG1002 for 8 weeks. SG1002 monotherapy was investigated in additional animals. Cardiac function (echocardiography and haemodynamics), exercise capacity, glucose handling and multiorgan pathology were monitored during experimental protocols. KEY RESULTS:SGLT2i treatment improved E/e' ratio and treadmill exercise in both models. Combination therapy afforded increases in cardiovascular sulphur bioavailability that coincided with improved left end-diastolic function (E/e' ratio), exercise capacity, metabolic state, cardiorenal fibrosis, and hepatic steatosis. Follow-up studies with SG1002 monotherapy revealed improvements in diastolic function, exercise capacity and multiorgan histopathology. CONCLUSIONS AND IMPLICATIONS:SGLT2i monotherapy remediated pathological complications exhibited by two well-established HFpEF models. Adjunctive H2S therapy resulted in further improvements of cardiometabolic perturbations beyond SGLT2i monotherapy. Follow-up SG1002 monotherapy studies inferred an improved phenotype with combination therapy beyond either monotherapy. These data demonstrate the differing effects of SGLT2i and H2S therapy while also revealing the superior efficacy of the combination therapy in cardiometabolic HFpEF.

Human cardiomyocytes from very obese patients with heart failure and preserved ejection fraction (HFpEF) have markedly depressed calcium-activated tension and increased resting stiffness. To test if either are recapitulated by obese-HFpEF animal models, tension‑calcium and tension-sarcomere length relations were measured in myocytes from mice on a high fat diet (HFD) with L-NAME, ZSF1 rats, and Göttingen minipigs on HFD + DOCA (MP). Only MP myocytes displayed reduced Ca2+-activated tension, and none exhibited increased resting stiffness versus respective controls. Consistent with prior myofibrillar data, crossbridge attachment and detachment rates at matched tension were slower in rodent models, and detachment slower in MP.

Originally, light-to-moderate alcohol consumption was believed to provide some protective effects on the cardiovascular system through its antioxidant properties and other constituents; this was illustrated through the longstanding J-shaped curve association between alcohol consumption and the incidence of cardiovascular disease (Gillman et al., 1995). However, more recent meta-analyses and genome-wide association studies reveal that a paradigm shift from the beneficial to harmful effects of alcohol consumption has occurred after taking into account other confounders, such as the participants' background and lifestyle (Biddinger et al., 2022). Furthermore, it has been well established that chronic heavy alcohol usage causes oxidative stress and systemic inflammation, which deteriorates cardiac function over time through a myriad of cellular and organ-level perturbations, resulting in fibrosis development (Bishehsari et al., 2017). As a result, inhibiting inflammation has become a treatment strategy to restore myocardial function and prevent fibrosis progression induced by alcohol consumption. Thromboxane A2 receptor (TPR), a G-protein-coupled receptor, is ubiquitously expressed in multiple organs and cell types including platelets, macrophages, monocytes, and vascular endothelial cells (Giannarelli et al., 2010). Upon activation through a myriad of ligands including 8-iso prostaglandin (8-iso) and/or thromboxane A2 (TXA2), produced primarily by platelets (Smyth, 2010), TPR signaling can result in cytoskeleton alterations, platelet adhesion, and vesicle trafficking to induce platelet activation (Ting et al., 2012). Thus, mutations and dysregulation of TPR signaling play a role in the pathogenesis of several cardiovascular diseases, including myocardial infarction, stroke, atherosclerosis, and thrombosis (Rucker & Dhamoon, 2019). As a result, TPR presents as a promising therapeutic target, and many believe that developing the appropriate TPR antagonists would provide significant efficacy in modulating cardiovascular-related diseases, including alcohol-induced cardiomyopathy. Ai et al. (2024) explored the role of TPR signaling in alcohol-mediated cardiac inflammation and TPR antagonism as a treatment for early cardiac injury. The group utilized a mouse model of chronic plus binge ethanol exposure that has been extensively studied and characterized to mimic alcohol use disorders in humans (NIAAA model) (Bertola et al., 2013). In this model, 8-week-old mice were fed ethanol for 10 days, followed by one binge of ethanol. A cohort of mice in the study received a TPR antagonist, SQ29,548, during their 10 days of ethanol feeding. Measurement of various inflammatory, oxidative stress, cardiac remodeling, and fibrosis biomarkers were the primary endpoints. The group confirmed the inflammatory and oxidative stress responses induced by alcohol using only rudimentary molecular techniques, limiting the evidence on which they based their conclusions. Additional studies using flow cytometric quantification of myocardial immune cell infiltrates and/or histological assessment could provide a more substantial set of results confirming the biomarker findings. The exclusion of functional cardiac measurements raises questions about the cardiac physiological state after a 10-day binge-on-chronic ethanol and the utility of this early cardiac inflammatory phenotype, as described by the authors, to predict future outcomes in the presence or absence of TPS antagonism. The addition of rigorous physiological testing, which could include invasive hemodynamics, endurance exercise testing, or cardiac ultrasound analysis, would allow for observing any phenotypical changes due to the augmentation of these early molecular markers. In the end, the investigators exhibited the attenuating effects of SQ29,548 in reducing markers for oxidative stress, inflammation, and fibrosis compared with ethanol-fed controls. These results suggest that TPR antagonism is potentially an effective treatment for alcoholic cardiomyopathy. Although researchers have successfully developed TPR antagonists, none are FDA-approved; targeting TPR in humans remains elusive (Jourdi et al., 2021). An upstream treatment strategy involving the thromboxane pathway includes using cyclooxygenase inhibitors such as aspirin, which relies on inhibiting the TPR agonist TXA2. Aspirin inhibits COX-1, which is expressed by platelets, to reduce the biosynthesis of TXA2 (Roth et al., 1975). However, aspirin can cause nonspecific COX inhibition to disrupt the maintenance of the endothelium, resulting in gastrointestinal erosions, renal, and hepatic insufficiency (Suleyman et al., 2007). This type of nonspecificity has limited the clinical utility of current TPR-related pharmacotherapies. Most discovered TPR antagonists remain pharmacological research tools for uncovering TPR-mediated signaling events in various pathological pathways (Ting et al., 2012). Most notably, the pharmacological compound SQ29,548 presents as a highly selective TPR antagonist that helped researchers' study TPR's role in human platelet aggregation and animal disease models of asthma, rhinitis, and brain ischemia (Ogletree et al., 1985; Ting et al., 2012). Yet, TPR antagonists fail to extend beyond stage I/II of clinical trials with concerns for toxicity and effectiveness in humans (Savage et al., 1995; Serruys et al., 1991). Ultimately, TPR antagonists persist as a powerful research tool, yet they currently lack clinical utility. Researchers continue exploring TPR's role in various pathological pathways and attempting to redesign therapeutics to improve its translational profile, especially in cardiovascular diseases in which TPR expression and signaling are known to induce cardiac inflammation. In vascular and atherothrombotic diseases, TPR stimulates endothelial production of superoxide anions and reactive oxygen/nitrogen species through eNOS uncoupling, leading to the generation of oxidative stress and endothelial dysfunction (Zhang et al., 2011). Furthermore, TXA2 binds to C-EL2, a ligand binding domain of TPR, to mediate platelet aggregation in humans in vitro and murine ex vivo (Murad et al., 2012). In contrast, TPR activation is also involved in oligodendrocyte maturation as TPRs localize to the nuclear compartment during oligodendrocyte differentiation (Ramamurthy et al., 2006). A renewal of interest in studying the pharmacophore features of TPR antagonists in humans and performing docking simulations of TPR antagonists through artificial intelligence could pave the way for novel and more effective antagonist designs (Hu et al., 2020). To solidify the role of the TPR antagonist as a viable treatment option, future studies need to improve its selectivity and cell/organ specificity. In addition, most TPR-related studies are currently done in rodent models (Xiao et al., 2001; Xie et al., 2017; Zuccollo et al., 2005). Given the evolutionary variation of TPR isoforms between rodents, large mammals, and humans, more relevant preclinical testing, which mimics the human variants and signaling, should be explored to produce more meaningful observations. With the renewed interest in drugging TPR signaling cascades, Ai et al. (2024) provides preliminary findings demonstrating, yet another disease pathology with which TPR antagonism may be a viable therapeutic target. In addition to the treatment of alcohol-induced inflammation and cardiomyopathy, successful implementation of TPR antagonism may lead to a new therapeutic strategy for modern-day cardiovascular-related diseases, including hypertension, obesity, diabetes, and heart failure. As this area of therapeutic interest attempts to resurrect itself from its historical stagnation (Ting et al., 2012), we hope that further and more rigorous studies will provide substantiation of the observation made by Ai et al. The authors (TES & TTV) have no conflicts of interest to disclose.

HomeJournal of the American Heart AssociationAhead of PrintCommon Heart Failure With Preserved Ejection Fraction Animal Models Yield Disparate Myofibril Mechanics Open AccessRapid CommunicationPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessRapid CommunicationPDF/EPUBCommon Heart Failure With Preserved Ejection Fraction Animal Models Yield Disparate Myofibril Mechanics Axel J. Fenwick, Vivek P. Jani, D. Brian Foster, Thomas E. Sharp, Traci T. Goodchild, Kyle LaPenna, Jake E. Doiron, David J. Lefer, Joseph A. Hill, David A. Kass and Anthony Cammarato Axel J. FenwickAxel J. Fenwick https://orcid.org/0000-0002-8296-537X , Division of Cardiology, Department of Medicine, , Johns Hopkins University, , Baltimore, , MD, , USA, , Vivek P. JaniVivek P. Jani https://orcid.org/0000-0002-3811-4973 , Division of Cardiology, Department of Medicine, , Johns Hopkins University, , Baltimore, , MD, , USA, , Department of Biomedical Engineering, , Johns Hopkins University, , Baltimore, , MD, , USA, , D. Brian FosterD. Brian Foster https://orcid.org/0000-0002-9290-9590 , Division of Cardiology, Department of Medicine, , Johns Hopkins University, , Baltimore, , MD, , USA, , Thomas E. SharpThomas E. Sharp https://orcid.org/0000-0001-8706-9825 , Department of Molecular Pharmacology and Physiology, , University of South Florida, , Tampa, , FL, , USA, , Traci T. GoodchildTraci T. Goodchild , Department of Cardiac Surgery, , Smidt Heart Institute, Cedars‐Sinai Medical Center, , Los Angeles, , CA, , USA, , Kyle LaPennaKyle LaPenna https://orcid.org/0000-0003-0470-0138 , Department of Medicine, Cardiovascular Center of Excellence, , Louisiana State University Health Sciences Center, , New Orleans, , LA, , USA, , Jake E. DoironJake E. Doiron https://orcid.org/0000-0002-4584-4819 , Department of Medicine, Cardiovascular Center of Excellence, , Louisiana State University Health Sciences Center, , New Orleans, , LA, , USA, , David J. LeferDavid J. Lefer https://orcid.org/0000-0003-2293-7278 , Department of Cardiac Surgery, , Smidt Heart Institute, Cedars‐Sinai Medical Center, , Los Angeles, , CA, , USA, , Joseph A. HillJoseph A. Hill https://orcid.org/0000-0002-5379-1614 , Department of Medicine, , UT Southwestern Medical Center, , Dallas, , TX, , USA, , Department of Molecular Biology, , UT Southwestern Medical Center, , Dallas, , TX, , USA, , David A. KassDavid A. Kass https://orcid.org/0000-0003-1596-2299 , Division of Cardiology, Department of Medicine, , Johns Hopkins University, , Baltimore, , MD, , USA, , Department of Biomedical Engineering, , Johns Hopkins University, , Baltimore, , MD, , USA, and Anthony CammaratoAnthony Cammarato * Correspondence to: Anthony Cammarato, PhD, Johns Hopkins University, Ross 1050, 720 Rutland Ave, Baltimore, MD 21205. Email: E-mail Address: [email protected] https://orcid.org/0000-0003-1104-4407 , Division of Cardiology, Department of Medicine, , Johns Hopkins University, , Baltimore, , MD, , USA, Originally published9 Jan 2024https://doi.org/10.1161/JAHA.123.032037Journal of the American Heart Association. 2024;0:e032037Heart failure with preserved ejection fraction (HFpEF) is a complex, multiorgan syndrome. Cardiac manifestations include diastolic stiffening and impaired relaxation, normal resting systolic function but depressed systolic reserve, and modest hypertrophy.1 Although diastolic dysfunction remains a benchmark of HFpEF, the extent to which myofibrils, the contractile organelles of myocytes, contribute to this behavior remains unknown. HFpEF animal models historically emphasized hypertension and ventricular hypertrophy to achieve diastolic dysfunction, and recently have incorporated obesity and diabetes as they are increasingly prevalent. Popular rodent models include Zucker obese/spontaneously hypertensive rats2 and mice given a high‐fat diet (HFD) and the constitutive NO synthase inhibitor, Nω‐nitro‐l‐arginine methyl ester (ʟ‐NAME) (HFD+ʟ‐NAME).3 However, neither model developed diastolic disease as severe as that observed in patients with HFpEF. Heightened diastolic pathology was achieved in larger Göttingen minipigs fed a HFD and treated with desoxycorticosterone acetate (DOCA) to induce volume retention/hypertension.4 Although each model exhibited gross‐scale diastolic dysfunction, albeit to different extents, there are no data yet reported from myofibrils on their mechanical activation and relaxation properties. Thus, it remains unclear whether the mechanistic basis of global, organ‐level diastolic impairments observed among the models involves common underlying myofibrillar deficiencies. This has become salient as newer pharmaceuticals are targeting sarcomeric proteins to treat such diseases. Therefore, to test if shared defects in subcellular mechanics exist, and thereby potentially contribute to chamber‐level pathophysiology, we resolved the kinetic parameters of contraction and relaxation of individual myofibrils from each HFpEF animal model and its respective control.The models, generated in independent laboratories, all had elevated ventricular diastolic filling pressure, normal‐range ejection fraction, myocardial hypertrophy and fibrosis, and obesity with glucose intolerance.2, 3, 4 Procedures followed were in accordance with institutional guidelines. Heart tissue from each model and control group was sectioned and frozen in liquid nitrogen. Frozen left ventricle tissue strips were incubated in a 4% triton skinning solution overnight at 4 °C and then homogenized to produce a concentrated suspension of myofibrils. Cell‐level measurements cannot time‐resolve relaxation kinetics, as even in single permeabilized cardiomyocytes, calcium diffusion is too slow to achieve this. However, given their small diameter, myofibrils promptly equilibrate with bathing solutions without significant diffusional constraints.5 Individual myofibrils were electrostatically tethered between a glass probe connected to a Piezo‐length controller and a glass cantilever of known stiffness (0.031 N/m). Sarcomere length was set to 2.1 μm from an average of ≈1.7 μm. Cantilever displacement was measured by deflection of its shadow, incident on a photodiode, and converted into tension (force/cross‐sectional area). A double‐barreled pipette attached to a fast‐step motor permitted switching between relaxing and activating bathing solutions in <1 ms. Myofibrillar relaxation kinetics following sudden calcium removal are uniquely biphasic, and each phase can inform about deficits linked to regulatory and motor proteins.5 Data supporting this study are available from the corresponding author on reasonable request.Myofibrils from HFpEF minipigs exhibited no difference in resting tension (Figure [A] and [B]) but produced significantly less maximal active tension compared with control (Figure [A] and [C]). By contrast, resting and active tension generated by myofibrils from Zucker obese/spontaneously hypertensive rats and HFD+ʟ‐NAME mice were similar to controls, although myofibrillar activation was slower in the obese versus lean rats (Figure [D]). With respect to relaxation kinetics, we observed no differences in the duration or rate of the initial slow linear (Figure [A], [E], and [F]) or subsequent fast exponential relaxation phase (Figure [A] and [G]) in minipig myofibrils. Nevertheless, the initial linear relaxation phase was prolonged and slower in rodent HFpEF models versus controls, with Zucker obese/spontaneously hypertensive rat myofibrils additionally displaying a slower subsequent fast exponential‐decay rate. Collectively, these data show depressed systolic myofibrillar function only in the pig model and reduced relaxation rates exclusively in the rodent models. Shifts from fast α‐myosin to slower β‐myosin could partially explain differences in relaxation kinetics.5 However, mass spectrometry analysis of the purified myofibrils revealed no significant increases in β‐myosin versus α‐myosin heavy chain protein content in any of the models, implying alternative underlying causes.Download figureDownload PowerPointFigure . Kinetic and mechanical properties of myofibrils from heart failure with preserved ejection fraction (HFpEF) models.A, Averaged myofibril tension over time (solid line) and SE (shaded region) for each model, aligned to the time of solution change (insets: dashed lines and arrows indicate the time of solution change; SP and FP highlight the initial, slow linear and subsequent fast, exponential phases of relaxation, respectively). B, Resting tension is steady‐state myofibril tension produced at pCa 8 (log10[Ca2+]) (gray bars represent mean values). C, Active tension is the difference between steady‐state maximal tension produced at pCa 4 and the resting tension. D, Myofibrillar activation rate as calculated from a monoexponential fit. E, The slow linear phase of relaxation is mediated by thin filament deactivation and cross‐bridge detachment.5 The start of the slow phase is provided by the trigger signal of the solution exchange motor, and its end is determined by the peak residual error of a progressive linear fit of the subsequent data. F, The rate of the slow linear phase is calculated as the slope of the slow phase, normalized to the active tension of the myofibril. G, The fast exponential phase is primarily mediated by passive sarcomeric elements and is independent of thin filament activation.5 The rate of the fast phase is determined by a monoexponential fit of the data starting from the end of the slow phase. N=3 animals for each group, and n=5 to 9 myofibrils from each animal. Significance was assessed using a 2‐tailed Welch t‐test between controls and experimental groups of each model (P values are listed above each comparison; P<0.05 in bold). HFD indicates high‐fat diet; ʟ‐NAME, Nω‐nitro‐l‐arginine methyl ester; WT, wild type; and ZSF1, Zucker obese/spontaneously hypertensive rat.Our findings demonstrate that myofibril mechanics vary significantly between HFpEF animal models, suggesting that some may uniquely recapitulate distinct aspects of the disorder and particular subphenotypes observed among patients.1 Although all 3 models had signs of diastolic dysfunction, impaired myofibril relaxation appears to contribute mechanistically to chamber‐level dysfunction, only in the rodent models. In the minipig model, because myofibril relaxation was unperturbed, global changes in diastole must involve other cellular‐ and tissue‐level pathologies. This discordance likely stems from physiological differences inherent among the hearts of small and large mammals and the unique regimens deployed to engender HFpEF‐like phenotypes. No single experimental system can perfectly mimic human disease, and this is especially true with HFpEF, because of its complex multiorgan pathology and distinct subphenotypes. These intricacies thus necessitate the judicious selection, or even combinatorial use, of distinct animal models to better simulate and understand various disease facets, to help refine therapeutic targeting, and to enhance the translational impact of preclinical HFpEF research.Sources of FundingThese studies were supported by National Institutes of Health (NIH) grant T32HL007227 and an American Heart Association/DC Women's Board grant 22POS915659 to Dr Fenwick; American Heart Association grant 23PRE1026275 and NIH grant F31HL168850 to V. P. Jani; NIH grant HL164478 to Dr Foster; NIH grant AA029984 to Dr Sharp; NIH grant HL159428 to Dr Goodchild; NIH grant TL1TR003106 to J. E. Doiron; NIH grants HL146098, HL146514, and HL151398 to Dr Lefer; NIH grants HL128215, HL147933, HL155765, and HL164586 to Dr Hill; NIH grants R35HL135827, R35HL166565, and support from Cytokinetics to Dr Kass; and NIH grant HL124091 to Dr Cammarato.DisclosuresDr Lefer is a consultant for Sulfagenix. Dr Kass receives research support from Amgen related to human heart failure with preserved ejection fraction studies. The remaining authors have no disclosures to report.Footnotes* Correspondence to: Anthony Cammarato, PhD, Johns Hopkins University, Ross 1050, 720 Rutland Ave, Baltimore, MD 21205. Email: acammar3@jhmi.eduThis article was sent to Julie K. Freed, MD, PhD, Associate Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 3.References1 Shah SJ, Borlaug BA, Kitzman DW, McCulloch AD, Blaxall BC, Agarwal R, Chirinos JA, Collins S, Deo RC, Gladwin MT, et al. Research priorities for heart failure with preserved ejection fraction: National Heart, Lung, and Blood Institute working group summary. Circulation. 2020; 141:1001–1026. doi: 10.1161/CIRCULATIONAHA.119.041886LinkGoogle Scholar2 Schauer A, Draskowski R, Jannasch A, Kirchhoff V, Goto K, Männel A, Barthel P, Augstein A, Winzer E, Tugtekin M, et al. ZSF1 rat as animal model for HFpEF: development of reduced diastolic function and skeletal muscle dysfunction. ESC Heart Fail. 2020; 7:2123–2134. doi: 10.1002/ehf2.12915CrossrefMedlineGoogle Scholar3 Schiattarella GG, Altamirano F, Tong D, French KM, Villalobos E, Kim SY, Luo X, Jiang N, May HI, Wang ZV, et al. Nitrosative stress drives heart failure with preserved ejection fraction. Nature. 2019; 568:351–356. doi: 10.1038/s41586-019-1100-zCrossrefMedlineGoogle Scholar4 Sharp TE, Scarborough AL, Li Z, Polhemus DJ, Hidalgo HA, Schumacher JD, Matsuura TR, Jenkins JS, Kelly DP, Goodchild TT, et al. Novel Göttingen miniswine model of heart failure with preserved ejection fraction integrating multiple comorbidities. JACC Basic Transl Sci. 2021; 6:154–170. doi: 10.1016/j.jacbts.2020.11.012CrossrefMedlineGoogle Scholar5 Stehle R, Solzin J, Iorga B, Poggesi C. Insights into the kinetics of Ca2+‐regulated contraction and relaxation from myofibril studies. Pflugers Arch. 2009; 458:337–357. doi: 10.1007/s00424-008-0630-2CrossrefMedlineGoogle Scholar eLetters(0) eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. 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Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.https://doi.org/10.1161/JAHA.123.032037PMID: 38193306 Manuscript receivedAugust 1, 2023Manuscript acceptedNovember 27, 2023Originally publishedJanuary 9, 2024 Keywordsanimal modelsdiastoleheart failureheart failure with preserved ejection fractionmyofibrilPDF download Subjects Animal Models of Human Disease Basic Science Research Contractile Function Heart Failure

Background The renal sympathetic nervous system modulates systemic blood pressure, cardiac performance, and renal function. Pathological increases in renal sympathetic nerve activity contribute to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). We investigated the effects of renal sympathetic denervation performed at early or late stages of HFpEF progression. Methods and Results Male ZSF1 obese rats were subjected to radiofrequency renal denervation (RF‐RDN) or sham procedure at either 8 weeks or 20 weeks of age and assessed for cardiovascular function, exercise capacity, and cardiorenal fibrosis. Renal norepinephrine and renal nerve tyrosine hydroxylase staining were performed to quantify denervation following RF‐RDN. In addition, renal injury, oxidative stress, inflammation, and profibrotic biomarkers were evaluated to determine pathways associated with RDN. RF‐RDN significantly reduced renal norepinephrine and tyrosine hydroxylase content in both study cohorts. RF‐RDN therapy performed at 8 weeks of age attenuated cardiac dysfunction, reduced cardiorenal fibrosis, and improved endothelial‐dependent vascular reactivity. These improvements were associated with reductions in renal injury markers, expression of renal NLR family pyrin domain containing 3/interleukin 1β, and expression of profibrotic mediators. RF‐RDN failed to exert beneficial effects when administered in the 20‐week‐old HFpEF cohort. Conclusions Our data demonstrate that early RF‐RDN therapy protects against HFpEF disease progression in part due to the attenuation of renal fibrosis and inflammation. In contrast, the renoprotective and left ventricular functional improvements were lost when RF‐RDN was performed in later HFpEF progression. These results suggest that RDN may be a viable treatment option for HFpEF during the early stages of this systemic inflammatory disease.

The mechanism(s) underlying gut microbial metabolite (GMM) contribution towards alcohol-mediated cardiovascular disease (CVD) is unknown. Herein we observe elevation in circulating phenylacetylglutamine (PAGln), a known CVD-associated GMM, in individuals living with alcohol use disorder. In a male murine binge-on-chronic alcohol model, we confirm gut microbial reorganization, elevation in PAGln levels, and the presence of cardiovascular pathophysiology. Fecal microbiota transplantation from pair-/alcohol-fed mice into naïve male mice demonstrates the transmissibility of PAGln production and the CVD phenotype. Independent of alcohol exposure, pharmacological-mediated increases in PAGln elicits direct cardiac and vascular dysfunction. PAGln induced hypercontractility and altered calcium cycling in isolated cardiomyocytes providing evidence of improper relaxation which corresponds to elevated filling pressures observed in vivo. Furthermore, PAGln directly induces vascular endothelial cell activation through induction of oxidative stress leading to endothelial cell dysfunction. We thus reveal that the alcohol-induced microbial reorganization and resultant GMM elevation, specifically PAGln, directly contributes to CVD.

Background:Recent reports suggest increased myocardial iNOS expression leads to excessive protein s -nitrosylation, contributing to the pathophysiology of HFpEF. However, the relationship between NO bioavailability, dynamic regulation of protein s -nitrosylation by trans- and de-nitrosylases, and HFpEF pathophysiology has not been elucidated. Here, we provide novel insights into the delicate interplay between NO bioavailability and protein s -nitrosylation in HFpEF. Methods:Plasma nitrite, nitrosothiols (RsNO), and 3-nitrotyrosine (3-NT) were measured in HFpEF patients and in controls. Studies in WKY or ZSF1 obese rats were performed to evaluate HFpEF severity, NO signaling, and total nitroso-species (Rx(s)NO) levels. snRNA sequencing was performed to identify key genes involved in NO signaling and s -nitrosylation regulation. Results:In HFpEF patients, circulating RsNO and 3-NT were significantly elevated while nitrite, a biomarker for NO bioavailability, remained unchanged. In ZSF1 obese rats, NO bioavailability was significantly reduced while Rx(s)NO levels exhibited an age-dependent increase as HFpEF progressed. snRNA seq highlighted significant upregulation of a trans-nitrosylase, hemoglobin-beta subunit (HBb), which was corroborated in human HFpEF hearts 1 . Subsequent experiments confirmed HBb upregulation and revealed significant reductions in enzyme activity of two major de-nitrosylases, Trx2 and GSNOR in ZSF1 obese hearts. Further, elevated RxNO levels, increased HBb expression, and reduced activity of Trx2 and GSNOR were identified in the kidney and liver of the ZSF1 obese rats. Conclusions:Our data reveal circulating markers of nitrosative stress (RsNO and 3-NT) are significantly elevated in HFpEF patients. Data from the ZSF1 obese rat model mirror the results from HFpEF patients and reveal that pathological accumulation of RxNO/nitrosative stress in HFpEF may be in part, due to the upregulation of the trans-nitrosylase, HBb, and impaired activity of the de-nitrosylases, Trx2 and GSNOR. Our data suggest that dysregulated protein nitrosylation dynamics in the heart, liver, and kidney contribute to the pathogenesis of cardiometabolic HFpEF. Translational Perspective:Our findings describe for the first time that circulating RsNO and 3-NT are significantly upregulated in HFpEF patients suggesting systemic nitrosative stress in HFpEF, and demonstrate a profound disconnect between insufficient physiological NO signaling and pathological nitrosative stress in HFpEF, which is in stark contrast to HFrEF in which both NO bioavailability and protein s -nitrosylation are attenuated. Further, this study provides novel mechanistic insights into a critical molecular feature of HFpEF in humans and animal models: nitrosative stress arises predominantly from imbalance of trans-nitrosylases and de-nitrosylases, thereby leading to impaired NO bioavailability concomitant with increased protein s -nitrosylation. Importantly, these perturbations extend beyond the heart to the kidney and liver, suggesting HFpEF is characterized by a systemic derangement in trans- and de-nitrosylase activity and providing a unifying molecular lesion for the systemic presentation of HFpEF pathophysiology. These findings have direct clinical implications for the modulation of NO levels in the HFpEF patient, and indicate that restoring the balance between trans- and denitrosylases may be novel therapeutic targets to ameliorate disease symptoms in HFpEF patients.

Background:Heart failure with preserved ejection fraction (HFpEF) accounts for ~50% of HF cases, with no effective treatments. The ZSF1-obese rat model recapitulates numerous clinical features of HFpEF including hypertension, obesity, metabolic syndrome, exercise intolerance, and LV diastolic dysfunction. Here, we utilized a systems-biology approach to define the early metabolic and transcriptional signatures to gain mechanistic insight into the pathways contributing to HFpEF development. Methods:Male ZSF1-obese, ZSF1-lean hypertensive controls, and WKY (wild-type) controls were compared at 14w of age for extensive physiological phenotyping and LV tissue harvesting for unbiased metabolomics, RNA-sequencing, and assessment of mitochondrial morphology and function. Utilizing ZSF1-lean and WKY controls enabled a distinction between hypertension-driven molecular changes contributing to HFpEF pathology, versus hypertension + metabolic syndrome. Results:ZSF1-obese rats displayed numerous clinical features of HFpEF. Comparison of ZSF1-lean vs WKY (i.e., hypertension-exclusive effects) revealed metabolic remodeling suggestive of increased aerobic glycolysis, decreased β-oxidation, and dysregulated purine and pyrimidine metabolism with few transcriptional changes. ZSF1-obese rats displayed worsened metabolic remodeling and robust transcriptional remodeling highlighted by the upregulation of inflammatory genes and downregulation of the mitochondrial structure/function and cellular metabolic processes. Integrated network analysis of metabolomic and RNAseq datasets revealed downregulation of nearly all catabolic pathways contributing to energy production, manifesting in a marked decrease in the energetic state (i.e., reduced ATP/ADP, PCr/ATP). Cardiomyocyte ultrastructure analysis revealed decreased mitochondrial area, size, and cristae density, as well as increased lipid droplet content in HFpEF hearts. Mitochondrial function was also impaired as demonstrated by decreased substrate-mediated respiration and dysregulated calcium handling. Conclusions:Collectively, the integrated omics approach applied here provides a framework to uncover novel genes, metabolites, and pathways underlying HFpEF, with an emphasis on mitochondrial energy metabolism as a potential target for intervention.

Cardiovascular diseases (CVD) are the leading cause of death worldwide, and smoking remains the most significant preventable risk factor. The contribution of nicotine, the addictive component of all tobacco products, to smoking-related CVD remains poorly understood. In addition to smoking, obesity is also a major risk factor for the development of CVD. Recent studies indicate that individuals who are overweight or obese are at greater risk of nicotine product usage. With more than 40% of the US population affected by obesity, the combined effects of obesity and nicotine on cardiovascular (CV) health warrant urgent investigation. In the present study we investigated the combined effects of nicotine exposure and a high fat diet (HFD) on CV function.Male C57BL/6N mice (8 wk-old) were placed on either a HFD (60 kcal% Fat) or standard chow (SC, 22 kcal% Fat) and exposed to room air (RA) or nicotine vapor (NIC, 12 hr/day during the active phase) for 10 weeks (n=10-15/group). Mice with HFD exposure alone exhibited significantly greater weight gain at 10 weeks vs. SC-RA controls (122.5±9.8% increase in body weight in HFD-RA vs. 40.7±3.3% in SC-RA, p < 0.0001), whereas concurrent nicotine inhalation significantly reduced this weight gain (66.5±5.8% in HFD-NIC group, p < 0.0001). In the HFD-NIC group, blood pressure (BP) monitoring via telemetry (24 hours) revealed non-significant increases in BP in the inactive phase, coupled with a significant (p < 0.05) reduction in the expected dipping in BP during the inactive compared to the active phase (HFD-NIC: 4.5±1.2; SC-RA: 11.3±2.2; HFD-RA: 12.4±1.0 and SC-NIC: 9.7±4.6 mmHg). LV catheterization showed that mice exposed to both nicotine and HFD had elevated LV end diastolic pressure (HFD-NIC: 14.1±0.7; SC-RA: 4.5±0.8; HFD-RA: 2.8±0.7 and SC-NIC: 3.9±1.3 mmHg; p<0.05) and prolonged time constant of LV relaxation (Tau). LV RNAseq analysis revealed that HFD-NIC resulted in 70 differentially expressed genes (36 upregulated and 34 downregulated) unique from nicotine or HFD alone, with multiple differentially regulated pathways including dilated cardiomyopathy signaling pathway (p = 2.8E-04). Plasma NT-pro-BNP levels were significantly increased in HFD-RA mice vs. SC-RA controls, whereas this response was absent in HFD-NIC. Finally, HFD+NIC led to greater impairment of endothelium-dependent vasorelaxation in thoracic aortas compared to either treatment alone.In conclusion, although nicotine reduced HFD-induced weight gain, combined exposure to inhaled nicotine and HFD led to greater cardiovascular dysfunction including dysregulation in physiological blood pressure responses, LV diastolic dysfunction, and greater impairment of endothelial-dependent vascular function. National Institute of Health [grant numbers HL135635 (to JDG, EL and XY); HL146098, HL146514, and HL151398 (to DJL.); and F30HL160071 (to AKW)]; Department of Veterans Affairs (BX004294 to EL) and the American Heart Association [Award 829761 (to AKW) and Award 20POST35200075 (to ZL)]. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diastolic dysfunction is a hallmark of heart failure with preserved ejection fraction (HFpEF) and its subphenotypes, including one that is observed comorbidly with obesity/diabetes (Ob/Dm) and the "classic" phenotype defined by hypertension/hypertrophy (Ht/Hp). Previous experiments revealed reduced maximal contractile force (Fmax) in human Ob/Dm cardiomyocytes compared to controls. While Fmax was not reduced in Ht/Hp cardiomyocytes, excessive tension under diastolic calcium was observed, suggesting unique subcellular mechanisms that elevate diastolic and preserve systolic force, and potentially impede relaxation in Ht/Hp samples. To investigate these mechanisms, we assessed force production and activation/relaxation kinetics in myofibrils from Ht/Hp-HFpEF patient biopsies. Ht/Hp myofibrils showed a prolonged linear relaxation phase, indicating excessive actomyosin interactions following calcium removal. A significant decrease in Fmax was again not observed, and the exponential relaxation rate remained unaltered. X-ray diffraction was performed to determine whether anomalous structural relationships between myofilament proteins contribute to the Ob/Dm or Ht/Hp subphenotypes. Ob/Dm samples had increased lattice spacing and reduced equatorial intensity ratio, I1,1/I1,0, (indicative of decreased actomyosin interaction) compared to control, while neither property differed in Ht/Hp biopsies. These findings suggest altered sarcomere structure or thick filament activity as potential contributors to the Ob/Dm phenotype, while other mechanisms, such as aberrant thin-filament regulation, may explain impaired relaxation in Ht/Hp myofibrils and elevated diastolic force in cardiomyocytes. Finally, we assessed myofibril mechanics of three common HFpEF animal models: Göttingen minipigs, ZSF1-obese rats, and mice exposed to a high-fat diet and ʟ-NAME. While all models showed organ-level diastolic dysfunction, only the rodent models exhibited impaired myofibril relaxation, most closely mimicking the human Ht/Hp phenotype. Ongoing studies on Ob/Dm patient samples aim to determine which models best replicate this subphenotype and to further understand the role of myofibril relaxation in HFpEF.

Heart failure with preserved ejection fraction (HFpEF) has few effective therapies yet exacts substantial mortality. Its multi-system nature has made animal modeling difficult, but recent efforts combining diet-induced obesity and hemodynamic stress are popular: mouse - L-NAME/high-fat diet (HFD) and ovariectomized (OVX) females with HFD-induced obesity/cardiac pressure overload (PO) (HFD+OVX+PO), ZSF1 rat, and obese/hypertensive Göttingen minipigs. We reported human HFpEF myocyte defects including reduced Ca 2+ -activated maximum tension (T max ) negatively correlate with body mass index, reduced Hill coefficient (n H, e.g., cooperativity) and Ca 2+ at 50% T max (EC 50 ), and higher resting tension. Here we tested whether such deficits are found in HFpEF animal models. The L-NAME/HFD mouse had no differences in Ca 2+ -activated (p=0.20) or resting (p=0.75) tension. In obese ZSF-1 rats, while T max (p=0.23) and resting tension (p=0.39) were not different, n H was reduced (2±1 vs. 4±2 p=0.01). In HFD+OVX+PO mice, T max (15±2 vs. 20±2 mN/mm 2 , p=0.006) and EC 50 (1.9±0.6 vs. 2.4±0.7 μM, p=0.008) were reduced, but resting tension (p=0.70) was not different. In the Göttingen minipig HFpEF model, EC 50 (p=0.93) and n H (p=0.35) were not different, whereas T max was reduced (17±3 vs. 27±3 mN/mm 2 , p=2x10 -6 ) and tension less at physiologic Ca 2+ . Resting tension was lower (not greater) in this model (p=0.001). The HFD+OVX+PO and minipig HFpEF models recapitulate depressed Ca 2+ -activated tension as in obese human HFpEF, whereas none manifested increased resting tension ( Table 1 ). Studies investigating HFpEF sarcomere dysfunction should consider these findings in model selection.