Model Of Heart Failure With Preserved Ejection Fraction Research Articles

Abstract 13724: Development of VS-041 as a Precision Medicine Targeting Cardiac-Relevant Matrix Metalloproteases (MMPs) in Heart Failure With Preserved Ejection Fraction (HFpEF)

Introduction. HFpEF is a heterogeneous syndrome, with several underlying etiologic and pathophysiologic factors. As “one size fits all” therapies are not equally efficacious across various HFpEF comorbidities, a precision medicine approach can offer a disease modifying treatment for HFpEF subpopulation stratified for specific molecular targets. Expression of MMP2, 9, and 13 in heart is elevated in HFpEF patients with hypertension, diabetes or coronary disease, and their levels in plasma correlate with survival, suggesting utility as predictive biomarkers. Genetic ablation of MMP2, 9, and 13 leads to a robust antifibrotic effect in the myocardium. Inhibition of MMP2, 9, and 13 directly regulates of Ca 2+ transients and protects sarcomere function. Hypothesis. We present preclinical profile of VS-041, a novel and narrow-spectrum MMP inhibitor, as a drug candidate for HFpEF patients with elevated levels of MMP2, 9, and 13. Methods and Results. VS-041, optimized with computer-aided drug design methods, shows single-digit IC 50 against MMP2 (1nM), MMP9 (2nM) and MMP13 (1nM), and maintains >300-fold selectivity against MMP1 and TACE. In isolated cardiomyocytes VS-041 reduces contractility deficit due to fast pacing. With high predicted metabolic bioavailability (MF%=90), >80% target occupancy by VS-041 is estimated after 50 mg/kg PO dose in rats. VS-041 demonstrated robust efficacy in Dahl Salt Sensitive rat model of hypertension and HFpEF after dosing at 25 or 50 mg/kg PO for 5 wks in a therapeutic mode (after 1-wk of high salt lead-in). VS-041 had no effect on blood pressure or ejection fraction confirming a direct effect on diastolic function. In Doppler mitral inflow, analysis of the isovolumic relaxation time and the E/A ratio of peak velocity of filling waves revealed 40% improvement in diastolic function. LV fibrosis was dose-proportionally decreased based on PSR morphometry. No adverse effects were observed after 5 weeks of dosing. Conclusions. Preclinical pharmacology and safety profile of VS-041 support its nomination as clinical candidate and initiation of IND-enabling studies. VS-041 has potential to be first-in-class precision medicine treatment for HFPEF patients with elevated levels of cardiac-relevant MMP2, 9 and 13.

Abstract 11965: Cellular Senescence Produces Diastolic Dysfunction and Cardiac Hypertrophy With Aging in Mice

Background: Age is a major risk factor for heart failure with preserved ejection fraction (HFpEF); however, the underlying mechanisms are poorly understood. Cellular senescence is an aging and stress related response; senescent cells (SCs) secrete bioactive substances that produce a variety of pathological changes. The role of senescence in HFpEF is unknown. Objectives: To study the effect of targeted deletion of SCs on cardiac function, structure and left ventricular (LV) pressure in an aging mouse model of HFpEF. Methods: We studied INK ATTAC mice, in which SCs undergo targeted clearance following treatment with a dimerizing agent, AP20187 (AP). Mice were treated with blinded AP or vehicle from 12 months to 18 months of age. Cardiac function and structure were studied with echocardiography. Contractility parameters were measured with a Millar catheter. The percentage of SCs in different cardiac-cell populations was analyzed with the use of Fluorescence-Activated Cell Sorting (FACS). Results: From 12 to 18 months, LV mass/LV diameter at end diastole (LVDd) and the LV anterior wall thickness at end diastole (LVAWd) increased in the vehicle group while they decreased in the AP group (LV mass/LVDd: %Δvehicle=+7.9± 3%, %ΔAP=-2±1.2%, P=0.005; LVAWd: %Δvehicle=+5.3±2.5%, %ΔAP=-3.5±1.6%, P=0.007) (Fig1A, B). E/e’ (echo) and isovolumic relaxation time indicated diastolic dysfunction in vehicle mice, and improved with AP (Fig1C, D). GFP bicistronic with p16 indicated SCs; FACS-purified fibroblasts (FBs) showed reduced GFP fluorescence (P=0.056) in the AP group, whereas immune and endothelial cells were not affected, suggesting that the anti-HFpEF effects of SC-clearance might be mediated through the clearance of senescent FBs (Fig1E). Conclusions: Our study points to a significant role of SCs, likely senescent FBs, in the pathophysiology of HFpEF. Modulating senescence might provide the basis for novel therapeutic approaches to HFpEF prevention and/or therapy.

Abstract 15337: Chronic Oral Finerenone Treatment Does Not Improve Left Ventricular Diastolic Compliance in Swine With Repetitive Pressure Overload-Induced HFpEF

Introduction: The non-steroidal mineralocorticoid receptor antagonist finerenone has emerged as a potential treatment for heart failure with preserved ejection fraction (HFpEF) based, in part, on initial evidence of its anti-fibrotic properties. However, it is unclear whether chronic oral finerenone therapy can reverse established cardiac fibrosis. Accordingly, we tested the efficacy of chronic oral finerenone in a porcine model of HFpEF characterized by interstitial fibrosis and reduced left ventricular (LV) diastolic compliance in the absence of co-morbidities or anatomic LV hypertrophy. Methods: Swine (n=12) were subjected to daily (1 hr) episodes of repetitive LV pressure overload (RPO) for 2 weeks using phenylephrine (24 mg/hr) to transiently elevate LVEDP to ~30 mm Hg. After 2 weeks, RPO was discontinued and animals were followed for an additional 4 weeks, during which a subset received oral finerenone (20 mg/day; n=6). LV end-diastolic volume (EDV) and pressure (EDP) were measured at normal and elevated preload to assess LV compliance (ΔLVEDV/ΔLVEDP) and post-mortem picrosirius red staining was performed to quantify interstitial fibrosis vs. normal controls (n=8). Results: Four-weeks after cessation of RPO, untreated animals exhibited a significant increase in interstitial fibrosis vs. normal controls that was attenuated in finerenone-treated animals ( Figure A ). Despite this regression of interstitial collagen, a persistent reduction in LV diastolic compliance was observed after finerenone treatment (1.18 ± 0.33 mL/mmHg vs. 2.11 ± 0.22 mL/mmHg in normal controls; p=0.04) that was comparable to that of untreated animals after RPO (0.97 ± 0.22 mL/mmHg; p=0.61; Figure B ). Conclusion: Four weeks of chronic oral finerenone treatment does not restore LV diastolic compliance in swine with RPO-induced HFpEF despite regression of interstitial collagen content, suggesting that other factors may contribute to diastolic stiffening in this model.

Abstract 14641: Diastolic Dysfunction “In a Dish“- A Novel Organoid Model of Heart Failure With Preserved Ejection Fraction

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a major cause of mortality. No therapies have shown a survival benefit in HFpEF. A potential reason may be the lack of an in-vitro model for identifying novel therapeutic targets. Here, we aimed at generating a model of HFpEF using engineered cardiac organoids. Methods: Human cardiomyocytes generated from human-induced pluripotent stem cells were used to form 3D cardiac organoids. The organoids were exposed to conditions mimicking the comorbid conditions (Diabetes, hypertension, obesity, inflammation) leading to HFpEF: high glucose media with insulin deprivation; pro-fibrotic and hypertrophic stimuli such as Angiotensin II and Endothelin-1; free fatty acids, and pro-inflammatory factors ( e.g., IL-1 ). The passive stiffness, active contractile forces, and tau (relaxation constant) were evaluated using a force-transducer and a length controller. Structural and molecular phenotyping were conducted as well. Results: Exposure to HFpEF-inducing media resulted in single cells hypertrophy with an increase of average cell size (6670.87±1363.86 um^2) in comparison to control media (2840.56±520.70 um^2). The generated organoids exposed to HFpEF media developed a significant increase in passive force of 7.35±2.47 N/m vs. 2.04±0.85 N/m for control. Relaxation was prolonged in the HFpEF media and resulted in an increased ratio of 1.87±0.21 of relaxation constant, tau, in comparison to baseline and no modulation of tau in the control (1.08± 0.08 ). Structural and molecular evaluation showed characteristic HFpEF phenotype. Discussion: Cardiac organoids can recapitulate HFpEF phenotypes such as the increase in passive force, elongation of diastole, hypertrophy, and no change in the active force. An HFpEF in-vitro model may provide an unprecedented insight into the early pathogenetic mechanisms involved in HFpEF and may serve as a high-throughput platform for drug discovery.

Abstract 9832: Liver-Heart Crosstalk Mediated by Coagulation Factor XI Protects Against Heart Failure

Introduction: Tissue-tissue crosstalk by endocrine factors is a vital mechanism to maintain proper physiologic homeostasis. The heart and the liver display multifaceted interactions and it is common to observe heart diseases affecting the liver and vice versa. Hypothesis: We hypothesized that the liver-heart interactions may be mediated by novel endocrine factors. Methods: Global transcriptomic data from the heart and the liver were generated across all 100 diverse inbred strains of mice, the Hybrid Mouse Diversity Panel (HMDP), and used to detect the correlation structure between secreted proteins from the liver and their downstream effects on the heart. Candidates were validated in a “two-hit” model of heart failure with preserved ejection fraction (HFpEF) and plasma samples from HFpEF patients and controls were used to examine the clinical relevance. Results: Using the bioinformatics framework, we predicted that coagulation factor XI (FXI) is a liver-derived protein that influences heart gene expression and diastolic dysfunction, a key trait of HFpEF. In a mouse model of HFpEF, mice overexpressing FXI in the liver showed improved diastolic function and reduced inflammation and fibrosis, whereas FXI knockout mice were sensitized for diastolic dysfunction. FXI overexpression activated the bone morphogenetic protein (BMP)-Smad1/5 pathway in the heart, thus inhibiting genes involved in inflammation and fibrosis. The action of FXI on heart requires proteolytic activity, as point mutations in its catalytic domain eliminated effects on BMP signaling and heart function. BMP7 is secreted as an inactive precursor and our results indicate that it is cleaved by FXI, releasing the active dimer and monomer growth factor from the prodomain that binds to the extracellular matrix. Further, plasma FXI was inversely correlated with E/e’ ratio in all human participants and HFpEF patients. Conclusions: Our studies indicate that liver-derived FXI specifically regulates cardiomyocytes through the BMP-Smad1/5 pathway, resulting in attenuation of fibrosis, inflammation, and diastolic dysfunction in the context of a HFpEF model. Our study reveals a protective role of FXI on heart injury, distinct from its role in coagulation.

Involvement of pyroptosis pathway in epicardial adipose tissue - myocardium axis in experimental heart failure with preserved ejection fraction

Epicardial adipose tissue (EAT) is a metabolically active organ which generates inflammatory cytokines. Thickness of EAT is associated with onset and development of heart failure with preserved ejection fraction (HFpEF). However, it is still unclear the specific mechanisms and pharmacological targets on EAT induced inflammation in HFpEF. A two-hit protocol with western diet and Nω-nitrol-arginine methyl ester (L-NAME) was used to establish HFpEF mouse model. In HFpEF mice, inflammatory biomarkers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and von willebrand factor (vWF) elevated in myocardium compared to control. Inflammatory cell infiltration in myocardium was increased. In HFpEF mice, inflammasome-mediated pyroptosis pathway was activated in the EAT. Suppression of pyroptosis-related protein gasdermin D (GSDMD) in cultured EAT could lower cardiomyocyte inflammation and autophagy. Furthermore, spironolactone and rosuvastatin, the two-hit anti-inflammatory agents, reduced NLR family pyrin domain containing 3 (NLRP3)/GSDMD pyroptosis in EAT and autophagy in myocardium of HFpEF mouse. The combination treatment also enhanced exercise tolerance and appeased inflammatory injuries in HFpEF mice. ConclusionPyroptosis signaling is involved in EAT-myocardium axis in mouse model of HFpEF. Targeting adipocyte-derived inflammation in EAT bears potential to treatment HFpEF.

OR19-2 Treatment of Heart Failure with Preserved Ejection Fraction (HFpEF) with Low and High Dose of Triiodothyronine: an Animal Model of Metabolic Syndrome with HFpEF

Abstract Introduction Low levels of triiodothyronine (T3) are common in patients with heart failure (HF). It has been proposed that the decreased levels of T3 contribute to metabolic and cardiac dysfunction in HF. We aimed to evaluate the effects of treatment with low dose and high dose of T3 in an animal model of HF with preserved ejection fraction (HFpEF). Materials and Methods We evaluated four groups: ZSF1 Lean (Lean-Ctrl, n=8), ZSF1 obese (rat model of metabolic syndrome with HFpEF, HFpEF-Ctrl, n=13), ZSF1 obese supplemented with a replacement dose of T3 (initially 0.04mg/ml, increased to 0.06mg/ml after 4 weeks; HFpEF-T3high, n=8), and ZSF1 Obese supplemented with a low-dose of T3 (0.03mg/mL; HFpEF-T3low, n=8). T3 was supplemented in drinking water from week 13 to 24. The animals were submitted to anthropometric and metabolic evaluation, echocardiography, VO2max evaluation, hemodynamic evaluation, single cardiomyocyte evaluation, and myocardial tissue collection at 24 weeks. Results HFpEF-Ctrl animals had lower serum and myocardial thyroid hormone levels than Lean-Ctrl. Treatment with low dose and high dose of T3 did not correct serum T3 levels, but increased myocardial T3 levels, with normalization of myocardial T3 levels in the HFpEF-T3high group. In comparison to HFpEF-Ctrl, body weight was significantly decreased in HFpEF-T3low and HFpEF-T3high groups (616±39g, 572±24g and 535±18g respectively, p<0.001). An improvement in glucose metabolism was observed only in HFpEF-T3high, with a decreased AUC of glucose during OGTT and insulin tolerance test. Both treated groups presented an improvement in diastolic function (with decreased isovolumetric relaxation time) and systolic function (with increased peak systolic velocity and decreased ejection time) comparing to HFpEF-Ctrl. These results were supported by enhanced Ca2+ transients during diastole and sarcomere relaxation. Comparing with HFpEF-Ctrl, animals treated with T3 had higher myocardial expression of the calcium transporter RYR2, of the transcriptional coactivator PGC-1α, and of the α-myosin heavy chain (MHC), with a lower expression of β-MHC. VO2max was improved with low-dose and high-dose T3 compared with HFpEF-Ctrl. Comparing with HFpEF-Ctrl, heart rate was increased with high-dose (277.9±30.2 vs 313.5±35.8, p=0.049) but not significantly with low-dose (277.9±30.2 vs 299.8±30.9 bpm, p=0.13). Mortality was increased in the HFpEF-T3high comparing with the other groups (no animals died during the protocol in the Lean-Ctrl, HFpEF-Ctrl and HFpEF-T3low groups; three animals had sudden death in the HFpEF-T3high group). Conclusion Treatment with T3 improves metabolic function, myocardial calcium handling, diastolic and systolic function, and exercise capacity in this animal model of HFpEF. While the low dose was well tolerated and safe, the high dose was associated with increased heart rate and a higher risk of sudden death. Modulation of thyroid hormones may be a potential therapeutic target in HFpEF; however such approaches must take into account a narrow therapeutic window of T3 in HFpEF. Presentation: Monday, June 13, 2022 11:15 p.m. - 11:30 a.m.

SIRT6 Mitigates Heart Failure With Preserved Ejection Fraction in Diabetes.

Heart failure with preserved ejection fraction (HFpEF) is a growing health problem without effective therapies. Epidemiological studies indicate that diabetes is a strong risk factor for HFpEF, and about 45% of patients with HFpEF are suffering from diabetes, yet the underlying mechanisms remain elusive. Using a combination of echocardiography, hemodynamics, RNA-sequencing, molecular biology, in vitro and in vivo approaches, we investigated the roles of SIRT6 (sirtuin 6) in regulation of endothelial fatty acid (FA) transport and HFpEF in diabetes. We first observed that endothelial SIRT6 expression was markedly diminished in cardiac tissues from heart failure patients with diabetes. We then established an experimental mouse model of HFpEF in diabetes induced by a combination of the long-term high-fat diet feeding and a low-dose streptozocin challenge. We also generated a unique humanized SIRT6 transgenic mouse model, in which a single copy of human SIRT6 transgene was engineered at mouse Rosa26 locus and conditionally induced with the Cre-loxP technology. We found that genetically restoring endothelial SIRT6 expression in the diabetic mice ameliorated diastolic dysfunction concurrently with decreased cardiac lipid accumulation. SIRT6 gain- or loss-of-function studies showed that SIRT6 downregulated endothelial FA uptake. Mechanistically, SIRT6 suppressed endothelial expression of PPARγ through SIRT6-dependent deacetylation of histone H3 lysine 9 around PPARγ promoter region; and PPARγ reduction mediated SIRT6-dependent inhibition of endothelial FA uptake. Importantly, oral administration of small molecule SIRT6 activator MDL-800 to diabetic mice mitigated cardiac lipid accumulation and diastolic dysfunction. The impairment of endothelial SIRT6 expression links diabetes to HFpEF through the alteration of FA transport across the endothelial barrier. Genetic and pharmacological strategies that restored endothelial SIRT6 function in mice with diabetes alleviated experimental HFpEF by limiting FA uptake and improving cardiac metabolism, thus warranting further clinical evaluation.

Morphological, functional, and biomechanical progression of LV remodelling in a porcine model of HFpEF

Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for about half of heart failure cases, but the progression of cardiac biomechanics during pathogenesis is not completely understood. We investigated a published porcine model of HFpEF, generated by progressive constriction of an aortic cuff causing progressive left ventricle (LV) pressure overload, and characterized by hypertrophy, diastolic dysfunction and overt HF with elevated plasma beta natriuretic peptide (BNP). We characterized morphological and functional features and performed image-based finite element modelling over multiple time points, so as to understand how biomechanics evolved with morphological and functional changes during pathogenesis, and to provide data for future growth and remodeling investigations. Results showed that the hypertrophic responses quickly manifested and were effective at preventing an elevation of systolic myocardial stresses, suggesting active compensated remodeling. Consequent to the hypertrophy, diastolic myocardial stresses decreased despite the elevations in diastolic pressures. The left ventricle hypertrophy (LVH) myocardium also exhibited a quick elevation of active tension at the onset of the disease. There was a progressive and significant decrease in myocardial strain, which was more significant in the longitudinal direction. Further, elevated myocardial stiffness and diastolic pressures, which reflected diastolic dysfunction, also manifested, but this was delayed from the onset of the disease. Correlation analysis showed that hypertrophy was closely correlated to systolic pressure, active tension and systolic myocardial stress, suggesting that these factors may play a role in initiating hypertrophy. Myocardial stiffness was weakly correlated to LV pressures and myocardial stresses.

PINK1 Phosphorylates Drp1S616 to Improve Mitochondrial Fission and Inhibit the Progression of Hypertension-Induced HFpEF.

(1) Background: Heart failure with preserved ejection fraction (HFpEF) is a major subtype of HF with no effective treatments. Mitochondrial dysfunctions relevant to the imbalance of fusion and fission occur in HFpEF. Drp1 is a key protein regulating mitochondrial fission, and PINK1 is the upstream activator of Drp1, but their relationship with HF has not been clarified. The aim of the study is to investigate molecular mechanisms of mitochondrial dysfunctions in animals with hypertension-induced HFpEF. (2) Methods and Results: The hypertension-induced HFpEF model was established by feeding Dahl/SS rats with high salt, showing risk factors such as hypertension, mitochondrial dysfunctions, and so on. Physiological and biological measurements showed a decrease in the expression of mitochondrial function-related genes, ATP production, and mitochondrial fission index. PINK1 knockout in H9C2 cardiomyocytes showed similar effects. Moreover, PINK1 myocardium-specific overexpression activated Drp1S616 phosphorylation and enhanced mitochondrial fission to slow the progression of hypertension-induced HFpEF. (3) Conclusions: PINK1 could phosphorylate Drp1S616 to improve mitochondrial fission and relieve mitochondrial dysfunctions, which highlights potential treatments of HFpEF.

Human epididymis protein 4 indicates progression of cardiac fibrosis in Heart Failure with preserved Ejection Fraction

Abstract Background Cardiac fibrosis is one of the main causes of diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF). Human epididymis protein 4 (HE4) is a novel pro-fibrotic protein expressed especially in activated fibroblast, so-call myofibroblast [1]. Although our previous study showed that HE4 functioned as a secretory factor promoting cardiac fibrosis in dilated cardiomyopathy [2], little is known about the role of HE4 in HFpEF. Purpose The aim of this study is to evaluate the pathophysiological role of HE4 in HFpEF and the association between serum HE4 levels and clinical course of HFpEF. Methods In basic research, we administered high-fat diet and NOS inhibitor (L-NAME) to mice for 5 weeks to induce HFpEF. We analyzed echocardiographic findings and mRNA expressions of fibrosis-related genes including HE4 by real time PCR. In clinical research, we measured serum HE4 levels of 79 patients with HFpEF and divided these patients into low and high HE4 group using median values of HE4 (102.0 pmol/L). Then, we evaluated their mortality and cardiovascular events retrospectively. Results In vivo, the administration of high fat diet and L-NAME induced significant increase in left ventricular mass on echocardiography compared with control group (p=0.006). The mRNA expressions of aSMA and collagen3a1 in heart tissue was upregulated in HFpEF model mice (p=0.008 and p=0.003, respectively). Furthermore, the mRNA expression of HE4 was also upregulated in HFpEF model mice (n=6) than in control mice (n=5), although these differences did not reach statistical significance (p=0.238). In vitro, recombinant HE4 upregulated mRNA expression of pro-fibrotic genes, such as periostin (p=0.001), collagen3a1 (p=0.037), aSMA (p=0.095), and PAI-1 (p=0.17), indicating HE4 facilitate the activation of fibroblast and cardiac fibrosis although some did not reach statistical significance. In clinical research, Kaplan-Meier curve revealed that the rate of all cause death and rehospitalization for worsening heart failure were significantly higher in the high than the low HE4 group, suggesting HE4 can predicts prognosis in patients with HFpEF. Conclusion HE4 induces the activation of cardiac fibroblasts and is upregulated in the heart of HFpEF model mice. Furthermore, it is a useful biomarker for predicting cardiovascular events in patients with HFpEF. Funding Acknowledgement Type of funding sources: None.

The role of endothelial adipose triglyceride lipase (ecATGL) in heart failure with preserved ejection fraction (HFpEF)

Abstract Background and objectives Obesity is one of the major drivers of heart failure with preserved ejection fraction (HFpEF). Endothelial cells (EC) have an important barrier function including the control of lipid transport from the coronary circulation to the myocardium. This EC lipid transport involves, in addition to fast transcellular transport, processes of EC-lipogenesis and -lipolysis, and is likely involved in the development of cardiac lipid accumulation in obese phenotypes of HFpEF. In order to determine the role of EC lipid metabolism/transport in HFpEF, we generated mice with an EC-specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, and subjected these mice to a hypertensive-obese two-hit HFpEF model. Methods and results Inducible endothelial-specific ATGL knockout (ecATGL-KO) mice were generated by crossbreeding Atgl fl/fl mice with Cdh5(PAC)-CreERT2 mice. For the induction of HFpEF, mice received a high fat diet (HFD, 60% kcal from fat) and L-NAME for 15 weeks, while control animals (Ctrl) received low fat diet (LFD, 10% kcal from fat) and water, as previously described. To analyse cardiac function, standard and speckle-tracking echocardiography were performed. After 15 weeks, Cre− littermates (wild type, wt) with HFpEF developed obesity and hypertension (body weight: wt-Ctrl: 33.8±2.5g vs. wt-HFpEF: 45.1±4.4g, p<0.0001; systolic blood pressure: wt-Ctrl: 99.9±3.7mmHg vs. wt-HFpEF: 135.5±5.6mmHg, p<0.0001), which did not significantly differ in ecATGL-KO mice. In parallel, wt-HFpEF mice developed established echocardiographic signs of HFpEF including diastolic dysfunction: E/é: (wt-Ctrl: −22.3±1.3 vs. wt-HFpEF: −45.2±3.8; p<0.0001), IVRT: (wt-Ctrl: 17.8±0.7ms vs. wt-HFpEF: 19.3±0.4ms; p= p<0.0001), and an impaired global longitudinal strain (GLS) (wt-Ctrl: −20.7±1.1% vs. wt-HFpEF 12.9±0.7%; p<0.0001). HFpEF parameters were significantly improved in ecATGL-KO mice after 15 weeks of HFD/L-NAME: (E/é: −28.9±2.8; p<0.0001 vs. wt-HFpEF; IVRT: 18.3±0.6; p<0.05 vs. wt-HFpEF; GLS: −19.1±1.7; p<0.0001 vs. wt-HFpEF). No changes in systolic function (EF) were observed. Electron microscopical investigations of cardiac (LV) tissue sections showed enhanced lipid droplet accumulation in coronary ECs of ecATGL-KO mice when compared to wt mice. Complementary in vitro experiments in HUVECs using an ATGL inhibitor or genetic deletion of Atgl using siRNA resulted in significant lipid droplet accumulation which did not affect EC survival and function. Conclusion This study demonstrates that the lack of ATGL in ECs attenuates the development of HFpEF. Enhanced lipid droplet accumulation in coronary ECs in the absence of ATGL may protect the myocardium from lipid overload, as seen in obese phenotypes of HFpEF. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Einstein Foundation Berlin;DZHK (German Centre for Cardiovascular Research), partner site Berlin;DFG (Deutsche Forschungsgemeinschaft)

Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction induced by abdominal aorta constriction through cyclic guanosine monophosphate-dependent protein kinase Signaling Pathway.

To determine whether Shunxin decoction improves diastolic function in rats with heart failure with preserved ejection fraction (HFpEF) by regulating the cyclic guanosine monophosphate-dependent protein kinase (cGMP-PKG) signaling pathway. Except for control group 8 and sham surgery group 8, the remaining 32 male Sprague-Dawlay rats were developed into HFpEF rat models using the abdominal aorta constriction method. These rats in the HFpEF model were randomly divided into the model group, the Shunxin high-dose group, the Shunxin low-dose group, and the Qiliqiangxin capsule group. The three groups received high-dose Shunxin decoction, low-dose Shunxin decoction, and Qiliqiangxin capsule by gavage, respectively, for 14 d. After the intervention, the diastolic function of each rat was evaluated by testing E/A, heart index, hematoxylin-eosin staining, Masson, myocardial ultrastructure, and N-terminal pro-brain natriuretic peptide (NT-proBNP). The Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) software was used to predict targets for which Shunxin decoction acts on the cGMP-PKG pathway. Natriuretic peptide receptor A (NPRA) and guanylate cyclase (GC) were detected by immunohistochemistry, and eNOS, phosphodiesterase 5A (PDE5A), and cGMP-dependent protein kinase 1(PKG I) were determined by Western blotting. Compared to the model group, the thickness of the interventricular septum at the end of diastole (IVSd) and the thickness of the posterior wall at the end of diastole (PWd) of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group were all significantly reduced ( < 0.01). Furthermore, Shunxin decoction high-dose group E/A value was decreased ( < 0.01). Compared to the model group, the expression of NPRA and GC increased in the Shunxin decoction low-dose group and the Qiliqiangxin capsule group ( < 0.01). Compared to the model group, the expressions of eNOS and PKG I increased ( < 0.05) in the Shunxin decoction high-dose group. The expression of PDE5A expression decreased in the myocardium of the Shunxin decoction high-dose group, Shunxin decoction low-dose group, and Qiliqiangxin capsule group compared to the model group ( < 0.01). Shunxin decoction can improve diastolic function in rats with HFpEF. It increases the expression of NPRA, GC, and eNOS in the myocardial cell cGMP-PKG signaling pathway, upregulates cGMP expression, decreases PDE5A expression to reduce the cGMP degradation. Thus, the cGMP continually stimulates PKG I, reversing myocardial hypertrophy and improving myocardial compliance in HFpEF rats.

Acetylation and phosphorylation changes to cardiac proteins in experimental HFpEF due to metabolic risk reveal targets for treatment

AimsDespite the high prevalence of heart failure with preserved ejection fraction (HFpEF), the pathomechanisms remain elusive and specific therapy is lacking. Disease-causing factors include metabolic risk, notably obesity. However, proteomic changes in HFpEF are poorly understood, hampering therapeutic strategies. We sought to elucidate how metabolic syndrome affects cardiac protein expression, phosphorylation and acetylation in the Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 (ZSF1) rat HFpEF model, and to evaluate changes regarding their potential for treatment. Main methodsZSF1 obese and lean rats were fed a Purina diet up to the onset of HFpEF in the obese animals. We quantified the proteome, phosphoproteome and acetylome of ZSF1 obese versus lean heart tissues by mass spectrometry and singled out targets for site-specific evaluation. Key findingsThe acetylome of ZSF1 obese versus lean hearts was more severely altered (21 % of proteins changed) than the phosphoproteome (9 %) or proteome (3 %). Proteomic alterations, confirmed by immunoblotting, indicated low-grade systemic inflammation and endothelial remodeling in obese hearts, but low nitric oxide-dependent oxidative/nitrosative stress. Altered acetylation in ZSF1 obese hearts mainly affected pathways important for metabolism, energy production and mechanical function, including hypo-acetylation of mechanical proteins but hyper-acetylation of proteins regulating fatty acid metabolism. Hypo-acetylation and hypo-phosphorylation of elastic titin in ZSF1 obese hearts could explain myocardial stiffening. SignificanceCardiometabolic syndrome alters posttranslational modifications, notably acetylation, in experimental HFpEF. Pathway changes implicate a HFpEF signature of low-grade inflammation, endothelial dysfunction, metabolic and mechanical impairment, and suggest titin stiffness and mitochondrial metabolism as promising therapeutic targets.

Tubulin expression and modification in heart failure with preserved ejection fraction (HFpEF)

Diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF) is characterised by increased left ventricular stiffness and impaired active relaxation. Underpinning pathomechanisms are incompletely understood. Cardiac hypertrophy and end stage heart disease are associated with alterations in the cardiac microtubule (MT) network. Increased amounts and modifications of α-tubulin associate with myocardial stiffness. MT alterations in HFpEF have not been analysed yet. Using ZSF1 obese rats (O-ZSF1), a validated HFpEF model, we characterised MT-modifying enzymes, quantity and tyrosination/detyrosination pattern of α-tubulin at 20 and 32 weeks of age. In the left ventricle of O-ZSF1, α-tubulin concentration (20 weeks: 1.5-fold, p = 0.019; 32 weeks: 1.7-fold, p = 0.042) and detyrosination levels (20 weeks: 1.4-fold, p = 0.013; 32 weeks: 1.3-fold, p = 0.074) were increased compared to lean ZSF1 rats. Tyrosination/α-tubulin ratio was lower in O-ZSF1 (20 weeks: 0.8-fold, p = 0.020; 32 weeks: 0.7-fold, p = 0.052). Expression of α-tubulin modifying enzymes was comparable. These results reveal new alterations in the left ventricle in HFpEF that are detectable during early (20 weeks) and late (32 weeks) progression. We suppose that these alterations contribute to diastolic dysfunction in HFpEF and that reestablishment of MT homeostasis might represent a new target for pharmacological interventions.

Empagliflozin Preserves Skeletal Muscle Function in a HFpEF Rat Model.

Besides structural alterations in the myocardium, heart failure with preserved ejection fraction (HFpEF) is also associated with molecular and physiological alterations of the peripheral skeletal muscles (SKM) contributing to exercise intolerance often seen in HFpEF patients. Recently, the use of Sodium-Glucose-Transporter 2 inhibitors (SGLT2i) in clinical studies provided evidence for a significant reduction in the combined risk of cardiovascular death or hospitalization for HFpEF. The present study aimed to further elucidate the impact of Empagliflozin (Empa) on: (1) SKM function and metabolism and (2) mitochondrial function in an established HFpEF rat model. At the age of 24 weeks, obese ZSF1 rats were randomized either receiving standard care or Empa in the drinking water. ZSF1 lean animals served as healthy controls. After 8 weeks of treatment, echocardiography and SKM contractility were performed. Mitochondrial function was assessed in saponin skinned fibers and SKM tissue was snap frozen for molecular analyses. HFpEF was evident in the obese animals when compared to lean—increased E/é and preserved left ventricular ejection fraction. Empa treatment significantly improved E/é and resulted in improved SKM contractility with reduced intramuscular lipid content. Better mitochondrial function (mainly in complex IV) with only minor modulation of atrophy-related proteins was seen after Empa treatment. The results clearly documented a beneficial effect of Empa on SKM function in the present HFpEF model. These effects were accompanied by positive effects on mitochondrial function possibly modulating SKM function.

Sex-specific responses to slow progressive pressure overload in a large animal model of HFpEF.

Approximately 50% of all heart failure (HF) diagnoses can be classified as HF with preserved ejection fraction (HFpEF). HFpEF is more prevalent in females compared with males, but the underlying mechanisms are unknown. We previously showed that pressure overload (PO) in male felines induces a cardiopulmonary phenotype with essential features of human HFpEF. The goal of this study was to determine if slow progressive PO induces distinct cardiopulmonary phenotypes in females and males in the absence of other pathological stressors. Female and male felines underwent aortic constriction (banding) or sham surgery after baseline echocardiography, pulmonary function testing, and blood sampling. These assessments were repeated at 2 and 4 mo postsurgery to document the effects of slow progressive pressure overload. At 4 mo, invasive hemodynamic studies were also performed. Left ventricle (LV) tissue was collected for histology, myofibril mechanics, extracellular matrix (ECM) mass spectrometry, and single-nucleus RNA sequencing (snRNAseq). The induced pressure overload (PO) was not different between sexes. PO also induced comparable changes in LV wall thickness and myocyte cross-sectional area in both sexes. Both sexes had preserved ejection fraction, but males had a slightly more robust phenotype in hemodynamic and pulmonary parameters. There was no difference in LV fibrosis and ECM composition between banded male and female animals. LV snRNAseq revealed changes in gene programs of individual cell types unique to males and females after PO. Based on these results, both sexes develop cardiopulmonary dysfunction but the phenotype is somewhat less advanced in females.NEW & NOTEWORTHY We performed a comprehensive assessment to evaluate the effects of slow progressive pressure overload on cardiopulmonary function in a large animal model of heart failure with preserved ejection fraction (HFpEF) in males and females. Functional and structural assessments were performed at the organ, tissue, cellular, protein, and transcriptional levels. This is the first study to compare snRNAseq and ECM mass spectrometry of HFpEF myocardium from males and females. The results broaden our understanding of the pathophysiological response of both sexes to pressure overload. Both sexes developed a robust cardiopulmonary phenotype, but the phenotype was equal or a bit less robust in females.

Proteomic and phosphoproteomic profiling in heart failure with preserved ejection fraction (HFpEF).

Although the prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, evidence-based therapies for HFpEF remain limited, likely due to an incomplete understanding of this disease. This study sought to identify the cardiac-specific features of protein and phosphoprotein changes in a murine model of HFpEF using mass spectrometry. HFpEF mice demonstrated moderate hypertension, left ventricle (LV) hypertrophy, lung congestion and diastolic dysfunction. Proteomics analysis of the LV tissue showed that 897 proteins were differentially expressed between HFpEF and Sham mice. We observed abundant changes in sarcomeric proteins, mitochondrial-related proteins, and NAD-dependent protein deacetylase sirtuin-3 (SIRT3). Upregulated pathways by GSEA analysis were related to immune modulation and muscle contraction, while downregulated pathways were predominantly related to mitochondrial metabolism. Western blot analysis validated SIRT3 downregulated cardiac expression in HFpEF vs. Sham (0.8 ± 0.0 vs. 1.0 ± 0.0; P < 0.001). Phosphoproteomics analysis showed that 72 phosphosites were differentially regulated between HFpEF and Sham LV. Aberrant phosphorylation patterns mostly occurred in sarcomere proteins and nuclear-localized proteins associated with contractile dysfunction and cardiac hypertrophy. Seven aberrant phosphosites were observed at the z-disk binding region of titin. Additional agarose gel analysis showed that while total titin cardiac expression remained unaltered, its stiffer N2B isoform was significantly increased in HFpEF vs. Sham (0.144 ± 0.01 vs. 0.127 ± 0.01; P < 0.05). In summary, this study demonstrates marked changes in proteins related to mitochondrial metabolism and the cardiac contractile apparatus in HFpEF. We propose that SIRT3 may play a role in perpetuating these changes and may be a target for drug development in HFpEF.

A pacing-controlled protocol for frequency-diastolic relations distinguishes diastolic dysfunction specific to a mouse HFpEF model.

Heart failure with preserved ejection fraction (HFpEF), characterized by diastolic dysfunction and insufficient exercise capacity, is a growing health problem worldwide. One major difficulty with experimental research on HFpEF is the lack of methods to consistently detect diastolic dysfunction in mouse models. We developed a pacing-controlled pressure-volume (PV) loop protocol for the assessment of diastolic function at different heart rates in mice and tested if the protocol could detect diastolic dysfunction specific to a HFpEF model. A HFpEF model was generated by high-fat diet (HFD) feeding with concomitant NG-nitro-l-arginine methyl ester administration, and a pressure-overload hypertrophy (PO) model was produced by surgical constriction of the transverse aorta (TAC). Heart rate (HR) was slowed below 400 beats/min by intraperitoneal injection of ivabradine. PV loop data were acquired and analyzed at HR incrementing from 400 to 700 beats/min via atrial pacing using a miniature pacing catheter inserted into the esophagus, and comparisons were made among control, HFpEF, and PO mice. At baseline without pacing, no diastolic abnormalities were detected in either PO or HFpEF models. Frequency-diastolic relations, however, revealed the significant diastolic impairment specific to the HFpEF model; both relaxation time constant (Tau) and end-diastolic pressure-volume relationship (EDPVR) were worsened as heart rate increased. Peak positive first derivative of left ventricular pressure (dP/dtmax) was significantly lower in HFpEF versus controls only at a high HR of 700 beats/min. A pacing-controlled protocol would be a feasible and potent method to detect diastolic dysfunction specific to a mouse HFpEF model.NEW & NOTEWORTHY We developed a pacing-controlled PV loop protocol for the assessment of diastolic function at different heart rates in mice, which is a feasible and potent method for the characterization of diastolic dysfunction in a murine HFpEF model whose diastolic dysfunction might be difficult to be detected under resting conditions without pacing.

Abstract P3015: Cite-seq Identifies A Novel Role For Macrophage Trem2 In A Murine Model Of Hfpef

Deoxycorticosterone acetate (DOCA)-salt is a common model of hypertension and has been used to study heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and to identify immune mediators of cardiac remodeling using unbiased methods. DOCA-salt mice underwent uninephrectomy, implantation of a DOCA pellet, and supplementation of the drinking water with 1%NaCl for three weeks. Control mice underwent a sham procedure and received normal water. Compared to control mice, DOCA-salt mice exhibited increased systolic blood pressure, cardiac hypertrophy, and decreased time to exhaustion upon treadmill exercise testing. In addition, DOCA-salt mice exhibited albuminuria, indicative of renal glomerular injury. On echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.70±.24, 8.22±.45; n=8,5) in DOCA-salt mice, consistent with diastolic dysfunction. CITE-seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, was performed on a total of 4,359 and 7,600 sorted leukocytes from four sham and four DOCA-salt left ventricles, respectively. Differential gene expression analysis revealed major transcriptional changes in cardiac macrophages between groups and identified Trem2 as being upregulated in cardiac macrophages. We validated a two-fold increase in Trem2 gene expression in left ventricles of DOCA-salt mice and observed a two-fold increase in cardiac Trem2+Mac2+ cells in DOCA-salt by immunofluorescence. To test whether Trem2 has a causal role in HFpEF, wild type and Trem2 deficient animals were subjected to DOCA-salt. After DOCA-salt, Trem2-deficient mice displayed significantly increased heart weight to tibia length ratios (9.39±0.20, 11.21±0.80; n=6-7) and albumin to creatinine ratios (5.5±1.2, 14.2±2.9; n=6-7) compared to wild-type controls. No difference in albuminuria was observed at baseline (0.36±0.05, 0.46±0.10; n=10). In conclusion, the DOCA-salt mouse model induces key features of HFpEF and identifies Trem2 as a potential novel modulator of cardiac remodeling and renal injury.