Obese ZSF1 Rats Research Articles

Vascular Rarefaction and Perivascular Fibrosis Contribute to the Development of Left Ventricular Diastolic Dysfunction in HFpEF

Coronary microvascular dysfunction (CMD) develops in patients with heart failure with preserved ejection fraction (HFpEF, also known as diastolic heart failure), but the nature of the underlying pathomechanisms behind this prevalent disease remain poorly understood. The hypothesis tested was that coronary microvascular rarefaction contributes to left ventricle (LV) diastolic function in HFpEF. The obese ZSF1 rat model of human HFpEF was employed and using transthoracic echocardiography it was found that 18-week-old male obese ZSF1 rats exhibited a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities) and increased DT (E wave deceleration time) with no change in ejection fraction, indicating diastolic dysfunction. In isolated myocardial papillary muscle preparations, the isometric force generation and myocardial relaxation kinetics were found to be similar in lean and obese ZSF1 rats, indicating no significant differences in contractility. Coronary arteriolar and capillary trees were labeled using Tomato Lectin (Lycopersicon esculentum) DyLight®594 and were imaged by fluorescent confocal microscopy to generate image stacks for 3D reconstruction. Unbiased automated tracing of the microvasculature was done using VesselLucida360 software (MBF) followed by a morphometric analysis (VesselLucida Explorer). It was found that total vessel length and the number of vessel's branching nodes were reduced in the obese ZSF1 rats, whereas the total vessel's volumes were increased, when compared to ZSF1 lean controls. These changes in the coronary microvasculature were accompanied by enhanced perivascular fibrosis within obese ZSF1 rats, as assessed by Masson's trichrome staining for collagen. From these results, it was concluded that vascular rarefaction and perivascular fibrosis both play a mechanistic role in the development of LV diastolic dysfunction in the obese ZSF1 rat model of human HFpEF.

Sacubitril/Valsartan Improves Diastolic Function But Not Skeletal Muscle Function in a Rat Model of HFpEF.

The angiotensin receptor/neprilysin inhibitor Sacubitril/Valsartan (Sac/Val) has been shown to be beneficial in patients suffering from heart failure with reduced ejection fraction (HFrEF). However, the impact of Sac/Val in patients presenting with heart failure with preserved ejection fraction (HFpEF) is not yet clearly resolved. The present study aimed to reveal the influence of the drug on the functionality of the myocardium, the skeletal muscle, and the vasculature in a rat model of HFpEF. Female obese ZSF-1 rats received Sac/Val as a daily oral gavage for 12 weeks. Left ventricle (LV) function was assessed every four weeks using echocardiography. Prior to organ removal, invasive hemodynamic measurements were performed in both ventricles. Vascular function of the carotid artery and skeletal muscle function were monitored. Sac/Val treatment reduced E/é ratios, left ventricular end diastolic pressure (LVEDP) and myocardial stiffness as well as myocardial fibrosis and heart weight compared to the obese control group. Sac/Val slightly improved endothelial function in the carotid artery but had no impact on skeletal muscle function. Our results demonstrate striking effects of Sac/Val on the myocardial structure and function in a rat model of HFpEF. While vasodilation was slightly improved, functionality of the skeletal muscle remained unaffected.

Linagliptin prevents left ventricular stiffening by reducing titin cleavage and hypophosphorylation.

The metabolic syndrome (MetS) is an escalating problem worldwide, causing left ventricular stiffening, an early characteristic of diastolic dysfunction for which no treatment exists. As diastolic dysfunction and stiffening in MetS patients are associated with increased circulating dipeptidyl peptidase‐4 (DPP‐4) levels, we investigated whether the clinically approved DPP‐4 inhibitor linagliptin reduces left ventricular stiffness in MetS‐induced cardiac disease. Sixteen‐week‐old obese ZSF1 rats, displaying the MetS and left ventricular stiffness, received linagliptin‐supplemented or placebo diet for four weeks. Linagliptin significantly reduced obesity, hyperlipidaemia, and hyperglycaemia and improved left ventricular relaxation. This improved relaxation was related to decreased cardiac fibrosis and cardiomyocyte passive stiffness (F passive). The reduced F passive was the result of titin isoform switching from the stiff N2B to the more flexible N2BA and increased phosphorylation of total titin and specifically its N2Bus region (S4080 and S3391). Importantly, DPP‐4 directly cleaved titin in vitro, resulting in an increased F passive, which was prevented by simultaneous administration of linagliptin. In conclusion, linagliptin improves left ventricular stiffness in obese ZSF1 rats by preventing direct DPP4‐mediated titin cleavage, as well as by modulating both titin isoform levels and phosphorylation. Reducing left ventricular stiffness by administering linagliptin might prevent MetS‐induced early diastolic dysfunction in human.

Abstract 15172: Murf1 Inhibitor Embl205 is a New Approach for Treatment of Heart Failure With Preserved Ejection Fraction in an Animal Model

Background: The novel MuRF1 inhibitor EMBL205 attenuates effectively developing skeletal muscle atrophy and dysfunction in animals with heart failure with preserved ejection fraction (HFpEF, ZSF1 rat model). The impact of EMBL205 on myocardial function in the HFpEF setting is currently unknown and was evaluated in ZSF1 rats. Methods: 20 wks-old female obese ZSF1 rats received EMBL205 (12 wks, conc. of 0.1% in chow; HFpEF-EMBL205). Age-matched untreated lean (con) and obese (HFpEF) ZSF1 rats served as controls. At 32 wks of age left ventricular (LV)-, aortic valve (AV) function and LV end diastolic pressure (LVEDP) was determined by echocardiography and invasive hemodynamic measurements. LV expression of collagen 1A (Col1A) and 3A (Col3A) was assessed by qRT-PCR, MMP2 expression was obtained by zymography and perivascular fibrosis was quantified in histological sections. Results: Development of HFpEF in ZSF1 obese animals is associated with cardiac enlargement and hypertrophy, as evident by increased myocardial weight, an increase in end diastolic volume (EDV) and LV anterior and posterior wall diameters. Diastolic LV-function is disturbed with elevation of E/é, an increased LVEDP and a preserved LV ejection fraction. AV peak velocity and peak gradient are significantly increased and AV opening area (AVA) significantly decreased. Col1A and Col3A expression are increased in HFpEF animals. EMBL205 treatment results in a significant reduction of myocardial weight and a trend towards lower EDV compared to HFpEF group. EMBL205 attenuates the increase in E/é, LVEDP, AV peak gradient and the decrease of AVA. EMBL205 significantly reduces Col3A expression and a trend for Col1A expression is seen. Increased perivascular fibrosis and MMP2 expression in HFpEF is extenuated by EMBL205 treatment (table 1). Conclusions: Application of EMBL205 attenuated the development of pathological myocardial alterations associated with HFpEF in ZSF1rats due to antifibrotic effects.

The effect of different anaesthetics on echocardiographic evaluation of diastolic dysfunction in a heart failure with preserved ejection fraction model

Heart failure with preserved ejection fraction (HFpEF) is currently untreated. Therapeutics development demands effective diagnosis of diastolic dysfunction in animal models mimicking human pathology, which requires appropriate anaesthetics. Here, we investigated which anaesthetic, ketamine/xylazine or isoflurane, could be used to reveal diastolic dysfunction in HFpEF-diseased obese ZSF1 rats by echocardiography. First, diastolic dysfunction was confirmed by pressure-volume loops in obese compared to lean control ZSF1 rats. In echocardiography, ketamine/xylazine, unlike isoflurane, was able to demonstrate impaired relaxation in obese ZSF1 rats, as reflected by impaired early (E) and late (A) filling peak velocities, decreased E/A ratio, and a prolonged deceleration and isovolumic relaxation time. Interestingly, ketamine/xylazine induced a wider separation of both tissue and pulsed wave Doppler-derived echocardiographic waves required for diastolic dysfunction diagnosis, potentially by reducing the heart rate (HR), while isoflurane resulted in merged waves. To assess whether HR-lowering alone explained the differences between the anaesthetics, echocardiography measurements under isoflurane with and without the HR-lowering drug ivabradine were compared. However, diastolic dysfunction could not be diagnosed in ivabradine-treated obese ZSF1 rats. In summary, ketamine/xylazine compared to isoflurane is the anaesthetic of choice to detect diastolic dysfunction by echocardiography in rodent HFpEF, which was only partly mediated by HR-lowering.

Glucosuria Interferes With Measurement of Effective Renal Plasma Flow Using para-Aminohippuric Acid, With a Focus on SGLT2 Inhibitors

Abstract Para-aminohippuric acid (PAH)-derived effective renal plasma flow (ERPF) is increasingly being used in parallel with the revived interest in kidney hemodynamic function due to the development of sodium glucose co-transporter 2 (SGLT2) inhibitors. In our trial (Kidney Int 2020;97: 202-12) we measured ERPF by urinary PAH clearance and calculated fractional PAH extraction ratios, which initially were very low during both the hyperglycemic clamps at baseline, as well as the euglycemic conditions following treatment with SGLT2 inhibitor dapagliflozin. Previous literature showed that hyperglycemic i.e. glucosuric conditions reduce urinary PAH concentrations, due to a Schiff base that is formed between the para-amino group of PAH and the aldehyde group of glucose. By using hydrochloric acid (HCl) any PAH that has been glycosylated can be reconverted, independent of storage time. After treatment with HCL we indeed found higher PAH extraction ratios in the expected range. In addition, a negative correlation between storage time and PAH extraction ratio in people treated with SGLT2 inhibitor dapagliflozin during a euglycemic clamp before treatment with HCl. Besides these human data, comparable results were observed in male obese ZSF1 rats with hyperglycemia. Therefore, researchers should be aware that even under euglycemic conditions, SGLT2 inhibitors induce glucosuria that interferes with accurate ERPF measurement due to glycosylation of PAH in stored urine samples. Pre-treatment of urine with NaOH or adding HCl prior to measurement will overcome this problem.

Praliciguat inhibits progression of diabetic nephropathy in ZSF1 rats and suppresses inflammation and apoptosis in human renal proximal tubular cells.

Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-β-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.

The evolving systemic biomarker milieu in obese ZSF1 rat model of human cardiometabolic syndrome: Characterization of the model and cardioprotective effect of GDF15.

Cardiometabolic syndrome has become a global health issue. Heart failure is a common comorbidity of cardiometabolic syndrome. Successful drug development to prevent cardiometabolic syndrome and associated comorbidities requires preclinical models predictive of human conditions. To characterize the heart failure component of cardiometabolic syndrome, cardiometabolic, metabolic, and renal biomarkers were evaluated in lean and obese ZSF1 19- to 32-week-old male rats. Histopathological assessment of kidneys and hearts was performed. Cardiac function, exercise capacity, and left ventricular gene expression were also analyzed. Obese ZSF1 rats exhibited multiple features of human cardiometabolic syndrome by pathological changes in systemic renal, metabolic, and cardiovascular disease circulating biomarkers. Hemodynamic assessment, echocardiography, and decreased exercise capacity confirmed heart failure with preserved ejection fraction. RNA-seq results demonstrated changes in left ventricular gene expression associated with fatty acid and branched chain amino acid metabolism, cardiomyopathy, cardiac hypertrophy, and heart failure. Twelve weeks of growth differentiation factor 15 (GDF15) treatment significantly decreased body weight, food intake, blood glucose, and triglycerides and improved exercise capacity in obese ZSF1 males. Systemic cardiovascular injury markers were significantly lower in GDF15-treated obese ZSF1 rats. Obese ZSF1 male rats represent a preclinical model for human cardiometabolic syndrome with established heart failure with preserved ejection fraction. GDF15 treatment mediated dietary response and demonstrated a cardioprotective effect in obese ZSF1 rats.

A directed network analysis of the cardiome identifies molecular pathways contributing to the development of HFpEF

AimsThe metabolic syndrome and associated comorbidities, like diabetes, hypertension and obesity, have been implicated in the development of heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms underlying the development of HFpEF remain to be elucidated. We developed a cardiome-directed network analysis and applied this to high throughput cardiac RNA-sequencing data from a well-established rat model of HFpEF, the obese and hypertensive ZSF1 rat. With this novel system biology approach, we explored the mechanisms underlying HFpEF. Methods and resultsUnlike ZSF1-Lean, ZSF1-Obese and ZSF1-Obese rats fed with a high-fat diet (HFD) developed diastolic dysfunction and reduced exercise capacity. The number of differentially expressed genes amounted to 1591 and 1961 for the ZSF1-Obese vs. Lean and ZSF1-Obese+HFD vs. Lean comparison, respectively. For the cardiome-directed network analysis (CDNA) eleven biological processes related to cardiac disease were selected and used as input for the STRING protein-protein interaction database. The resulting STRING network comprised 3.460 genes and 186.653 edges. Subsequently differentially expressed genes were projected onto this network. The connectivity between the core processes within the network was assessed and important bottleneck and hub genes were identified based on their network topology.Classical gene enrichment analysis highlighted many processes related to mitochondrial oxidative metabolism. The CDNA indicated high interconnectivity between five core processes: endothelial function, inflammation, apoptosis/autophagy, sarcomere/cytoskeleton and extracellular matrix. The transcription factors Myc and Peroxisome Proliferator-Activated Receptor-α (Ppara) were identified as important bottlenecks in the overall network topology, with Ppara acting as important link between cardiac metabolism, inflammation and endothelial function. ConclusionsThis study presents a novel systems biology approach, directly applicable to other cardiac disease-related transcriptome data sets. The CDNA approach enabled the identification of critical processes and genes, including Myc and Ppara, that are putatively involved in the development of HFpEF.

Both male and female obese ZSF1 rats develop cardiac dysfunction in obesity-induced heart failure with preserved ejection fraction.

Heart failure with a preserved ejection fraction (HFpEF) is associated with multiple comorbidities, such as old age, hypertension, type 2 diabetes and obesity and is more prevalent in females. Although the male obese ZSF1 rat has been proposed as a suitable model to study the development of diastolic dysfunction and early HFpEF, studies in female animals have not been performed yet. Therefore, we aimed to characterize the cardiac phenotype in female obese ZSF1 rats and their lean counterparts. Additionally, we aimed to investigate whether differences exist in disease progression in obese male and female ZSF1 rats. Therefore, male and female ZSF1 rats, lean as well as obese (N = 6-9/subgroup), were used. Every two weeks, from 12 to 26 weeks of age, systolic blood pressure and echocardiographic measurements were performed, and venous blood was sampled. Female obese ZSF1 rats, as compared to female lean ZSF1 rats, developed diastolic dysfunction with cardiac hypertrophy and fibrosis in the presence of severe dyslipidemia, increased plasma growth differentiation factor 15 and mild hypertension, and preservation of systolic function. Although obese female ZSF1 rats did not develop hyperglycemia, their diastolic dysfunction was as severe as in the obese males. Taken together, the results from the present study suggest that the female obese ZSF1 rat is a relevant animal model for HFpEF with multiple comorbidities, suitable for investigating novel therapeutic interventions.

Fat Quality Matters: Distinct Proteomic Signatures Between Lean and Obese Cardiac Visceral Adipose Tissue Underlie its Differential Myocardial Impact.

Heart failure with preserved ejection fraction (HFpEF) is recognised as an important cause of cardiovascular mortality and morbidity, accounting for approximately 50% of heart failure cases. Metabolic-related complications, such as obesity, have been associated with the pathophysiology of this complex syndrome. The anatomic proximity between cardiac visceral adipose tissue (CVAT) and the myocardium has been drawing attention due to its potential pathogenic role in cardiac diseases. Thus, we aimed to characterise the phenotypic and proteomic differences between CVAT from ZSF1 lean (control) and ZSF1 obese (HFpEF) rats as well as to evaluate the myocardial impact of conditioned media derived from CVAT of these 2 groups. CVAT of 20-weeks-old lean and obese ZSF1 rats was collected for: 1) 24h DMEM incubation to obtain conditioned media, 2) separation of proteins to mass spectrometry identification, 3) adipokines' expression, 4) adipocytes cross-sectional area assessment. Organotypic cultures were prepared from 7 days-old Wistar Han cardiac explants and incubated for 24h with the conditioned media. After incubation, cross-section area of cardiomyocytes and fibrosis were evaluated. Cardiomyocytes were isolated from Wistar Han and incubated with conditioned media for viability studies. CVAT from lean rats presented a higher expression of uncoupling protein-1 (UCP-1) protein, associated with a multilocular appearance and an increased expression of brown adipose tissue markers. Contrarily, CVAT from obese rats revealed a white adipose tissue-like phenotype accompanied by hypertrophy of adipocytes. The analysis of the CVAT proteome reinforced the phenotypic differences between lean and obese CVAT, showing enrichment of proteins involved in triglyceride metabolic processes in obese CVAT. In contrast, mitochondrial proteins were prominent in lean CVAT, further suggesting a brown adipose tissue-like phenotype. The twenty-four hours-long incubation of myocardial organo-cultures with conditioned media obtained from CVAT obese (CM-obese) rats significantly reduced cell viability, induced cardiomyocytes hypertrophy and fibrosis, in stark contrast with the incubation with the conditioned media from lean rats CVAT (CM-lean). Furthermore, the deleterious effect imposed by CM-obese was associated with a pro-inflammatory profile, characterised by an increased expression of several pro-inflammatory adipokines. Obesity promotes alterations in CVAT proteome signature, structure, composition and secretome, translating into dramatic myocardial consequences.

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat

BackgroundEmpagliflozin (empa), a selective sodium–glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its’ lean control.MethodsLean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis.ResultsEmpa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats.ConclusionEmpa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

P6341Impact of left-ventricular hemodynamics on treadmill exercise intolerance in conscious rats: pilot evaluation in animals with diastolic dysfunction

Abstract Introduction Exercise intolerance is a clinical hallmark of patients with hypertrophic cardiomyopathy and/or impaired diastolic function. Elevated LV filling pressures, particularly in response to acute exercise bouts, are thought to play a role limiting exercise capacity in ventricles with abnormal relaxation/compliance. However, it is experimentally difficult to obtain in vivo hemodynamic measures necessary for the evaluation of centrally mediated dysfunction. Leveraging radio-telemetry, we evaluated central hemodynamic parameters as mediators of exercise intolerance in obese ZSF1 rats with diabetes, hypertension, and diastolic dysfunction. Methods Both ZSF1 (637+12g, n=8) and age-matched (28 weeks) healthy control (CTRL, 543+14g, n=4, P<0.05) rats were instrumented for telemetric left-ventricular pressure (LVP) recordings. Following surgical recovery rats were familiarized to treadmill running and subsequently challenged with an exercise protocol aimed at increasing heart rate by 200 beats/min (5° incline, 15 m/min, increased by 3 m/min every 2-min) until volitional exhaustion. LV pressures were collected continuously during cage resting, treadmill resting, and post-exercise until heart rate returned to baseline. Additionally, resting echocardiographic and blood glucose measures were collected. Results At rest, ZSF1 rats had preserved ejection fraction (73+6 vs 79+9%), elevated (P<0.05) blood glucose (237+83 vs 94+23 mg/dL), end-systolic (147+18 vs 103+13 mmHg), and end-diastolic pressures (16+3 vs 9+3 mmHg), with preserved indexed end-diastolic volumes (670+95 vs 741+89 μL/kg), suggesting impaired diastolic compliance. ZSF1 rats terminated exercise prematurely (8:26+1:20 vs 10:27+1:18 min, P<0.05), indicating a limitation in exercise capacity. This early volitional exhaustion was noted while end-diastolic pressures were not further increased (17+7 mmHg), suggesting that other pathological derangement may play a role modulating exercise capacity. For instance, ZSF1 rats tended to have a blunted increase in the systolic index dP/dt40 (+2589+1450 vs +3938+749 mmHg/s, P<0.1) despite achieving comparable increases in HR (193+34 vs 196+38 bpm) with exercise. Conclusion This pilot study demonstrates the feasibility for evaluation of left-ventricular hemodynamics during exercise in rodents with diastolic dysfunction, establishing a platform to evaluate both the mechanisms of exercise intolerance as well as potential therapeutic approaches to rescue exercise capacity. Acknowledgement/Funding MyoKardia

P5995A novel biased S1P1 agonist improves renal and cardiac functions in ZSF1 rats, a model of metabolic syndrome-associated Heart Failure with preserved Ejection Fraction

Abstract Background/Introduction Heart Failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with currently no approved treatment. Diastolic dysfunction, dyspnea, intolerance to effort, high cardiac filling pressure, and lung congestion coexist with normal ejection fraction in this clinical syndrome. Ageing, obesity, type 2 diabetes, hypertension and renal dysfunction are the main comorbidities found in this heterogeneous group of patients. Microvascular endothelial dysfunction, driven by these risk factors, may be a common link with other aspects of the HFpEF pathogenesis that include oxidative stress, inflammation, cardiomyocyte stiffness/hypertrophy, and myofibroblast accumulation. Sphingosine-1-phosphate type-1 receptor (S1P1), a G protein–coupled receptor highly expressed in endothelial cells, regulates vascular integrity, vascular development and immune cell trafficking. Compound A is a novel G protein-biased S1P1 agonist that lacks functional antagonism and has endothelial-protective properties. Purpose S1P1 activation could promote phosphorylation of endothelial nitric oxide synthase, restoration of endothelial structure and function, and thus diminish cardiac and vascular stiffness, hypertrophy and fibrosis. The aim of this study was to investigate if compound A could improve renal and cardiac functions in a rat model of HFpEF with metabolic syndrome. Methods 65-week-old obese ZSF1 rats were fed a chow diet containing compound A (8 mg/kg/day) or no compound for 4 weeks. Lean ZSF1 and Wistar rats were included in the study as control groups. Urinary protein/creatinine ratio was measured as an index of glomerular injury. Cardiac hypertrophy and function were assessed by two-dimensional and Doppler echocardiography. Total cardiac and atrial weights and pulmonary edema were assessed. Results The obese ZSF1 rat was confirmed as a relevant model of HFpEF with advanced renal dysfunction. These rats showed severe glomerular filtration impairment, left ventricular and atrial hypertrophy and pulmonary edema. Cardiac systolic function, cardiac output and chamber volumes were preserved, diastolic function was impaired, and left ventricular posterior walls and septal thicknesses were increased compared to control groups. Four weeks of compound A treatment reduced urinary protein/creatinine ratio, blunted cardiac and atrial hypertrophy, and partially restored diastolic function. Circulating lymphocytes were not reduced by compound A, confirming that these pharmacological effects were not associated with S1P1 desensitization. Conclusion Compound A, a novel S1P1 agonist with endothelial properties, improves cardiac and renal functions in a rat model of metabolic syndrome-associated HFpEF. Sustained S1P1 activation with compound A may be a promising strategy for HFpEF treatment.

Adenosine Kinase Inhibition Augments Conducted Vasodilation and Prevents Left Ventricle Diastolic Dysfunction in Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is often manifested as impaired cardiovascular reserve. We sought to determine if conducted vasodilation, which coordinates microvascular resistance longitudinally to match tissue metabolic demand, becomes compromised in HFpEF. We hypothesized that the metabolic vasodilator adenosine facilitates and that inhibition of ADK (adenosine kinase) augments conducted vasodilation for a more efficient myocardial perfusion and improved left ventricle (LV) diastolic function in HFpEF. We assessed conducted vasodilation in obese ZSF1 rats that develop LV diastolic dysfunction and is used to model human HFpEF. Additionally, conducted vasodilation was measured in arterioles isolated from the right atrial appendages of patients with HFpEF. We found a markedly reduced conducted vasodilation both in obese ZSF1 rats and in patients with HFpEF. Impaired conducted vasodilation was accompanied by increased vascular ADK expression. Isolated rat and human arterioles incubated with adenosine (10 nmol/L) or ADK inhibitor ABT-702 (0.1 µmol/L) both displayed augmented conducted vasodilation. Treatment of obese ZSF1 rats with ABT-702 (1.5 mg/kg, IP for 8 weeks) prevented LV diastolic dysfunction, and in a crossover design augmented conducted vasodilation and improved LV diastolic function. ABT-702 treated obese ZSF1 rats exhibited reduced expression of myocardial carbonic anhydrase 9 and collagen, surrogate markers of myocardial hypoxia. Upregulation of vascular ADK mitigates adenosine-facilitated conducted vasodilation in obese ZSF1 rats and in patients with HFpEF. We propose that pharmacological inhibition of ADK could be beneficial for therapeutic augmentation of conducted vasodilation, thereby improving tissue perfusion and LV diastolic function in HFpEF.

Limited synergy of obesity and hypertension, prevalent risk factors in onset and progression of heart failure with preserved ejection fraction.

Obesity and hypertension are prevalent comorbidities in heart failure with preserved ejection fraction. To clarify if and how interaction between these comorbidities contributes to development of diastolic dysfunction, lean and obese ZSF1 rats were treated with deoxycorticosterone acetate implants and a high‐salt diet (DS) to induce severe hypertension, or with placebo. In addition to echocardiographic, metabolic and hemodynamic analyses, immunohistochemistry and RNAseq were performed on left ventricular tissue. Obesity negatively affected cardiac output, led to an elevated E/e’ ratio and mildly reduced ejection fraction. DS‐induced hypertension did not affect cardiac output and minimally elevated E/e’ ratio. Diastolic derangements in placebo‐treated obese rats developed in absence of inflammation and fibrosis, yet in presence of oxidative stress and hypertrophic remodelling. In contrast, hypertension triggered apoptosis, inflammation and fibrosis, with limited synergy of the comorbidities observed for inflammation and fibrosis. Transcriptional data suggested that these comorbidities exerted opposite effects on mitochondrial function. In placebo‐treated obese rats, genes involved in fatty acid metabolism were up‐regulated, whereas DS‐induced a down‐regulation of genes involved in oxidative phosphorylation. Overall, limited interaction was observed between these comorbidities in development of diastolic dysfunction. Importantly, differences in obesity‐ and hypertension‐induced cardiac remodelling emphasize the necessity for comorbidity‐specific phenotypical characterization.

A Novel Dual Soluble Epoxide Hydrolase Inhibitor/Cyclooxygenase‐2 Inhibitor Treats Type 2 Diabetic Complications in Obese ZSF1 Rats

Diabetes is a highly prevalent chronic disease worldwide that represents a global healthcare burden. A devastating type 2 diabetes complication is diabetic nephropathy. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxyganse‐2 inhibitor (sEHi/COXi) to combat diabetic nephropathy. The objective for the current investigation is to determine if PTUPB can treat diabetic nephropathy in obese ZSF1 rats. PTUPB was compared to an angiotensin converting enzyme inhibitor that is the current standard of care for diabetic nephropathy. Obese ZSF‐1 rats at 16 weeks of age received PTUPB (10mg/kg/d i.p.) or enalapril (10mg/kg/d, p/o.) for 8 weeks. Type 2 diabetes was assessed in obese ZSF1 rats. Renal injury was determined using several biochemical, histological, and immunohistological techniques. Obese ZSF1 rats were diabetic with fasting blood glucose (19±0.4 vs. 4.8±0.1 mmol/l in lean ZSF1) . Neither PTUPB nor enalapril treated diabetes and did not reduce hyperglycemia or HbA1c in obese ZSF1 rats. Obese ZSF1 rats developed diabetic nephropathy with elevated albuminuria (49±6 vs. 1.3±0.2 mg/mg creatinine in lean ZSF1), tubular cast formation (cortex, 14±1 vs. 1.0±0.1%; medulla, 22.4±2 vs. 0.9±0.2%), renal fibrosis (cortex, 11±1 vs. 4.4±0.3%; medulla, 10±1.2 vs. 4.0±0.8%) and glomerular injury (3.4±0.6 vs. 0.6±0.08 a.u.) compared to lean ZSF1 lean (all P<0.05). PTUPB treated diabetic nephropathy by reducing albuminuria (by 50%), renal tubular cast formation (by 60% in cortex; 70% in medulla), renal fibrosis (by 50% in cortex; 36% in medulla), and glomerular injury (by 55%) compared to vehicle. Enalapril treatment reduced diabetic nephropathy in obese ZSF1 rats like PTUPB and reduced the renal injury parameters by 30 to 50% compared to vehicle. Diabetic renal injury in obese ZSF1 rats was accompanied by inflammation with elevated urinary MCP‐1 levels (29±2 vs. 5±0.6 ng/mg creatinine in lean ZSF1 rat, P<0.05) and renal infiltration of CD‐68 cells (95±10 vs. 16±1.2 cell/mm2, P<0.05). PTUPB or enalapril treated renal inflammation and reduced MCP‐1 excretion (PTUPB reduced by 50% and enalapril reduced by 20%) and renal CD‐68 positive cell infiltration (by 60% and 50%, respectively by PTUPB or enalapril). These findings demonstrate that PTUPB is not anti‐diabetic but markedly treat diabetic nephropathy. We also demonstrate that PTUPB has renal actions comparable to enalapril which is currently the standard of care to treat diabetic nephropathy.Support or Funding InformationNational Institute of Health (NIH) grant (DK103616) and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee grant to JDI; NIEHS/R01 ES002710 (HAM1400) and NIEHS/Superfund Research Program P42 ES004699 (SUPR?30) to BDHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The adenosine kinase inhibitor ABT‐702 improves coronary vasodilator and left ventricle diastolic dysfunction in obese ZSF1 rats

Coronary microvascular dysfunction contributes to the development of heart failure with preserved ejection fraction (HFpEF) for which no effective therapies are currently available. We tested the hypothesis that pharmacological inhibition of adenosine kinase (ADK), the major adenosine metabolizing enzyme, augments coronary artery vasodilation and improves left ventricle (LV) diastolic function in HFpEF. We employed the obese ZSF1 rat model of HFpEF. We assessed agonist‐induced coronary artery dilation and performed echocardiographic assessment of LV diastolic function before and after treatment with selective ADK inhibitor, ABT‐702 (1.5 mg/kg, i.p. for 8‐week). We found that obese ZSF1 rats exhibited impaired acetylcholine (ACh)‐induced coronary artery dilation and LV diastolic dysfunction, as indicated by reduced E/A ratio and increased deceleration time of mitral flow velocity. Treatment with ABT‐702 augmented ACh‐induced coronary vasodilation in obese ZSF1 rats, which was not affected by inhibition of NO synthase, with N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME). ABT‐702 treatment also improved LV diastolic function in obese ZSF‐1 rats, but had no effect on lean controls. Collectively, these data suggest that a pharmacological inhibition of ADK provides beneficial effects on both coronary vasodilator and LV diastolic function in HFpEF.Support or Funding InformationAmerican Heart Association 17GRNT33680171, NIH F31 HL142183‐01 and NIH R01AG054651This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of Synergy between Major Risk Factors, Obesity and Hypertension, in Development of Heart Failure with Preserved Ejection Fraction

BackgroundObesity and hypertension are highly associated with diastolic dysfunction. The current consensus is that metabolic‐ and hypertensive disease, like other comorbidities, contribute synergistically to a systemic state of inflammation, leading to coronary microvascular dysfunction, oxidative stress, cardiomyocyte stiffness and myocardial fibrosis. As unambiguous evidence for such a synergistic relation is currently lacking, we aimed to clarify if and how obesity and hypertension interact in development of diastolic dysfunction.MethodsMale lean and obese ZSF1 rats, of which the latter were previously shown to develop heart failure with preserved ejection fraction, were studied from 13 till 26 weeks of age. To assess the impact of severe hypertension, both lean and obese rats were treated with Deoxycorticosterone acetate implants plus 6% dietary salt (DS) or vehicle, from 19 to 26 weeks of age. In addition to echocardiographic, metabolic, and hemodynamic analyses, RNAseq and histochemistry were done on left ventricle tissue.ResultsBody weight, cholesterol, and triglycerides were elevated in obese rats (table 1). Systolic blood pressure (SBP) was increased by DS treatment. Ejection fraction (EF) was slightly reduced in both vehicle‐ and DS‐treated obese rats, though all animals remained within the spectrum of preserved EF (>50%). Illustrative for diastolic dysfunction, the ratio between peak velocity of early mitral inflow, and early diastolic mitral annulus velocity (E/e′), was increased in obese animals. Importantly, no synergistic effects were observed of obesity and hypertension (p=0.55). In the vehicle‐treated obese rats, diastolic dysfunction developed in absence of significant fibrosis. Computational pathway analysis of the RNAseq data using Ingenuity Pathway Analysis however, predicted a steep elevation in obesity‐induced mitochondrial fatty acid oxidation (p=1.99E‐09). Both DS‐treated lean and obese rats on the other hand, showed severe mitochondrial dysfunction, as evidenced by an overall downregulation of enzymes involved in oxidative phosphorylation, and elevated cardiac fibrosis, macrophage presence, and apoptosis.ConclusionIn this study, no synergistic effect was observed of obesity‐ and hypertension in development of diastolic dysfunction. Our findings indicate that the metabolic derangements had a more pronounced contribution to diastolic dysfunction, whereas DS‐induced hypertension mainly triggered loss of cardiomyocytes, macrophage influx, and fibrosis.Support or Funding InformationThis study was supported by a grant from the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11 (RECONNECT)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PDE9A inhibitor PF04447943 improves coronary arteriole vasodilation and left ventricular diastolic dysfunction in HFpEF

Coronary microvascular dysfunction develops in patients with heart failure with preserved ejection fraction (HFpEF, also known as diastolic heart failure). Phosphodiesterase 9A (PDE9A) was shown to be upregulated in the hypertrophic myocardium in HFpEF patients. The hypothesis tested was that PDE9A inhibition improves coronary arteriole vasodilation and left ventricle (LV) diastolic function in HFpEF. The obese ZSF1 rat, a model of human HFpEF, was employed in which it was found that LV diastolic dysfunction develops by 20 weeks of age. 18‐week old male obese ZSF‐1 rats were treated with PDE9A inhibitor, PF04447943 (3 mg/kg) or vehicle by daily oral gavage for 2 weeks and LV diastolic function was assessed by transthoracic echocardiography (E/A ratio and deceleration time) and vasodilation was examined in isolated and pressurized (70 mmHg) small coronary arteries (150–200 μm in diameter) using videomicroscopy. Results showed that LV relaxation and coronary artery dilations to acetylcholine (ACh) and also to the direct nitric oxide donor, sodium nitroprusside (SNP) were significantly reduced in the 20‐week old vehicle treated obese ZSF‐1 rats, when compared to lean controls. In the PDE9A inhibitor, PF04447943 treated obese ZSF1 rats both LV diastolic function and coronary artery dilations to ACh and SNP were improved significantly. Further results revealed that ex vivo exposure of coronary arteries to PF04447943 (18 nmol/l for 30 minutes) also augmented vasodilation in response to ACh and SNP in the obese ZSF‐1 rats. It was concluded that PDE9A inhibition improves LV diastolic function and increases agonist‐induced coronary arteriole dilation in an animal model of HFpEF.Support or Funding InformationAmerican Heart Association 17GRNT33680171, NIH F31 HL142183‐01 and NIH R01AG054651This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.