Obese ZSF1 Rats Research Articles

12380 Point Of Care Microscale Magnetic Resonance (µNMR) Effectively Monitors Mitigation Of Oxidative Stress By GLP-1 Treatment In A Preclinical Model Of Diabetic Nephropathy

Abstract Disclosure: A.T. Alves: ; co-founder. ; LarmorBio. S.S. Thamarath: ; co-founder. ; LarmorBio. S. Fjordside: Employee; Self; Novo Nordisk. S. Sassower, MS.EE: ; Co-founder. ; LarmorBio. R. Rohr: ; co-founder,CEO. ; LarmorBio. A.P. Chambers: Employee; Self; Novo Nordisk. Oxidative stress is a major driver in the pathogenesis of diabetes, obesity, and related (cardio-renal) disorders. Early detection and prevention of an oxidative stress overload can improve and potentially modify the long-term harmful outcomes associated with chronic hyperglycaemia. A novel assay was developed, that uses µNMR technology to directly monitor oxidative stress from 5 µL of plasma at the point of collection in less than 10 minutes. This assay is highly sensitive to redox changes in the blood microenvironment, including increased ferric iron (Fe3+), protein oxidation and lipid peroxidation. Here, we show the oxidative stress profiling of three groups of ZSF1 rats in a twelve week (11th -23rd week of age) observational study. Vehicle lean, vehicle obese, and glucagon-like-peptide-1 (GLP-1) treated obese ZSF1 rats (n = 8/group) were studied with the assay along with conventional diabetic/metabolic markers, blood glucose (BG), HbA1c, Albumin Creatinine Ratio (ACR). The kidney (immuno)histology studies, alpha-smooth muscle actin (aSMA) and leucocyte common antigen (CD45) were performed at the termination phase of the ZSF1 rats (23rd week of age). The results demonstrated that the assay distinguished obese rats from lean rats with statistical significance (P ≤ 0.01) independent of other in-life diabetic/metabolic markers. Significant correlations with urinary Albumin Creatinine Ratio (ACR, ρ = 0.64, P ≤ 0.0001) were established throughout the study. A positive quantitative association is highlighted with classic histological end points of organ damage, kidney fibrogenesis (aSMA, ρ = 0.23), and inflammation (CD45, ρ = 0.45). Moreover, the assay detected significantly reduced oxidative stress levels (e.g., P ≤ 0.01) in the GLP-1 drug treatment group at five weeks of follow-up. In summary, the µNMR assay effectively monitored oxidative stress and captured disease-modifying therapeutics. Presentation: 6/1/2024

PDE9A Inhibition Improves Coronary Microvascular Rarefaction and Left Ventricular Diastolic Dysfunction in the ZSF1 Rat Model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) commonly arises from comorbid diseases, such as hypertension, obesity, and diabetes mellitus. Selective inhibition of phosphodiesterase 9A (PDE9A) has emerged as a potential therapeutic approach for treating cardiometabolic diseases. Coronary microvascular disease (CMD) is one of the key mechanisms contributing to the development of left ventricular (LV) diastolic dysfunction in HFpEF. Our study aimed to investigate the mechanisms by which PDE9A inhibition could ameliorate CMD and improve LV diastolic function in HFpEF. The obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model of HFpEF was employed in which it was found that a progressively developing coronary microvascular rarefaction is associated with LV diastolic dysfunction when compared to lean, nondiabetic hypertensive controls. Obese ZSF1 rats had an increased cardiac expression of PDE9A. Treatment of obese ZSF1 rats with the selective PDE9A inhibitor, PF04447943 (3 mg/kg/day, oral gavage for 2 weeks), improved coronary microvascular rarefaction and LV diastolic dysfunction, which was accompanied by reduced levels of oxidative and nitrosative stress markers, hydrogen peroxide, and 3-nitrotyrosine. Liquid chromatography-mass spectrometry (LC-MS) proteomic analysis identified peroxiredoxins (PRDX) as downregulated antioxidants in the heart of obese ZSF1 rats, whereas Western immunoblots showed that the protein level of PRDX5 was significantly increased by the PF04447943 treatment. Thus, in the ZSF1 rat model of human HFpEF, PDE9A inhibition improves coronary vascular rarefaction and LV diastolic dysfunction, demonstrating the usefulness of PDE9A inhibitors in ameliorating CMD and LV diastolic dysfunction through augmenting PRDX-dependent antioxidant mechanisms.

Interactions between the gut microbiome, associated metabolites and the manifestation and progression of heart failure with preserved ejection fraction in ZSF1 rats

BackgroundHeart failure with preserved ejection fraction (HFpEF) is associated with systemic inflammation, obesity, metabolic syndrome, and gut microbiome changes. Increased trimethylamine-N-oxide (TMAO) levels are predictive for mortality in HFpEF. The TMAO precursor trimethylamine (TMA) is synthesized by the intestinal microbiome, crosses the intestinal barrier and is metabolized to TMAO by hepatic flavin-containing monooxygenases (FMO). The intricate interactions of microbiome alterations and TMAO in relation to HFpEF manifestation and progression are analyzed here.MethodsHealthy lean (L-ZSF1, n = 12) and obese ZSF1 rats with HFpEF (O-ZSF1, n = 12) were studied. HFpEF was confirmed by transthoracic echocardiography, invasive hemodynamic measurements, and detection of N-terminal pro-brain natriuretic peptide (NT-proBNP). TMAO, carnitine, symmetric dimethylarginine (SDMA), and amino acids were measured using mass-spectrometry. The intestinal epithelial barrier was analyzed by immunohistochemistry, in-vitro impedance measurements and determination of plasma lipopolysaccharide via ELISA. Hepatic FMO3 quantity was determined by Western blot. The fecal microbiome at the age of 8, 13 and 20 weeks was assessed using 16s rRNA amplicon sequencing.ResultsIncreased levels of TMAO (+ 54%), carnitine (+ 46%) and the cardiac stress marker NT-proBNP (+ 25%) as well as a pronounced amino acid imbalance were observed in obese rats with HFpEF. SDMA levels in O-ZSF1 were comparable to L-ZSF1, indicating stable kidney function. Anatomy and zonula occludens protein density in the intestinal epithelium remained unchanged, but both impedance measurements and increased levels of LPS indicated an impaired epithelial barrier function. FMO3 was decreased (− 20%) in the enlarged, but histologically normal livers of O-ZSF1. Alpha diversity, as indicated by the Shannon diversity index, was comparable at 8 weeks of age, but decreased by 13 weeks of age, when HFpEF manifests in O-ZSF1. Bray–Curtis dissimilarity (Beta-Diversity) was shown to be effective in differentiating L-ZSF1 from O-ZSF1 at 20 weeks of age. Members of the microbial families Lactobacillaceae, Ruminococcaceae, Erysipelotrichaceae and Lachnospiraceae were significantly differentially abundant in O-ZSF1 and L-ZSF1 rats.ConclusionsIn the ZSF1 HFpEF rat model, increased dietary intake is associated with alterations in gut microbiome composition and bacterial metabolites, an impaired intestinal barrier, and changes in pro-inflammatory and health-predictive metabolic profiles. HFpEF as well as its most common comorbidities obesity and metabolic syndrome and the alterations described here evolve in parallel and are likely to be interrelated and mutually reinforcing. Dietary adaption may have a positive impact on all entities.Graphical abstract

Empagliflozin Improves Diastolic Function in HFpEF by Restabilizing the Mitochondrial Respiratory Chain.

Clinical studies demonstrated beneficial effects of sodium-glucose-transporter 2 inhibitors on the risk of cardiovascular death in patients with heart failure with preserved ejection fraction (HFpEF). However, underlying processes for cardioprotection remain unclear. The present study focused on the impact of empagliflozin (Empa) on myocardial function in a rat model with established HFpEF and analyzed underlying molecular mechanisms. Obese ZSF1 (Zucker fatty and spontaneously hypertensive) rats were randomized to standard care (HFpEF, n=18) or Empa (HFpEF/Empa, n=18). ZSF1 lean rats (con, n=18) served as healthy controls. Echocardiography was performed at baseline and after 4 and 8 weeks, respectively. After 8 weeks of treatment, hemodynamics were measured invasively, mitochondrial function was assessed and myocardial tissue was collected for either molecular and histological analyses or transmission electron microscopy. In HFpEF Empa significantly improved diastolic function (E/é: con: 17.5±2.8; HFpEF: 24.4±4.6; P<0.001 versus con; HFpEF/Empa: 19.4±3.2; P<0.001 versus HFpEF). This was accompanied by improved hemodynamics and calcium handling and by reduced inflammation, hypertrophy, and fibrosis. Proteomic analysis demonstrated major changes in proteins involved in mitochondrial oxidative phosphorylation. Cardiac mitochondrial respiration was significantly impaired in HFpEF but restored by Empa (Vmax complex IV: con: 0.18±0.07 mmol O2/s/mg; HFpEF: 0.13±0.05 mmol O2/s/mg; P<0.041 versus con; HFpEF/Empa: 0.21±0.05 mmol O2/s/mg; P=0.012 versus HFpEF) without alterations of mitochondrial content. The expression of cardiolipin, an essential stability/functionality-mediating phospholipid of the respiratory chain, was significantly decreased in HFpEF but reverted by Empa (con: 15.9±1.7 nmol/mg protein; HFpEF: 12.5±1.8 nmol/mg protein; P=0.002 versus con; HFpEF/Empa: 14.5±1.8 nmol/mg protein; P=0.03 versus HFpEF). Transmission electron microscopy revealed a reduced size of mitochondria in HFpEF, which was restored by Empa. The study demonstrates beneficial effects of Empa on diastolic function, hemodynamics, inflammation, and cardiac remodeling in a rat model of HFpEF. These effects were mediated by improved mitochondrial respiratory capacity due to modulated cardiolipin and improved calcium handling.

Estrogen and the estrogen receptor in ZSF1 rats with heart failure with preserved ejection fraction

Abstract Funding Acknowledgements None. Background Heart failure with preserved ejection fraction (HFpEF) is more prevalent in women, so declining estrogen levels after menopause may be involved in genesis of the disease. Estrogen is cardio-protective, affecting blood pressure and vasodilatation and endothelial dysfunction is discussed as HFpEF pathomechanism. However, the potential connection between HFpEF and estrogen levels remains poorly understood. The well-established HFpEF ZSF1 rat model was used to analyze endothelial dysfunction, and a pronounced imbalance of the NO synthase substrates arginine and homoarginine was observed. Of note, estrogen levels in woman are directly and positively associated to homoarginine levels. Purpose We characterized serum estrogen levels and the expression of the estrogen receptor alpha (ERα) in the hearts of female ZSF1 HFpEF rats to investigate a possible contribution of estrogen signaling in HFpEF manifestation and to evaluate the suitability of this animal model in the context of estrogen and HFpEF. Methods In total, 12 obese ZSF1 rats with HFpEF (O-ZSF1) and 12 lean healthy ZSF1 rats (L-ZSF1) at 20 weeks of age and 5 animals of either phenotype aged 32 weeks were analyzed. Further, six O-ZSF1 rats were supplemented with homoarginine (hArg) when HFpEF was manifest and were observed up to 32 weeks of age. The manifestation of HFpEF was confirmed by echocardiography and determination of NT-proBNP by ELISA. Circulating estradiol (E2) in serum was determined using ELISA. Expression of the estrogen receptor alpha was determined by Western Blot analysis in the left ventricle (LV), septum and right ventricle (RV). Results Serum estradiol levels were similar in age-matched L-ZSF1 and O-ZSF1, but were ~2-fold increased at an age of 32 weeks compared to 20 weeks (p=0.002). The canonical ERα-66kDa was very weakly expressed in all tissues. ERα-45kDa expression in the LV was lower in O-ZSF1 at an age of 20 weeks (-46%, p=0.008) and at 32 weeks (-46%, p=0.002). ERα-45kDa expression in LV was 2-fold higher than in septum and 5-fold higher than in RV in rats aged 20 weeks (p&amp;lt;0.001). In RV, no differences in ERα-45kDa expression were observed. Supplementation of O-ZSF1 with hArg reestablished the ERα-45kDa expression in the LV (p=0.005 vs. O-ZSF1 without supplementation). Conclusion The manifestation and progression of HFpEF in ZSF1 rats is accompanied by a decrease of the ERα-45kDa expression in the LV, whereas the RV is not affected. Supplementation with homoarginine normalizes ERα-45kDa expression. The ERα-45kDa isoform was found to suppress the transcriptional activity of the classical ERα-66kDa isoform and to be an acute regulator of endothelial NO synthase in response to estrogen. These results suggest that altered ERα expression in the LV may represent a novel pathomechanism in HFpEF. In combination with decreasing estrogen levels in woman, adverse consequences for NO production in the LV seem likely.

Characterization of cardiac fibrosis in older lean vs. obese male ZSF1 rats

The ZSF1 rat is a novel strain produced by crossing a ZDF (Zucker diabetic fatty) female and SHHF (Spontaneously Hypertensive Heart Failure) male rat. Offspring of this hybrid have two phenotypes: healthy lean or obese prone to diabetes and heart failure. The metabolic syndrome and hypertension displayed by the obese ZSF1 rat mimics that seen in individuals who are predisposed to heart failure with preserved ejection fraction (HFpEF). Historically, HFpEF is a diffcult disease to accurately model with rodents and the ZSF1 rat is a promising model for the spontaneous progression to HFpEF. In recent studies, the oldest age at which ZSF1 rats were studied was 32 weeks and these obese rats displayed diastolic dysfunction, cardiac fibrosis, and hypertrophy. Since the prevalence of HFpEF increases with advanced age, the aim of the current study is to evaluate the extent of fibrosis in older ZSF1 rats (40-42 weeks) and determine whether this age group is more appropriate for the study of fibrotic cardiac remodeling related to HFpEF. We hypothesize that left ventricular (LV) fibrosis and collagen gene expression will be increased in obese compared to lean counterparts. Furthermore, we expect that the extent of fibrosis in this age group will exceed that reported in 32-week-old rats; thus, providing an improved model for the study of fibrotic heart disease. Lean and obese male ZSF1 rats (n=8/group) were aged to 40-42 weeks. Rats were anesthetized with a ketamine/xylazine cocktail and cardiac function was evaluated by invasive hemodynamics. The heart was removed, and the LV was trimmed and weighed. A mid-myocardial section from the LV was fixed and embedded in paraffn for histology. Remaining LV tissue was frozen for extraction of RNA and protein for biochemical analysis. LV collagen was assessed by Sirius red stain and collagen I ( Col1a1) and III ( Col3a1) gene expression were assessed by RTqPCR. With no significant difference in heart rate, obese ZSF1 rats had higher LV developed pressure compared to lean (191.1 vs. 122.0 mmHg, p&lt;0.05) and increased maximum and minimum dP/dT (p&lt;0.05). Obese ZSF1 rats exhibited cardiac hypertrophy as measured by LV weight normalized to tibial length (30.21 vs. 23.61 mg/mm, p&lt;0.05, n=7/group) and increased fibrosis (Sirius red) compared to lean counterparts. Furthermore, the fibrosis in obese ZSF1 rats was concentrated in the endocardial and perivascular regions, and qualitatively more than previously published in the 32-week-old ZSF1. Preliminarily, obese ZSF1 rats exhibit a 3.5-fold increase in Col1a1 (p=0.02) and a 19-fold increase in C ol3a1 (p=0.06) gene expression compared to lean (n=3/group). Interestingly, not only is this a larger increase than that reported in 32-week-old old ZSF1s, but the relative increase in Col3a1 vs. Col1a1 is larger. Taken together, these data suggest a progression of cardiac fibrosis beyond what has been reported in the 32-week-old ZSF1 rats. Use of aged ZSF1 rats may be valuable for the study of fibrogenic mediators in HFpEF for the elucidation of therapeutic agents to reduce or reverse cardiac fibrosis. Future studies will include female rats to evaluate sex-specific progression of fibrosis. North Carolina Biotechnology Center Flash Grant (JH) and ECU REDE startup program (AG). This is the full abstract presented at the American Physiology Summit 2024 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.

(065) Characterization of Cardiovascular Function, Metabolic Status and Erectile Function in Preclinical Model of Cardiometabolic Syndrome

Abstract Introduction Cardiometabolic syndrome is a widespread health issue and common cause of erectile dysfunction (ED). Cardiometabolic syndrome encompasses a group of indications including abdominal obesity, insulin-resistant glucose metabolism, dyslipidemia, and hypertension. To date, studies have looked at high fructose or high fat diets to induce cardiometabolic syndrome and ED in rodents, but these models do not closely model the clinical phenotype. Furthermore, the molecular basis of ED in cardiometabolic syndrome needs to be elucidated. Objective Our objective is to characterize the cardiovascular and erectile function, and metabolic status in the ZSF-1 obese rat. This hybrid rat is a cross between a Zucker diabetic fatty female and a spontaneously hypertensive heart failure male rat. We hypothesize that with age the ZSF-1 obese rats will demonstrate similar disease phenotype as seen clinically and develop ED. Methods We used male lean and obese ZSF-1 rats (n=8/group). We measured body weight weekly, and performed apomorphine erectile testing, MRI to assess body composition and glucose tolerance tests at 10, 20, 30 weeks. At 30 weeks, we assessed erectile function using ICP/MAP and vascular stiffness was measured via laser doppler pulse wave velocity (PWV). Additionally, acetylcholine (Ach)-mediated vasodilation, vasodilation to sodium nitroprusside (SNP), and contraction to phenylephrine (PE) was measured in thoracic and abdominal aortas. Aorta and penile segments were collected for histological analyses. Results On average, obese rats were 60% larger (230g) than controls, had greater percent body fat and had significantly impaired glucose tolerance at all time points (p&amp;lt;0.05). Over time, ZSF-1 obese rats developed ED as there was a significant decrease in apomorphine-induced erections by 30 weeks (p&amp;lt;0.05). ED was confirmed by significantly reduced ICP/MAP in ZSF-1 obese compared to lean rats (p&amp;lt;0.05). Obese rats were significantly more hypertensive compared to lean rats (MAP: 140.2 mmHg vs 99.6mmHg, p&amp;lt;0.05). Lean and obese rats had no difference in vascular stiffness indicated by PWV. In our tissue bath experiments, ACh-mediated vasodilation was significantly decreased in the thoracic aorta of obese rats (p&amp;lt;0.05) while the abdominal aorta was unchanged. SNP-mediated vasodilation was similar between groups in both thoracic and abdominal aortas. At high concentrations, PE-mediated vasoconstriction was significantly increased in the thoracic and decreased in the abdominal aortas of obese rats (p&amp;lt;0.05). Conclusions To our knowledge, this is the first report on the erectile function in the ZSF-1 rat model of cardiometabolic syndrome. The ZSF-1 obese rats had marked adiposity, hypertension, hyperglycemia and presented with aortic endothelial dysfunction and ED. We are currently assessing testosterone levels and penile fibrosis. We believe that the ZSF-1 rat is useful for studying ED as they gradually develop metabolic disease and ED over time like the human disease. Disclosure No.

An updated review of experimental rodent models of pulmonary hypertension and left heart disease.

Left heart disease (LHD) is the leading cause of pulmonary hypertension (PH). Its recent growth has not been matched by the design of therapeutic agents directly targeting the disease. Effective therapies approved for pulmonary arterial hypertension (PAH) have been shown to be inefficient in patients with PH-LHD. Hence, there is a need for an animal model that would closely mimic PH-LHD in preclinical experiments. The current study describes and compares a number of rodent models of left ventricular failure and their potential to induce PH. It also evaluates whether, and to what extent, common PH models could develop LV failure. Articles were identified in the Pubmed/Medline and Web of Science online electronic databases following the PRISMA Protocol between 1992 and 2022. Quality assessment was carried out using the SYRCLE risk-of-bias tool for animal studies. Publication bias across studies using Egger's regression test statistic, was performed together with sensitivity analysis. A wide spectrum of protocols-135 studies and 207 interventions, was examined, including systemic hypertensive models, pressure-overload-induced HF, model of ischemic heart failure, and metabolic approaches based on high fat diet or metabolic syndrome. The most pronounced alterations in PH-related parameters were demonstrated for the common PH models, but were also seen in animals with LV failure induced by ischemic conditions, pressure overload or metabolic conditions. Models based on aortic banding, transverse aortic constriction (TAC), or with myocardial infarction (MI) caused by coronary artery ligation, demonstrated more pronounced worsening in PH due to LV failure; however, they also demonstrated poor survival, especially the ischemic-HF model. Common PH models, excluding prolonged exposure to monocrotaline, do not promote LV hypertrophy. Prolonged exposure to a high-fat diet, or a two-hit model of an obese ZSF1 rat combined with SU5416-induced pulmonary endothelial impairment (a VEGF receptor antagonist) worsened PH and impaired diastolic dysfunction. Due to the limited number of protocols, further trials are needed to confirm the utility of such approaches for modeling PH in subjects with metabolic syndrome. This would provide a clearer insight into the complexity of LHD, PH and metabolic disorders in PH-LHD, and thus accelerate the development of new therapies in clinical trials.

Unveiling the Therapeutic Potential of SGLT2 inhibitors and Oral Ketone Ester in Heart Failure with Preserved Ejection Fraction: A Comprehensive Exploration of Cardiac Function, Structure, and Metabolism in a Rat Model

BackgroundThis study investigated the potential efficacy of SGLT2 inhibitors empagliflozin and oral ketone supplementation as treatments for heart failure with preserved ejection fraction (HFpEF), which is often associated with obesity and type 2 diabetes mellitus. Methods20-week-old obese ZSF-1 rats were given different treatments for 12 weeks, including vehicle, empagliflozin, ketone ester (KE), or a combination of empagliflozin and KE. Cardiac magnetic resonance imaging (CMR) and hyperpolarized carbon-13 (13C) labeled pyruvate magnetic resonance spectroscopy imaging were used to evaluate cardiac function and metabolism, while histological and molecular markers of cardiac remodeling were assessed in the left ventricle. ResultsThe untreated HFpEF rats exhibited obesity, diabetes, cardiac hypertrophy, atrial enlargement, lung congestion, and increased cardiomyocyte size and cardiac expression of atrial natriuretic peptide (ANP) and fibrosis marker col1a1 and TIMP1. The treatment groups exhibited decreased body weight, increased circulating ketone levels, and reduced blood glucose levels. All treatment regimens significantly attenuated ventricular hypertrophy, atrial enlargement, and lung congestion. Furthermore, all treatments significantly reduced cardiomyocyte size and the expression of ANP and fibrosis marker. The combination of empagliflozin and KE showed the greatest improvement in diastolic function and cardiac ATP levels. Interestingly, hyperpolarized [1-13C] pyruvate detected a decrease in lactate/pyruvate ratio in the hearts treated with empagliflozin. ConclusionObese ZSF1 rats exhibit features of clinical HFpEF with predominant background of obesity and diabetes. A combination of empagliflozin and KE has greater benefits in treating HFpEF. Targeting cardiometabolic dysregulation may be a promising approach for HFpEF treatment.

Empagliflozin improves diastolic function in obese ZSF1 rats by restoring the cardiac mitochondrial respiratory capacity

Abstract Background Clinical studies demonstrated beneficial effects of Sodium-Glucose-Transporter 2 inhibitors (SGLT2i) on the combined risk of cardiovascular death or heart failure hospitalization in patients with heart failure with preserved ejection fraction (HFpEF). However, underlying processes leading to cardioprotection remain unclear. Purpose The present study aimed to elucidate the impact of Empagliflozin on myocardial function and metabolism in a rat model (ZSF1 rat) with established HFpEF and to analyze underlying mechanisms. Methods 24 week old, obese ZSF1 rats were randomized either receiving standard care (obese) or Empagliflozin (Empa, 30 mg/kg/d p.o.). ZSF1 lean rats (lean) served as healthy controls. All animals underwent a baseline, intermediate and final echocardiography. After 8 weeks of treatment, hemodynamics were measured invasively, blood samples were taken, cardiac mitochondrial function was assessed and left ventricular tissue was collected for molecular and histological analyses or for transmission electron microscopy (TEM). Results Compared to the obese control Empa treated obese rats demonstrated a significantly improved diastolic function already at 4 weeks of treatment (E/é: lean: 17.9 ± 0.8; obese: 23.7 ± 0.7, p&amp;lt;0.001 vs. lean; Empa: 21.0 ± 1.1, p&amp;lt;0.05 vs. lean; p&amp;lt;0.05 vs. obese), which reached the level of healthy controls after 8 weeks of treatment (E/é: lean: 17.5 ± 0.7; obese: 24.4 ± 1.1, p&amp;lt;0.001 vs. lean; Empa: 19.4 ± 0.8, p&amp;lt;0.001 vs. obese). This was accompanied by reduced hypertrophy, fibrosis, left ventricular stiffness and inflammation as well as by improved hemodynamics. Except for a slightly improved myocardial ketone body utilization in Empa treated rats, there was no shift in metabolic substrate preference between groups. Cardiac mitochondrial respiratory function observed in untreated obese rats was significantly impaired compared to healthy controls but restored by Empa treatment (Vmax of complex II: lean: 0.15 ± 0.007 nmol O2/s/mg, obese: 0.12 ± 0.004 nmol O2/s/mg, p&amp;lt;0.01 vs. lean; Empa: 0.17 ± 0.006 nmol O2/s/mg, p&amp;lt;0.05 vs. lean, p&amp;lt;0.001 vs. obese). Moreover, mitochondrial respiratory capacity correlated with diastolic function, suggesting a pivotal role of restored respiratory function for improved diastolic relaxation. TEM revealed reduced size of cardiac mitochondria in obese rats, which was significantly restored by Empa. Proteomic analysis demonstrated major changes in proteins involved in mitochondrial oxidative phosphorylation and antioxidant metabolism. Conclusions The present study demonstrates for the first time beneficial effects of Empaglifozin on cardiac remodeling, diastolic function and hemodynamics in an obese rat model of HFpEF. These effects were accompanied by improved mitochondrial respiratory function, enhanced ketone body utilization and improved antioxidant capacity.

Abstract 13962: Exploring the Therapeutic Potential of SGLT2 Inhibitors and Oral Ketone Ester in HFpEF: A Comprehensive Exploration of Cardiac Function, Structure, and Metabolism in a Rat Model

Introduction: This study aimed to assess the potential efficacy of the SGLT2 inhibitor empagliflozin and oral ketone ester supplementation as treatments for heart failure with preserved ejection fraction (HFpEF), which is commonly associated with obesity and type 2 diabetes mellitus. Methods: Twenty-week-old obese ZSF-1 rats were initially fed a high-fat diet (HFD) to establish and maintain the phenotype. Subsequently, the rats were randomly assigned to different treatment groups. These groups consisted of a control group receiving only the high-fat diet (HFD), as well as groups receiving empagliflozin, ketone ester (DeltaG®, KE), or a combination of both in addition to the HFD. Cardiac magnetic resonance imaging (CMR) and hyperpolarized carbon-13 (13C) labeled pyruvate magnetic resonance spectroscopy imaging were used to evaluate cardiac function and metabolism. Histological and molecular markers of cardiac remodeling were assessed in the left ventricle. Results: Untreated HFpEF rats exhibited obesity, diabetes, cardiac hypertrophy, atrial enlargement, lung congestion, increased cardiomyocyte size and fibrosis, as well as elevated expression of atrial natriuretic peptide (ANP), col1a1 (fibrosis marker), and TIMP1. The treatment groups displayed decreased body weight, increased circulating ketone levels, and reduced blood glucose levels. All treatment regimens significantly mitigated ventricular hypertrophy, atrial enlargement, and lung congestion. Furthermore, they reduced cardiomyocyte size, ANP expression, fibrosis marker expression, and increased cardiac ATP levels. CMR revealed that the combination of empagliflozin and KE resulted in improvement in diastolic function. Hyperpolarized [1-13C] pyruvate imaging revealed a decrease in lactate/pyruvate ratio in empagliflozin-treated hearts, indicating improved oxidative metabolism. Conclusions: The findings of this study suggest that targeting cardiometabolic dysregulation holds promise as an effective approach for the treatment of HFpEF. While both empagliflozin or KE alone demonstrated improvements in cardiac energetics and remodeling, the combination of empagliflozin and KE yielded the most notable benefits in enhancing cardiac diastolic function.

Systemic Effects of Homoarginine Supplementation on Arginine Metabolizing Enzymes in Rats with Heart Failure with Preserved Ejection Fraction.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.

The sGC Activator Runcaciguat Has Kidney Protective Effects and Prevents a Decline of Kidney Function in ZSF1 Rats.

Chronic kidney disease (CKD) progression is associated with persisting oxidative stress, which impairs the NO-sGC-cGMP signaling cascade through the formation of oxidized and heme-free apo-sGC that cannot be activated by NO. Runcaciguat (BAY 1101042) is a novel, potent, and selective sGC activator that binds and activates oxidized and heme-free sGC and thereby restores NO-sGC-cGMP signaling under oxidative stress. Therefore, runcaciguat might represent a very effective treatment option for CKD/DKD. The potential kidney-protective effects of runcaciguat were investigated in ZSF1 rats as a model of CKD/DKD, characterized by hypertension, hyperglycemia, obesity, and insulin resistance. ZSF1 rats were treated daily orally for up to 12 weeks with runcaciguat (1, 3, 10 mg/kg/bid) or placebo. The study endpoints were proteinuria, kidney histopathology, plasma, urinary biomarkers of kidney damage, and gene expression profiling to gain information about relevant pathways affected by runcaciguat. Furthermore, oxidative stress was compared in the ZSF1 rat kidney with kidney samples from DKD patients. Within the duration of the 12-week treatment study, kidney function was significantly decreased in obese ZSF1 rats, indicated by a 20-fold increase in proteinuria, compared to lean ZSF1 rats. Runcaciguat dose-dependently and significantly attenuated the development of proteinuria in ZSF1 rats with reduced uPCR at the end of the study by -19%, -54%, and -70% at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo treatment. Additionally, average blood glucose levels measured as HbA1C, triglycerides, and cholesterol were increased by five times, twenty times, and four times, respectively, in obese ZSF1 compared to lean rats. In obese ZSF1 rats, runcaciguat reduced HbA1c levels by -8%, -34%, and -76%, triglycerides by -42%, -55%, and -71%, and cholesterol by -16%, -17%, and -34%, at 1, 3, and 10 mg/kg/bid, respectively, compared to placebo. Concomitantly, runcaciguat also reduced kidney weights, morphological kidney damage, and urinary and plasma biomarkers of kidney damage. Beneficial effects were accompanied by changes in gene expression that indicate reduced fibrosis and inflammation and suggest improved endothelial stabilization. In summary, the sGC activator runcaciguat significantly prevented a decline in kidney function in a DKD rat model that mimics common comorbidities and conditions of oxidative stress of CKD patients. Thus, runcaciguat represents a promising treatment option for CKD patients, which is in line with recent phase 2 clinical study data, where runcaciguat showed promising efficacy in CKD patients (NCT04507061).

Abstract P1024: Cardiomyocyte Mechanics In Animal Models Of Heart Failure With Preserved Ejection Fraction

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.

840-P: Dual Amylin and Calcitonin Receptor Agonist Treatment Improves Blood Pressure and Glucose Control in ZSF1 Rats

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for the treatment of type 2 diabetes and obesity due to their beneficial effects on both body weight, glucose control and insulin action. However, whether DACRAs affect hypertension, and diabetes-related cardiovascular complications remain unknown. In this study, the cardiovascular protective effect of the DACRA KBP-336 was evaluated in obese Zucker diabetic fatty-Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats. Obese ZSF1 rats were treated for 60 days with the KBP-336 (4.5 nmol/kg Q3D) and treatment efficacy on glucose control and blood pressure measured by tail cuff was evaluated. Furthermore, serum biomarkers of cardiovascular complications were evaluated. KBP-336 treatment significantly lowered fasting blood glucose levels compared to vehicle (P&amp;lt;0.001). This was also illustrated in the HbA1c levels at study end, where KBP-336 resulted in significantly (P&amp;lt;0.001) lower levels than vehicle. Importantly, KBP-336 significantly reduced both systolic (AUC levels reduced by 13.3%, P&amp;lt;0.001) and diastolic (AUC levels reduced by 17.0%, P&amp;lt;0.001) blood pressure compared to vehicle without affecting the heart rate. Notably, the beneficial effects on both glucose control and blood pressure were obtained independent of weight loss as KBP-336 only resulted in a small and transient weight reduction. Furthermore, serum levels of the marker of cardiovascular disease, rPRO-C6 (collagen type VI formation marker) were 6.4% lower in KBP-336 treated rats compared to vehicle, supporting improvement in cardiovascular parameters. In summary, we show for the first time that KBP-336 benefits blood pressure in a metabolic rat model suffering from hypertension. In addition, KBP-336 improved glucose control independent of weight loss. These data highlight KBP-336 as a promising candidate for treating obesity and related comorbidities including type 2 diabetes and hypertension. Disclosure S.A.Melander: None. A.T.Larsen: Employee; Nordic Bioscience A/S. K.Mohamed: Employee; Nordic Bioscience. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. Funding Danish Research Foundation; Innovation Fund Denmark; European Union’s Horizon 2020 Research and Innovation Program (814244)

#5907 RENAL INJURY IN RELATION TO OBESITY AND THE ADDITIVE EFFECT OF HYPERTENSION IN FEMALE AND MALE OBESE AND LEAN ZSF1 RATS

Abstract Background and Aims Chronic kidney disease co-exists with heart failure with preserved ejection fraction (HFpEF), which is characterized by the presence of obesity, hypertension, and diabetes mellitus type 2. Obese ZSF1 rats, a model of HFpEF, exhibit multiple of these comorbidities that can disturb cardiac function. Little attention has been paid to how these comorbidities affect renal disease in the ZSF1 rats. HFpEF is found predominantly in women in whom obesity and hypertension are particularly prevalent. Therefore, we aimed to characterize the renal phenotype in female and male, lean and obese, ZSF1 rats and investigated additional effects of worsened hypertension on disease severity. Method Systolic blood pressure and renal function were assessed biweekly in female and male, lean and obese, ZSF1 rats from 12 to 26 weeks of age by tail-cuff plethysmography, urine collection via metabolic cages and plasma collection, respectively. From 19 weeks of age, rats were implanted with either a deoxycorticosterone acetate pellet (DOCA) and fed high salt diet (6% NaCl) or with a placebo pellet and fed a normal salt diet. At 26 weeks of age, terminal GFR was assessed via inulin clearance under isoflurane. Renal sections were processed for histological analysis. Results Lean and obese placebo ZSF1 rats, both female and male, were mildly hypertensive (systolic blood pressure 140-150 mmHg). All obese rats showed HFpEF, evidenced by elevated E/e’ ratio. In female normoglycemic ZSF1 rats, obesity associates with mild proteinuria, decreased GFR and glomerular hypertrophy. The addition of DOCA-salt worsened hypertension in female obese ZSF1 rats, but not in their lean counterparts. DOCA-salt-worsened hypertension enhanced proteinuria and triggered glomerulosclerosis in female obese rats. Male obese placebo ZSF1 rats were hyperglycemic and showed proteinuria, glomerular hypertrophy and sclerosis, and tubulo-interstitial damage. DOCA-salt worsened hypertension in both male lean and obese rats and further aggravated proteinuria, tubulo-interstitial damage, glomerular hypertrophy and sclerosis in obese male rats. No effect of DOCA-salt was observed on GFR in both male and female ZSF1 rats. Conclusion Female obese ZSF1 rats developed mild renal disease and DOCA-salt-worsened hypertension deteriorated renal function and structure in normoglycemic female obese ZSF1 rats similar to hyperglycemic male obese ZSF1 rats.

Renal injury in relation to obesity and the additive effect of hypertension in female and male obese and lean ZSF1 rats.

Heart failure with preserved ejection fraction (HFpEF) is characterized by obesity, hypertension, diabetes mellitus and chronic kidney disease. Obese ZSF1 rats, a model of HFpEF, exhibit multiple such comorbidities that can disturb cardiac function. Little attention has been paid to how these comorbidities affect renal disease in ZSF1 rats. HFpEF is found predominantly in women in whom obesity and hypertension are particularly prevalent. Therefore, we characterized renal phenotype in female and male, lean and obese, ZSF1 rats and investigated additional effects of worsened hypertension on disease severity. Systolic blood pressure and renal function were assessed biweekly from 12 to 26 weeks. From 19 weeks, rats were implanted with either a deoxycorticosterone acetate pellet and fed high salt diet (DS) or a placebo pellet and fed normal salt diet. At 26 weeks, terminal GFR was assessed via inulin clearance under isoflurane. Renal sections were processed for histological analysis. Lean and obese ZSF1 rats, both female and male, were mildly hypertensive (systolic blood pressure 140-150 mmHg). All obese ZSF1 rats showed HFpEF. In female normoglycemic ZSF1 rats, obesity associates with mild proteinuria, decreased glomerular filtration rate and glomerular hypertrophy. DS-worsened hypertension enhanced proteinuria and triggered glomerulosclerosis. Male obese ZSF1 rats were hyperglycemic and showed proteinuria, glomerular hypertrophy and sclerosis, and tubulo-interstitial damage. DS-worsened hypertension aggravated this phenotype in male ZSF1 rats. In conclusion, female obese ZSF1 rats develop mild renal dysfunction and DS-worsened hypertension compromises renal function and structure in normoglycemic female obese ZSF1 rats as in hyperglycemic male obese ZSF1 rats.

Paradoxically reduced pulmonary vascular resistance and abnormal vascular remodeling in the obese spontaneously hypertensive heart failure ZSF1 rats

Pulmonary arterial dysfunction and remodeling often develop secondary to left heart disease and systemic hypertension. Excessive body weight is a well-established independent risk factor for the development of cardiovascular disease. However, data have emerged that for heart failure associated pulmonary complications obesity displays a lower long-term mortality. The impact of obesity on hypertension-related heart failure in affecting pulmonary vascular changes is incompletely understood. Here, we examined pulmonary vascular resistance and pulmonary artery function and remodeling in obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats. 20-week-old male obese ZSF1 rats display increased left ventricle (LV) hypertrophy and diastolic dysfunction, when compared to age-matched lean spontaneously hypertensive heart failure (SHHR). Pulmonary vascular resistance, pulmonary arterial function and vascular wall remodeling were examined in isolated and perfused, ventilated lung preparations and isolated small pulmonary artery pressure and wire myography, respectively. We found that the pulmonary vascular resistance (normalized to lung weight) was significantly reduced in obese ZSF1 rats compared to SHHR (ZSF1: 0.2±0.02 mmHg·mL -1 ·min·g-1 vs SHHR: 0.29±0.018 mmHg·mL -1 ·min·g-1, p=0.01). In isolated pulmonary arteries the acetylcholine-induced relaxation was maintained, whereas the nitric oxide donor, sodium nitroprusside-induced relaxation was reduced in obese ZSF1 rats (SNP -log(IC50) ZSF1: 4.185±0.166 vs SHHR: 4.96±0.136, p=0.005). The wall thickness and wall-to-lumen ratio of pulmonary arteries were higher in the obese ZSF1 rats when compared to SHHR. Collectively, we found that obesity is associated with a reduced pulmonary vascular resistance and abnormal pulmonary artery remodeling in rats with systemic hypertension and diastolic heart failure, which may have implications in the clinically observed obesity paradox. 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.

Exercise training impacts skeletal muscle remodelling induced by metabolic syndrome in ZSF1 rats through metabolism regulation

Metabolic syndrome (MetS), characterized by a set of conditions that include obesity, hypertension, and dyslipidemia, is associated with increased cardiovascular risk. Exercise training (EX) has been reported to improve MetS management, although the underlying metabolic adaptations that drive its benefits remain poorly understood. This work aims to characterize the molecular changes induced by EX in skeletal muscle in MetS, focusing on gastrocnemius metabolic remodelling. 1H NMR metabolomics and molecular assays were employed to assess the metabolic profile of skeletal muscle tissue from lean male ZSF1 rats (CTL), obese sedentary male ZSF1 rats (MetS-SED), and obese male ZF1 rats submitted to 4 weeks of treadmill EX (5 days/week, 60 min/day, 15 m/min) (MetS-EX). EX did not counteract the significant increase of body weight and circulating lipid profile, but had an anti-inflammatory effect and improved exercise capacity. The decreased gastrocnemius mass observed in MetS was paralleled with glycogen degradation into small glucose oligosaccharides, with the release of glucose-1-phosphate, and an increase in glucose-6-phosphate and glucose levels. Moreover, sedentary MetS animals' muscle exhibited lower AMPK expression levels and higher amino acids' metabolism such as glutamine and glutamate, compared to lean animals. In contrast, the EX group showed changes suggesting an increase in fatty acid oxidation and oxidative phosphorylation. Additionally, EX mitigated MetS-induced fiber atrophy and fibrosis in the gastrocnemius muscle. EX had a positive effect on gastrocnemius metabolism by enhancing oxidative metabolism and, consequently, reducing susceptibility to fatigue. These findings reinforce the importance of prescribing EX programs to patients with MetS.

Restoration of mitochondrial Ca2+ and redox homeostasis by enhancement of SK channels rescues Ca2+cycling in HFpEF.

Heart failure is the leading cause of death in the postindustrial world. While many effective therapies are available to treat heart failure with reduced ejection fraction (HFrEF), these approaches fail to improve outcomes in heart failure with preserved ejection fraction (HFpEF). Small conductance Ca2+-activated K+ (SK) channels have recently emerged as an attractive target to improve defective mitochondrial function and reduce emission of damaging reactive oxygen species (ROS) by the organelle. The goal of the present study was to test enhancement of SK as a new strategy to restore abnormal intracellular Ca2+ cycling and mitochondrial redox and Ca2+ homeostasis in ventricular myocytes from HFpEF rat hearts. To achieve this goal as a model of HFpEF we employed ZSF1 obese rats that demonstrate diastolic dysfunction, unchanged fractional shortening and ejection fraction. The confocal Ca2+ and ROS imaging experiments were carried out in ventricular myocytes isolated from lean and obese ZSF1 rats. Mitochondrial matrix [Ca2+] and ROS were assessed in ventricular myocytes expressing matrix-targeted biosensors mtRCamp1h and MLS-HyPer-7, respectively. To increase SK channel activity, we employed adenovirus-mediated overexpression of rat SK channel type 2 and pharmacological SK channel enhancer NS309 . Periodically paced ventricular myocytes isolated from obese ZSF1 rat hearts exhibited enhanced propensity to spontaneous sarcoplasmic reticulum (SR) Ca2+ release, increased mito-ROS production and a dramatic increase in mitochondrial [Ca2+] when exposed to beta-adrenergic agonist isoproterenol. Enhancement of SK channels in ZSF1 obese myocytes prevented mitochondrial Ca2+ overload, reduced mito-ROS emission to the control levels, and improved cytosolic Ca2+ cycling, reducing diastolic SR Ca2+ release. In summary, enhancement of SK channels shows high therapeutic potential in HFpEF by limiting mitochondrial Ca2+ uptake, thereby reducing oxidative stress restraining excessive SR Ca2+ release during diastole.