ZSF1 Rats Research Articles

Effects of homoarginine supplementation on heart and skeletal muscle of rats with heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is associated with left ventricular stiffness, impaired diastolic relaxation, and severe exercise intolerance. Decreased homoarginine (hArg) levels are an independent predictor of mortality in cardiovascular disease and correlate with impaired exercise performance. We recently reported alterations in arginine, hArg, and related amino acids in obese ZSF1 rats (O-ZSF1), with a HFpEF phenotype. Although low hArg is associated with diastolic dysfunction in humans, potential effects of hArg supplementation were not tested yet. At an age of 6weeks, 12 O-ZSF1 were randomized into two groups: (1) O-ZSF1 rats supplemented with hArg in their drinking water (sO-ZSF1) or (2) O-ZSF1 rats receiving no hArg supplementation (O-ZSF1). At an age of 32weeks, effects of primary prevention by hArg supplementation on echocardiographic, histological, and functional parameters of heart and skeletal muscle were determined. Lean ZSF1 rats (L-ZSF1) served as controls. hArg supplementation did not prevent impairment of diastolic relaxation (E/e': O-ZSF1 21±3 vs. sO-ZSF1 22±3, P=0.954, L-ZSF1 18±5) but resulted in more cardiac fibrosis (histological collagen staining: +57% in sO-ZSF1 vs. O-ZSF1, P=0.027) and increased collagen gene expression (Col1a1: +48% in sO-ZSF1 vs. O-ZSF1, P=0.026). In contrary, right ventricular function was preserved by hArg supplementation (TAPSE (mm): O-ZSF1 1.2±0.3 vs. sO-ZSF1 1.7±0.3, P=0.020, L-ZSF1 1.8±0.4). Musculus soleus maximal specific muscle force (N/cm2 ) in O-ZSF1 (30.4±0.8) and sO-ZSF1 (31.9±0.9) was comparable but significantly reduced compared with L-ZSF1 (36.4±0.7; both P<0.05). Maximal absolute muscle force (g) (O-ZSF1: 177.6±7.8, sO-ZSF1: 187.8±5.0, L-ZSF1: 181.5±7.9, all P>0.05) and cross-sectional fibre area (arbitrary units) (O-ZSF1: 1697±57, sO-ZSF1: 1965±121, L-ZSF1: 1691±104, all P>0.05) were not altered. Preservation of physiological hArg level in HFpEF may not be suited to prevent alterations in left ventricular and skeletal muscle function and structure. However, hArg supplementation may be beneficial for right ventricular function especially in pulmonary hypertension in HFpEF. We may speculate that clinically observed decreased hArg level are not the cause but the consequence of a yet unrecognized pathomechanism that underpins HFpEF.

Both short- and long-term treatment with the direct AMP kinase activator PXL770 improves cardiac function in ZSF-1 rats

AMP kinase activation is a target for the treatment of metabolic syndrome-related cardiac dysfunction PXL770 is a direct AMP kinase activator under clinical development for the treatment of metabolic diseases. Since potential cardiac effects of PXL770 have not been previously assessed we investigated whether PXL770 treatment exerts protective cardiac effects in rats with metabolic syndrome. ZSF-1 rats were treated with PXL770 (150 mg/kg orally BID for 90 days initiated at the age of 12 weeks, or for 7 days initiated at the age of 23 weeks) in order to determine left ventricular (LV) function and remodeling. After 90 days, untreated ZSF-1 rats showed signs of LV diastolic dysfunction, illustrated by the increases in LV end-diastolic pressure (LV EDP; 5.58 ± 0.57 and 8.28 ± 1.02 mmHg in lean and ZSF-1 rats, respectively) and in LV end-diastolic pressure volume-relation (LV EDPVR; 1.10 ± 0.23 and 5.44 ± 0.65 mmHg/RVU in lean and ZSF-1 rats, respectively) with preserved LV systolic function, illustrated by the slight decrease in LV fractional shortening (LV FS; 46 ± 1 and 42 ± 1% in lean and ZSF-1 rats, respectively; P < 0.05) and similar LV end-systolic pressure (LV ESP; 173 ± 10 and 197 ± 6 mmHg, respectively) or LV end-systolic pressure volume-relation (LV ESPVR; 32.7 ± 4.2 to 28.6 ± 1.4 mmHg/RVU, respectively), associated with a significant increase in LV tissue collagen density (2.62 ± 0.17 and 4.03 ± 0.13%, respectively) without an alteration in LV weight (1.27 ± 0.02 to 1.27 ± 0.02 g; respectively). Short- and long-term PXL770 improved LV diastolic function, illustrated by the reduced LV EDP in short- and long-term PXL treated ZSF-1 (5.98 ± 0.94 and 5.34 ± 0.93 mmHg, respectively) and reduced LV EDPVR (4.22 ± 0.30 and 3.35 ± 0.12 mmHg/RVU, respectively). Simultaneously LV FS was significantly increased (52 ± 1 and 50 ± 1%; respectively), as well as LV ESPVR (34.6 ± 1.4 and 33.0 ± 0.9 mmHg/RVU). The improvement of diastolic function was associated with a slight reduction in LV weight (1.19 ± 0.04 and 1.17 ± 0.02 g) and a significant reduction in collagen density long-term PXL770 (3.35 ± 0.12%; P < 0.05) but not short-term PXL770 (4.22 ± 0.30) ( Table 1 ). These results suggest that PXL770 exerts protective effects on cardiac function and structure in developing or established cardiomyopathy. Thus, direct AMP kinase activation appears promising for the treatment of cardiac abnormalities associated with metabolic diseases.

Role of S‐nitrosoglutathione reductase (GSNOR) Dysregulation and Nitrosative Stress in Heart Failure With Preserved Ejection Fraction

BackgroundHeart failure with preserved ejection fraction (HFpEF) is the most predominant form of heart failure in the United States. This multi‐system pathology results in a 5‐year mortality rate for which there are very few treatment options. The obese ZSF1 rat model has previously been shown to recapitulate much of the human phenotype including obesity, metabolic syndrome, diabetes, and hypertension. In this study, we investigated the complex and largely unknown role of abnormal nitric oxide signaling and nitrosative stress in the pathobiology of HFpEF.Materials and MethodsMale ZSF1 rats and normotensive controls of Wistar Kyoto (WKY) background strain (n=7 each group) were studied at 10, 14 and 18 weeks of age to monitor disease progression and alterations in nitric oxide signaling. Cardiac protein nitrosylation (RxNO) was measured by chemiluminescence. Analysis of circulating plasma nitrite bioavailability was performed using high performance liquid chromatography. GSNOR, one of the enzymes that degrade nitrosothiols, mRNA expression was measured and plotted as relative fold difference to WKY expression in this model of cardiometabolic HFpEF.ResultsZSF1 rats exhibited significantly increased cardiac protein nitrosylation as the HFpEF phenotype progressed from 10 to 18 weeks of age as compared to the WKY rat group. GSNOR mRNA expression was significantly decreased in the ZSF1 rat relative to the WKY at all time points. During the later 18‐week timepont, cardiac GSNOR activity was found to be decreased compared to that of the WKY. Circulating plasma nitrite, a measure of eNOS function, was decreased in the ZSF1 rat at 14 and 18 weeks compared to the WKY at subsequent aging timepoints. These data correlate to an increasing left ventricular end diastolic pressure (LVEDP) and decreased vascular relaxation as the ZSF1 animals age and develop more severe HFpEF disease pathology.ConclusionOur data suggests that nitric oxide signaling is dysregulated in the setting of HFpEF resulting in a significant increase in cardiac protein nitrosylation coupled with a reduction in circulating nitrite and NO signaling in the vasculature. Excessive cardiac nitrosative stress resulting in pathological myocardial signaling has been previously implicated as a driver of HFpEF. Reduced vascular nitrite bioavailability resulting in attenuated physiological NO signaling could account for the profound vascular dysfunction observed in HFpEF patients. Future studies are aimed at the further elucidation of the role of various NO storage pools in HFpEF.

Insulin is Not Driving Urinary Bladder Enlargement in Rodent Models of Diabetes

The urinary bladder is enlarged in all models of type 1 and several of type 2 diabetes (doi 10.1002/nau.23786). We have previously reported that such enlargement is correlated to insulin, but not glucose levels in fructose‐fed rats (doi 10.1096/fasebj.2020.34.s1.00542). Because this was surprising, we have now repeated that study and expanded our analysis to other models of diabetes and obesity.Five studies were performed in models of type 2 diabetes and obesity, one in fructose‐fed rats (n = 16), one each in ZSF1 rats at age 20 and 28 weeks (a cross between a female Zucker Diabetic Fatty and a male spontaneously hypertensive SHHF rat; n = 30 and 45, respectively), and two in mice on a high‐fat diet (n = 23 and 24, respectively). Bladder weight at study end and blood insulin concentrations were subjected to linear correlation analysis based on individual animal data, and data expressed a squared Pearsson correlation coefficient (r2).Animals in the diabetic/obese groups were hyperinsulinemic, whereas bladder enlargement was observed in both studies with ZSF1 rats, the old but not the new in fructose‐fed rats, and in neither study with mice on a high‐fat diet. Bladder weight and insulin levels were not correlated in the new study in fructose‐fed rats or any of the other four studies; the numerical correlation coefficient was negative in two of these studies. When data from all six studies were pooled, a very weak inverse correlation was observed (r2 0.0682, descriptive p‐value 0.0094; Figure 1).We conclude that, contrary to our previous observation, bladder weight is not positively correlated with insulin levels in rodent models of type 2 diabetes and obesity.

FABP1: A Potential Biomarker for Nephropathy in Type II Diabetes

Aims of the studyNephropathy is an important and frequent complication of diabetes mellitus. Diabetic nephropathy (DN) is also the leading cause of end stage renal disease in most parts of the world. Albuminuria is the hallmark of DN although recent observations suggest that non‐albuminuric nephropathy accounts for as much as 40% of all cases of DN. There is a need for additional and more accurate biomarkers for diagnosis and prognosis of DN. Fatty acid binding protein 1 (FABP1) is normally expressed in the liver but also in the kidney in human and animal DN models. However, it has not been detected in normal rat kidneys. We hypothesized that FABP1 is consistently elevated in the kidneys of experimental nephropathy of type II diabetes and could be a diagnostic biomarker and may have a potential pathogenic role.MethodsMale and female ZSF1 rats were used as an experimental model for nephropathy in type II diabetes and CD rats (non‐diabetic rats) were used as controls. Animals were studied from 8th week and sacrificed at 40 weeks. Kidneys were harvested and tissue homogenates were used for total RNA extraction for Next Gen sequencing and protein extraction for western blots. Serum and urine samples were also collected at the start and end of the study to quantitate the renal function and excretion of markers.ResultsThe ZSF1 rats spontaneously developed obesity, diabetes and hyperlipidemia as expected. Male rats developed more severe hyperglycemia, renal failure and proteinuria (data previously communicated) than females. Next Gen Sequencing of kidney tissue revealed amongst other findings a 1396‐fold increased expression of FABP1. Examination of kidney tissue protein homogenates for the expression of FABP1 showed increased expression especially in the male ZSF1 rats. Examination of urine samples for the presence of FABP1 by ELISA showed remarkably increased levels (12 to15‐fold in males and 6 to 7‐fold in females) increase compared to CD rats. (See figure 1)ConclusionsBoth male and female ZSF1 rats developed metabolic syndrome and nephropathy although the disease was much more severe in male rats. FABP1 was expressed more in male rat kidneys compared to female and control rat kidneys. Additionally, the urine excretion of FABP1 was increased in both male and female ZSF1 rats compared to control rats. These data suggest FABP1 may be a potential biomarker in DN. Additional studies are warranted to examine the mechanisms driving the robust renal FABP1 production/excretion and the possible pathogenic role and potential of FABP1 as a therapeutic target in DN.

Abstract 334: Gut Microbiome Derived Trimethyl-n-oxide Exacerbates Inflammation And Induces Cardiac Fibrosis In Human Cardiac Fibroblasts And In Rodent Model Of Cardiorenal Disease

Trimethlyamine N-oxide (TMAO), a gut-microbiome derived metabolite of dietary choline and other trimethylamine containing nutrients, has been consistently associated with poor prognosis with cardiovascular diseases (CVD) in recent years. Clinical studies have demonstrated that systemic circulating TMAO levels are independently associated with adverse CVD events such as MI, heart failure (HF), stroke, CKD and death. Previous studies have reported elevated TMAO both in mice models of atherosclerosis and in patients with atherosclerosis, and association between increased TMAO levels with adverse outcomes in HF patients, with significant correlation between increased TMAO levels and heart failure risk. The role and underlying mechanisms of TMAO in mediating disease pathophysiology remains elusive. Here, we use primary human cardiac fibroblasts and rodent models of heart failure with TMAO challenge to study its role in the heart and in cardiac fibroblasts. C57BL/6 mice were administered with TMAO (0.2%) in the drinking water for 2, 4 and 8 weeks. Heart tissues were collected at the end of the study to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. For heart failure studies, ZSF1 rats that underwent uninephrectomy surgery were fed high salt diet and were used as a model for heart failure with preserved ejection fracture (HFpEF), and were followed for 12 weeks. ZSF1 rats were also fed with high choline diet and cardiac function was measured using echocardiography. We demonstrated an elevation in blood TMAO levels, with a concomitant increase in circulating pro-inflammatory cytokine levels and upregulation in fibrosis markers in hearts from mice administered TMAO. In a ZSF1 rat model of HFpEF, we showed that increased TMAO levels correlated with deteriorating cardiac dysfunction. In human cardiac fibroblasts, stimulation with TMAO promoted upregulation of key fibrosis markers such as TGF-β1 in a dose-dependent manner. Inflammation, cardiac function, and cardiac fibrosis were exacerbated in mice administered with TMAO in their drinking water. Furthermore, in ZSF1 rat model for HFpEF, there was a significant association of high TMAO levels to worsening of cardiac and renal dysfunction.

The Role of CD44v6 in Vascular Rarefaction and Left Ventricular Diastolic Dysfunction in HFpEF

CD44 is differentially spliced to form specific CD44 variants, but little is known regarding their specific contribution to cardiovascular pathologies. Specifically, CD44 variant 6 (CD44v6) is involved in abnormal angiogenesis. The hypothesis tested was that CD44v6 leads to coronary microvascular rarefaction whereby it contributes to left ventricle (LV) diastolic function in heart failure with preserved ejection fraction (HFpEF, also known as diastolic heart failure). The obese ZSF1 rat model of human HFpEF was employed and using transthoracic echocardiography, it was found that 21‐week‐old male obese ZSF1 rats exhibited diastolic dysfunction, accompanied with increased myocardial expression of CD44v6. 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 remained unchanged, when compared to the lean ZSF1 controls. These changes in the microvasculature were accompanied by decreased angiogenesis in the coronary arteries in the obese ZSF1 rats when compared to the lean ZSF1 rats. From these results, it was concluded that upregulation of CD44v6 plays a role in the impaired angiogenesis underlying the development of LV diastolic dysfunction in HFpEF.

Long Non‐coding RNAs Mediate Heart Failure with Preserved Ejection Fraction Associated with Thyroid Dysfunction

IntroductionHeart failure with preserved ejection fraction (HEpEF) carries significantly high morbidity and mortality. HFpEF is associated with preserved contractile function and can present with diastolic dysfunction, cardiac fibrosis and hypothyroidism (low levels of thyroid hormones, THs). We previously demonstrated safe, cardioprotective effects with treatment of active form of THs in multiple rodent models of cardiovascular disorders. Long‐noncoding ribonucleic acids (lncRNAs; &gt;200 nucleotides long) account for large majority of the genome with significant transcriptional potential. However, their role in HFpEF associated with TH dysfunction is unclear. We hypothesized that lncRNAs act as mediators in development of hypothyroid HFpEF.MethodsHearts were isolated from obese ZSF1 rats that presented with HFpEF phenotype. Wild type and ZSF1 lean rats served as controls. Cardiac ultrasound and morphometric analyses were performed. RNA was isolated from left ventricular (LV) tissues and studied using Agilent LncRNA Microarray platform and analyzed using Feature Extraction and GeneSpring. Fibroblasts were isolated from wild type adult rat hearts and treated with TH receptor inhibitor. Real‐time quantitative polymerase chain reaction was used to study lncRNA expression.ResultsStudies revealed that total thyroxine TH levels significantly decreased in five‐six‐month old (early; p&lt;0.05) ZSF1 obese rats compared to ZSF1 lean controls (45% decrease) and wild type controls (42% decrease), and this remained low at 13 months (late; p&lt;0.05). Total tri‐iodothyronine TH levels also followed similar pattern in the early time‐point (37%‐42% decrease). ZSF1 obese rats showed increased normalized heart weight, LV weight and liver weight indicating cardiac hypertrophy and development of HF. In early obese rats, echocardiography showed preserved LV EF and significantly elevated E/E’ indicating diastolic dysfunction (p&lt;0.05). LncRNA Microarray analysis including unsupervised hierarchical clustering showed that several unique lncRNAs were significantly altered (p&lt;0.05; &gt;2‐fold change) in obese LVs compared to the lean or wild type control counterparts and select lncRNA expression was validated with qPCR. Furthermore, isolated wild type adult rat cardiac fibroblasts subjected to TH receptor inhibition (mimicking hypothyroidism) in vitro showed significant increase in lncRNA expression that was also increased in obese ZSF1 hearts presenting with hypothyroidism (p&lt;0.05).ConclusionsThese results indicate that lncRNAs may play important roles in in HFpEF pathogenesis associated with hypothyroidism. These studies will serve as significant step in identification of key lncRNAs in cardiac‐thyroid axis offering potentially valuable translational opportunities.

Targeting MuRF1 by small molecules in a HFpEF rat model improves myocardial diastolic function and skeletal muscle contractility.

BackgroundAbout half of heart failure (HF) patients, while having preserved left ventricular function, suffer from diastolic dysfunction (so‐called HFpEF). No specific therapeutics are available for HFpEF in contrast to HF where reduced ejection fractions (HFrEF) can be treated pharmacologically. Myocardial titin filament stiffening, endothelial dysfunction, and skeletal muscle (SKM) myopathy are suspected to contribute to HFpEF genesis. We previously described small molecules interfering with MuRF1 target recognition thereby attenuating SKM myopathy and dysfunction in HFrEF animal models. The aim of the present study was to test the efficacy of one small molecule (MyoMed‐205) in HFpEF and to describe molecular changes elicited by MyoMed‐205.MethodsTwenty‐week‐old female obese ZSF1 rats received the MuRF1 inhibitor MyoMed‐205 for 12 weeks; a comparison was made to age‐matched untreated ZSF1‐lean (healthy) and obese rats as controls. LV (left ventricle) function was assessed by echocardiography and by invasive haemodynamic measurements until week 32. At week 32, SKM and endothelial functions were measured and tissues collected for molecular analyses. Proteome‐wide analysis followed by WBs and RT‐PCR was applied to identify specific genes and affected molecular pathways. MuRF1 knockout mice (MuRF1‐KO) SKM tissues were included to validate MuRF1‐specificity.ResultsBy week 32, untreated obese rats had normal LV ejection fraction but augmented E/e′ ratios and increased end diastolic pressure and myocardial fibrosis, all typical features of HFpEF. Furthermore, SKM myopathy (both atrophy and force loss) and endothelial dysfunction were detected. In contrast, MyoMed‐205 treated rats had markedly improved diastolic function, less myocardial fibrosis, reduced SKM myopathy, and increased SKM function. SKM extracts from MyoMed‐205 treated rats had reduced MuRF1 content and lowered total muscle protein ubiquitination. In addition, proteomic profiling identified eight proteins to respond specifically to MyoMed‐205 treatment. Five out of these eight proteins are involved in mitochondrial metabolism, dynamics, or autophagy. Consistent with the mitochondria being a MyoMed‐205 target, the synthesis of mitochondrial respiratory chain complexes I + II was increased in treated rats. MuRF1‐KO SKM controls also had elevated mitochondrial complex I and II activities, also suggesting mitochondrial activity regulation by MuRF1.ConclusionsMyoMed‐205 improved myocardial diastolic function and prevented SKM atrophy/function in the ZSF1 animal model of HFpEF. Mechanistically, SKM benefited from an attenuated ubiquitin proteasome system and augmented synthesis/activity of proteins of the mitochondrial respiratory chain while the myocardium seemed to benefit from reduced titin modifications and fibrosis.

Urinary miRNA Profiles in Chronic Kidney Injury-Benefits of Extracellular Vesicle Enrichment and miRNAs as Potential Biomarkers for Renal Fibrosis, Glomerular Injury, and Endothelial Dysfunction.

Micro-RNAs (miRNAs) are regulators of gene expression and play an important role in physiological homeostasis and disease. In biofluids, miRNAs can be found in protein complexes or in extracellular vesicles (EVs). Altered urinary miRNAs are reported as potential biomarkers for chronic kidney disease (CKD). In this context, we compared established urinary protein biomarkers for kidney injury with urinary miRNA profiles in obese ZSF1 and hypertensive renin transgenic rats. Additionally, the benefit of urinary EV enrichment was investigated in vivo and the potential association of urinary miRNAs with renal fibrosis in vitro. Kidney damage in both rat models was confirmed by histopathology, proteinuria, and increased levels of urinary protein biomarkers. In total, 290 miRNAs were elevated in obese ZSF1 rats compared with lean controls, whereas 38 miRNAs were altered in obese ZSF1 rats during 14-26 weeks of age. These 38 miRNAs correlated better with disease progression than established urinary protein biomarkers. MiRNAs increased in obese ZSF1 rats were associated with renal inflammation, fibrosis, and glomerular injury. Eight miRNAs were also changed in urinary EVs of renin transgenic rats, including one which might play a role in endothelial dysfunction. EV enrichment increased the number and detection level of several miRNAs implicated in renal fibrosis in vitro and in vivo. Our results show the benefit of EV enrichment for miRNA detection and the potential of total urine and urinary EV-associated miRNAs as biomarkers of altered kidney physiology, renal fibrosis and glomerular injury, and disease progression in hypertension and obesity-induced CKD.

Microtubule detyrosination contributes to mechanical dysfunction in a rat model of HFpEF

Characterized by ventricular stiffening and associated with diabetes, obesity and hypertension, heart failure with preserved ejection fraction (HFpEF) is emerging as the prevalent form of heart failure. Stabilized microtubules stiffen failing myocardium and therefore present a potential therapeutic target for HFpEF, but microtubule network remodeling remains largely uncharacterized in HFpEF. Thus we aimed to quantify microtubule stabilization and its impact on myocyte contractility and mechanics in an established model of HFpEF, the ZSF1 rat.

Effects of soluble guanylate cyclase stimulator on renal function in ZSF-1 model of diabetic nephropathy.

BackgroundDiabetic nephropathy is associated with endothelial dysfunction and oxidative stress, in which the nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway is impaired. We hypothesize that sGC stimulator Compound 1 can enhance NO signaling, reduce proteinuria in a diabetic nephropathy preclinical model with diminished NO bioavailability and increased oxidized sGC. Therefore, we evaluated the effect of sGC stimulator Compound 1 on the renal effect in obese ZSF1 (ZSF1 OB) rats.Materials and methodsThe sGC stimulator Compound 1, the standard of care agent Enalapril, and a combination of Compound 1 and Enalapril were administered chronically to obese ZSF1 rats for 6 months. Mean arterial pressure, heart rate, creatinine clearance for glomerular filtration rate (eGFR), urinary protein excretion to creatinine ratio (UPCR), and urinary albumin excretion ratio (UACR) were determined during the study. The histopathology of glomerular and interstitial lesions was assessed at the completion of the study.ResultsWhile both Compound 1 and Enalapril significantly reduced blood pressure, the combination of Compound 1 and Enalapril normalized blood pressure levels. Compound 1 improved eGFR and reduced UPCR and UACR. A combination of Enalapril and Compound 1 resulted in a marked reduction in UPCR and UACR and improved GFR.ConclusionThe sGC stimulator Compound 1 as a monotherapy slowed renal disease progression, and a combination of the sGC stimulator with Enalapril provided greater renal protection in a rodent model of diabetic nephropathy.

A G-protein-biased S1P1 agonist, SAR247799, improved LVH and diastolic function in a rat model of metabolic syndrome.

AimHeart failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with no approved treatment. Left ventricular hypertrophy (LVH) and diastolic dysfunction represent the structural and functional components of HFpEF, respectively. Endothelial dysfunction is prevalent in HFpEF and predicts cardiovascular events. We investigated if SAR247799, a G-protein-biased sphingosine-1-phosphate receptor 1 (S1P1) agonist with endothelial-protective properties, could improve cardiac and renal functions in a rat model of metabolic syndrome LVH and diastolic function.Methods31- and 65-week-old obese ZSF1 (Ob-ZSF1) rats, representing adult and aged animals with LVH and diastolic dysfunction, were randomized to a chow diet containing 0.025% (w/w) of SAR247799, or control (CTRL) chow for 4 weeks. Age-matched lean ZSF1 (Le-ZSF1) rats were fed control chow. Echocardiography, telemetry, biochemical and histological analysis were performed to evaluate the effect of SAR247799.ResultsEchocardiography revealed that Ob-ZSF1 rats, in contrast to Le-ZSF1 rats, developed progressive diastolic dysfunction and cardiac hypertrophy with age. SAR247799 blunted the progression of diastolic dysfunction in adult and aged animals: in adult animals E/e’ was evaluated at 21.8 ± 1.4 for Ob-ZSF1-CTRL, 19.5 ± 1.2 for Ob-ZSF1-SAR247799 p<0.01, and 19.5 ± 2.3 for Le-ZSF1-CTRL (median ± IQR). In aged animals E/e’ was evaluated at 23.15 ± 4.45 for Ob-ZSF1-CTRL, 19.5 ± 5 for Ob-ZSF1-SAR247799 p<0.01, and 16.69 ± 1.7 for Le-ZSF1-CTRL, p<0.01 (median ± IQR). In aged animals, SAR247799 reduced cardiac hypertrophy (g/mm mean ± SEM of heart weight/tibia length 0.053 ± 0.001 for Ob-ZSF1-CTRL vs 0.046 ± 0.002 for Ob-ZSF1-SAR247799 p<0.01, Le-ZSF1-CTRL 0.035 ± 0.001) and myocardial perivascular collagen content (p<0.001), independently of any changes in microvascular density. In adult animals, SAR247799 improved endothelial function as assessed by the very low frequency bands of systolic blood pressure variability (mean ± SEM 67.8 ± 3.41 for Ob-ZSF1-CTRL 55.8 ± 4.27 or Ob-ZSF1-SAR247799, p<0.05 and 57.3 ± 1.82 Le-ZSF1-CTRL), independently of any modification of arterial blood pressure. In aged animals, SAR247799 reduced urinary protein/creatinine ratio, an index of glomerular injury, (10.3 ± 0.621 vs 8.17 ± 0.231 for Ob-ZSF1-CTRL vs Ob-ZSF1-SAR247799, respectively, p<0.05 and 0.294 ± 0.029 for Le-ZSF1-CTRL, mean ± SEM) and the fractional excretion of electrolytes. Circulating lymphocytes were not decreased by SAR247799, confirming lack of S1P1 desensitization.ConclusionsThese experimental findings suggest that S1P1 activation with SAR247799 may be considered as a new therapeutic approach for LVH and diastolic dysfunction, major components of HFpEF.

In silico identification of potential calcium dynamics and sarcomere targets for recovering left ventricular function in rat heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a complex disease associated with multiple co-morbidities, where impaired cardiac mechanics are often the end effect. At the cellular level, cardiac mechanics can be pharmacologically manipulated by altering calcium signalling and the sarcomere. However, the link between cellular level modulations and whole organ pump function is incompletely understood. Our goal is to develop and use a multi-scale computational cardiac mechanics model of the obese ZSF1 HFpEF rat to identify important biomechanical mechanisms that underpin impaired cardiac function and to predict how whole-heart mechanical function can be recovered through altering cellular calcium dynamics and/or cellular contraction. The rat heart was modelled using a 3D biventricular biomechanics model. Biomechanics were described by 16 parameters, corresponding to intracellular calcium transient, sarcomere dynamics, cardiac tissue and hemodynamics properties. The model simulated left ventricular (LV) pressure-volume loops that were described by 14 scalar features. We trained a Gaussian process emulator to map the 16 input parameters to each of the 14 outputs. A global sensitivity analysis was performed, and identified calcium dynamics and thin and thick filament kinetics as key determinants of the organ scale pump function. We employed Bayesian history matching to build a model of the ZSF1 rat heart. Next, we recovered the LV function, described by ejection fraction, peak pressure, maximum rate of pressure rise and isovolumetric relaxation time constant. We found that by manipulating calcium, thin and thick filament properties we can recover 34%, 28% and 24% of the LV function in the ZSF1 rat heart, respectively, and 39% if we manipulate all of them together. We demonstrated how a combination of biophysically based models and their derived emulators can be used to identify potential pharmacological targets. We predicted that cardiac function can be best recovered in ZSF1 rats by desensitising the myofilament and reducing the affinity to intracellular calcium concentration and overall prolonging the sarcomere staying in the active force generating state.

Abstract 12203: Sotatercept Analog RAP-011 is More Effective Than Sildenafil in Improving Pulmonary Hypertension and Reducing Right Ventricular Hypertrophy in a ZSF1 Rat Model of Heart Failure With Preserved Ejection Fraction

Introduction: Pulmonary hypertension (PH) due to heart failure with preserved ejection fraction (PH-HFpEF) is the most prevalent form of WHO Group 2 PH but lacks effective treatment options. We previously found that sequestration of activins and growth differentiation factors with the sotatercept analog RAP-011, an activin receptor type IIA-Fc fusion protein, reverses pathological remodeling of pulmonary arteries and the right ventricle (RV) in experimental models of pulmonary arterial hypertension (PAH, Group 1 PH). This strategy of selective multi-ligand neutralization offers a promising new approach for treating patients with PAH and is under active clinical evaluation (PULSAR, SPECTRA, and STELLAR). Here, we compared the cardiopulmonary effects of therapeutic treatment with RAP-011 to those of sildenafil in a rat model of PH-HFpEF. Methods: PH was induced in adult male obese ZSF1 rats by a single subcutaneous injection of SU5416 (100 mg/kg). To model the timeline of diagnosis and treatment for Group 2 PH patients, we initiated treatment with RAP-011 and sildenafil 8 weeks post SU5416 administration—at which point RV dysfunction is prominent—and continued treatment for 8 weeks. RV systolic pressure (RVSP), pulmonary artery acceleration time (PAAT), RV hypertrophy, and tricuspid annular plane systolic excursion (TAPSE) were evaluated by right heart catheterization and echocardiography following the 8-week treatment regimen. Results: Obese ZSF1 rats treated with SU5416 displayed significantly increased RVSP (P &lt; 0.0001) and decreased PAAT (P &lt; 0.0001). RAP-011 treatment fully reversed changes in RVSP (P &lt; 0.0001) and PAAT (P &lt; 0.0001), whereas sildenafil treatment improved RVSP by 19% (P &lt; 0.05) and PAAT by 36% (P &lt; 0.001). Treatment with RAP-011, but not sildenafil, normalized RV hypertrophy (P &lt; 0.0001) and increased TAPSE (P &lt; 0.0001). Conclusions: Therapeutic treatment with the sotatercept analog RAP-011 exerted beneficial cardiopulmonary effects in a ZSF1 rat model of PH-HFpEF that were significantly greater than those produced by sildenafil, fully normalizing PH and RV hypertrophy and improving RV function. These findings support evaluation of sotatercept in patients with PH-HFpEF (Group 2 PH).

Abstract 12968: mitoTEMPO Improves Expression of Pulmonary Artery Soluble Guanylate Cyclase β1 and Lowers Exercise Associated Elevations in Pulmonary Artery Pressures in a Rat Model of Combined Pre- and Post-Capillary Pulmonary Hypertension

Introduction: Exercise induced pulmonary hypertension (EIPH) portends worse prognosis compared to isolated resting PH. Exercise increases mitochondrial reactive oxygen species (mROS) production in peripheral blood mononuclear cells of patients with heart failure. Metabolic syndrome related mROS contribute to EIPH by decreasing the expression of the vasodilatory enzyme soluble guanylate cyclase (sGC) via downregulating an sGC transcription factor, namely nuclear factor Y alpha (NFYA; Satoh et al in press). Hypothesis: mROS scavenging with mitoTEMPO will restore sGC expression and ameliorate EIPH in a rat model of combined pre-and post-capillary pulmonary hypertension (CpcPH). Methods: Obese ZSF1 rats treated with sugen were used to model CpcPH. Half received mitoTEMPO (2mM) for 2 weeks. CpcPH rats treated with vehicle and lean ZSF1 rats served as controls (n=6/group). Hemodynamics were evaluated at rest and during treadmill exercise. Cardiac function was assessed using Doppler-echocardiography. We cultured pulmonary artery vascular smooth muscle cells (PAVSMCs) from lean rats in palmitate, glucose and insulin (PGI) containing media to simulate the metabolic syndrome milieu. Cells were treated with vehicle or mitoTEMPO. Expression of sGCβ1 and NFYA in pulmonary arteries of animals and in cultured cells was quantified by Western Blot. Data were analyzed with repeated measures two-way ANOVA. Results: MitoTEMPO significantly improved EIPH by reducing pulmonary vascular resistance (fig1A), significantly improved right ventricular function (fig1B) and significantly restored NFYA/sGCβ1 expression in pulmonary arteries (fig1C) and PAVSMCs of rats with CpcPH (fig1D). MitoTEMPO significantly blunted PGI-induced downregulation of NFYA and sGCβ1 in PAVSMCs from lean rats (fig1E). Conclusions: Mitochondria targeted antioxidants can improve EIPH in CpcPH by restoring NFYA/sGCβ1 expression, identifying a potential therapeutic target for CpcPH.

Histological and haemodynamic characterization of right ventricle in sedentary and trained rats with heart failure with preserved ejection fraction.

What is the central question of this study? Right ventricle (RV) dysfunction is highly prevalent in heart failure with preserved ejection fraction (HFpEF), nearly doubling the risk of death: what are the RV functional and structural changes in HFpEF and how does aerobic exercise impact them? What is the main finding and its importance? The HFpEF ZSF1 rat model presents RV structural and functional changes mimicking the human condition. Aerobic exercise prevented the decline in , lowered surrogate markers of RV overload (e.g., higher mean and maximum systolic pressure) and improved diastolic dysfunction (e.g., end-diastolic pressure and relaxation time constant). This emphasizes the importance of using exercise to manage HFpEF. Right ventricle (RV) dysfunction is highly prevalent in heart failure with preserved ejection fraction (HFpEF) and is a marker of poor prognosis. We assessed the obese ZSF1 rat model of HFpEF to ascertain if these animals also develop RV dysfunction and evaluated whether aerobic exercise could prevent this. Obese ZSF1 rats were randomly allocated to an aerobic exercise training group (n=7; treadmill running, 5days/week, 60min/day, 15m/min for 5weeks) or to a sedentary group (n=7). We used lean ZSF1 rats (n=7) as the control group. After 5weeks, rats were submitted to an exercise tolerance test and invasive haemodynamic evaluation, killed and samples from the RV collected for histological analysis. Obese sedentary ZSF1 rats showed lower , RV pressure overload (e.g., higher mean and maximum systolic pressure) and diastolic dysfunction (e.g., higher minimum and end-diastolic pressure and relaxation time constant), paralleled by RV cardiomyocyte hypertrophy. Except for cardiomyocyte hypertrophy, aerobic exercise prevented these functional changes. Our data support that this model of HFpEF shows functional and structural changes in the RV that resemble the human HFpEF phenotype, reinforcing its utility to understand this pathophysiology and to adress novel therapeutic targets to manage HFpEF. In addition, we showed that aerobic exercise is cardioprotective for the RV. A deeper knowledge of the mechanisms underlying the benefits of aerobic exercise could also lead to the identification of therapeutic targets to be further explored.

Arginine metabolism and nitric oxide turnover in the ZSF1 animal model for heart failure with preserved ejection fraction

Endothelial dysfunction and altered nitric oxide (NO) metabolism are considered causal factors in heart failure with preserved ejection fraction (HFpEF). NO synthase activity depends on the availability of arginine and its derivatives. Thus, we analyzed arginine, associated metabolites, arginine-metabolizing enzymes and NO turnover in 20-week-old female healthy lean (L-ZSF1) and obese ZSF1 rats (O-ZSF1) with HFpEF. Serum, urine and lysates of liver, kidney and heart were analyzed. There were significantly lower lysine (− 28%), arginine (− 31%), homoarginine (− 72%) and nitrite (− 32%) levels in serum of O-ZSF1 rats. Ornithine (+ 60%) and citrulline (+ 20%) levels were higher. Similar results were found in the heart. Expression of arginine consuming enzymes in liver and kidney was unchanged. Instead, we observed a 5.8-fold higher arginase 1 expression, presumably of granulocyte origin, in serum and > fourfold increased cardiac macrophage invasion in O-ZSF1. We conclude that inflammatory cells in blood and heart consume arginine and probably homoarginine via arginase 1 and inducible NO synthase and release ornithine and citrulline. In combination with evidence for decreased NO turnover in O-ZSF1 rats, we assume lower arginine bioavailability to endothelial NO synthase.

Both short-and long-term treatment with the direct AMP kinase activator PXL770 improves cardiac function in ZSF-1 rats

Abstract Introduction AMPK is a central energy sensor with cardioprotective effects, and its activation has emerged as a target for the treatment of metabolic syndrome-related cardiac dysfunction. PXL770 is the first direct AMPK activator under clinical development, currently investigated for the treatment of NASH. Since potential cardiac benefits of PXL770 have not been previously assessed we investigated whether short- and long-term PXL770 treatment exerts protective cardiac effects in rats with metabolic syndrome. Methods ZSF-1 rats were treated with PXL770 (150 mg/kg orally BID for 90 days initiated at the age of 12 weeks, or for 7 days initiated at the age of 23 weeks) in order to determine left ventricular (LV) function and remodeling. Results After 90 days, untreated ZSF-1 rats showed signs of LV diastolic dysfunction, illustrated by the increase in LV end-diastolic pressure (LV EDP; 5.58±0.57 and 8.28±1.02 mmHg in lean and ZSF-1 rats, respectively; p&amp;lt;0.05) and in LV end-diastolic pressure volume-relation (LV EDPVR; 1.10±0.23 and 5.44±0.65 mmHg/RVU in lean and ZSF-1 rats, respectively; p&amp;lt;0.05) with preserved LV systolic function, illustrated by the slight decrease in LV fractional shortening (LV FS; 46±1 and 42±1% in lean and ZSF-1 rats, respectively; p&amp;lt;0.05) and similar LV end-systolic pressure (LV ESP; 173±10 and 197±6 mmHg, respectively) or LV end-systolic pressure volume-relation (LV ESPVR; 32.7±4.2 to 28.6±1.4 mmHg/RVU, respectively). LV diastolic dysfunction was associated with a significant increase in LV tissue collagen density (2.62±0.17 and 4.03±0.13%, respectively) without an alteration in LV weight (1.27±0.02 to 1.22±0.03 g; respectively) Short- and long-term treatment with PXL770 improved LV diastolic function in treated ZSF-1, illustrated by the reduced LV EDP (5.34±0.93 and 5.98±0.94 mmHg, short- and long-term PXL770, respectively) and the reduced LV EDPVR (3.31±0.43 and 2.73±0.16 mmHg/RVU, respectively; p&amp;lt;0.05 vs untreated ZSF-1). Simultaneously LV FS was significantly increased (52±1 and 50±1%, respectively; p&amp;lt;0.05 vs untreated ZSF-1), as well as LV ESPVR (34.6±1.4 and 33.0±0.9 mmHg/RVU, respectively; p&amp;lt;0.05 vs untreated ZSF-1). The improvement of diastolic function was associated with a reduction in LV weight (1.19±0.04 and 1.17±0.02 g, respectively; long-term PXL770 p&amp;lt;0.0.5 vs untreated ZSF-1) and a significant reduction in collagen density after long-term PXL770 (3.35±0.12%; p&amp;lt;0.05) but not short-term PXL770 (4.22±0.30%). Conclusion These results suggest that PXL770 exerts protective effects on cardiac function and structure in developing or established cardiomyopathy. Thus, by directly activating AMPK, the PXL770 appears promising for the treatment of cardiac dysfunction associated with metabolic diseases. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Poxel SA

Contributions of Obesity and Hypertension to Progression of Cardiorenal Syndrome in Non-Diabetic Obese Female ZSF1 Rats

Contributions of Obesity and Hypertension to Progression of Cardiorenal Syndrome in Non-Diabetic Obese Female ZSF1 Rats