Introduction and Objective: The high prevalence of cardiovascular-kidney-metabolic (CKM) syndrome in the population requires novel therapeutics to reduce cardiovascular death, and to this aim, predictive animal models are needed. Here we evaluated the effects of the GLP-1 receptor agonist semaglutide (SEMA) on kidney dysfunction and left ventricle diastolic dysfunction in the Spontaneously Diabetic Torii (SDT) fatty rat, a new type 2 diabetic animal model of CKM syndrome. Methods: Male, 6-week-old SDT fatty rats were treated with vehicle or SEMA 15 nmol/kg s.c. Q3D for 12 weeks. At 6 weeks and 12 weeks of treatment, urine parameters and glomerular filtration rate (GFR) were measured, and cardiac function was investigated by echocardiography. Results: Compared to vehicle, SEMA significantly reduced body weight, %HbA1c and blood glucose levels. Vehicle-treated SDT fatty rats showed hyperfiltration with high GFR values at treatment start and at 6 weeks of treatment, followed by a marked GFR decline (-47%) at 12 weeks. After 6 weeks of treatment, SEMA significantly improved hyperfiltration with a 25 % reduction in GFR (p<0.05 vs. vehicle), while it limited the GFR decline (-27%) at 12 weeks. Additionally, SEMA significantly lowered albuminuria and proteinuria at both 6 and 12 weeks of treatment. Echocardiography performed at 6 and 12 weeks of treatment demonstrated that SEMA did not alter left ventricle ejection fraction but improved diastolic dysfunction of SDT fatty rats. SEMA reduced left ventricle dilation with lower left ventricle internal diameter and significantly reduced end-diastolic volume. A positive effect was observed on cardiac hypertrophy with significant reduction in left ventricle wall thickness and heart weight. Additionally, SEMA significantly lowered the E/E’ ratio, increased E’/A’ ratio, and reduced isovolumic relaxation time. Conclusion: SEMA demonstrates cardiorenal benefits in the SDT fatty rat. This model will be useful to evaluate novel therapies targeting CKM syndrome versus SEMA as standard of care. Disclosure F. Briand: Employee; Current; PHYSIOGENEX. Stock/Shareholder; Current; PHYSIOGENEX. G. Bourdier: None. E. Grasset: None. M. Shinohara: None. R. Assaly: Employee; Current; Cardiomedex. C. Dubroca: None. T. Sulpice: None.

Heart failure with preserved ejection fraction (HFpEF) is complex clinical phenotype associated with multiple comorbidities; type2 diabetes, obesity, metabolic syndrome, Metabolic Dysfunction associated Steatohepatitis (MASH). No animal model captures all HFpEF variables. Thus, it is important to develop different models each replicating some features to optimize bench-to-bed translation. We developed 3 models each representing specific phenogroup of patients. Empagliflozin (EMPA), clinically proven SGLT-2i was used for validation. Methods: MASH related HFpEF was induced in male Golden Syrian hamsters fed free choice diet (free access to control chow/water or high fat/high cholesterol diet+10% fructose water). HFpEF associated with metabolic disorders was induced in C57Bl6N mice fed high-fat diet (60% kcal from fat) and water with L-NAME (0.5 g/L) for 5 weeks. Spontaneously Diabetic Torii (SDT) fatty rat was characterized as a model of diabetes/obesity-related HFpEF. EMPA or vehicle was administered orally at 10mg/kg for at least 3 weeks before evaluation of cardiac function by echocardiography and invasive hemodynamics. Results: While the systolic function was preserved in all models, different stages of diastolic dysfunction were observed. In HFD+L-NAME model, mild diastolic dysfunction characterized by pseudo-normal profiles was observed (E/A=1.2±0.04 E/E’= 16.2±0.9 in HFD/L-NAME vs E/A=1.4 ± 0.02 E/E’= 14.4±0.4 in control). Restrictive pattern (i.e. grade 3 diastolic dysfunction) was predominant in MASH hamster model (E/A= 2.16±0.09 E/E’=20.50±1.05 vs E/A 1.44±0.02 E/E’=14.23±0.9 in control) and in 12-week old SDT ( E/A=1.5±0.1 E/E’=19.5± 0.9 vs E/A= 1.2±0.04 E/E’=12.1±0.1 in control). EMPA alleviated partially the diastolic dysfunction and had positive outcome on cardiac remodeling in terms of left ventricle hypertrophy and enlargement (p<0.05). Conclusion: These results suggest that each of these models help features a specific phenogroup of HFpEF patients. One model does not fil all, thus it is probable that new therapies have to be tested in several models, each replicating some features of the disease to increase the success rate in clinical trials.

Obesity is associated with several comorbidities including Heart Failure with preserved Ejection Fraction (HFpEF), Metabolic dysfunction-Associated SteatoHepatitis (MASH), also termed MetALD when MASH patients have increased alcohol intake. The worldwide epidemic of obesity creates a constant need for new therapies and animal models to test their effectiveness. In the present study, we evaluated the effects of the GLP-1 receptor agonist semaglutide and the pan-PPAR agonist lanifibranor in the free choice diet induced obese MASH hamster, a preclinical model with human-like lipoprotein metabolism, MASH and HFpEF. The same model was exposed to ethanol to evaluate the effects of both semaglutide and lanifibranor on chronic alcohol intake. To set up a model of MetALD, obese MASH hamsters were also challenged to alcohol binge drinking and the preventative effects of lanifibranor were evaluated. Compared with vehicle, semaglutide transiently reduced food intake and significantly reduced fructose and alcohol consumption. This effect was associated with significant body weight loss, lower HOMA-IR index of insulin resistance, improved dyslipidemia and HFpEF, but semaglutide only reduced hepatic fat content. Lanifibranor showed the same cardiometabolic benefits but had superior effects in the liver, with significant improvement in MASH and MetALD. As observed in humans, lanifibranor and semaglutide showed multiple metabolic benefits in free-choice diet induced obese hamster models of MASH and MetALD. These hamster models demonstrated good translability regarding the effects observed in clinical trials and will be helpful to evaluate novel therapies targeting obesity and associated comorbidities, including MetALD.

Intro: The 2-hit HFD/L-NAME model is extensively used to develop new therapies for patients suffering from Heart failure with Preserved Ejection Fraction (HFpEF). It captures the features of one of the leading phenogroups of patients; the cardiometabolic and mild hypertension associated HFpEF. Hypothesis: We assessed the effect of the duration of HFD+L-NAME on the progression of diastolic dysfunction. Then, we chose the optimal timing to enable time-effective proof-of-concept studies for drugs targeting this phenotype. Cardiac, metabolic and renal aspects were assessed in the chosen optimal model, in addition to the mitochondria state in cardiac tissue. Empagliflozin (EMPA), the clinical benchmark, was used for pharmacological validation. Methods: C57BL6N mice were fed HFD (60% Kcal from fat)+water with L-NAME (0.5g/l) up to 20 weeks. Control mice (Ctrl) received normal chow+water. Cardiac function was evaluated at several timepoints to set the optimal timing. In the 2nd set of experiments, mice were randomized after 10 weeks of diet to receive vehicle or EMPA (10mg/kg) QD for 6 weeks followed by treadmill exercise tolerance test, echocardiography, invasive hemodynamic and quantification of metabolic and renal biomarkers. Cardiac tissue was used to assess cardiac fibrosis using sirius red and mitochondrial functional defects in this HFpEF model using Oroboros® Results: HFD/L-NAME mice showed preserved systolic function and progressive diastolic dysfunction reaching a moderate phenotype characterized by inverted or pseudonormal profiles (E’/A’=0.8±0.02 E/E’=33.2±1.1 in HFD/L-NAME vs E’/A’=1.5±0.02 E/E’=26.9±2.1 in Ctrl). These mice showed left ventricular concentric hypertrophy, increased arterial and left ventricular end-diastolic pressure, cardiac fibrosis (p<0.05 vs. Ctrl, respectively), and altered exercise capacity (p<0.01 for running distance and time vs. Ctrl). A decrease in complex I activity was also observed. Insulin resistant state, increased blood glucose, cholesterol, ALT and FGF-21 were noted but not urea/creatinine ratio. EMPA alleviated cardiac dysfunction (E’/A’=1.3±0.03 E/E’=28.0±0.9) and had positive outcome on cardiac remodeling and metabolic biomarkers. Conclusion: These data demonstrated that HFD/L-NAME captures several cardiac and metabolic aspects of patients suffering from moderate HFpEF associated with obesity/metabolic syndrome and mild hypertension. This model showed also cardiac fibrosis and impairment of metabolic flexibility.

Intro: Drug Development for Heart failure with Preserved Ejection Fraction (HFpEF) is a major challenge facing cardiovascular research due to its complex pathophysiology and existence of comorbidities, leading to recognize distinct HFpEF phenogroups. Animal model development should consider this heterogeneity and each model capturing features of specific phenogroups. Hypothesis: We established a 2-hit model consistent with one of the leading phenogroups, the cardiometabolic and mild hypertension associated HFpEF, by combining a high fat diet to trigger obesity/metabolic syndrome and L-NAME to induce mild hypertension (HFD/L-NAME). We evaluated the presence of HFpEF hallmark and corroborated our data with literature. Empagliflozin (EMPA), the clinical benchmark, was used to confirm the relevance of the model. Methods: To induce obesity/metabolic syndrome, mild and HFpEF, C57BL6N mice were fed HFD (60% Kcal from fat) and water with L-NAME (0.5g/l) for 8 weeks. Control mice (Ctrl) were fed normal chow and water. At 5weeks, mice were randomized based on E/A ratio and ejection fraction and were treated QD for 3weeks with vehicle or EMPA (10mg/kg). Then, treadmill exercise tolerance test was performed, cardiac geometry, systolic and diastolic function were evaluated by echocardiography and heart and lungs were harvested. Longitudinal blood pressure was evaluated by tail cuff. Results: Compared with Ctrl, HFD/L-NAME mice showed cardiac remodeling, preserved systolic function and moderate diastolic dysfunction characterized by inverted or pseudonormal profiles and higher filling pressure (E/A=1.2±0.04 E’/A’=1.1±0.02 E/E’=16.4±0.6 in HFD/L-NAME vs E/A=1.4 ± 0.02 E’/A’=1.3±0.01 E/E’=14.4±0.4 in Ctrl). HFD/L-NAME mice showed altered exercise capacity (p<0.05 for running distance and time vs. Ctrl). Cardiac hypertrophy was confirmed by increased heart weight (p<0.01 vs. Ctrl). Consistent with elevated filling pressure, lung weight was increased in HFD/L-NAME mice (p<0.01 vs. Ctrl), indicative of pulmonary congestion. Mean blood pressure was consistently increased in HFD/L-NAME mice (p<0.05 vs. Ctrl). EMPA had positive outcome on cardiac remodeling in terms of left ventricle hypertrophy and enlargement (p<0.05 vs. Ctrl) and alleviated diastolic dysfunction. Conclusion: The 8-week HFD diet/L-NAME captures several aspects of patients suffering from moderate HFpEF associated to obesity/metabolic syndrome and mild hypertension allowing cost and time effective POC studies.

Introduction: Sodium-glucose cotransporter 2 inhibitor empagliflozin (EMPA) has shown significant benefits for patients with diabetic nephropathy and heart failure with preserved ejection fraction (HFpEF), but the effects on nonalcoholic steatohepatitis (NASH) and liver fibrosis remain unclear. To further investigate the benefits of EMPA on these comorbidities, we established a novel diet-induced obese diabetic hamster model with human-like NASH and lipoprotein cholesterol metabolism, that also develops nephropathy and HFpEF. Methods: Obesity, NASH, and HFpEF were induced with a 20-week free choice diet, which presents hamsters with a choice between control chow or high fat/cholesterol diet, and normal water or 10% fructose water. After 20 weeks of diet, obese hamsters were intraperitoneally injected with streptozotocin (STZ) to induce diabetes and were then treated orally once daily for 5 weeks with vehicle or EMPA 30mg/kg. Results: Compared with vehicle, EMPA reduced hyperglycemia (vehicle: 373 +/- 43 mg/dL; EMPA: 182 +/- 30 mg/dL, p<0.01), increased pancreatic insulin content (+146%, p<0.05), significantly reduced plasma triglycerides and moderately raised LDL-cholesterol levels, as expected. However, EMPA did not improve NASH (including hepatocyte ballooning score) and liver fibrosis. EMPA reduced glomerular filtration rate and proteinuria (p=0.06 for both vs. vehicle), and significantly reduced urine albumin-to-creatinine ratio (-35%, p<0.01). In addition, EMPA showed significant improvement in HFpEF with reduced E/A and E’/E ratios with no alteration in ejection fraction, as measured by echocardiography. Conclusion: EMPA improves kidney parameters and HFpEF despite limited effects on NASH in obese diabetic hamsters. This preclinical model with multiple comorbidities will be useful to evaluate the combination of EMPA or other antidiabetic therapies with drugs targeting NASH. Disclosure F. Briand: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. C. Dubroca: Employee; CARDIOMEDEX. T. Sulpice: Employee; PHYSIOGENEX. Stock/Shareholder; PHYSIOGENEX. Employee; CARDIOMEDEX. Stock/Shareholder; CARDIOMEDEX.

Background: Sodium-glucose cotransporter 2 inhibitor empagliflozin improves heart failure with preserved ejection fraction (HFpEF), but the effects on non-alcoholic steatohepatitis (NASH) and liver fibrosis remain unclear.

Intro: Heart failure with preserved ejection fraction (HFpEF) is frequently associated with multiple comorbidities including nonalcoholic steatohepatitis (NASH) and obesity. Drug development for HFpEF is partly hampered by the limitations of preclinical models, such as diet-induced obese mouse and rat. Hypothesis: To overcome these limitations, we recently established a nutritional hamster model developing obesity, with human features for dyslipidemia NASH, and HFpEF. To confirm the relevance of our model, we tested whether the clinical benchmarks empagliflozin (EMPA, a SGLT2 inhibitor), vardenafil (VAR, a PDE5 inhibitor) and semaglutide (SEMA, a GLP-1r agonist) would improve HFpEF. Methods: To induce obesity, NASH and HFpEF, hamsters were fed with a free choice diet, which presents hamsters with a choice between control chow or high fat/cholesterol diet, and normal or 10% fructose water for 20 weeks. Obese hamsters were then randomized based on echocardiography parameters, i.e., E/A ratio and ejection fraction (EF) and were treated QD for 5 weeks with vehicle, EMPA (30mg/kg), VAR (10mg/kg) or SEMA (0.06mg/kg). Chow fed hamsters were used as control. Results: Compared with control, echocardiography in obese hamsters showed cardiac remodeling, preserved systolic function and normal EF. Free choice fed hamsters had impaired diastolic function with higher E/A (2.0±0.1 vs 1.41±0.03 in control, p<0.001), while the inverted E’/A’ (0.8± 0.1 vs 1.3±0.04, p<0.001), a sign of relaxation default, suggested restrictive filling pattern. Increased E/E’ suggested higher filling pressure (18-20 vs 12-14 in control, p<0.01). Plasma NT-proBNP levels were also increased (p<0.05 vs. control). Compared with vehicle, all drug treatments alleviated diastolic dysfunction: E/A returned to control values (E/A=1.4-1.5) with VAR (p<0.001 vs. vehicle), EMPA (p<0.01) and SEMA (p<0.05). As well, E’/A’ was normalized (p<0.01 for all), and E/E’ was also reduced (p<0.05 for all). EMPA and VAR reduced NT-proBNP levels (p<0.05 vs. vehicle). Conclusion: Our data indicate that, VAR, EMPA and SEMA improved diastolic function in the obese NASH hamster. This novel model provides a unique alternative to mice and rats limitations for evaluating drugs targeting HFpEF.

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.

Objective: Myogenic tone, which has a major role in the regulation of local blood flow, refers to the ability of vascular smooth muscle to adapt its contractility to changes in transmural pressure. Although Rho-kinase is involved in myogenic tone, the pathway involved remains unclear, especially concerning translocation to the plasma membrane and activation of RhoA. As caveolae have a key role in the signal transduction of membrane-bound proteins, we tested the hypothesis that RhoA might be activated by pressure and that its activation might involve caveolin-1, which has been shown to be involved in vascular functions. Methods: Myogenic tone was studied in isolated rat mesenteric resistance arteries (118 +/- 15 mu m internal diameter with a pressure of 75 mmHg) submitted to pressure steps (25, 75, and 150 mmHg). Pharmacological blockade of caveolae or RhoA-Rho-kinase pathway was assessed by confocal microscopy in pressurized arteries to analyze protein co-localization and by co-immunoprecipitation in order to confirm protein interactions. Caveolin-1-deficient mice were used to confirm the role of the protein in myogenic tone. Results: Pressure-induced myogenic tone was significantly reduced by RhoA inactivation with TAT-C3 (90.5% inhibition at 150 mmHg) and by the Rho-kinase inhibitor Y27632 (91.8% inhibition at 150 mmHg). In arteries pressurized at 150 mmHg, RhoA was localized to the plasma membrane (localization by confocal microscopy and increased quantity of RhoA in the membrane fraction after protein extraction). Thus, translocation of RhoA to the plasma membrane was associated with pressure-induced tone. In addition, caveolae disruption with methyl-beta-cyclodextrin reduced myogenic tone by 66% at 150 mmHg. Further, myogenic tone was significantly reduced to 24% of control in caveolin-1-deficient mice (active tone was 32.3 +/- 2.8 mu m and 9.1 +/- 3.7 mu m in +/+ and -/- mice, respectively, n=5 per group), suggesting a key role of caveolin-1 in myogenic tone. Finally, RhoA and caveolin-1 co-immunoprecipitation and co-localization significantly increased when myogenic tone developed at 150 mmHg (co-localization showed 26 +/- 13% merging at 25 mmHg versus 97 +/- 21% at 150 mmHg, n=5). Co-immunoprecipitation was prevented by TAT-C3 and by methyl beta-cyclodextrin. Conclusion: RhoA activation is critical for the development of myogenic tone in resistance arteries. This activation induced translocation of RhoA to the plasma membrane within caveolae, where the interaction of RhoA with caveolin-1 leads selectively to the activation of a Rho-kinase-dependent force development. (c) 2006 European Society of Cardiology. Published by Elsevier B.V.

RATIONALE In addition to its typical rod-shape, the mammalian adult cardiomyocyte (CM) harbors a unique lateral membrane surface architecture with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) the role of which is still unknown. OBJECTIVE To investigate the development and functional role of CM crests during the postnatal period. METHODS AND RESULTS Electron/confocal microscopy and western-blot of left ventricular tissues from rat hearts indicated a late CM surface crest maturation, between postnatal day 20 (P20) and P60, as shown by substantial SSM swelling and increased claudin-5 cell surface expression. The P20-P60 postnatal stage also correlates with an ultimate maturation of the T-Tubules and the intercalated disk. At the cellular level, we identified an atypical CM hypertrophy characterized by an increase in long- and short-axes without myofibril addition and with sarcomere lateral stretching, indicative of lateral stretch-based CM hypertrophy. We confirmed the P20-P60 hypertrophy at the organ level by echocardiography but also demonstrated a transcriptomic program after P20 targeting all the cardiac cell populations. At the functional level, using Doppler echocardiography, we found that the P20-P60 period is specifically dedicated to the improvement of relaxation. Mechanistically, using CM-specific knock-out mice, we identified ephrin-B1 as a determinant of CM crest maturation after P20 controlling lateral CM stretch-hypertrophy and relaxation. Interestingly, while young adult Efnb1 CMspe−/− mice essentially show a relaxation impairment with exercise intolerance, they progressively switch toward heart failure with 100% KO mice dying after 13 months. CONCLUSIONS This study highlights a new late P20-P60 postnatal developmental stage of the heart in rodents during which the CM surface crests mature through an ephrin-B1-dependant mechanism and regulate the diastolic function. Moreover, we demonstrate for the first time that the CM crest architecture is cardioprotective.

Abstract BACKGROUND AND AIMS Beyond the glucose control, anti-diabetic drugs need to demonstrate benefits on metabolic comorbidities. An animal model that recapitulates obesity and type 2 diabetes (T2D) comorbidities [i.e. non-alcoholic steato-hepatitis (NASH), nephropathy and heart failure with preserved ejection fraction (HFpEF)] is still needed for preclinical drug development. To overcome this limitation, we evaluated the effects of a high fat/cholesterol/fructose (HFCF) diet in the Lund MetS rat, a congenic BBDR.cg-lepr.cp model generated by introgression of the Koletsky leptin receptor mutation into the BioBreeding Diabetes Resistant (BBDR) rat. METHOD 17-week-old, male, lean control (ctrl) or Lund MetS obese T2D rats were fed a control chow (CC) diet or a HFCF diet, respectively for 8 weeks. Blood biochemistry was measured at 0, 4 and 8 weeks of diet. Kidney, heart and liver parameters were assessed at the end of the 8-week diet period. RESULTS Compared with ctrl, Lund MetS rats were obese (56% higher body weight) and diabetic with significantly higher %HbA1c (up to + 3%) and blood glucose levels (up to 3-fold higher) during the 8-week HFCF diet period. Significantly higher plasma insulin (up to 11-fold), total cholesterol (up to 5-fold), triglycerides (up to 8-fold), transaminases (up to 10-fold higher) levels were also observed in Lund MetS rats, as compared with ctrl. Hepatic total cholesterol, triglycerides and fatty acids levels were significantly higher in Lund Mets rats (14-, 7.3- and 6.7-fold higher as compared to ctrl, respectively). Liver histopathological scoring confirmed a NASH phenotype in Lund MetS rats fed the HFCF diet with strong liver steatosis, hepatic inflammation and portal to bridging fibrosis. Kidney function was substantially altered with a 60% decline in glomerular filtration rate and a 16-fold increase in urine albumin-to-creatinine ratio (both P < 0.0001 versus ctrl). Finally, echocardiography indicated diastolic dysfunction with significantly reduced E/A and greater E/e’ ratios, along with a preserved ejection fraction in Lund MetS rats, as compared with lean ctrl. CONCLUSION The present data demonstrate that the Lund MetS rat fed an HFCF diet that recapitulates the major metabolic comorbidities of obesity/T2D and may be a useful model for preclinical drug development.

Abstract BACKGROUND AND AIMS The Glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide (LIRA) has cardioprotective effects and may reduce the development of kidney disease in type 2 diabetic patients. We here evaluated the effects of LIRA on both kidney and heart function in the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic cardiorenal model. METHOD SDT fatty male rats were treated subcutaneously with vehicle or LIRA 0.4 mg/kg QD for 10 weeks. To measure the effects of LIRA on glomerular hyperfiltration, rats were injected with FITC-sinistrin to measure glomerular filtration rate (GFR) at 4 weeks of treatment. At 5 weeks of treatment, rats underwent unilateral nephrectomy and were put on a 0.3% salt diet to induce a GFR decline. GFR was then measured at 10 weeks of treatment, before hemodynamics measurement and echocardiography. RESULTS Compared with vehicle, LIRA induced significant body weight loss, as well as blood glucose levels reduction by up to ∼20%. During the hyperfiltration phase, LIRA attenuated hyperfiltration, with a 19% lower GFR versus vehicle (P < 0.05). In vehicle-treated animals, unilateral nephrectomy and 0.3% salt diet induced a major reduction (−56%) in GFR, as expected. After 10 weeks of treatment, LIRA markedly attenuated this GFR decline (vehicle: 9.3 ± 0.5 mL/min/kg; LIRA: 13.3 ± 0.9 mL/min/kg, P < 0.001 versus vehicle). Compared with vehicle, LIRA also lowered urine albumin-to-creatinine ratio both in the hyperfiltration and GFR decline phases, and significantly reduced kidney inflammation and fibrosis histology scores. LIRA showed significant reduction in cardiac hypertrophy, as well as both arterial and left ventricle end-systolic pressures. Moreover, LIRA normalized the diastolic dysfunction with preserved ejection fraction observed in SDT fatty rats (P < 0.001 versus vehicle). CONCLUSION In the SDT fatty rat, LIRA shows significant benefits by reducing renal hyperfiltration, preventing GFR decline, and improving cardiac hypertrophy, blood pressure and diastolic dysfunction. This preclinical model will be useful to evaluate drugs targeting the cardiorenal axis in type 2 diabetes.

Background Nitric oxide (NO) as a vaso- and cardio-protective agent could reduce vasomotor dysfunction in different cardiovascular diseases. One of the current therapeutics targeted at NO availability in the vascular wall are highly diluted antibodies to endothelial NO-synthase (eNOS). This drug has previously shown its endothelium-protective effect and effectiveness in reducing hypertension. Current study was dedicated to evaluate the direct impact of highly diluted antibodies to eNOS on the vessel constriction and dilation ex vivo. Methods For that purpose, we used thoracic aortas dissected from spontaneously hypertensive (SHR) rats. Endothelium-dependent relaxation in the presence of highly diluted antibodies to eNOS (1 mL) was examined after phenylephrine-induced pre-constriction of the aorta rings in response to gradually increased acetylcholine concentration (1 nM to 10 µM). Results Highly diluted antibodies to eNOS enhanced acetylcholine-induced relaxation in a statistically significant manner. Moreover, it was demonstrated that observed effect was similar to perindopril, a well-known angiotensin-converting-enzyme inhibitor, which works through relaxing and widening blood vessels. Conclusions Our findings indicate that highly diluted antibodies to eNOS restored impaired endothelium function, as demonstrated by increased relaxation of SHR rats aorta rings. The revealed results suggest beneficial effect of highly diluted antibodies to eNOS to ameliorate hypertension and related diseases.

Background: Cardiovascular disease is the leading cause of deaths in nonalcoholic steatohepatitis (NASH) patients. Mouse models, while widely used for drug development, do not fully replicate human NASH nor integrate the associated cardiac dysfunction, i.e. heart failure with preserved ejection fraction (HFpEF). To overcome these limitations, we established a nutritional hamster model developing both NASH and HFpEF. We then evaluated the effects of the dual peroxisome proliferator activated receptor alpha/delta agonist elafibranor developed for the treatment of NASH patients. Methods: Male Golden Syrian hamsters were fed for 10 to 20 weeks with a free choice diet, which presents hamsters with a choice between control chow diet with normal drinking water or a high fat/high cholesterol diet with 10% fructose enriched drinking water. Biochemistry, histology and echocardiography analysis were performed to characterize NASH and HFpEF. Once the model was validated, elafibranor was evaluated at 15 mg/kg/day orally QD for 5 weeks. Results: Hamsters fed a free choice diet for up to 20 weeks developed NASH, including hepatocyte ballooning (as confirmed with cytokeratin-18 immunostaining), bridging fibrosis, and a severe diastolic dysfunction with restrictive profile, but preserved ejection fraction. Elafibranor resolved NASH, with significant reduction in ballooning and fibrosis scores, and improved diastolic dysfunction with significant reduction in E/A and E/E ' ratios. Conclusion: Our data demonstrate that the free choice diet induced NASH hamster model replicates the human phenotype and will be useful for validating novel drug candidates for the treatment of NASH and associated HFpEF. (c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Lorcaserin (LORCA) and liraglutide (LIRA) were evaluated in a novel diet-induced obese (DIO) rat model fed a free choice (FC) diet, that presents rats with the options between control chow (CC) or high fat/cholesterol (HFC) diet, and normal water (NW) or 10% fructose water (FW). After 8 weeks of FC diet-induced obesity/insulin resistance, rats were maintained on FC diet and treated daily for 5 weeks with vehicle, LORCA 18 mg/kg orally or LIRA 0.4 mg/kg subcutaneously. Compared to CC diet, FC diet resulted in higher intake of HFC and FW, and significantly higher caloric intake and overweight. LIRA induced a lower HFC/FW and higher CC/NW intake, a 12% body weight loss (P < 0.01 vs. FC) and 40% lower visceral fat mass (P < 0.001). LORCA only reduced HFC intake and body weight gain (P < 0.001 vs. FC). FC diet raised HOMA-IR index and plasma leptinemia by 66% and 165% (both P < 0.05 vs. CC), which were 50% and 70% lower with LIRA (both P < 0.05 vs. FC), but unchanged by LORCA. LIRA and LORCA significantly improved FC diet-induced glucose intolerance. Only LIRA reduced liver fatty acids, triglycerides, and cholesterol by 68, 71 and 51% (all P < 0.001). FC diet also induced a diastolic dysfunction with reduced E/A ratio (P < 0.01 vs. CC), which was improved by LIRA and LORCA (both P < 0.01 vs. FC). LIRA also raised fractional shortening (P < 0.01 vs. FC). Overall, LIRA showed superior cardiometabolic benefits than LORCA in DIO rats under the FC diet, a model that will be useful to evaluate novel drugs targeting obesity and co-morbidities.