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Key points Heart failure with preserved ejection fraction (HFpEF) is seen more frequently in older women; risk factors include age, hypertension and excess weight. No female animal models of early stage remodelling (pre‐HFpEF) have examined the effects that the convergence of such factors have on cardiac structure and function. In this study, we demonstrate that ageing can lead to the development of mild chamber remodelling, diffuse fibrosis and loss of diastolic function. The loss of oestrogens further aggravates such changes by leading to a notable drop in cardiac output (while preserving normal ejection fraction) in the presence of diffuse fibrosis that is more predominant in endocardium and is accompanied by papillary fibrosis. Excess weight did not markedly aggravate such findings. This animal model recapitulates many of the features recognized in older, female HFpEF patients and thus, may serve to examine the effects of candidate therapeutic agents. Abstract Two‐thirds of patients with heart failure with preserved ejection fraction (HFpEF) are older women, and risk factors include hypertension and excess weight/obesity. Pathophysiological factors that drive early disease development (before heart failure ensues) remain obscure and female animal models are lacking. The study evaluated the intersecting roles of ageing, oestrogen depletion and excess weight on altering cardiac structure/function. Female, 18‐month‐old, Fischer F344 rats were divided into an aged group, aged + ovariectomy (OVX) and aged + ovariectomy + 10% fructose (OVF) in drinking water (n = 8–16/group) to induce weight gain. Left ventricular (LV) structure/function was monitored by echocardiography. At 22 months of age, animals were anaesthetized and catheter‐based haemodynamics evaluated, followed by histological measures of chamber morphometry and collagen density. All aged animals developed hypertension. OVF animals increased body weight. Echocardiography only detected mild chamber remodelling with ageing while intraventricular pressure–volume loop analysis showed significant (P < 0.05) decreases vs. ageing in stroke volume (13% OVX and 15% for OVF), stroke work (34% and 52%) and cardiac output (29% and 27%), and increases in relaxation time (10% OVX) with preserved ejection fraction. Histology indicated papillary and interstitial fibrosis with ageing, which was higher in the endocardium of OVX and OVF groups. With ageing, ovariectomy leads to the loss of diastolic and global LV function while preserving ejection fraction. This model recapitulates many cardiovascular features present in HFpEF patients and may help understand the roles that ageing and oestrogen depletion play in early (pre‐HFpEF) disease development.

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Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity.

Evidence has implicated oxidative stress (OS) and inflammation as drivers of neurodegenerative pathologies. We previously reported on the beneficial effects of (-)-epicatechin (Epi) treatment, on aging-induced OS and its capacity...

Gulf War illness (GWI) afflicts military personnel who served during the Persian Gulf War and is notable for cognitive deficits, depression, muscle pain, weakness, intolerance to exercise, and fatigue. Suspect causal agents include the chemicals pyridostigmine (PB), permetrim (PM) and N,N-diethylm-toluamide (DEET) used as protectants against insects and nerve gases. No pre-clinical studies have explored the effects on skeletal muscle (SkM). Young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks followed a 3-week recovery. GWI gastrocnemius weight was ~ 35% lower versus controls, which correlated with decreases in myofiber area, limb strength, and treadmill time/distance. In GWI rats, SkM fiber type relative abundance changed towards slow type I. Muscle wasting pathway proteins were upregulated while those that promote growth decreased as did mitochondrial endpoints and muscle ATP levels. Proteomic analysis of SkM also documented unique alterations in mitochondrial and metabolic pathways. Thus, exposure to GWI chemicals/stress adversely impacts key metabolic pathways leading to muscle atrophy and loss of function. These changes may account for GWI Veterans symptoms. Gulf War Illness (GWI) afflicts ~ 30% of the US military personnel who served in the 1990-1991 first Persian Gulf War. GWI comprises a gathering of symptoms that prominently affect the nervous and skeletal muscle (SkM) systems leading to cognitive deficits, muscle pain, weakness, exercise intolerance and fatigue 1,2. GWI affected Veterans continue to experience symptoms and altered function after 25+ years. However, the clinical presentation of GWI is unique to the 1990-1991 conflict, with no similar illness being reported in any other military campaign, indicating that the etiology cannot solely be attributed to combat-related stress 2. While the precise etiology is unknown, several hypotheses have been proposed most prominently, co-exposure to specific chemical agents and stress 2. Military personnel stationed in the battlefield are believed to have consumed the acetylcholinesterase inhibitor (AChEi) pyridostigmine bromide (PB) pills as a daily prophylactic treatment to protect against nerve gas 1,2. In addition, to reduce the risk of infections transmitted by vectors, personnel were also exposed to insecticides and insect repellants most commonly permethrin (PM) and N,Ndietyl-m-toluamide (DEET). PM is a widely used insecticide and intoxication leads to the opening of voltagegated sodium channels 3. DEET is also widely used and its target is unknown, but human poisoning can occur. AChEi, organophosphate toxicity and lethality have been related to the development of oxidative stress (OS) and mitochondrial dysfunction (MD) 2-4. Rodent models have been developed to examine the effects that GWI associated chemicals and stress have on physiological systems. A significant amount of pre-clinical work has focused on the nervous system as GWI

We examined in a rat model of Gulf War illness (GWI), the potential of (−)-epicatechin (Epi) to reverse skeletal muscle (SkM) atrophy and dysfunction, decrease mediators of inflammation and normalize metabolic perturbations. Male Wistar rats (n = 15) were provided orally with pyridostigmine bromide (PB) 1.3 mg/kg/day, permethrin (PM) 0.13 mg/kg/day (skin), DEET 40 mg/kg/day (skin) and were physically restrained for 5 min/day for 3 weeks. A one-week period ensued to fully develop the GWI-like profile followed by 2 weeks of either Epi treatment at 1 mg/kg/day by gavage (n = 8) or water (n = 7) for controls. A normal, control group (n = 15) was given vehicle and not restrained. At 6 weeks, animals were subjected to treadmill and limb strength testing followed by euthanasia. SkM and blood sampling was used for histological, biochemical and plasma pro-inflammatory cytokine and metabolomics assessments. GWI animals developed an intoxication profile characterized SkM atrophy and loss of function accompanied by increases in modulators of muscle atrophy, degradation markers and plasma pro-inflammatory cytokine levels. Treatment of GWI animals with Epi yielded either a significant partial or full normalization of the above stated indicators relative to normal controls. Plasma metabolomics revealed that metabolites linked to inflammation and SkM waste pathways were dysregulated in the GWI group whereas Epi, attenuated such changes. In conclusion, in a rat model of GWI, Epi partially reverses detrimental changes in SkM structure including modulators of atrophy, inflammation and select plasma metabolites yielding improved function.

We previously reported on the development of left ventricular (LV) structural and functional changes in an aged, female rat model where the effects of ovariectomy and excess weight (stimulated by fructose in water) were also explored. Ovariectomy and/or excess weight led to a prolongation of active relaxation, loss of cardiac output, and LV fibrosis in the setting of preserved ejection fraction. In this follow-up study, we wished to characterize the possible role of LV inflammation, oxidative stress (OS), and cell death in inducing such changes. Four experimental groups were studied: young (3 months old), aged (18 months old), aged + ovariectomy (OVX), and aged + ovariectomy + 10% fructose (OVF). Using conventional histology and immunohistochemistry of myocardium as well as biochemical assays of plasma samples, we document the presence of inflammatory cell aggregates in LV myocardium which are associated to high levels of plasma inflammatory cytokines (IL-1β, TNF-α, IFN-γ, TGF-β1) and OS (carbonyl proteins) in aged, OVX, and OVF vs. young animals. In the inflammatory areas, normal cardiac tissue was substituted by replacement and interstitial fibrosis and M1 macrophages, (as per by CD68 immunostaining) as we all as by co-localization with TGF-β1. We also document increases in plasma troponin I levels, loss of capillary density, cardiomyocyte hypertrophy, and death. Select changes were further aggravated by ovariectomy and/or excess weight. In conclusion, aging in the female rat heart, when compounded with estrogen depletion and excess weight promotes the development of greater levels inflammation, OS, fibrosis, capillary rarefaction, cardiomyocyte hypertrophy, and injury/death. These factors likely play an important role in the development of LV remodeling that leads to the development of a “pre-HFpEF” phenotype. Impact statement The incidence of HFpEF continues to increase and ∼2/3 of the patient population are post-menopausal women. Unfortunately, most studies focus on the use of male animal models of remodeling. In this study, however, using female rats to set a model of pre-HFpEF, we provide insights to possible mechanisms that contribute to HFpEF development in humans that will lead us to a better understanding of the underlying pathophysiology of HFpEF.