(-)-Epicatechin Inhibits Metastatic-Associated Proliferation, Migration, and Invasion of Murine Breast Cancer Cells In Vitro.

Breast cancer, due to its high incidence and mortality, is a public health problem worldwide. Current chemotherapy uses non-specific cytotoxic drugs, which inhibit tumor growth but cause significant adverse effects. (-)-Epicatechin (EC) is part of a large family of biomolecules called flavonoids. It is widely distributed in the plant kingdom; it can be found in green tea, grapes, and cocoa. Several studies in animals and humans have shown that EC induces beneficial effects in the skeletal muscle and the cardiovascular system, reducing risk factors such as arterial hypertension, endothelial dysfunction, damage to skeletal muscle structure, and mitochondrial malfunction by promoting mitochondrial biogenesis, with no adverse effects reported. Recently, we reported that EC had an antitumor effect in a murine triple-negative mammary gland tumor model, decreasing tumoral size and volume and increasing survival by 44%. This work aimed to characterize the effects of flavanol EC on proliferation, migration, and metastasis markers of triple-negative murine breast (4T1) cancer cells in culture. We found proliferation diminished and Bax/Bcl2 ratio increased. When the migration of culture cells was evaluated, we observed a significant reduction in migration. Also, the relative expression of the genes associated with metastasis, Cdh1, Mtss1, Pten, Bmrs, Fat1, and Smad4, was increased. In conclusion, these results contribute to understanding molecular mechanisms activated by EC that can inhibit metastatic-associated proliferation, migration, and invasion of murine breast cancer cells.

Foetal lipoprotein oxidation and preeclampsia.

Preeclampsia (PE) is a multisystemic syndrome specific to pregnancy. Although PE is the leading cause of death from complications associated with pregnancy, its aetiology is still unknown. In PE, lipid metabolism is altered. When lipids are damaged, both the mother and the foetus may be at risk. Lipoproteins contain apolipoproteins, triacylglycerols, free and esterified cholesterol, and phospholipids, all of which are susceptible to oxidative stress when high levels of oxygen and nitrogen free radicals are present. Lipoperoxidation can occur in three stages: mild, moderate, and severe. In severe lipid damage, highly toxic products such as malondialdehyde (MDA) can be generated; under these conditions, low-density lipoprotein (LDL) proteins can be oxidized (oxLDL). oxLDL is a biomolecule that can affect the production of nitric oxide (NO), the main vasodilator derived from the endothelium. oxLDL can interfere with the transduction of the signals responsible for triggering the activation of endothelial nitric oxide synthase (eNOS), causing reduced vasodilation and endothelial dysfunction, which are the main characteristics of preeclampsia. The objective of the review was to analyse the information the current information about exists about the impact generated by the oxidation of LDL and HDL lipoproteins in neonates of women with preeclampsia and how these alterations can predispose the neonate to develop diseases in adulthood.PE can cause foetal loss, intrauterine growth restriction, or developmental complications. Neonates of mothers with PE have a high risk of cardiovascular diseases, stroke, mental retardation, sensory deficiencies and an increased risk of developing metabolic diseases. PE not only affects the foetus, generating complications during pregnancy but also predisposes them to chronic diseases in adulthood.

(-)-Epicatechin Is a Biased Ligand of Apelin Receptor.

(-)-Epicatechin (EC) is part of a large family of biomolecules called flavonoids and is widely distributed in the plant kingdom. Several studies have shown the beneficial effects of EC consumption. Many of these reported effects are exerted by activating the signaling pathways associated with the activation of two specific receptors: the G protein-coupled estrogen receptor (GPER), a transmembrane receptor, and the pregnane X receptor (PXR), which is a nuclear receptor. However, the effects of EC are so diverse that these two receptors cannot describe the complete phenomenon. The apelin receptor or APLNR is classified within the G protein-coupled receptor (GPCR) family, and is capable of activating the G protein canonical pathways and the ß-arrestin transducer, which participates in the phenomenon of receptor desensitization and internalization. ß-arrestin gained interest in selective pharmacology and mediators of the so-called "biased agonism". With molecular dynamics (MD) and in vitro assays, we demonstrate how EC can recruit the ß-arrestin in the active conformation of the APLN receptor acting as a biased agonist.

Markers of oxidative stress in postmenopausal women with metabolic syndrome.

During the postmenopausal period, there are metabolic alterations that predispose individuals to metabolic syndrome (MS), oxidative stress (OS), and the risk of developing cardiovascular diseases. We aimed to compare the concentrations of OS markers in postmenopausal women with and without MS. Malondialdehyde, carbonyl groups, and total antioxidant capacity (TAC) were quantified. We conducted a cross-sectional study: Group 1 (n = 42) included women without MS, and Group 2 (n = 58) comprised women with MS. Participants' age was similar between groups. Glucose, insulin, the homeostasis model assessment of insulin resistance, triglycerides, uric acid, and body mass index were significantly lower in postmenopausal women without MS. OS markers were significantly lower in Group 1 vs. Group 2: malondialdehyde, 31.32 ± 14.93 vs. 40.27 ± 17.62 pmol MDA/mg dry weight (p = .01); protein carbonylation, 6325 ± 1551 vs. 7163 ± 1029 pmol PC/mg protein (p = .0003); and TAC, 1497 ± 297.3 vs. 1619 ± 278.8 pmol Trolox equivalent/mg protein (p = .041). OS markers were significantly higher in postmenopausal women with MS. Impact statementWhat is already known on this subject? Oxidative stress has been implicated in numerous disease processes; however, information on the relationship between oxidative stress and metabolic syndrome among postmenopausal women remains limited.What do the results of this study add? Our results indicate that in postmenopausal Mexican women, oxidative stress markers were significantly lower in those without metabolic syndrome, whereas total antioxidant capacity was higher in those with metabolic syndrome, which could be explained as an antioxidant defense mechanism capable of neutralising excess oxidative damage markers.What are the implications of these findings for clinical practice and/or further research? This study is of interest to a broad audience because it compares the concentrations of oxidative stress markers in postmenopausal women with and without metabolic syndrome. Our study could support intervention with supplements or foods rich in antioxidants as lifestyle modifications in postmenopausal women with metabolic syndrome.

(-)-Epicatechin induces mitochondrial biogenesis and markers of muscle regeneration in adults with Becker muscular dystrophy.

INTRODUCTION: We conducted an open-label study to examine the effects of the flavonoid (-)-epicatechin in seven ambulatory adult patients with Becker muscular dystrophy (BMD). METHODS: Seven participants received (-)-epicatechin 50 mg twice per day for 8 weeks. Pre- and postprocedures included biceps brachii biopsy to assess muscle structure and growth-relevant endpoints by western blotting, mitochondria volume measurement, and cristae abundance by electron microscopy, graded exercise testing, and muscle strength and function tests. RESULTS: Western blotting showed significantly increased levels of enzymes modulating cellular bioenergetics (liver kinase B1 and 5'-adenosine monophosphate-activated protein kinase). Peroxisome proliferator-activated receptor gamma coactivator-1alpha, a transcriptional coactivator of genes involved in mitochondrial biogenesis and cristae-associated mitofilin levels, increased as did cristae abundance. Muscle and plasma follistatin increased significantly while myostatin decreased. Markers of skeletal muscle regeneration myogenin, myogenic regulatory factor-5, myoblast determination protein 1, myocyte enhancer factor-2, and structure-associated proteins, including dysferlin, utrophin, and intracellular creatine kinase, also increased. Exercise testing demonstrated decreased heart rate, maximal oxygen consumption per kilogram, and plasma lactate levels at defined workloads. Tissue saturation index improved in resting and postexercise states. DISCUSSION: (-)-Epicatechin, an exercise mimetic, appears to have short-term positive effects on tissue biomarkers indicative of mitochondrial biogenesis and muscle regeneration, and produced improvements in graded exercise testing parameters in patients with BMD.

Antihyperglycemic and Lipid Profile Effects of Salvia amarissima Ortega on Streptozocin-Induced Type 2 Diabetic Mice.

Salvia amarissima Ortega was evaluated to determinate its antihyperglycemic and lipid profile properties. Petroleum ether extract of fresh aerial parts of S. amarissima (PEfAPSa) and a secondary fraction (F6Sa) were evaluated to determine their antihyperglycemic activity in streptozo-cin-induced diabetic (STID) mice, in oral tolerance tests of sucrose, starch, and glucose (OSTT, OStTT, and OGTT, respectively), in terms of glycated hemoglobin (HbA1c), triglycerides (TG), and high-density lipoprotein (HDL). In acute assays at doses of 50 mg/kg body weight (b.w.), PEfAPSa and F6Sa showed a reduction in hyperglycemia in STID mice, at the first and fifth hour after of treatment, respectively, and were comparable with acarbose. In the sub-chronic test, PEfAPSa and F6Sa showed a reduction of glycemia since the first week, and the effect was greater than that of the acarbose control group. In relation to HbA1c, the treatments prevented the increase in HbA1c. In the case of TG and HDL, PEfAPSa and F6Sa showed a reduction in TG and an HDL increase from the second week. OSTT and OStTT showed that PEfAPSa and F6Sa significantly lowered the postprandial peak at 1 h after loading but only in sucrose or starch such as acarbose. The results suggest that S. amarissima activity may be mediated by the inhibition of disaccharide hydrolysis, which may be associated with an α-glucosidase inhibitory effect.

11-ß-hydroxysterols as possible endogenous stimulators of mitochondrial biogenesis as inferred from epicatechin molecular mimicry.

Currently, there is great interest in identifying endogenous (i.e. physiological) stimulators of mitochondrial biogenesis (MB), in particular, those that may mediate the effects of exercise. The molecular size of the cacao flavanols (epicatechin and catechin) highly resembles that of sterols and epicatechin has been reported to activate cells surface receptors leading to the stimulation of MB in endothelial and skeletal muscle cells translating into enhanced exercise capacity. We therefore hypothesize, that epicatechin may be acting as a structural mimic of an as yet unknown sterol capable of stimulating MB. We developed a new synthetic process for obtaining enantiomerically pure preparations of (-)-epicatechin and (+)-epicatechin. Applying spatial analytics and molecular modeling, we found that the two isoforms of epicatechin, (-) and (+), have a structural resemblance to 11-ß-hydroxypregnenolone, a sterol with no previously described biological activity. As reported in this proof-of-concept study performed in primary cultures of endothelial and muscle cells, 11-ß-hydroxypregnenolone is one of the most potent inducers of MB as significant activity can be detected at femtomolar levels. The relative potency of (-)/(+)-epicatechin isoforms and on inducing MB correlates with their degree of spatial homology towards the 11-ß-hydroxypregnenolone. On the basis of these results, the detailed in vivo characterization of the potential for these sterols to act as endogenous modulators of MB is warranted.

(-)-Epicatechin reduces muscle waste after complete spinal cord transection in a murine model: role of ubiquitin-proteasome system.

The skeletal muscle mass reduces 30-60% after spinal cord injury, this is mostly due to protein degradation through ubiquitin-proteasome system. In this work, we propose that the flavanol (-)-epicatechin, due its widespread biological effects on muscle health, can prevent muscle mass decrease after spinal cord injury. Thirty-six female Long Evans rats were randomized into 5 groups: (1) Spinal cord injury 7 days, (2) Spinal cord injury + (-)-epicatechin 7 days, (3) Spinal cord injury 30 days, (4) Spinal cord injury + (-)-epicatechin 30 days and (5) Sham (Only laminectomy). Hind limb perimeter, muscle cross section area, fiber cross section area and ubiquitin-proteasome system protein expression together with total protein ubiquitination were assessed. At 30 days Spinal cord injury group lost 49.52 ± 2.023% of muscle cross section area (-)-epicatechin treated group lost only 24.28 ± 15.45% being a significant difference. Ubiquitin-proteasome markers showed significant changes. FOXO1a increased in spinal cord injury group vs Sham (-)-epicatechin reduced this increase. In spinal cord injury group MAFbx increased significantly vs Sham but decrease in (-)-epicatechin treatment group at 30 days. At 7 and 30 days MuRF1 increased in the spinal cord injury and decreased in the (-)-epicatechin group. The global protein ubiquitination increases after spinal cord injury, epicatechin treatment induce a significant decrease in protein ubiquitination. These results suggest that (-)-epicatechin reduces the muscle waste after spinal cord injury through down regulation of the ubiquitin-proteasome system.

Randomized Trial of Deep Vein Thrombosis Chemoprophylaxis with Bemiparin and Enoxaparin in Patients with Moderate to High Thrombogenic Risk Undergoing Plastic and Reconstructive Surgery Procedures.

BACKGROUND: Deep vein thrombosis (DVT) is a common complication during postoperative convalescence characterized by hypercoagulability, vascular endothelium damage and blood stasis. It increases noticeably in peri/postoperative phases of surgery procedures. Pulmonary embolism secondary to iliofemoral DVT is a frequent cause of death. METHODS: Adult patients scheduled for plastic and reconstructive surgery (PRSx) with moderate to high thrombogenic risk were selected. We evaluated the efficacy and safety of bemiparin compared to enoxaparin as chemoprophylaxis for DVT. Following balanced general anesthesia techniques, patients were randomly assigned for subcutaneous enoxaparin 40 IU (Group-E) or bemiparin 3500 IU (Group-B) q24h starting 6 h after procedure conclusion for at least 10 days. All patients were evaluated for DVT through Doppler ultrasound mapping of the lower limbs. RESULTS: Seventy-eight patients were evaluated, mostly women (83%), physical status ASA II (59%), ASA III (10%); Caprini's thrombogenic risk score 3-4 (moderate) 58%, 5-6 (high) 29%, > 6 (too high) 13%; demographics, clinical variables and scores were similar between groups. Median drainage time in breast surgery was 4 days in both groups (p = 0.238). In the case of abdominal surgery, median was 14 days in Group-E versus 13 days in Group-B (p = 0.059). No DVT was detected in either group. CONCLUSIONS: DVT was prevented with bemiparin, without significant bleeding increase nor adverse events; moreover, the cost of bemiparin is lower than enoxaparin. Bemiparin can be considered as alternative drug for DVT chemoprophylaxis in PRSx procedures. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Unmasking of oestrogen-dependent changes in left ventricular structure and function in aged female rats: a potential model for pre-heart failure with preserved ejection fraction.

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.

Synthesis of novel (-)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects.

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (-)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity.

The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection.

Role for high-glucose-induced protein O-GlcNAcylation in stimulating cardiac fibroblast collagen synthesis.

Excess enzyme-mediated protein O-GlcNAcylation is known to occur with diabetes mellitus. A characteristic of diabetic cardiomyopathy is the development of myocardial fibrosis. The role that enhanced protein O-GlcNAcylation plays in modulating the phenotype of cardiac fibroblasts (CF) is unknown. To address this issue, rat CF were cultured in normal glucose (NG; 5 mM glucose) or high-glucose (HG; 25 mM) media for 48 h. Results demonstrate that CF cultured in HG have higher levels (~50%) of overall protein O-GlcNAcylation vs. NG cells. Key regulators of collagen synthesis such as transforming-growth factor-ß1 (TGF-ß1), SMADs 2/3, and SMAD 7 protein levels, including those of arginase I and II, were altered, leading to increases in collagen levels. The nuclear transcription factor Sp1 and arginase II evidence excess O-GlcNAcylation in HG cells. Expression in CF of an adenovirus coding for the enzyme N-acetylglucosaminidase, which removes O-GlcNAc moieties from proteins, decreased Sp1 and arginase II O-GlcNAcylation and restored HG-induced perturbations in CF back to NG levels. These findings may have important pathophysiological implications for the development of diabetes-induced cardiac fibrosis.

Cell membrane mediated (-)-epicatechin effects on upstream endothelial cell signaling: evidence for a surface receptor.

The consumption of cacao-derived products, particularly in the form of dark chocolate is known to provide beneficial cardiovascular effects in normal individuals and in those with vascular dysfunction (reduced nitric oxide [NO] bioavailability and/or synthesis). Upstream mechanisms by which flavonoids exert these effects are poorly understood and may involve the participation of cell membrane receptors. We previously demonstrated that the flavanol (-)-epicatechin (EPI) stimulates NO production via Ca(+2)-independent eNOS activation/phosphorylation. We wished to investigate the plausible participation of a cell surface receptor using a novel cell-membrane impermeable EPI-Dextran conjugate (EPI-Dx). Under Ca(2+)-free conditions, human coronary artery endothelial cells (HCAEC) were treated for 10min with EPI or EPI-Dx at equimolar concentrations (100nM). Results demonstrate that both EPI and EPI-Dx induced the phosphorylation/activation of PI3K, PDK-1, AKT and eNOS. Interestingly, EPI-Dx effects were significantly higher in magnitude than those of EPI alone. The capacity of EPI-Dx to stimulate cell responses supports the existence of an EPI cell membrane receptor mediating eNOS activation.

Obesity, Metabolic Syndrome, and Dietary Therapeutical Approaches with a Special Focus on Nutraceuticals (Polyphenols): A Mini-Review.

More than half of all global deaths in 2010 were related to non-communicable diseases, including obesity, cancers, diabetes, and cardiovascular illnesses. It has been suggested that the alarming increase in the incidence of cardiovascular disease is the epidemiologic result of a nutrition transition characterized by dietary patterns featuring an increase in the intake of total fat, cholesterol, sugars, and other refined carbohydrates, concomitant with low consumption of polyunsaturated fatty acids and fiber. Although traditional dietary approaches have proven successful as part of the treatment for obesity and cardiometabolic derangements within clinical trial scenarios, they lack effectiveness in the long term, mainly due to poor compliance. Research has thus turned its attention to nutraceutics, nutrients that have the ability to modulate physiological and pathophysiological molecular mechanisms, thus resulting in favorable health outcomes. Polyphenols have been considered as among the bioactive molecules as they are thought to yield beneficial effects by exerting antioxidant activity; however, there are other--and even more robust--metabolic pathways through which polyphenols enhance cardiovascular health, such as via promoting vasodilatory, anti-atherogenic, antithrombotic, and anti-inflammatory effects. No standard dose has yet been determined, as the effects greatly vary among polyphenols and food sources; thus, there is an imperative need to generate more evidence in order to support dietary recommendations aimed at the prevention and therapeutics of obesity and its associated cardiometabolic diseases.

Overweight, Obesity, and Age Are the Main Determinants of Cardiovascular Risk Aggregation in the Current Mexican Population: The FRIMEX III Study.

Background: The Mexican population exhibits several cardiovascular risk factors (CVRF) including high blood pressure (HBP), dysglycemia, dyslipidemia, overweight, and obesity. This study is an extensive observation of the most important CVFRs in six of the most populated cities in Mexico. Methods: In a cohort of 297,370 participants (54% female, mean age 43 ± 12.6 years), anthropometric (body mass index (BMI)), metabolic (glycemia and total cholesterol (TC)), and blood pressure (BP) data were obtained. Results: From age 40, 40% and 30% of the cohort's participants were overweight or obese, respectively. HBP was found in 27% of participants. However, only 8% of all hypertensive patients were controlled. Fifty percent of the subjects 50 years and older were hypercholesterolemic. Glycemia had a constant linear relation with age. BMI had a linear correlation with SBP, glycemia, and TC, with elevated coefficients in all cases and genders. The ß1 coefficient for BMI was more significant in all equations than the other ß, indicating that it greatly influences the other CVRFs. Conclusions: TC, glycemia, and SBP, the most critical atherogenic factors, are directly related to BMI.

Restorative effects of (+)-epicatechin in a rodent model of aging induced muscle atrophy: underlying mechanisms.

Sarcopenia is a progressive and generalized age-related skeletal muscle (SkM) disorder characterized by the accelerated loss of muscle mass (atrophy) and function. SkM atrophy is associated with increased incidence of falls, functional decline, frailty and mortality. In its early stage, SkM atrophy is associated with increased pro-inflammatory cytokine levels and proteasome-mediated protein degradation. These processes also link to the activation of atrophy associated factors and signaling pathways for which, there is a lack of approved pharmacotherapies. The objective of this study, was to characterize the capacity of the flavanol (+)-epicatechin (+Epi) to favorably modulate SkM mass and function in a rat model of aging induced sarcopenia and profile candidate mechanisms. Using 23 month old male Sprague-Dawley rats, an 8 weeks oral administration of the +Epi (1 mg per kg per day in water by gavage) was implemented while control rats only received water. SkM strength (grip), treadmill endurance, muscle mass, myofiber area, creatine kinase, lactate dehydrogenase, troponin, α-actin, tumor necrosis factor (TNF)-α and atrophy related endpoints (follistatin, myostatin, NFκB, MuRF 1, atrogin 1) were quantified in plasma and/or gastrocnemius. We also evaluated effects on insulin growth factor (IGF)-1 levels and downstream signaling (AKT/mTORC1). Treatment of aged rats with +Epi, led to significant increases in front paw grip strength, treadmill time and SkM mass vs. controls as well as beneficial changes in makers of myofiber integrity. Treatment significantly reversed adverse changes in plasma and/or SkM TNF-α, IGF-1, atrophy and protein synthesis related endpoints vs. controls. In conclusion, +Epi has the capacity to reverse sarcopenia associated detrimental changes in regulatory pathways leading to improved SkM mass and function. Given these results and its recognized safety and tolerance profile, +Epi warrants consideration for clinical trials.

Perturbations in skeletal muscle sarcomere structure in patients with heart failure and type 2 diabetes: restorative effects of (-)-epicatechin-rich cocoa.

HF (heart failure) and T2D (Type 2 diabetes) associate with detrimental alterations in SkM (skeletal muscle) structure/function. We have demonstrated recently that (-)-ERC (epicatechin-rich cocoa) improves SkM mitochondrial structure [Taub, Ramirez-Sanchez, Ciaraldi, Perkins, Murphy, Naviaux, Hogan, Ceballos, Maisel, Henry et al. (2012) Clin. Trans. Sci. 5, 43-47]. We hypothesized that an improved mitochondrial structure may facilitate the reversal of detrimental alterations in sarcomeric microstructure. In a pilot study, five patients with HF and T2D consumed ERC for 3 months; treadmill testing [VO2max (maximum oxygen consumption)] and SkM biopsies were performed. Western blot analysis, immunohistochemistry and electron microscopy were used. We report severe perturbations in components of the DAPC (dystrophin-associated protein complex) as well as sarcomeric microstructure at baseline. ERC induced recovery/enhancement of DAPC protein levels, sarcomeric microstructure and, in a co-ordinated fashion, alterations in markers of SkM growth/differentiation consistent with myofibre regeneration. VO2max increased (~24%) but did not reach statistical significance. These initial results warrant further rigorous investigation, since the use of ERC (or pure epicatechin) may represent a safe and novel means of improving muscle function.

Effects of (-)-epicatechin and derivatives on nitric oxide mediated induction of mitochondrial proteins.

Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial function and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and thus, reduce CVD. In this study we examined the capacity of the flavanol (-)-epicatechin and two novel derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria effects (including biogenesis), we also examined the dependence of responses on this molecule using an inhibitor of nitric oxide synthase. Results indicate that the flavanol (-)-epicatechin and derivatives are capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in mediating these effects. The results observed indicate that the three agents are effective in enhancing mitochondria function and protein content. The effects noted for (-)-epicatechin may serve to explain the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products.

Effects of aging and type 2 diabetes on cardiac structure and function: Underlying mechanisms.

We characterized long-term changes in cardiac structure and function in a high-fat diet/streptozotocin mouse model of aging and type 2 diabetes mellitus (T2D) and examined how the intersection of both conditions alters plasma metabolomics. We also evaluated the possible roles played by oxidative stress, arginase activity and pro-inflammatory cytokines. C57BL/6 male mice (13-month-old) were used. Control animals (n = 13) were fed regular chow for 10 months (aged group). T2D animals (n = 25) were provided a single injection of streptozotocin and fed a high fat diet for 10 months. In select endpoints, young animals were used for comparison. To monitor changes in left ventricular (LV) structure and function, echocardiography was used. At the terminal study (23 months), blood was collected and hearts processed for biochemical or histological analysis. Echo yielded diminished diastolic function with aging and T2D. LV fractional shortening and ejection fraction decreased with T2D by 16 months peaking at 23 months. Western blots noted increases in fibronectin and type I collagen with aging/T2D and greater levels with T2D in α-smooth muscle actin. Increases in plasma and/or myocardial protein carbonyls, arginase activity and pro-inflammatory cytokines occurred with aging and T2D. Untargeted metabolomics and cheminformatics revealed differences in the plasma metabolome of T2D vs. aged mice while select classes of lipid metabolites linked to insulin resistance, were dysregulated. We thus, document changes in LV structure and function with aging that in select endpoints, are accentuated with T2D and link them to increases in OS, arginase activity and pro-inflammatory cytokines.