Echocardiographic predictors of cardiovascular outcome in heart failure with preserved ejection fraction.

AIMS: The optimal echocardiographic predictors of cardiovascular outcome in heart failure (HF) with preserved ejection fraction (HFpEF) are unknown. We aimed to identify independent echocardiographic predictors of cardiovascular outcome in patients with HFpEF. METHODS AND RESULTS: Systematic literature search of three electronic databases was conducted from date of inception until November 2022. Hazard ratios (HRs) and their 95% confidence intervals (CIs) for echocardiographic variables from multivariate prediction models for the composite primary endpoint of cardiovascular death and HF hospitalization were pooled using a random effects meta-analysis. Specific subgroup analyses were conducted for studies that enrolled patients with acute versus chronic HF, and for those studies that included E/e', pulmonary artery systolic pressure (PASP), renal function, natriuretic peptides and diuretic use in multivariate models. Forty-six studies totalling 20 056 patients with HFpEF were included. Three echocardiographic parameters emerged as independent predictors in all subgroup analyses: decreased left ventricular (LV) global longitudinal strain (HR 1.24, 95% CI 1.10-1.39 per 5% decrease), decreased left atrial (LA) reservoir strain (HR 1.30, 95% CI 1.13-1.1.50 per 5% decrease) and lower tricuspid annular plane systolic excursion (TAPSE) to PASP ratio (HR 1.17, 95% CI 1.07-1.25 per 0.1 unit decrease). Other independent echocardiographic predictors of the primary endpoint were a higher E/e', moderate to severe tricuspid regurgitation, LV mass index and LA ejection fraction, although these variables were less robust. CONCLUSIONS: Impaired LV global longitudinal strain, lower LA reservoir strain and lower TAPSE/PASP ratio predict cardiovascular death and HF hospitalization in HFpEF and are independent of filling pressures, clinical characteristics and natriuretic peptides. These echocardiographic parameters reflect key functional changes in HFpEF, and should be incorporated in future prospective risk prediction models.

Cardiac Substrate Utilization and Relationship to Invasive Exercise Hemodynamic Parameters in HFpEF.

The authors conducted transcardiac blood sampling in healthy subjects and subjects with heart failure with preserved ejection fraction (HFpEF) to compare cardiac metabolite and lipid substrate use. We demonstrate that fatty acids are less used by HFpEF hearts and that lipid extraction is influenced by hemodynamic factors including pulmonary pressures and cardiac index. The release of many products of protein catabolism is apparent in HFpEF compared to healthy myocardium. In subgroup analyses, differences in energy substrate use between female and male hearts were identified.

Indole-3-Propionic Acid Protects Against Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a common but poorly understood form of heart failure, characterized by impaired diastolic function. It is highly heterogeneous with multiple comorbidities, including obesity and diabetes, making human studies difficult. METHODS: Metabolomic analyses in a mouse model of HFpEF showed that levels of indole-3-propionic acid (IPA), a metabolite produced by gut bacteria from tryptophan, were reduced in the plasma and heart tissue of HFpEF mice as compared with controls. We then examined the role of IPA in mouse models of HFpEF as well as 2 human HFpEF cohorts. RESULTS: The protective role and therapeutic effects of IPA were confirmed in mouse models of HFpEF using IPA dietary supplementation. IPA attenuated diastolic dysfunction, metabolic remodeling, oxidative stress, inflammation, gut microbiota dysbiosis, and intestinal epithelial barrier damage. In the heart, IPA suppressed the expression of NNMT (nicotinamide N-methyl transferase), restored nicotinamide, NAD+/NADH, and SIRT3 (sirtuin 3) levels. IPA mediates the protective effects on diastolic dysfunction, at least in part, by promoting the expression of SIRT3. SIRT3 regulation was mediated by IPA binding to the aryl hydrocarbon receptor, as Sirt3 knockdown diminished the effects of IPA on diastolic dysfunction in vivo. The role of the nicotinamide adenine dinucleotide circuit in HFpEF was further confirmed by nicotinamide supplementation, Nnmt knockdown, and Nnmt overexpression in vivo. IPA levels were significantly reduced in patients with HFpEF in 2 independent human cohorts, consistent with a protective function in humans, as well as mice. CONCLUSIONS: Our findings reveal that IPA protects against diastolic dysfunction in HFpEF by enhancing the nicotinamide adenine dinucleotide salvage pathway, suggesting the possibility of therapeutic management by either altering the gut microbiome composition or supplementing the diet with IPA.

The first human normative ranges and biomarker performance of dimethylguanidino valeric acid isoforms in fatty liver disease.

We have recently determined dimethylguanidino valeric acid (DMGV) to be a novel biomarker of liver injury in non-alcoholic fatty liver disease (NAFLD) and an independent predictor of incident diabetes over a decade in advance. DMGV consists of two stereo-isomers, asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV). Here we report, for the first time, the upper limits of normal of both isomers in humans at the accepted 5.56% liver fat threshold for NAFLD, determined using in vivo magnetic resonance spectroscopy. We performed independent and blinded comparative analyses of ADGV and SDGV levels using two different liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in (A) our laboratory, and (B) the New South Wales Chemical Pathology state laboratory, using unique columns, LC-MS/MS equipment, extraction protocols and normalisation approaches. Despite these differences, each laboratory reported consistent absolute concentrations across a range of liver fat percentages. We next determined the diagnostic performance of SDGV compared to ADGV in a cohort of 268 individuals with liver fat measurements. In derivation-validation analyses we determined rule-in/rule-out thresholds and the concentration of SDGV that provides optimal performance across sensitivity and specificity for the identification of NAFLD. In conclusion, we have herein determined for the first time the true human plasma reference range of both isoforms of an emerging novel biomarker of NAFLD, at the accepted upper normal threshold of liver fat. We have also identified that SDGV is the isoform with the best diagnostic performance and determined the optimal cut-point for its detection of NAFLD.

Metabolomics and Lipidomics Signatures of Insulin Resistance and Abdominal Fat Depots in People Living with Obesity.

The liver, skeletal muscle, and adipose tissue are major insulin target tissues and key players in glucose homeostasis. We and others have described diverse insulin resistance (IR) phenotypes in people at risk of developing type 2 diabetes. It is postulated that identifying the IR phenotype in a patient may guide the treatment or the prevention strategy for better health outcomes in populations at risk. Here, we performed plasma metabolomics and lipidomics in a cohort of men and women living with obesity not complicated by diabetes (mean [SD] BMI 36.0 [4.5] kg/m2, n = 62) to identify plasma signatures of metabolites and lipids that align with phenotypes of IR (muscle, liver, or adipose tissue) and abdominal fat depots. We used 2-step hyperinsulinemic-euglycemic clamp with deuterated glucose, oral glucose tolerance test, dual-energy X-ray absorptiometry and abdominal magnetic resonance imaging to assess muscle-, liver- and adipose tissue- IR, beta cell function, body composition, abdominal fat distribution and liver fat, respectively. Spearman's rank correlation analyses that passed the Benjamini−Hochberg statistical correction revealed that cytidine, gamma-aminobutyric acid, anandamide, and citrate corresponded uniquely with muscle IR, tryptophan, cAMP and phosphocholine corresponded uniquely with liver IR and phenylpyruvate and hydroxy-isocaproic acid corresponded uniquely with adipose tissue IR (p < 7.2 × 10−4). Plasma cholesteryl sulfate (p = 0.00029) and guanidinoacetic acid (p = 0.0001) differentiated between visceral and subcutaneous adiposity, while homogentisate correlated uniquely with liver fat (p = 0.00035). Our findings may help identify diverse insulin resistance and adiposity phenotypes and enable targeted treatments in people living with obesity.

Intersection of Diet and Exercise with the Gut Microbiome and Circulating Metabolites in Male Bodybuilders: A Pilot Study.

Diet, exercise and the gut microbiome are all factors recognised to be significant contributors to cardiometabolic health. However, diet and exercise interventions to modify the gut microbiota to improve health are limited by poor understanding of the interactions between them. In this pilot study, we explored diet-exercise-microbiome dynamics in bodybuilders as they represent a distinctive group that typically employ well-defined dietary strategies and exercise regimes to alter their body composition. We performed longitudinal characterisation of diet, exercise, the faecal microbial community composition and serum metabolites in five bodybuilders during competition preparation and post-competition. All participants reduced fat mass while conserving lean mass during competition preparation, corresponding with dietary energy intake and exercise load, respectively. There was individual variability in food choices that aligned to individualised gut microbial community compositions throughout the study. However, there was a common shift from a high protein, low carbohydrate diet during pre-competition to a more macronutrient-balanced diet post-competition, which was associated with similar changes in the gut microbial diversity across participants. The circulating metabolite profiles also reflected individuality, but a subset of metabolites relating to lipid metabolism distinguished between pre- and post-competition. Changes in the gut microbiome and circulating metabolome were distinct for each individual, but showed common patterns. We conclude that further longitudinal studies will have greater potential than cross-sectional studies in informing personalisation of diet and exercise regimes to enhance exercise outcomes and improve health.

Myocardial fibrosis in Type 2 Diabetes is associated with functional and metabolomic parameters.

AIMS: To identify biomarkers of cardiomyopathy in patients with type 2 diabetes mellitus (T2DM) using cardiovascular magnetic resonance (CMR) and to identify associations between functional status, metabolomic profile and myocardial fibrosis. METHODS: In this prospective case control study, patients (n = 49) with T2DM without significant coronary artery disease, and matched controls (n = 18) underwent CMR, cardiopulmonary exercise testing, and plasma metabolomic analyses. RESULTS: Patients with T2DM (n = 49, median [interquartile range] age 61 [56-63] years, 61% male, diabetes duration 11 [7-20] years), historical HbA1c 7.6% (60 mmol/mol) (6.9-8.6) and matched controls (n = 18) were examined. Study patients had increased myocardial extracellular volume (ECV) (26.9 [23.8-30.0] vs 23.4 [22.4-25.5) %, p < 0.001). Increased ECV was associated with male sex (p = 0.04), time with T2DM (p = 0.02), reduced peak VO2 (R2 = 0.48, p = 0.01), increased circulating choline (p = 0.002) and cysteamine (p = 0.002) both of which were also associated with reduced peak VO2 (p < 0.025 and 0.014 respectively). CONCLUSIONS: Patients with well-controlled T2DM without significant coronary disease exhibit focal and diffuse myocardial fibrosis and diffuse myocardial fibrosis is associated with reduced exercise tolerance and metabolites. Plasma metabolites may provide mechanistic insights into diffuse myocardial fibrosis, and cardiopulmonary fitness.

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Multi-omics of a pre-clinical model of diabetic cardiomyopathy reveals increased fatty acid supply impacts mitochondrial metabolic selectivity.

The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic ß-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in ß-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete ß-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess ß-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.

Models of cardiovascular surgery biobanking to facilitate translational research and precision medicine.

Biobanking in health care has evolved over the last few decades from simple biological sample repositories to complex and dynamic units with multi-organizational infrastructure networks and has become an essential tool for modern medical research. Cardiovascular tissue biobanking provides a unique opportunity to utilize cardiac and vascular samples for translational research into heart failure and other related pathologies. Current techniques for diagnosis, classification, and treatment monitoring of cardiac disease relies primarily on interpretation of clinical signs, imaging, and blood biomarkers. Further research at the disease source (i.e. myocardium and blood vessels) has been limited by a relative lack of access to quality human cardiac tissue and the inherent shortcomings of most animal models of heart disease. In this review, we describe a model for cardiovascular tissue biobanking and databasing, and its potential to facilitate basic and translational research. We share techniques to procure endocardial samples from patients with hypertrophic cardiomyopathy, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction, in addition to aortic disease samples. We discuss some of the issues with respect to data collection, privacy, biobank consent, and the governance of tissue biobanking. The development of tissue biobanks as described here has significant scope to improve and facilitate translational research in multi-omic fields such as genomics, transcriptomics, proteomics, and metabolomics. This research heralds an era of precision medicine, in which patients with cardiovascular pathology can be provided with optimized and personalized medical care for the treatment of their individual phenotype.

Metabolite signatures of heart failure, sleep apnoea, their interaction, and outcomes in the community.

AIMS: Sleep apnoea and congestive heart failure (CHF) commonly co-exist, but their interaction is unclear. Metabolomics may clarify their interaction and relationships to outcome. METHODS AND RESULTS: We assayed 372 circulating metabolites and lipids in 1919 and 1524 participants of the Framingham Heart Study (FHS) (mean age 54 ± 10 years, 53% women) and Women's Health Initiative (WHI) (mean age 67 ± 7 years), respectively. We used linear and Cox regression to relate plasma concentrations of metabolites and lipids to echocardiographic parameters; CHF and its subtypes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF); and sleep indices. Adenine dinucleotide phosphate (ADP) associated with left ventricular (LV) fractional shortening; phosphocreatine with LV wall thickness; lysosomal storage molecule sphingomyelin 18:2 with LV mass; and nicotine metabolite cotinine with time spent with an oxygen saturation less than 90% (ß = 2.3 min, P = 2.3 × 10-5 ). Pro-hypertrophic metabolite hydroxyglutarate partly mediated the association between LV wall thickness and HFpEF. Central sleep apnoea was significantly associated with HFpEF (P = 0.03) but not HFrEF (P = 0.5). There were three significant metabolite canonical variates, one of which conferred protection from cardiovascular death [hazard ratio = 0.3 (0.11, 0.81), P = 0.02]. CONCLUSIONS: Energetic metabolites were associated with cardiac function; energy- and lipid-storage metabolites with LV wall thickness and mass; plasma levels of nicotine metabolite cotinine were associated with increased time spent with a sleep oxygen saturation less than 90%, a clinically significant marker of outcome, indicating a significant hazard for smokers who have sleep apnoea.

Optimizing the discovery and assessment of therapeutic targets in heart failure with preserved ejection fraction.

There is an urgent need for models that faithfully replicate heart failure with preserved ejection fraction (HFpEF), now recognized as the most common form of heart failure in the world. In vitro approaches have several shortcomings, most notably the immature nature of stem cell-derived human cardiomyocytes [induced pluripotent stem cells (iPSC)] and the relatively short lifespan of primary cardiomyocytes. Three-dimensional 'organoids' incorporating mature iPSCs with other cell types such as endothelial cells and fibroblasts are a significant advance, but lack the complexity of true myocardium. Animal models can replicate many features of human HFpEF, and rodent models are the most common, and recent attempts to incorporate haemodynamic, metabolic, and ageing contributions are encouraging. Differences relating to species, physiology, heart rate, and heart size are major limitations for rodent models. Porcine models mitigate many of these shortcomings and approximate human physiology more closely, but cost and time considerations limit their potential for widespread use. Ex vivo analysis of failing hearts from animal models offer intriguing possibilities regarding cardiac substrate utilisation, but are ultimately subject to the same constrains as the animal models from which the hearts are obtained. Ex vivo approaches using human myocardial biopsies can uncover new insights into pathobiology leveraging myocardial energetics, substrate turnover, molecular changes, and systolic/diastolic function. In collaboration with a skilled cardiothoracic surgeon, left ventricular endomyocardial biopsies can be obtained at the time of valvular surgery in HFpEF patients. Critically, these tissues maintain their disease phenotype, preserving inter-relationship of myocardial cells and extracellular matrix. This review highlights a novel approach, where ultra-thin myocardial tissue slices from human HFpEF hearts can be used to assess changes in myocardial structure and function. We discuss current approaches to modelling HFpEF, describe in detail the novel tissue slice model, expand on exciting opportunities this model provides, and outline ways to improve this model further.

A hierarchical approach to removal of unwanted variation for large-scale metabolomics data.

Liquid chromatography-mass spectrometry-based metabolomics studies are increasingly applied to large population cohorts, which run for several weeks or even years in data acquisition. This inevitably introduces unwanted intra- and inter-batch variations over time that can overshadow true biological signals and thus hinder potential biological discoveries. To date, normalisation approaches have struggled to mitigate the variability introduced by technical factors whilst preserving biological variance, especially for protracted acquisitions. Here, we propose a study design framework with an arrangement for embedding biological sample replicates to quantify variance within and between batches and a workflow that uses these replicates to remove unwanted variation in a hierarchical manner (hRUV). We use this design to produce a dataset of more than 1000 human plasma samples run over an extended period of time. We demonstrate significant improvement of hRUV over existing methods in preserving biological signals whilst removing unwanted variation for large scale metabolomics studies. Our tools not only provide a strategy for large scale data normalisation, but also provides guidance on the design strategy for large omics studies.

Deletion of the diabetes candidate gene Slc16a13 in mice attenuates diet-induced ectopic lipid accumulation and insulin resistance.

Genome-wide association studies have identified SLC16A13 as a novel susceptibility gene for type 2 diabetes. The SLC16A13 gene encodes SLC16A13/MCT13, a member of the solute carrier 16 family of monocarboxylate transporters. Despite its potential importance to diabetes development, the physiological function of SLC16A13 is unknown. Here, we validate Slc16a13 as a lactate transporter expressed at the plasma membrane and report on the effect of Slc16a13 deletion in a mouse model. We show that Slc16a13 increases mitochondrial respiration in the liver, leading to reduced hepatic lipid accumulation and increased hepatic insulin sensitivity in high-fat diet fed Slc16a13 knockout mice. We propose a mechanism for improved hepatic insulin sensitivity in the context of Slc16a13 deficiency in which reduced intrahepatocellular lactate availability drives increased AMPK activation and increased mitochondrial respiration, while reducing hepatic lipid content. Slc16a13 deficiency thereby attenuates hepatic diacylglycerol-PKCε mediated insulin resistance in obese mice. Together, these data suggest that SLC16A13 is a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease.

The effect of peer modelling and discussing modelled feedback principles on medical students' feedback skills: a quasi-experimental study.

BACKGROUND: Teaching is an important professional skill for physicians and providing feedback is an important part of teaching. Medical students can practice their feedback skills by giving each other peer feedback. Therefore, we developed a peer feedback training in which students observed a peer that modelled the use of good feedback principles. Students then elaborated on the modelled feedback principles through peer discussion. This combination of peer modelling and discussing the modelled feedback principles was expected to enhance emulation of the feedback principles compared to (1) only peer modelling and (2) discussing the feedback principles without previous modelling. METHODS: In a quasi-experimental study design, 141 medical students were assigned randomly to three training conditions: peer modelling plus discussion (MD), non-peer modelled example (NM) or peer modelling without discussion (M). Before and after the training, they commented on papers written by peers. These comments served as a pre- and a post-measure of peer feedback. The comments were coded into different functions and aspects of the peer feedback. Non-parametrical Kruskall-Wallis tests were used to check for pre- and post-measure between-group differences in the functions and aspects. RESULTS: Before the training, there were no significant between-group differences in feedback functions and aspects. After the training, the MD-condition gave significantly more positive peer feedback than the NM-condition. However, no other functions or aspects were significantly different between the three conditions, mainly because the within-group interquartile ranges were large. CONCLUSIONS: The large interquartile ranges suggest that students differed substantially in the effort placed into giving peer feedback. Therefore, additional incentives may be needed to motivate students to give good feedback. Teachers could emphasise the utility value of peer feedback as an important professional skill and the importance of academic altruism and professional accountability in the peer feedback process. Such incentives may convince more students to put more effort into giving peer feedback.

Impact of dietary carbohydrate type and protein-carbohydrate interaction on metabolic health.

Reduced protein intake, through dilution with carbohydrate, extends lifespan and improves mid-life metabolic health in animal models. However, with transition to industrialised food systems, reduced dietary protein is associated with poor health outcomes in humans. Here we systematically interrogate the impact of carbohydrate quality in diets with varying carbohydrate and protein content. Studying 700 male mice on 33 isocaloric diets, we find that the type of carbohydrate and its digestibility profoundly shape the behavioural and physiological responses to protein dilution, modulate nutrient processing in the liver and alter the gut microbiota. Low (10%)-protein, high (70%)-carbohydrate diets promote the healthiest metabolic outcomes when carbohydrate comprises resistant starch (RS), yet the worst outcomes were with a 50:50 mixture of monosaccharides fructose and glucose. Our findings could explain the disparity between healthy, high-carbohydrate diets and the obesogenic impact of protein dilution by glucose-fructose mixtures associated with highly processed diets.

Metabolic Signatures in Coronary Artery Disease: Results from the BioHEART-CT Study.

Despite effective prevention programs targeting cardiovascular risk factors, coronary artery disease (CAD) remains the leading cause of death. Novel biomarkers are needed for improved risk stratification and primary prevention. To assess for independent associations between plasma metabolites and specific CAD plaque phenotypes we performed liquid chromatography mass-spectrometry on plasma from 1002 patients in the BioHEART-CT study. Four metabolites were examined as candidate biomarkers. Dimethylguanidino valerate (DMGV) was associated with presence and amount of CAD (OR) 1.41 (95% Confidence Interval [CI] 1.12-1.79, p = 0.004), calcified plaque, and obstructive CAD (p < 0.05 for both). The association with amount of plaque remained after adjustment for traditional risk factors, ß-coefficient 0.17 (95% CI 0.02-0.32, p = 0.026). Glutamate was associated with the presence of non-calcified plaque, OR 1.48 (95% CI 1.09-2.01, p = 0.011). Phenylalanine was associated with amount of CAD, ß-coefficient 0.33 (95% CI 0.04-0.62, p = 0.025), amount of calcified plaque, (ß-coefficient 0.88, 95% CI 0.23-1.53, p = 0.008), and obstructive CAD, OR 1.84 (95% CI 1.01-3.31, p = 0.046). Trimethylamine N-oxide was negatively associated non-calcified plaque OR 0.72 (95% CI 0.53-0.97, p = 0.029) and the association remained when adjusted for traditional risk factors. In targeted metabolomic analyses including 53 known metabolites and controlling for a 5% false discovery rate, DMGV was strongly associated with the presence of calcified plaque, OR 1.59 (95% CI 1.26-2.01, p = 0.006), obstructive CAD, OR 2.33 (95% CI 1.59-3.43, p = 0.0009), and amount of CAD, ß-coefficient 0.3 (95% CI 0.14-0.45, p = 0.014). In multivariate analyses the lipid and nucleotide metabolic pathways were both associated with the presence of CAD, after adjustment for traditional risk factors. We report novel associations between CAD plaque phenotypes and four metabolites previously associated with CAD. We also identified two metabolic pathways strongly associated with CAD, independent of traditional risk factors. These pathways warrant further investigation at both a biomarker and mechanistic level.

Nicotinamide riboside supplementation does not alter whole-body or skeletal muscle metabolic responses to a single bout of endurance exercise.

KEY POINTS: Acute nicotinamide riboside (NR) supplementation does not alter substrate metabolism at rest, during or in recovery from endurance exercise. NR does not alter NAD+ -sensitive signalling pathways in human skeletal muscle. NR supplementation and acute exercise influence the NAD+ metabolome. ABSTRACT: Oral supplementation of the NAD+ precursor nicotinamide riboside (NR) has been reported to alter metabolism alongside increasing sirtuin (SIRT) signalling and mitochondrial biogenesis in rodent skeletal muscle. However, whether NR supplementation can elicit a similar response in human skeletal muscle is unclear. This study assessed the effect of 7-day NR supplementation on whole-body metabolism and exercise-induced mitochondrial biogenic signalling in skeletal muscle. Eight male participants (age: 23 ± 4 years, V̇O2peak 46.5 ± 4.4 ml kg-1  min-1 ) received 1 week of NR or cellulose placebo (PLA) supplementation (1000 mg day-1 ). Muscle biopsies were collected from the medial vastus lateralis prior to supplementation and pre-, immediately post- and 3 h post-exercise (1 h of 60% Wmax cycling) performed following the supplementation period. There was no effect of NR supplementation on substrate utilisation at rest or during exercise or on skeletal muscle mitochondrial respiration. Global acetylation, auto-PARylation of poly ADP-ribose polymerase 1 (PARP1), acetylation of Tumour protein 53 (p53)Lys382 and Manganese superoxide dismutase (MnSOD)Lys122 were also unaffected by NR supplementation or exercise. NR supplementation did not increase skeletal muscle NAD+ concentration, but it did increase the concentration of deaminated NAD+ precursors nicotinic acid riboside (NAR) and nicotinic acid mononucleotide (NAM) and methylated nicotinamide breakdown products (Me2PY and Me4PY), demonstrating the skeletal muscle bioavailability of NR supplementation. In summary, 1 week of NR supplementation does not alter whole-body metabolism or skeletal muscle signal transduction pathways implicated in the mitochondrial adaptation to endurance exercise.

Plasma levels of trimethylamine-N-oxide can be increased with 'healthy' and 'unhealthy' diets and do not correlate with the extent of atherosclerosis but with plaque instability.

AIMS: The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has attracted major interest and controversy both as a diagnostic biomarker and therapeutic target in atherothrombosis. METHODS AND RESULTS: Plasma TMAO increased in mice on 'unhealthy' high-choline diets and notably also on 'healthy' high-fibre diets. Interestingly, TMAO was found to be generated by direct oxidation in the gut in addition to oxidation by hepatic flavin-monooxygenases. Unexpectedly, two well-accepted mouse models of atherosclerosis, ApoE-/- and Ldlr-/- mice, which reflect the development of stable atherosclerosis, showed no association of TMAO with the extent of atherosclerosis. This finding was validated in the Framingham Heart Study showing no correlation between plasma TMAO and coronary artery calcium score or carotid intima-media thickness (IMT), as measures of atherosclerosis in human subjects. However, in the tandem-stenosis mouse model, which reflects plaque instability as typically seen in patients, TMAO levels correlated with several characteristics of plaque instability, such as markers of inflammation, platelet activation, and intraplaque haemorrhage. CONCLUSIONS: Dietary-induced changes in the microbiome, of both 'healthy' and 'unhealthy' diets, can cause an increase in the plasma level of TMAO. The gut itself is a site of significant oxidative production of TMAO. Most importantly, our findings reconcile contradictory data on TMAO. There was no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability we demonstrated an association of TMAO plasma levels with atherosclerotic plaque instability. The latter confirms TMAO as being a marker of cardiovascular risk.