Trimethylamine N-oxide: heart of the microbiota-CVD nexus?

We critically review potential involvement of trimethylamine N-oxide (TMAO) as a link between diet, the gut microbiota and CVD. Generated primarily from dietary choline and carnitine by gut bacteria and hepatic flavin-containing mono-oxygenase (FMO) activity, TMAO could promote cardiometabolic disease when chronically elevated. However, control of circulating TMAO is poorly understood, and diet, age, body mass, sex hormones, renal clearance, FMO3 expression and genetic background may explain as little as 25 % of TMAO variance. The basis of elevations with obesity, diabetes, atherosclerosis or CHD is similarly ill-defined, although gut microbiota profiles/remodelling appear critical. Elevated TMAO could promote CVD via inflammation, oxidative stress, scavenger receptor up-regulation, reverse cholesterol transport (RCT) inhibition, and cardiovascular dysfunction. However, concentrations influencing inflammation, scavenger receptors and RCT (≥100 µm) are only achieved in advanced heart failure or chronic kidney disease (CKD), and greatly exceed pathogenicity of <1-5 µm levels implied in some TMAO-CVD associations. There is also evidence that CVD risk is insensitive to TMAO variance beyond these levels in omnivores and vegetarians, and that major TMAO sources are cardioprotective. Assessing available evidence suggests that modest elevations in TMAO (≤10 µm) are a non-pathogenic consequence of diverse risk factors (ageing, obesity, dyslipidaemia, insulin resistance/diabetes, renal dysfunction), indirectly reflecting CVD risk without participating mechanistically. Nonetheless, TMAO may surpass a pathogenic threshold as a consequence of CVD/CKD, secondarily promoting disease progression. TMAO might thus reflect early CVD risk while providing a prognostic biomarker or secondary target in established disease, although mechanistic contributions to CVD await confirmation.

Complex Effects of Putative DRP-1 Inhibitors on Stress Responses in Mouse Heart and Rat Cardiomyoblasts.

Dynamin-related protein-1 (DRP-1)-dependent mitochondrial fission may influence cardiac tolerance to ischemic or oxidative stress, presenting a potential "cardioprotective" target. Effects of dynamin inhibitors [mitochondrial division inhibitor 1 (MDIVI-1) and dynasore] on injury, mitochondrial function, and signaling proteins were assessed in distinct models: ischemia-reperfusion (I-R) in mouse hearts and oxidative stress in rat H9c2 cardiomyoblasts. Hearts exhibited substantial cell death [approx. 40 IU lactate dehydrogenase (LDH) efflux] and dysfunction (approx. 40 mmHg diastolic pressure, approx. 40% contractile recovery) following 25 minutes' ischemia. Pretreatment with 1 µM MDIVI-1 reduced dysfunction (30 mmHg diastolic pressure, approx. 55% recovery) and delayed without reducing overall cell death, whereas 5 µM MDIVI-1 reduced overall death at the same time paradoxically exaggerating dysfunction. Postischemic expression of mitochondrial DRP-1 and phospho-activation of ERK1/2 were reduced by MDIVI-1. Conversely, 1 µM dynasore worsened cell death and reduced nonmitochondrial DRP-1. Postischemic respiratory fluxes were unaltered by MDIVI-1, although a 50% fall in complex-I flux control ratio was reversed. In H9c2 myoblasts stressed with 400 µM H2O2, treatment with 50 µM MDIVI-1 preserved metabolic (MTT assay) and mitochondrial (basal respiration) function without influencing survival. This was associated with differential signaling responses, including reduced early versus increased late phospho-activation of ERK1/2, increased phospho-activation of protein kinase B (AKT), and differential changes in determinants of autophagy [reduced microtubule-associated protein 1 light chain 3b (LC3B-II/I) vs. increased Parkinson juvenile disease protein 2 (Parkin)] and apoptosis [reduced poly-(ADP-ribose) polymerase (PARP) cleavage vs. increased BCL2-associated X (BAX)/B-cell lymphoma 2 (BCL2)]. These data show MDIVI-1 (not dynasore) confers some benefit during I-R/oxidative stress. However, despite mitochondrial and metabolic preservation, MDIVI-1 exerts mixed effects on cell death versus dysfunction, potentially reflecting differential changes in survival kinase, autophagy, and apoptosis pathways. SIGNIFICANCE STATEMENT: Inhibition of mitochondrial fission is a novel approach to still elusive cardioprotection. Assessing effects of fission inhibitors on responses to ischemic or oxidative stress in hearts and cardiomyoblasts reveals mitochondrial division inhibitor 1 (MDIVI-1) and dynasore induce complex effects and limited cardioprotection. This includes differential impacts on death and dysfunction, survival kinases, and determinants of autophagy and apoptosis. Although highlighting the interconnectedness of fission and these key processes, results suggest MDIVI-1 and dynasore may be of limited value in the quest for effective cardioprotection.

Synergistic effects of low-level stress and a Western diet on metabolic homeostasis, mood, and myocardial ischemic tolerance.

How low-level psychological stress and overnutrition interact in influencing cardiometabolic disease is unclear. Mechanistic overlaps suggest potential synergies; however, findings are contradictory. We test whether low-level stress and Western diet (WD) feeding synergistically influence homeostasis, mood, and myocardial ischemic tolerance. Male C57BL6/J mice were fed a control diet or WD (32%/57%/11% calories from fat/carbohydrates/protein) for 12 wk, with subgroups restrained for 30 min/day over the final 3 wk. Metabolism, behavior, tolerance of perfused hearts to ischemia-reperfusion (I/R), and cardiac "death proteins" were assessed. The WD resulted in insignificant trends toward increased body weight (+5%), glucose (+40%), insulin (+40%), triglycerides (+15%), and cholesterol (+20%) and reduced leptin (-20%) while significantly reducing insulin sensitivity [100% rise in homeostasis model assessment of insulin resistance (HOMA-IR), P < 0.05]. Restraint did not independently influence metabolism while increasing HOMA-IR a further 50% (and resulting in significant elevations in insulin and glucose to 60-90% above control) in WD mice (P < 0.05), despite blunting weight gain in control and WD mice. Anxiogenesis with restraint or WD was nonadditive, whereas anhedonia (reduced sucrose consumption) only arose with their combination. Neuroinflammation markers (hippocampal TNF-α, Il-1b) were unchanged. Myocardial I/R tolerance was unaltered with stress or WD alone, whereas the combination worsened dysfunction and oncosis [lactate dehydrogenase (LDH) efflux]. Apoptosis (nucleosome accumulation) and death protein expression (BAK, BAX, BCL-2, RIP-1, TNF-α, cleaved caspase-3, and PARP) were unchanged. We conclude that mild, anxiogenic yet cardio-metabolically "benign" stress interacts synergistically with a WD to disrupt homeostasis, promote anhedonia (independently of neuroinflammation), and impair myocardial ischemic tolerance (independently of apoptosis and death protein levels).

Chronic type 2 but not type 1 diabetes impairs myocardial ischaemic tolerance and preconditioning in C57Bl/6 mice.

NEW FINDINGS: • What is the central question of this study? What is the impact of chronic adult-onset diabetes on cardiac ischaemic outcomes and preconditioning? • What is the main finding and its importance? Chronic adult-onset type 2 but not type 1 diabetes significantly impairs myocardial ischaemic tolerance and ischaemic preconditioning. Preconditioning may be detrimental in type 2 diabetes, exaggerating nitrosative stress and apoptotic protein expression. ABSTRACT: Effects of diabetes on myocardial responses to ischaemia-reperfusion (I-R) and cardioprotective stimuli remain contentious, potentially reflecting influences of disease duration and time of onset. Chronic adult-onset type 1 diabetes (T1D) and type 2 diabetes (T2D) were modelled non-genetically in male C57Bl/6 mice via 5 × 50 mg kg-1 daily streptozotocin (STZ) injections + 12 weeks' standard chow or 1 × 75 mg kg-1 STZ injection + 12 weeks' obesogenic diet (32% calories as fat, 57% carbohydrate, 11% protein), respectively. Systemic outcomes were assessed and myocardial responses to I-R ± ischaemic preconditioning (IPC; 3 × 5 min I-R) determined in Langendorff perfused hearts. Uncontrolled T1D was characterised by pronounced hyperglycaemia (25 mm fasting glucose), glucose intolerance and ∼10% body weight loss, whereas T2D mice exhibited moderate hyperglycaemia (15 mm), hyperinsulinaemia, glucose intolerance and 17% weight gain. Circulating ghrelin, resistin and noradrenaline were unchanged with T1D, while leptin increased and noradrenaline declined in T2D mice. Ischaemic tolerance and IPC were preserved in T1D hearts. In contrast, T2D worsened post-ischaemic function (∼40% greater diastolic and contractile dysfunction) and cell death (100% higher troponin efflux), and abolished IPC protection. Whereas IPC reduced post-ischaemic nitrotyrosine and pro-apoptotic Bak and Bax levels in non-diabetic hearts, these effects were reduced in T1D and IPC augmented Bax and nitrosylation in T2D hearts. The data demonstrate chronic T1D does not inhibit myocardial I-R tolerance or IPC, whereas metabolic and endocrine disruption in T2D is associated with ischaemic intolerance and inhibition of IPC. Indeed, normally protective IPC may exaggerate damage mechanisms in T2D hearts.

Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells.

Wound healing is an important outcome of tissue damage and can be stimulated by adenosine released from cells during events such as tissue injury, ischaemia or tumour growth. The aim of this research was to determine the potency and efficacy of adenosine A1, A2A and A2B receptor agonists on the rate of wound healing and cell proliferation in human EA.hy926 endothelial cells. Real-time PCR data showed that only adenosine A1, A2A and A2B receptor mRNA were expressed in this cell line. All three adenosine receptor agonists, CPA, CGS21680 and NECA, significantly increased the rate of wound healing in human EAhy926 endothelial cells with the following order of potency CGS21680>CPA>NECA and efficacy CPA>NECA>CGS21680. The selective adenosine A1, A2A and A2B receptor antagonists, DPCPX, ZM241385 and MRS1754 (all at 10 nM), reversed the effects of their respective agonists. EAhy926 endothelial cell proliferation was also significantly increased with the adenosine A1 and A2B receptor agonists, CPA and NECA. Western blot analysis demonstrated that adenosine A2A and A1 receptor protein levels were highly expressed compared with the adenosine A2B receptors in the EAhy926 endothelial cell lines. While all three adenosine A1, A2A and A2B receptor subtypes contribute to cell proliferation and wound healing in human EAhy926 endothelial cells, treatments selectively targeting receptor subtypes may further enhance wound healing.

Bilirubin acts as a multipotent guardian of cardiovascular integrity: more than just a radical idea.

Bilirubin, a potentially toxic catabolite of heme and indicator of hepatobiliary insufficiency, exhibits potent cardiac and vascular protective properties. Individuals with Gilbert's syndrome (GS) may experience hyperbilirubinemia in response to stressors including reduced hepatic bilirubin excretion/increased red blood cell breakdown, with individuals usually informed by their clinician that their condition is of little consequence. However, GS appears to protect from all-cause mortality, with progressively elevated total bilirubin associated with protection from ischemic heart and chronic obstructive pulmonary diseases. Bilirubin may protect against these diseases and associated mortality by reducing circulating cholesterol, oxidative lipid/protein modifications, and blood pressure. In addition, bilirubin inhibits platelet activation and protects the heart from ischemia-reperfusion injury. These effects attenuate multiple stages of the atherosclerotic process in addition to protecting the heart during resultant ischemic stress, likely underpinning the profound reduction in cardiovascular mortality in hyperbilirubinemic GS. This review outlines our current knowledge of and uses for bilirubin in clinical medicine and summarizes recent progress in revealing the physiological importance of this poorly understood molecule. We believe that this review will be of significant interest to clinicians, medical researchers, and individuals who have GS.

The brain-adipocyte-gut network: Linking obesity and depression subtypes.

Major depressive disorder (MDD) and obesity are dominant and inter-related health burdens. Obesity is a risk factor for MDD, and there is evidence MDD increases risk of obesity. However, description of a bidirectional relationship between obesity and MDD is misleading, as closer examination reveals distinct unidirectional relationships in MDD subtypes. MDD is frequently associated with weight loss, although obesity promotes MDD. In contrast, MDD with atypical features (MDD-AF) is characterised by subsequent weight gain and obesity. The bases of these distinct associations remain to be detailed, with conflicting findings clouding interpretation. These associations can be viewed within a systems biology framework-the psycho-immune neuroendocrine (PINE) network shared between MDD and metabolic disorders. Shared PINE subsystem perturbations may underlie increased MDD in overweight and obese people (obesity-associated depression), while obesity in MDD-AF (depression-associated obesity) involves more complex interactions between behavioural and biomolecular changes. In the former, the chronic PINE dysfunction triggering MDD is augmented by obesity-dependent dysregulation in shared networks, including inflammatory, leptin-ghrelin, neuroendocrine, and gut microbiome systems, influenced by chronic image-associated psychological stress (particularly in younger or female patients). In MDD-AF, behavioural dysregulation, including hypersensitivity to interpersonal rejection, fundamentally underpins energy imbalance (involving hyperphagia, lethargy, hypersomnia), with evolving obesity exaggerating these drivers via positive feedback (and potentially augmenting PINE disruption). In both settings, sex and age are important determinants of outcome, associated with differences in emotional versus cognitive dysregulation. A systems biology approach is recommended for further research into the pathophysiological networks underlying MDD and linking depression and obesity.

Effect of GPR84 deletion on obesity and diabetes development in mice fed long chain or medium chain fatty acid rich diets.

PURPOSE: Although there is good evidence showing that diets rich in medium chain fatty acids (MCFAs) have less marked obesogenic and diabetogenic effects than diets rich in long chain fatty acids (LCFAs), the role of the pro-inflammatory, medium chain fatty acid receptor (GPR84) in the aetiology of obesity and glucose intolerance is not well characterised. We set out to determine whether GPR84 expression influences obesity and glucose intolerance susceptibility in MCFA and LCFA rich diet fed mice. METHODS: Wild type (WT) and GPR84 knockout (KO) mice were fed a control, MCFA or LCFA diet, and body mass, heart, liver and epididymal fat mass was assessed, as well as glucose tolerance and adipocyte size. RESULTS: LCFA diets increased body mass and decreased glucose tolerance in both WT and GPR84 KO animals while MCFA diets had no effect on these parameters. There were no differences in body weight when comparing WT and GPR84 KO mice on the respective diets. Glucose tolerance was also similar in WT and GPR84 KO mice irrespective of diet. Liver mass was increased following LCFA feeding in WT but not GPR84 KO mice. Hepatic triglyceride content was increased in GPR84 KO animals fed MCFA, and myocardial triglyceride content was increased in GPR84 KO animals fed LCFA. CONCLUSIONS: GPR84 deletion had no effects on body weight or glucose tolerance in mice fed either a high MCFA or LCFA diet. GPR84 may influence lipid metabolism, as GPR84 KO mice had smaller livers and increased myocardial triglyceride accumulation when fed LCFA diets, and increased liver triglyceride accumulation in responses to increased dietary MCFAs.

Analysis of MCQ and distractor use in a large first year Health Faculty Foundation Program: assessing the effects of changing from five to four options.

BACKGROUND: Multiple choice questions are commonly used in summative assessment. It is still common practice for tertiary institutions and accrediting bodies to use five-option single best answer multiple choice questions, despite a substantial body of evidence showing that multiple choice questions with only three or four options provide effective and discriminatory assessment. METHODS: In this study we investigated the distribution of distractor efficacy in exams from four large first-year undergraduate courses in chemistry and in anatomy and physiology in a Health Faculty; assessed the impact on overall student score after changing from five-option to four-option single best answer multiple choice questions; and assessed the impact of changing from five options to four options on item difficulty and discrimination. RESULTS: For the five-option questions analysed, 19% had four effective distractors, which is higher than previous studies, but still a minority of questions. After changing from five to four options, the overall student performance on all multiple choice questions was slightly lower in the second offering of one course, slightly higher in the second offering of another course, and similar in the second offering for two courses. For a subset of questions that were used in both offerings, there were negligible differences in item difficulty and item discrimination between offerings. CONCLUSIONS: These results provide further evidence that five-option questions are not superior to four-option questions, with reduction to four options making little if any difference to overall performance, particularly when MCQ is used in conjunction with other assessment types (including short answer questions, and practical or laboratory assessment). Further areas of study that arise from these findings are: to investigate the reasons for resistance to changing established assessment practice within institutions and by accrediting bodies; and to analyse student perceptions of the impact of a reduced number of options in MCQ-based assessment.

Faster Blood Flow Rate Does Not Improve Circuit Life in Continuous Renal Replacement Therapy: A Randomized Controlled Trial.

OBJECTIVES: To determine whether blood flow rate influences circuit life in continuous renal replacement therapy. DESIGN: Prospective randomized controlled trial. SETTING: Single center tertiary level ICU. PATIENTS: Critically ill adults requiring continuous renal replacement therapy. INTERVENTIONS: Patients were randomized to receive one of two blood flow rates: 150 or 250 mL/min. MEASUREMENTS AND MAIN RESULTS: The primary outcome was circuit life measured in hours. Circuit and patient data were collected until each circuit clotted or was ceased electively for nonclotting reasons. Data for clotted circuits are presented as median (interquartile range) and compared using the Mann-Whitney U test. Survival probability for clotted circuits was compared using log-rank test. Circuit clotting data were analyzed for repeated events using hazards ratio. One hundred patients were randomized with 96 completing the study (150 mL/min, n = 49; 250 mL/min, n = 47) using 462 circuits (245 run at 150 mL/min and 217 run at 250 mL/min). Median circuit life for first circuit (clotted) was similar for both groups (150 mL/min: 9.1 hr [5.5-26 hr] vs 10 hr [4.2-17 hr]; p = 0.37). Continuous renal replacement therapy using blood flow rate set at 250 mL/min was not more likely to cause clotting compared with 150 mL/min (hazards ratio, 1.00 [0.60-1.69]; p = 0.68). Gender, body mass index, weight, vascular access type, length, site, and mode of continuous renal replacement therapy or international normalized ratio had no effect on clotting risk. Continuous renal replacement therapy without anticoagulation was more likely to cause clotting compared with use of heparin strategies (hazards ratio, 1.62; p = 0.003). Longer activated partial thromboplastin time (hazards ratio, 0.98; p = 0.002) and decreased platelet count (hazards ratio, 1.19; p = 0.03) were associated with a reduced likelihood of circuit clotting. CONCLUSIONS: There was no difference in circuit life whether using blood flow rates of 250 or 150 mL/min during continuous renal replacement therapy.

Myocyte membrane and microdomain modifications in diabetes: determinants of ischemic tolerance and cardioprotection.

Cardiovascular disease, predominantly ischemic heart disease (IHD), is the leading cause of death in diabetes mellitus (DM). In addition to eliciting cardiomyopathy, DM induces a 'wicked triumvirate': (i) increasing the risk and incidence of IHD and myocardial ischemia; (ii) decreasing myocardial tolerance to ischemia-reperfusion (I-R) injury; and (iii) inhibiting or eliminating responses to cardioprotective stimuli. Changes in ischemic tolerance and cardioprotective signaling may contribute to substantially higher mortality and morbidity following ischemic insult in DM patients. Among the diverse mechanisms implicated in diabetic impairment of ischemic tolerance and cardioprotection, changes in sarcolemmal makeup may play an overarching role and are considered in detail in the current review. Observations predominantly in animal models reveal DM-dependent changes in membrane lipid composition (cholesterol and triglyceride accumulation, fatty acid saturation vs. reduced desaturation, phospholipid remodeling) that contribute to modulation of caveolar domains, gap junctions and T-tubules. These modifications influence sarcolemmal biophysical properties, receptor and phospholipid signaling, ion channel and transporter functions, contributing to contractile and electrophysiological dysfunction, cardiomyopathy, ischemic intolerance and suppression of protective signaling. A better understanding of these sarcolemmal abnormalities in types I and II DM (T1DM, T2DM) can inform approaches to limiting cardiomyopathy, associated IHD and their consequences. Key knowledge gaps include details of sarcolemmal changes in models of T2DM, temporal patterns of lipid, microdomain and T-tubule changes during disease development, and the precise impacts of these diverse sarcolemmal modifications. Importantly, exercise, dietary, pharmacological and gene approaches have potential for improving sarcolemmal makeup, and thus myocyte function and stress-resistance in this ubiquitous metabolic disorder.

Dependence of Cardiac Systolic Function on Elevated Fatty Acid Availability in Obese, Insulin-Resistant Rats.

BACKGROUND: Clinical data advocating an adverse effect of obesity on left ventricular (LV) systolic function independent of comorbidities is controversial. We hypothesized that in obesity with prediabetic insulin resistance, circulating fatty acids (FAs) become a valuable fuel source in the maintenance of normal systolic function. METHODS: Male Wistar rats were fed a high caloric diet for 32 weeks to induce obesity. Myocardial LV systolic function was assessed using echocardiography and isolated heart preparations. RESULTS: Aortic output was reduced in obese rat hearts over a range of filling pressures (for example: 15 cmH2O, obese: 32.6 ± 1.2 ml/min vs control: 46.2 ± 0.9 ml/min, P < .05) when perfused with glucose alone. Similarly, the slope of the LV end-systolic pressure-volume relationship decreased, and there was a right shift in the LV end-systolic stress-strain relationship as determined in Langendorff perfused, isovolumic rat heart preparations in the presence of isoproterenol (10(-8)M) (LV systolic stress-strain relationship and a reduced load-independent intrinsic systolic myocardial function, obese: 791 ± 62 g/cm(2) vs control: 1186 ± 74 g/cm(2), P < .01). The addition of insulin to the perfusion buffer improved aortic output, whereas the addition of FAs completely normalized aortic output. LV function was maintained in obese animals in vivo during an inotropic challenge. CONCLUSIONS: Elevated circulating FA levels may be important to maintain myocardial systolic function in the initial stages of obesity and insulin resistance.

A refined high carbohydrate diet is associated with changes in the serotonin pathway and visceral obesity.

Consumption of palatable foods high in refined carbohydrate has been implicated as a contributing factor to the epidemic levels of obesity. Such foods may disrupt appetite regulation in the hypothalamus through alterations in hunger and satiety signalling. This investigation examined whether a palatable high refined carbohydrate (HRC) diet with the potential to induce obesity was linked to modulation of serotonin and dopamine signalling within the hypothalamus of rats. Male Wistar rats were allowed ad libitum access to either a palatable refined carbohydrate enriched (HRC) diet or standard chow (SC). Visceral fat percentage was used as a measure of the animals' weight gain during the trial. Real-time PCR was applied to determine any variation in levels of expression of the serotonin (Slc6A4 or Sert) and dopamine transporter (Slc6A3 or Dat) genes. After 29 weeks, the HRC group showed a significant increase in visceral fat percentage accompanied by increased expression of Sert. Higher levels of circulating triglycerides were also seen. This investigation determined that a refined high carbohydrate diet is associated with visceral obesity, increased circulating lipids in the blood and distorted serotonergic signalling, which possibly alters satiety and hunger signals.

Hyperbilirubinemia modulates myocardial function, aortic ejection, and ischemic stress resistance in the Gunn rat.

Mildly elevated circulating unconjugated bilirubin (UCB) is associated with protection against hypertension and ischemic heart disease. We assessed whether endogenously elevated bilirubin in Gunn rats modifies cardiovascular function and resistance to ischemic insult. Hearts were assessed ex vivo (Langendorff perfusion) and in vivo (Millar catheterization and echocardiography), and left ventricular myocardial gene expression was measured via quantitative real-time PCR. Ex vivo analysis revealed reduced intrinsic contractility in the Gunn myocardium (+dP/dt: 1,976 ± 622 vs. 2,907 ± 334 mmHg/s, P < 0.01; -dP/dt: -1,435 ± 372 vs. -2,234 ± 478 mmHg/s, P < 0.01), which correlated positively with myocardial UCB concentration (P < 0.05). In vivo analyses showed no changes in left ventricular contractile parameters and ejection (fractional shortening and ejection fraction). However, Gunn rats exhibited reductions in the rate of aortic pressure development (3,008 ± 461 vs. 4,452 ± 644 mmHg/s, P < 0.02), mean aortic velocity (439 ± 64 vs. 644 ± 62 mm/s, P < 0.01), and aortic volume time integral pressure gradient (2.32 ± 0.65 vs. 5.72 ± 0.74 mmHg, P < 0.01), in association with significant aortic dilatation (12-24% increase in aortic diameter, P < 0.05). Ex vivo Gunn hearts exhibited improved ventricular function after 35 min of ischemia and 90 min of reperfusion (63 ± 14 vs. 35 ± 12%, P < 0.01). These effects were accompanied by increased glutathione peroxidase and reduced superoxide dismutase and phospholamban gene expression in Gunn rat myocardium (P < 0.05). These data collectively indicate that hyperbilirubinemia in Gunn rats 1) reduces intrinsic cardiac contractility, which is compensated for in vivo; 2) induces aortic dilatation, which may beneficially influence aortic ejection velocities and pressures; and 3) may improve myocardial stress resistance in association with beneficial transcriptional changes. These effects may contribute to protection from cardiovascular disease with elevated bilirubin.

Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection.

Cholesterol-rich caveolar microdomains and associated caveolins influence sarcolemmal ion channel and receptor function and protective stress signaling. However, the importance of membrane cholesterol content to cardiovascular function and myocardial responses to ischemia-reperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-ß-cyclodextrin (MßCD) and lifelong knockout (KO) or overexpression (OE) of caveolin-3 (Cav-3) on cardiac function, I/R tolerance, and opioid receptor (OR)-mediated protection. Langendorff-perfused hearts from young male C57Bl/6 mice were untreated or treated with 0.02-1.0 mM MßCD for 25 min to deplete membrane cholesterol and disrupt caveolae. Hearts were subjected to 25-min ischemia/45-min reperfusion, and the cardioprotective effects of morphine applied either acutely or chronically [sustained ligand-activated preconditioning (SLP)] were assessed. MßCD concentration dependently reduced normoxic contractile function and postischemic outcomes in association with graded (10-30%) reductions in sarcolemmal cholesterol. Cardioprotection with acute morphine was abolished with ≥20 µM MßCD, whereas SLP was more robust and only inhibited with ≥200 µM MßCD. Deletion of Cav-3 also reduced, whereas Cav-3 OE improved, myocardial I/R tolerance. Protection via SLP remained equally effective in Cav-3 KO mice and was additive with innate protection arising with Cav-3 OE. These data reveal the membrane cholesterol dependence of normoxic myocardial and coronary function, I/R tolerance, and OR-mediated cardioprotection in murine hearts (all declining with cholesterol depletion). In contrast, baseline function appears insensitive to Cav-3, whereas cardiac I/R tolerance parallels Cav-3 expression. Novel SLP appears unique, being less sensitive to cholesterol depletion than acute OR protection and arising independently of Cav-3 expression.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Effect of chronic CPT-1 inhibition on myocardial ischemia-reperfusion injury (I/R) in a model of diet-induced obesity.

PURPOSE: By increasing circulating free fatty acids and the rate of fatty acid oxidation, obesity decreases glucose oxidation and myocardial tolerance to ischemia. Partial inhibition of fatty acid oxidation may improve myocardial tolerance to ischemia/reperfusion (I/R) in obesity. We assessed the effects of oxfenicine treatment on post ischemic cardiac function and myocardial infarct size in obese rats. METHODS: Male Wistar rats were fed a control diet or a high calorie diet which resulted in diet induced obesity (DIO) for 16 weeks. Oxfenicine (200 mg/kg/day) was administered to control and DIO rats for the last 8 weeks. Isolated hearts were perfused and infarct size and post ischemic cardiac function was assessed after regional or global ischemia and reperfusion. Cardiac mitochondrial function was assessed and myocardial expression and activity of CPT-1 (carnitine palmitoyl transferase-1) and IRS-1 (insulin receptor substrate-1) was assessed using Western blot analysis. RESULTS: In the DIO rats, chronic oxfenicine treatment improved post ischemic cardiac function and reduced myocardial infarct size after I/R but had no effect on the cardiac mitochondrial respiration. Chronic oxfenicine treatment worsened post ischemic cardiac function, myocardial infarct size and basal mitochondrial respiration in control rat hearts. Basal respiratory control index (RCI) values, state 2 and state 4 respiration rates and ADP phosphorylation rates were compromised by oxfenicine treatment. CONCLUSION: Chronic oxfenicine treatment improved myocardial tolerance to I/R in the obese rat hearts but decreased myocardial tolerance to I/R in control rat hearts. This decreased tolerance to ischemia of oxfenicine treated controls was associated with adverse changes in basal and reoxygenation mitochondrial function. These changes were absent in oxfenicine treated hearts from obese rats.

Effect of chronic restraint stress and western-diet feeding on colonic regulatory gene expression in mice.

BACKGROUND: Diet-induced obesity (DIO) and psychological stress are significant independent regulators of gastrointestinal physiology; however, our understanding of how these two disorders influence the host-microbe interface is still poorly characterized. The aim of this study was to assess the combined influences of diet-induced obesity and psychological stress on microbiome composition and colonic gene expression. METHODS: C57BL/6J mice (n = 48) were subject to a combination of 22 weeks of Western diet (WD) feeding and a chronic restraint stressor (CRS) for the last 4 weeks of feeding. At the end of the combined intervention, microbiome composition was determined from cecal contents, and colonic tissue gene expression was assessed by multiplex analysis using NanoString nCounter System and real-time qPCR. RESULTS: WD feeding induced a DIO phenotype with increased body weight, worsened metabolic markers, and alterations to microbiome composition. CRS reduced body weight in both dietary groups while having differential effects on glucose metabolism. CRS improved the Firmicutes/Bacteroidetes ratio in WD-fed animals while expanding the Proteobacteria phyla. Significantly lower expression of colonic Tlr4 (p = 0.008), Ocln (p = 0.004), and Cldn3 (p = 0.004) were noted in WD-fed animals compared to controls with no synergistic effects observed when combined with CRS. No changes to colonic expression of downstream inflammatory mediators were observed. Interestingly, higher levels of expression of Cldn2 (p = 0.04) and bile acid receptor Nr1h4 (p = 0.02) were seen in mice exposed to CRS. CONCLUSION: Differential but not synergistic effects of WD and CRS were noted at the host-microbe interface suggesting multifactorial responses that require further investigation.

Stress-induced body weight loss and improvements in cardiometabolic risk factors do not translate to improved myocardial ischemic tolerance in western diet-fed mice.

Although both diet-induced obesity and psychological stress are recognized as significant independent contributors to cardiometabolic and behavioral disorders, our understanding of how these two disorders interact and influence cardiometabolic risk and myocardial ischemic tolerance is limited. The aim of this study was to assess the combined effects of an obesogenic diet and psychological stress on cardiometabolic risk factors (body weight, dyslipidemia, insulin sensitivity) and postischemic cardiovascular outcomes. C57Bl/6J mice (n = 48) were subject to a combination of 22 weeks of western diet (WD) feeding and chronic restraint stress (CRS) for the last 4 weeks. Metabolic and behavioral changes were assessed using glucose tolerance tests and open field tests (OFTs), respectively. After 22 weeks, cardiac function and ischemic tolerance were assessed in Langendorff perfused hearts. WD feeding increased body weight and worsened blood lipids and insulin sensitivity. WD-fed mice also exhibited reduced exploratory behavior within the OFT. CRS reduced body weight and increased locomotion in both dietary groups and had differential effects on fasting glucose metabolism in the two dietary groups while not impacting non-fasting insulin. Although the WD only marginally reduced reperfusion left ventricular developed pressure recovery, CRS worsened reperfusion diastolic dysfunction in both dietary groups. Interestingly, despite WD+CRS animals exhibiting improved cardiometabolic parameters compared to the WD group, these changes did not translate to marked improvements to postischemic cardiac outcomes. In conclusion, in this study, combined WD feeding and CRS did not act synergistically to worsen cardiometabolic risk factors but instead improved them. Despite these cardiometabolic improvements, WD+CRS increased reperfusion end diastolic pressure which may be indicative of worsened ischemia/reperfusion injury.

Behavioural disruption in diabetic mice: Neurobiological correlates and influences of dietary α-linolenic acid.

AIM: Evolving type 2 diabetes (T2D) may influence locomotion and affective state, promoting metabolic dysfunction. We examined behaviour and neurobiology in a model of T2D, testing for benefits with dietary n-3 polyunsaturated fatty acid (PUFA). METHODS: Male C57Bl/6 mice received vehicle or 75 mg/kg streptozotocin (STZ) and 21 wks of control or Western diets (43 % fat, 40 % carbohydrate, 17 % protein). Sub-sets received dietary α-linolenic acid (ALA; 10 % of fat intake) for 6 wks. Behaviour was examined via open field and sucrose preference tests, and hippocampal and frontal cortex (FC) leptin and dopamine levels and inflammatory signalling assessed. KEY FINDINGS: T2D mice exhibited weight gain (+15 %), hyperglycemia (+35 %), hyperinsulinemia (+60 %) and insulin-resistance (+80 % higher HOMA-IR), together with anxiety-like behaviour (without anhedonia) that appeared independent of body weight and glycemic status. Cortical leptin declined whereas receptor mRNA increased. Supplementation with ALA did not influence metabolic state, while enhancing locomotion and reducing anxiety-like behaviours in healthy but not T2D mice. Hippocampal dopamine was selectively increased by ALA in T2D mice, with a trend to reduced circulating leptin in both groups. Across all groups, anxiety-like behaviour was associated with declining cortical and hippocampal leptin levels and increasing receptor mRNA, while declining dopamine levels were accompanied by decreased dopamine/serotonin receptor transcripts. SIGNIFICANCE: Chronic T2D induced anxiogenesis in mice appears to be independent of metabolic homeostasis but linked to central leptin-resistance, together with disturbed dopamine and serotonin signalling. Despite anxiolytic effects of ALA in healthy mice, no metabolic or behavioural benefits were evident in T2D.