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.

A gut microbiome metabolite paradoxically depresses contractile function while activating mitochondrial respiration.

Trimethylamine-N-oxide (TMAO) is an end-product of gut microbiome metabolism linked to cardiovascular disease (CVD). However, precise cardiovascular influences of the TMAO concentrations reported in early or severe disease remain to be detailed. We investigated acute effects of TMAO on cardiac contractile, coronary and mitochondrial function. Male C57Bl/6 mouse hearts were Langendorff perfused to assess concentration-dependent effects of TMAO (1-300 µM) on left ventricular (LV) function, coronary flow and select protein expression. Effects of 10 µM and 100 µM TMAO on LV mitochondrial function were examined via respirometry. TMAO at 10-300 µM concentration-dependently depressed LV contractile function, with coronary flow paralleling changes in isovolumic pressure development. Direct coronary effects were evident at >30 µM TMAO in hearts performing minimal isovolumic work, although this response was reduced by >65%. In contrast, exposure to 10 µM or 100 µM TMAO increased mitochondrial complex I, II and maximal respiratory fluxes while appearing to reduce outer membrane integrity. Expression of phosphorylated AMPKα and total GSK-3ß declined. Thus, acute exposure of mouse hearts to TMAO levels reported in advanced CVD significantly inhibits cardiac contractility and induces modest coronary constriction while paradoxically overactivating mitochondrial respiration.

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.

Sex-specific behavioral, neurobiological, and cardiovascular responses to chronic social stress in mice.

Psychosocial stress promotes and links mood and cardiovascular disorders in a sex-specific manner. However, findings in animal models are equivocal, in some cases opposing human dimorphisms. We examined central nervous system (CNS), behavioral, endocrine, cardiac, and hepatic outcomes in male or female C57Bl/6 mice subjected to chronic social stress (56 days of social isolation, with intermittent social confrontation encounters twice daily throughout the final 20 days). Females exhibited distinct physiological and behavioral changes, including relative weight loss, and increases in coronary resistance, hepatic inflammation, and thigmotaxic behavior in the open field. Males evidence reductions in coronary resistance and cardiac ischemic tolerance, with increased circulating and hippocampal monoamine levels and emerging anhedonia. Shared CNS gene responses include reduced hippocampal Maoa and increased Htr1b expression, while unique responses include repression of hypothalamic Ntrk1 and upregulation of cortical Nrf2 and Htr1b in females; and repression of hippocampal Drd1 and hypothalamic Gabra1 and Oprm in males. Declining cardiac stress resistance in males was associated with repression of cardiac leptin levels and metabolic, mitochondrial biogenesis, and anti-inflammatory gene expression. These integrated data reveal distinct biological responses to social stress in males and females, and collectively evidence greater biological disruption or allostatic load in females (consistent with propensities to stress-related mood and cardiovascular disorders in humans). Distinct stress biology, and molecular to organ responses, emphasize the importance of sex-specific mechanisms and potential approaches to stress-dependent disease.

Central and cardiac stress resilience consistently linked to integrated immuno-neuroendocrine responses across stress models in male mice.

Stress resilience, and behavioural and cardiovascular impacts of chronic stress, are theorised to involve integrated neuro-endocrine/inflammatory/transmitter/trophin signalling. We tested for this integration, and whether behaviour/emotionality, together with myocardial ischaemic tolerance, are consistently linked to these pathways across diverse conditions in male C57Bl/6 mice. This included Restraint Stress (RS), 1 h restraint/day for 14 days; Chronic Unpredictable Mild Stress (CUMS), seven stressors randomised over 21 days; Social Stress (SS), 35 days social isolation with brief social encounters in final 13 days; and Control conditions (CTRL; un-stressed mice). Behaviour was assessed via open field (OFT) and sucrose preference (SPT) tests, and neurobiology from frontal cortex (FC) and hippocampal transcripts. Endocrine factors, and function and ischaemic tolerance in isolated hearts, were also measured. Model characteristics ranged from no behavioural or myocardial changes with homotypic RS, to increased emotionality and cardiac ischaemic injury (with apparently distinct endocrine/neurobiological profiles) in CUMS and SS models. Highly integrated expression of HPA axis, neuro-inflammatory, BDNF, monoamine, GABA, cannabinoid and opioid signalling genes was confirmed across conditions, and consistent/potentially causal correlations identified for (i) locomotor activity (noradrenaline, ghrelin; FC Crhr1, Tnfrsf1b, Il33, Nfkb1, Maoa, Gabra1; hippocampal Il33); (ii) thigmotaxis (adrenaline, leptin); (iii) anxiety-like behaviour (adrenaline, leptin; FC Tnfrsf1a; hippocampal Il33); (iv) depressive-like behaviour (ghrelin; FC/hippocampal s100a8); and (v) cardiac stress-resistance (noradrenaline, leptin; FC Il33, Tnfrsf1b, Htr1a, Gabra1, Gabrg2; hippocampal Il33, Tnfrsf1a, Maoa, Drd2). Data support highly integrated pathway responses to stress, and consistent adipokine, sympatho-adrenergic, inflammatory and monoamine involvement in mood and myocardial disturbances across diverse conditions.

Piezo1 regulates shear-dependent nitric oxide production in human erythrocytes.

Mature circulating red blood cells (RBCs) are classically viewed as passive participants in circulatory function, given erythroblasts eject their organelles during maturation. Endogenous production of nitric oxide (NO) and its effects are of particular significance; however, the integration between RBC sensation of the local environment and subsequent activation of mechano-sensitive signaling networks that generate NO remain poorly understood. The present study investigated endogenous NO production via the RBC-specific nitric oxide synthase isoform (RBC-NOS), connecting membrane strain with intracellular enzymatic processes. Isolated RBCs were obtained from apparently healthy humans. Intracellular NO was compared at rest and following shear (cellular deformation) using semiquantitative fluorescent imaging. Concurrently, RBC-NOS phosphorylation at its serine1177 (Ser1177) residue was measured. The contribution of cellular deformation to shear-induced NO production in RBCs was determined by rigidifying RBCs with the thiol-oxidizing agent diamide; rigid RBCs exhibited significantly impaired (up to 80%) capacity to generate NO via RBC-NOS during shear. Standardizing membrane strain of rigid RBCs by applying increased shear did not normalize NO production, or RBC-NOS activation. Calcium imaging with fluo-4 revealed that diamide-treated RBCs exhibited a 42% impairment in Piezo1-mediated calcium movement when compared with untreated RBCs. Pharmacological inhibition of Piezo1 with GsMTx4 during shear inhibited RBC-NOS activation in untreated RBCs, whereas Piezo1 activation with Yoda1 in the absence of shear stimulated RBC-NOS activation. Collectively, a novel, mechanically activated signaling pathway in mature RBCs is described. Opening of Piezo1 and subsequent influx of calcium appear to be required for endogenous production of NO in response to mechanical shear, which is accompanied by phosphorylation of RBC-NOS at Ser1177.NEW & NOTEWORTHY The mechano-sensitive ion channel Piezo1 is expressed in enucleated red blood cells and provides a mechanism of shear-induced red cell nitric oxide production via nitric oxide synthase phosphorylation. Thiol oxidation of red cells decreases Piezo1-dependent calcium movement and thus impairs nitric oxide generation in response to mechanical force. The emerging descriptions of exclusively posttranslational signaling networks in circulating red cells as acute regulators of cell function support that these cells play an important role in cardiovascular physiology that extends beyond passive oxygen transport.

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.

Increased release of serotonin from rat primary isolated adult cardiac myofibroblasts.

Elevated blood serotonin levels have been observed in patients with heart failure and serotonin has a role in pathological cardiac function. The serotonin receptor system was examined in adult rat isolated cardiac fibroblast and myofibroblast cells. This is one of the first studies that has investigated serotonin receptors and other proteins involved in the serotonin receptor system in rat cardiac fibroblast and myofibroblast cells. Rat primary cardiac fibroblasts were isolated and transformed into myofibroblasts using 5 ng/ml TGF-ß1. Transformation of cells to myofibroblasts was confirmed with the presence of α-smooth muscle actin using Western blot. Serotonin metabolism and receptor protein expression was assessed using Western blot techniques and serotonin levels measured using ELISA. The 5-HT1A, 5-HT2A and 5-HT2B receptors were found to be present in both rat cardiac fibroblasts and myofibroblast cells, however no significance in protein expression between the two cell types was found (P > 0.05). In this study a significant increase in the serotonin transporter (SERT), tryptophan hydroxylase 1 and extracellular serotonin levels was observed in rat cardiac myofibroblasts when compared to fibroblasts (P < 0.05). These results suggest that serotonin levels may rise in parallel with cardiac myofibroblast populations and contribute to the pathogenesis of heart failure via serotonin receptors.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

A YOLO based software for automated detection and analysis of rodent behaviour in the open field arena.

Rodent models are important in mechanistic studies of the physiological and pathophysiological determinants of behaviour. The Open Field Test (OFT) is one of the most commonly utilised tests to assess rodent behaviour in a novel open environment. The key variables assessed in an OFT are general locomotor activity and exploratory behaviours and can be assessed manually or by automated systems. Although several automated systems exist, they are often expensive, difficult to use, or limited in the type of video that can be analysed. Here we describe a machine-learning algorithm - dubbed Cosevare - that uses a trained YOLOv3 DNN to identify and track movement of mice in the open-field arena. We validated Cosevare's capacity to accurately track locomotive and exploratory behaviour in 10 videos, comparing outputs generated by Cosevare with analysis by 5 manual scorers. Behavioural differences between control mice and those with diet-induced obesity (DIO) were also documented. We found the YOLOv3 based tracker to be accurate at identifying and tracking the mice within the open-field arena and in instances with variable backgrounds. Additionally, kinematic and spatial-based analysis demonstrated highly consistent scoring of locomotion, centre square duration (CSD) and entries (CSE) between Cosevare and manual scorers. Automated analysis was also able to distinguish behavioural differences between healthy control and DIO mice. The study found that a YOLOv3 based tracker is able to easily track mouse behaviour in the open field arena and supports machine learning as a potential future alternative for the assessment of animal behaviour in a wide range of species in differing environments and behavioural tests.

Compromised right ventricular contractility in an ovine model of heart transplantation following 24 h donor brain stem death.

BACKGROUND: Heart failure is an inexorably progressive disease with a high mortality, for which heart transplantation (HTx) remains the gold standard treatment. Currently, donor hearts are primarily derived from patients following brain stem death (BSD). BSD causes activation of the sympathetic nervous system, increases endothelin levels, and triggers significant inflammation that together with potential myocardial injury associated with the transplant procedure, may affect contractility of the donor heart. We examined peri-transplant myocardial catecholamine sensitivity and cardiac contractility post-BSD and transplantation in a clinically relevant ovine model. METHODS: Donor sheep underwent BSD (BSD, n = 5) or sham (no BSD) procedures (SHAM, n = 4) and were monitored for 24h prior to heart procurement. Orthotopic HTx was performed on a separate group of donor animals following 24h of BSD (BSD-Tx, n = 6) or SHAM injury (SH-Tx, n = 5). The healthy recipient heart was used as a control (HC, n = 11). A cumulative concentration-effect curve to (-)-noradrenaline (NA) was established using left (LV) and right ventricular (RV) trabeculae to determine ß1-adrenoceptor mediated potency (-logEC50 [(-)-noradrenaline] M) and maximal contractility (Emax). RESULTS: Our data showed reduced basal and maximal (-)-noradrenaline induced contractility of the RV (but not LV) following BSD as well as HTx, regardless of whether the donor heart was exposed to BSD or SHAM. The potency of (-)-noradrenaline was lower in left and right ventricles for BSD-Tx and SH-Tx compared to HC. CONCLUSION: These studies show that the combination of BSD and transplantation are likely to impair contractility of the donor heart, particularly for the RV. For the donor heart, this contractile dysfunction appears to be independent of changes to ß1-adrenoceptor sensitivity. However, altered ß1-adrenoceptor signalling is likely to be involved in post-HTx contractile dysfunction.

Effects of voluntary exercise duration on myocardial ischaemic tolerance, kinase signaling and gene expression.

AIM: Exercise is cardioprotective, though optimal interventions are unclear. We assessed duration dependent effects of exercise on myocardial ischemia-reperfusion (I-R) injury, kinase signaling and gene expression. METHODS: Responses to brief (2 day; 2EX), intermediate (7 and 14 day; 7EX and 14EX) and extended (28 day; 28EX) voluntary wheel running (VWR) were studied in male C57Bl/6 mice. Cardiac function, I-R tolerance and survival kinase signaling were assessed in perfused hearts. KEY FINDINGS: Mice progressively increased running distances and intensity, from 2.4 ± 0.2 km/day (0.55 ± 0.04 m/s) at 2-days to 10.6 ± 0.4 km/day (0.72 ± 0.06 m/s) after 28-days. Myocardial mass and contractility were modified at 14-28 days VWR. Cardioprotection was not 'dose-dependent', with I-R tolerance enhanced within 7 days and not further improved with greater VWR duration, volume or intensity. Protection was associated with AKT, ERK1/2 and GSK3ß phosphorylation, with phospho-AMPK selectively enhanced with brief VWR. Gene expression was duration-dependent: 7 day VWR up-regulated glycolytic (Pfkm) and down-regulated maladaptive remodeling (Mmp2) genes; 28 day VWR up-regulated caveolar (Cav3), mitochondrial biogenesis (Ppargc1a, Sirt3) and titin (Ttn) genes. Interestingly, I-R tolerance in 2EX/2SED groups improved vs. groups subjected to longer sedentariness, suggesting transient protection on transition to housing with running wheels. SIGNIFICANCE: Cardioprotection is induced with as little as 7 days VWR, yet not enhanced with further or faster running. This protection is linked to survival kinase phospho-regulation (particularly AKT and ERK1/2), with glycolytic, mitochondrial, caveolar and myofibrillar gene changes potentially contributing. Intriguingly, environmental enrichment may also protect via similar kinase regulation.

Morphine induces physiological, structural, and molecular benefits in the diabetic myocardium.

The obesity epidemic has increased type II diabetes mellitus (T2DM) across developed countries. Cardiac T2DM risks include ischemic heart disease, heart failure with preserved ejection fraction, intolerance to ischemia-reperfusion (I-R) injury, and refractoriness to cardioprotection. While opioids are cardioprotective, T2DM causes opioid receptor signaling dysfunction. We tested the hypothesis that sustained opioid receptor stimulus may overcome diabetes mellitus-induced cardiac dysfunction via membrane/mitochondrial-dependent protection. In a murine T2DM model, we investigated effects of morphine on cardiac function, I-R tolerance, ultrastructure, subcellular cholesterol expression, mitochondrial protein abundance, and mitochondrial function. T2DM induced 25% weight gain, hyperglycemia, glucose intolerance, cardiac hypertrophy, moderate cardiac depression, exaggerated postischemic myocardial dysfunction, abnormalities in mitochondrial respiration, ultrastructure and Ca2+ -induced swelling, and cell death were all evident. Morphine administration for 5 days: (1) improved glucose homeostasis; (2) reversed cardiac depression; (3) enhanced I-R tolerance; (4) restored mitochondrial ultrastructure; (5) improved mitochondrial function; (6) upregulated Stat3 protein; and (7) preserved membrane cholesterol homeostasis. These data show that morphine treatment restores contractile function, ischemic tolerance, mitochondrial structure and function, and membrane dynamics in type II diabetic hearts. These findings suggest potential translational value for short-term, but high-dose morphine administration in diabetic patients undergoing or recovering from acute ischemic cardiovascular events.

Peritransplant Cardiometabolic and Mitochondrial Function: The Missing Piece in Donor Heart Dysfunction and Graft Failure.

Primary graft dysfunction is an important cause of morbidity and mortality after cardiac transplantation. Donor brain stem death (BSD) is a significant contributor to donor heart dysfunction and primary graft dysfunction. There remain substantial gaps in the mechanistic understanding of peritransplant cardiac dysfunction. One of these gaps is cardiac metabolism and metabolic function. The healthy heart is an "omnivore," capable of utilizing multiple sources of nutrients to fuel its enormous energetic demand. When this fails, metabolic inflexibility leads to myocardial dysfunction. Data have hinted at metabolic disturbance in the BSD donor and subsequent heart transplantation; however, there is limited evidence demonstrating specific metabolic or mitochondrial dysfunction. This review will examine the literature surrounding cardiometabolic and mitochondrial function in the BSD donor, organ preservation, and subsequent cardiac transplantation. A more comprehensive understanding of this subject may then help to identify important cardioprotective strategies to improve the number and quality of donor hearts.

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.

Dietary α-Linolenic Acid Counters Cardioprotective Dysfunction in Diabetic Mice: Unconventional PUFA Protection.

Whether dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) confers cardiac benefit in cardiometabolic disorders is unclear. We test whether dietary -linolenic acid (ALA) enhances myocardial resistance to ischemia-reperfusion (I-R) and responses to ischemic preconditioning (IPC) in type 2 diabetes (T2D); and involvement of conventional PUFA-dependent mechanisms (caveolins/cavins, kinase signaling, mitochondrial function, and inflammation). Eight-week male C57Bl/6 mice received streptozotocin (75 mg/kg) and 21 weeks high-fat/high-carbohydrate feeding. Half received ALA over six weeks. Responses to I-R/IPC were assessed in perfused hearts. Localization and expression of caveolins/cavins, protein kinase B (AKT), and glycogen synthase kinase-3 ß (GSK3ß); mitochondrial function; and inflammatory mediators were assessed. ALA reduced circulating leptin, without affecting body weight, glycemic dysfunction, or cholesterol. While I-R tolerance was unaltered, paradoxical injury with IPC was reversed to cardioprotection with ALA. However, post-ischemic apoptosis (nucleosome content) appeared unchanged. Benefit was not associated with shifts in localization or expression of caveolins/cavins, p-AKT, p-GSK3ß, or mitochondrial function. Despite mixed inflammatory mediator changes, tumor necrosis factor-a (TNF-a) was markedly reduced. Data collectively reveal a novel impact of ALA on cardioprotective dysfunction in T2D mice, unrelated to caveolins/cavins, mitochondrial, or stress kinase modulation. Although evidence suggests inflammatory involvement, the basis of this "un-conventional" protection remains to be identified.

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).

Calcium dynamically alters erythrocyte mechanical response to shear.

Red blood cells (RBC) are constantly exposed to varying mechanical forces while traversing the cardiovascular system. Upon exposure to mechanical stimuli (e.g., shear stress), calcium enters the cell and prompts potassium-efflux. Efflux of potassium is accompanied by a loss of intracellular fluid; thus, the volume of RBC decreases proportionately (i.e., 'Gárdos effect'). The mechanical properties of the cell are subsequently impacted due to complex interactions between cytosolic viscosity (dependent on cell hydration), the surface-area-to-volume ratio, and other molecular processes. The dynamic effects of calcium on RBC mechanics are yet to be elucidated, although accumulating evidence suggests a vital role. The present study thus examined the effects of calcium on contemporary biomechanical properties of RBC in conjunction with high-precision geometrical analyses with exposure to shear. Mechanical stimulation of RBC was performed using a co-axial Couette shearing system to deform the cell membrane; intracellular signaling events were observed via fluorescent imaging. Calcium was introduced into RBC using ionophore A23187. Increased intracellular calcium significantly impaired RBC deformability; these impairments were mediated by a calcium-induced reduction of cell volume through the Gárdos channel. Extracellular calcium in the absence of the ionophore only had an effect under shear, not at stasis. Under low shear, the presence of extracellular calcium induced progressive lysis of a sub-population of RBC; all remaining RBC exhibited exceptional capacity to deform, implying preferential removal of potentially aged cells. Collectively, we provide evidence of the mechanism by which calcium acutely regulates RBC mechanical properties.

Active modulation of human erythrocyte mechanics.

The classic view of the red blood cell (RBC) presents a biologically inert cell that upon maturation has limited capacity to alter its physical properties. This view developed largely because of the absence of translational machinery and inability to synthesize or repair proteins in circulating RBC. Recent developments have challenged this perspective, in light of observations supporting the importance of posttranslational modifications and greater understanding of ion movement in these cells, that each regulate a myriad of cellular properties. There is thus now sufficient evidence to induce a step change in understanding of RBC: rather than passively responding to the surrounding environment, these cells have the capacity to actively regulate their physical properties and thus alter flow behavior of blood. Specific evidence supports that the physical and rheological properties of RBC are subject to active modulation, primarily by the second-messenger molecules nitric oxide (NO) and calcium-ions (Ca2+). Furthermore, an isoform of nitric oxide synthase is expressed in RBC (RBC-NOS), which has been recently demonstrated to have an active role in regulating the physical properties of RBC. Mechanical stimulation of the cell membrane activates RBC-NOS, leading to NO generation, which has several intracellular effects, including the S-nitrosylation of integral membrane components. Intracellular concentration of Ca2+ is increased upon mechanical stimulation via the recently identified mechanosensitive cation channel piezo1. Increased intracellular Ca2+ modifies the physical properties of RBC by regulating cell volume and potentially altering several important intracellular proteins. A synthesis of recent advances in understanding of molecular processes within RBC thus challenges the classic view of these cells and rather indicates a highly active cell with self-regulated mechanical properties.

Cardiomyoblast caveolin expression: Effects of simulated diabetes, α-linolenic acid and cell signaling pathways.

Caveolins regulate myocardial substrate handling, survival signaling and stress-resistance, however control of expression is incompletely defined. We test how metabolic features of type 2 diabetes (T2D), and modulation of cell signaling, influence caveolins in H9c2 cardiomyoblasts. Cells were exposed to glucose (25 vs. 5 mM), insulin (100 nM) or palmitate (0.1 mM), individually or combined, and effects of adenylate cyclase (AC) activation (50 µM forskolin), focal adhesion kinase (FAK) or protein kinase C b2 (PKCß2) inhibition (1 µM FAK Inhibitor 14 or CGP-53353, respectively), or the polyunsaturated fatty acid (PUFA) α-linolenic acid (ALA; 10 µM) were tested. Simulated T2D (elevated glucose+insulin+palmitate) depressed caveolin-1 and -3 without modifying caveolin-2. Caveolin-3 repression was primarily palmitate dependent, whereas high glucose (HG) and insulin independently increased caveolin-3 (yet reduced expression when combined). Differential control was evident: baseline caveolin-3 was suppressed by FAK/PKCß2 and insensitive to AC activities, with baseline caveolin-1 and -2 suppressed by AC and insensitive to FAK/PKCß2. Forskolin and ALA selectively preserved caveolin-3 in T2D cells, whereas PKCb2 and FAK inhibition increased caveolin-3 under all conditions. Despite preservation of caveolin-3, ALA did not modify nucleosome content (apoptosis marker) or transcription of pro-inflammatory mediators in T2D cells. In summary: caveolin-1 and -3 are strongly repressed with simulated T2D, with caveolin-3 particularly sensitive to palmitate; intrinsic PKCb2 and FAK activities repress caveolin-3 in healthy and stressed cells; ALA, AC activation and PKCß2 inhibition preserve caveolin-3 under T2D conditions; and caveolin-3 changes with T2D and ALA appear unrelated to inflammatory signaling and extent of apoptosis.