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.

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.

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.

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.

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.

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.

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.

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).

Biomarkers for the identification of cardiac fibroblast and myofibroblast cells.

Experimental research has recognized the importance of cardiac fibroblast and myofibroblast cells in heart repair and function. In a normal healthy heart, the cardiac fibroblast plays a central role in the structural, electrical, and chemical aspects within the heart. Interestingly, the transformation of cardiac fibroblast cells to cardiac myofibroblast cells is suspected to play a vital part in the development of heart failure. The ability to differentiate between the two cells types has been a challenge. Myofibroblast cells are only expressed in the stressed or failing heart, so a better understanding of cell function may identify therapies that aid repair of the damaged heart. This paper will provide an outline of what is currently known about cardiac fibroblasts and myofibroblasts, the physiological and pathological roles within the heart, and causes for the transition of fibroblasts into myoblasts. We also reviewed the potential markers available for characterizing these cells and found that there is no single-cell specific marker that delineates fibroblast or myofibroblast cells. To characterize the cells of fibroblast origin, vimentin is commonly used. Cardiac fibroblasts can be identified using discoidin domain receptor 2 (DDR2) while α-smooth muscle actin is used to distinguish myofibroblasts. A known cytokine TGF-ß1 is well established to cause the transformation of cardiac fibroblasts to myofibroblasts. This review will also discuss clinical treatments that inhibit or reduce the actions of TGF-ß1 and its contribution to cardiac fibrosis and heart failure.

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.

Delta Opioid Receptors and Cardioprotection.

The opioid receptor family, with associated endogenous ligands, has numerous roles throughout the body. Moreover, the delta opioid receptor (DORs) has various integrated roles within the physiological systems, including the cardiovascular system. While DORs are important modulators of cardiovascular autonomic balance, they are well-established contributors to cardioprotective mechanisms. Both endogenous and exogenous opioids acting upon DORs have roles in myocardial hibernation and protection against ischaemia-reperfusion (I-R) injury. Downstream signalling mechanisms governing protective responses alternate, depending on the timing and duration of DOR activation. The following review describes models and mechanisms of DOR-mediated cardioprotection, the impact of co-morbidities and challenges for clinical translation.

The heartbreak of depression: 'Psycho-cardiac' coupling in myocardial infarction.

Ample evidence identifies strong links between major depressive disorder (MDD) and both risk of ischemic or coronary heart disease (CHD) and resultant morbidity and mortality. The molecular mechanistic bases of these linkages are poorly defined. Systemic factors linked to MDD, including vascular dysfunction, atherosclerosis, obesity and diabetes, together with associated behavioral changes, all elevate CHD risk. Nonetheless, experimental evidence indicates the myocardium is also directly modified in depression, independently of these factors, impairing infarct tolerance and cardioprotection. It may be that MDD effectively breaks the heart's intrinsic defense mechanisms. Four extrinsic processes are implicated in this psycho-cardiac coupling, presenting potential targets for therapeutic intervention if causally involved: sympathetic over-activity vs. vagal under-activity, together with hypothalamic-pituitary-adrenal (HPA) axis and immuno-inflammatory dysfunctions. However, direct evidence of their involvement remains limited, and whether targeting these upstream mediators is effective (or practical) in limiting the cardiac consequences of MDD is unknown. Detailing myocardial phenotype in MDD can also inform approaches to cardioprotection, yet cardiac molecular changes are similarly ill defined. Studies support myocardial sensitization to ischemic insult in models of MDD, including worsened oxidative and nitrosative damage, apoptosis (with altered Bcl-2 family expression) and infarction. Moreover, depression may de-sensitize hearts to protective conditioning stimuli. The mechanistic underpinnings of these changes await delineation. Such information not only advances our fundamental understanding of psychological determinants of health, but also better informs management of the cardiac consequences of MDD and implementing cardioprotection in this cohort.

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.

Bitter taste receptor agonists elicit G-protein-dependent negative inotropy in the murine heart.

G-protein-coupled receptors (GPCRs) are key mediators in cardiovascular physiology, yet frontline therapies for heart disease target only a small fraction of the cardiac GPCR repertoire. Moreover, there is emerging evidence that GPCRs implicated in taste (Tas1r and Tas2rs) have specific functions beyond the oral cavity. Our recent description of these receptors in heart tissue foreshadows a potential novel role in cardiac cells. In this study, we identified novel agonist ligands for cardiac-Tas2rs to enable the functional investigation of these receptors in heart tissue. Five Tas2rs cloned from heart tissue were screened against a panel of 102 natural or synthetic bitter compounds in a heterologous expression system. We identified agonists for Tas2r108, Tas2r137, and Tas2r143 that were then tested in Langendorff-perfused mouse hearts (from 8-wk-old male C57BL/6 mice). All Tas2r agonists tested exhibited concentration-dependent effects, with agonists for Tas2r108 and Tas2r137, leading to a ∼40% decrease in left ventricular developed pressure and an increase in aortic pressure, respectively. These responses were abrogated in the presence of Gαi and Gßγ inhibitors (pertussis toxin and gallein). This study represents the first demonstration of profound Tas2r agonist-induced, G protein-dependent effects on mouse heart function.

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.

Postnatal shifts in ischemic tolerance and cell survival signaling in murine myocardium.

The immature heart is known to be resistant to ischemia-reperfusion (I/R) injury; however, key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the postnatal changes in myocardial tolerance to I/R, including baseline expression of key proteins governing I/R tolerance and their phosphorylation during I/R. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 wk of age, n = 6/group) were assayed for survival signaling/effectors [Akt, p38MAPK, glycogen synthase kinase-3ß (GSK-3ß), heat shock protein 27 (HSP27), connexin-43, hypoxia-inducible factor-1α (HIF-1α), and caveolin-3] and regulators of apoptosis (Bax and Bcl-2) and autophagy (LC3B, Parkin, and Beclin1). The effect of I/R on ventricular function was measured in isolated perfused hearts from immature (4 wk) and adult (12 wk) mice. The neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phospho-inhibition of GSK-3ß; and high expression of caveolin-3, HIF-1α, LC3B, Beclin1, Bax, and Bcl-2. Immature hearts sustained less dysfunction and infarction following I/R than adults. Emergence of I/R intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax, and Bcl-2; increased GSK-3ß, connexin-43, HIF-1α, LC3B, and Bax:Bcl-2; enhanced postischemic HIF-1α, caveolin-3, Bax, and Bcl-2; and greater postischemic GSK-3ß and HSP27 phosphorylation. Neonatal myocardial stress resistance reflects high expression of prosurvival and autophagy proteins and apoptotic regulators. Notably, there is high phosphorylation of GSK-3ß, p38MAPK, and HSP27 and low phosphorylation of Akt (high Akt "reserve"). Subsequent maturation-related reductions in I/R tolerance are associated with reductions in Akt, Bcl-2, LC3B, and Beclin1, despite increased expression and reduced phospho-inhibition of GSK-3ß.