Louise See Hoe

Opioid receptors and cardioprotection – ‘opioidergic conditioning’ of the heart

Ischaemic heart disease (IHD) remains a major cause of morbidity/mortality globally, firmly established in Westernized or ‘developed’ countries and rising in prevalence in developing nations. Thus, cardioprotective therapies to limit myocardial damage with associated ischaemia–reperfusion (I–R), during infarction or surgical ischaemia, is a very important, although still elusive, clinical goal. The opioid receptor system, encompassing the δ (vas deferens), κ (ketocyclazocine) and μ (morphine) opioid receptors and their endogenous opioid ligands (endorphins, dynorphins, enkephalins), appears as a logical candidate for such exploitation. This regulatory system may orchestrate organism and organ responses to stress, induces mammalian hibernation and associated metabolic protection, triggers powerful adaptive stress resistance in response to ischaemia/hypoxia (preconditioning), and mediates cardiac benefit stemming from physical activity. In addition to direct myocardial actions, central opioid receptor signalling may also enhance the ability of the heart to withstand I–R injury. The δ‐ and κ‐opioid receptors are strongly implicated in cardioprotection across models and species (including anti‐infarct and anti‐arrhythmic actions), with mixed evidence for μ opioid receptor‐dependent protection in animal and human tissues. A small number of clinical trials have provided evidence of cardiac benefit from morphine or remifentanil in cardiopulmonary bypass or coronary angioplasty patients, although further trials of subtype‐specific opioid receptor agonists are needed. The precise roles and utility of this GPCR family in healthy and diseased human myocardium, and in mediating central and peripheral survival responses, warrant further investigation, as do the putative negative influences of ageing, IHD co‐morbidities, and relevant drugs on opioid receptor signalling and protective responses.

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 Gα1 and Gβγ inhibitors (pertussis toxin and gallein). This study represents the first demonstration of profound Tas2r agonist‐induced, G protein‐dependent effects on mouse heart function.—Foster, S. R., Blank, K., See Hoe, L. E., Behrens, M., Meyerhof, W., Peart, J. N., Thomas, W. G., Bitter taste receptor agonists elicit G‐protein‐dependent negative inotropy in the murine heart. FASEB J. 28, 4497–4508 (2014). www.fasebj.org

Essential role of EGFR in cardioprotection and signaling responses to A1 adenosine receptors and ischemic preconditioning

Transactivation of epidermal growth factor receptor (EGFR) may contribute to specific protective responses (e.g. mediated by δ-opioid, bradykinin, or muscarinic receptors). No studies have assessed EGFR involvement in cardioprotection mediated by adenosine receptors (ARs), and the role of EGFR in ischemic preconditioning (IPC) is unclear. We tested EGFR, matrix metalloproteinase (MMP), and heparin-binding EGF (HB-EGF) dependencies of functional protection via A(1)AR agonism or IPC. Pretreatment of mouse hearts with 100 nM of A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) or IPC (3 × 1.5-min ischemia/2-min reperfusion) substantially improved recovery from 25-min ischemia, reducing left ventricular diastolic dysfunction up to 50% and nearly doubling pressure development and positive change in pressure over time (+dP/dt). Benefit with both CCPA and IPC was eliminated by inhibitors of EGFR tyrosine kinase (0.3 μM AG1478), MMP (0.3 μM GM6001), or HB-EGF ligand (0.3 ng/ml CRM197), none of which independently altered postischemic outcome. Phosphorylation of myocardial EGFR, Erk1/2, and Akt increased two- to threefold during A(1)AR agonism, with responses blocked by AG1478, GM6001, and CRM197. Studies in HL-1 myocytes confirm A(1)AR-dependent Erk1/2 phosphorylation is negated by AG1478 or GM6001, and reduced with CRM197 (as was Akt activation). These data collectively reveal that A(1)AR- and IPC-mediated functional protection is entirely EGFR and MMP dependent, potentially involving the HB-EGF ligand. Myocardial survival kinase activation (Erk1/2, Akt) by A(1)AR agonism is similarly MMP/HB-EGF/EGFR dependent. Thus MMP-mediated EGFR activation appears essential to cardiac protection and signaling via A(1)ARs and preconditioning.

Sustained Ligand-Activated Preconditioning via δ-Opioid Receptors

We have previously described novel cardioprotection in response to sustained morphine exposure, efficacious in young to aged myocardium and mechanistically distinct from conventional opioid or preconditioning (PC) responses. We further investigate opioid-dependent sustained ligand-activated preconditioning (SLP), assessing duration of protection, opioid receptor involvement, additivity with conventional responses, and signaling underlying preischemic induction of the phenotype. Male C57BL/6 mice were treated with morphine (75-mg subcutaneous pellet) for 5 days followed by morphine-free periods (0, 3, 5, or 7 days) before ex vivo assessment of myocardial tolerance to 25-min ischemia/45-min reperfusion. SLP substantially reduced infarction (by ∼50%) and postischemic contractile dysfunction (eliminating contracture, doubling force development). Cardioprotection persisted for 5 to 7 days after treatment. SLP was induced specifically by δ-receptor and not κ- or μ-opioid receptor agonism, was eliminated by δ-receptor and nonselective antagonism, and was additive with adenosinergic but not acute morphine- or PC-triggered protection. Cotreatment during preischemic morphine exposure with the phosphoinositide-3 kinase (PI3K) inhibitor wortmannin, but not the protein kinase A (PKA) inhibitor myristoylated PKI-(14-22)-amide, prevented induction of SLP. This was consistent with shifts in total and phospho-Akt during the induction period. In summary, data reveal that SLP triggers sustained protection from ischemia for up to 7 days after stimulus, is δ-opioid receptor mediated, is induced in a PI3K-dependent/PKA-independent manner, and augments adenosinergic protection. Mechanisms underlying SLP may be useful targets for manipulation of ischemic tolerance in young or aged myocardium.

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-β-cyclodextrin (MβCD) 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 MβCD 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. MβCD 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 MβCD, whereas SLP was more robust and only inhibited with ≥200 μM MβCD. 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.

Unique Transcriptional Profile of Sustained Ligand-Activated Preconditioning in Pre- and Post-Ischemic Myocardium

Background Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3–5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. Methodology/Principal Findings Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip. Conclusions Protection via SLP is associated with transcriptional repression of inflammation/immunity, up-regulation of sarcomeric elements and natriuretic peptides, and modulation of cell stress, growth and development, while conventional protective molecules are unaltered.

Opposing effects of age and calorie restriction on molecular determinants of myocardial ischemic tolerance

We test the hypothesis that moderate calorie restriction (CR) reverses negative influences of age on molecular determinants of myocardial stress resistance. Postischemic contractile dysfunction, cellular damage, and expression of regulators of autophagy/apoptosis and of prosurvival and prodeath kinases were assessed in myocardium from young adult (YA; 2- to 4-month-old) and middle-aged (MA; 12-month-old) mice, and MA mice subjected to 14 weeks of 40% CR (MA-CR). Ventricular dysfunction after 25%±2%), as was cell death indicated by troponin I (TnI) efflux (1,701±214 ng vs. 785±102 ng in YA). MA hearts exhibited 30% and 65% reductions in postischemic Beclin1 and Parkin, respectively, yet 50% lower proapoptotic Bax and 85% higher antiapoptotic Bcl2, increasing the Bcl2/Bax ratio. Age did not influence Akt or p38-mitogen-activated protein kinase (MAPK) expression; reduced expression of increasingly phosphorylated ribosomal protein S6 kinase (p70S6K), increased expression of dephosphorylated glycogen synthase kinase 3β (GSK3β) and enhanced postischemic p38-MAPK phosphorylation. CR countered the age-related decline in ischemic tolerance, improving contractile recovery (60%±4%) and reducing cell death (123±22 ng of TnI). Protection was not associated with changes in Parkin or Bax, whereas CR partially limited the age-related decline in Beclin1 and further increased Bcl2. CR counteracted age-related changes in p70S6K, increased Akt levels, and reduced p38-MAPK (albeit increasing preischemic phosphorylation), and paradoxically reduced postischemic GSK3β phosphorylation. In summary, moderate age worsens cardiac ischemic tolerance; this is associated with reduced expression of autophagy regulators, dysregulation of p70S6K and GSK3β, and postischemic p38-MAPK activation. CR counters age effects on postischemic dysfunction/cell death; this is associated with reversal of age effects on p70S6K, augmentation of Akt and Bcl2 levels, and preischemic p38-MAPK activation. Age and CR thus impact on distinct determinants of ischemic tolerance, although p70S6K signaling presents a point of convergence.

Cardio-protective effects of combined l-arginine and insulin: Mechanism and therapeutic actions in myocardial ischemia-reperfusion injury.

Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l-arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l-arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l-arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l-arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1nM insulin+1mM l-arginine exhibited significant (all P<0.05) improvements in NO generation and mitochondrial membrane potential, with a concomitant fall in reactive oxygen species production and LDH release. Insulin also increased l-arginine uptake following hypoxia-reoxygenation (P<0.05; n=4-6). In langendorff perfused isolated mouse hearts, combined l-arginine-insulin treatment upon reperfusion significantly (all P<0.05; n=9-11) improved recovery of left ventricular developed pressure, rate pressure product and end diastolic pressure following ischemia, independent of any changes in post-ischemic coronary flow, together with significantly lower LDH release. The observed improvements were greater than l-arginine or insulin treatment alone. In isolated cardiomyocytes (n=3-5), 1nM insulin caused cationic amino acid transporter-1 to redistribute to the cellular membrane from the cytosol and the effects of insulin on l-arginine uptake were partially dependent on the PI3K/Akt pathway. l-arginine-insulin treatment may be a novel strategy to ameliorate I-R injury.

An Ovine Model of Hyperdynamic Endotoxemia and Vital Organ Metabolism

Background: Animal models of endotoxemia are frequently used to understand the pathophysiology of sepsis and test new therapies. However, important differences exist between commonly used experimental models of endotoxemia and clinical sepsis. Animal models of endotoxemia frequently produce hypodynamic shock in contrast to clinical hyperdynamic shock. This difference may exaggerate the importance of hypoperfusion as a causative factor in organ dysfunction. This study sought to develop an ovine model of hyperdynamic endotoxemia and assess if there is evidence of impaired oxidative metabolism in the vital organs. Methods: Eight sheep had microdialysis catheters implanted into the brain, heart, liver, kidney, and arterial circulation. Shock was induced with a 4 h escalating dose infusion of endotoxin. After 3 h vasopressor support was initiated with noradrenaline and vasopressin. Animals were monitored for 12 h after endotoxemia. Blood samples were recovered for hemoglobin, white blood cell count, creatinine, and proinflammatory cytokines (IL-1Beta, IL-6, and IL-8). Results: The endotoxin infusion was successful in producing distributive shock with the mean arterial pressure decreasing from 84.5 ± 12.8 mm Hg to 49 ± 8.03 mm Hg (P < 0.001). Cardiac index remained within the normal range decreasing from 3.33 ± 0.56 L/min/m2 to 2.89l ± 0.36 L/min/m2 (P = 0.0845). Lactate/pyruvate ratios were not significantly abnormal in the heart, brain, kidney, or arterial circulation. Liver microdialysis samples demonstrated persistently high lactate/pyruvate ratios (mean 37.9 ± 3.3). Conclusions: An escalating dose endotoxin infusion was successful in producing hyperdynamic shock. There was evidence of impaired oxidative metabolism in the liver suggesting impaired splanchnic perfusion. This may be a modifiable factor in the progression to multiple organ dysfunction and death.

Unintended Consequences: Fluid Resuscitation Worsens Shock in an Ovine Model of Endotoxemia

Rationale: Fluid resuscitation is widely considered a life‐saving intervention in septic shock; however, recent evidence has brought both its safety and efficacy in sepsis into question. Objectives: In this study, we sought to compare fluid resuscitation with vasopressors with the use of vasopressors alone in a hyperdynamic model of ovine endotoxemia. Methods: Endotoxemic shock was induced in 16 sheep, after which they received fluid resuscitation with 40 ml/kg of 0.9% saline or commenced hemodynamic support with protocolized noradrenaline and vasopressin. Microdialysis catheters were inserted into the arterial circulation, heart, brain, kidney, and liver to monitor local metabolism. Blood samples were recovered to measure serum inflammatory cytokines, creatinine, troponin, atrial natriuretic peptide, brain natriuretic peptide, and hyaluronan. All animals were monitored and supported for 12 hours after fluid resuscitation. Measurements and Main Results: After resuscitation, animals that received fluid resuscitation required significantly more noradrenaline to maintain the same mean arterial pressure in the subsequent 12 hours (68.9 mg vs. 39.6 mg; P = 0.04). Serum cytokines were similar between groups. Atrial natriuretic peptide increased significantly after fluid resuscitation compared with that observed in animals managed without fluid resuscitation (335 ng/ml [256‐382] vs. 233 ng/ml [144‐292]; P = 0.04). Cross‐sectional time‐series analysis showed that the rate of increase of the glycocalyx glycosaminoglycan hyaluronan was greater in the fluid‐resuscitated group over the course of the study (P = 0.02). Conclusions: Fluid resuscitation resulted in a paradoxical increase in vasopressor requirement. Additionally, it did not result in improvements in any of the measured microcirculatory‐ or organ‐specific markers measured. The increase in vasopressor requirement may have been due to endothelial/glycocalyx damage secondary to atrial natriuretic peptide‐mediated glycocalyx shedding.