Background: Fluid resuscitation is widely considered a life saving intervention in septic

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.

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 ϳ40% decrease in left ventricular developed pressure and an increase in aortic pressure, respectively. These responses were abrogated in the presence of G␣ i and G␤␥ 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

Changes in cytoprotective signaling may influence cardiac aging, and underpin sensitization to ischemic insult and desensitization to ‘anti-ischemic’ therapies. We tested whether age-dependent shifts in ischemia-reperfusion (I-R) tolerance in murine and human myocardium are associated with reduced efficacies and coupling of membrane, cytoplasmic and mitochondrial survival-signaling. Hormesis (exemplified in ischemic preconditioning; IPC) and expression of proteins influencing signaling/stress-resistance were also assessed in mice. Mouse hearts (18 vs. 2–4 mo) and human atrial tissue (75±2 vs. 55±2 yr) exhibited profound age-dependent reductions in I-R tolerance. In mice aging negated cardioprotection via IPC, G-protein coupled receptor (GPCR) agonism (opioid, A1 and A3 adenosine receptors) and distal protein kinase C (PKC) activation (4 nM phorbol 12-myristate 13-acetate; PMA). In contrast, p38-mitogen activated protein kinase (p38-MAPK) activation (1 μM anisomycin), mitochondrial ATP-sensitive K+ channel (mKATP) opening (50 μM diazoxide) and permeability transition pore (mPTP) inhibition (0.2 μM cyclosporin A) retained protective efficacies in older hearts (though failed to eliminate I-R tolerance differences). A similar pattern of change in protective efficacies was observed in human tissue. Murine hearts exhibited molecular changes consistent with altered membrane control (reduced caveolin-3, cholesterol and caveolae), kinase signaling (reduced p70 ribosomal s6 kinase; p70s6K) and stress-resistance (increased G-protein receptor kinase 2, GRK2; glycogen synthase kinase 3β, GSK3β; and cytosolic cytochrome c). In summary, myocardial I-R tolerance declines with age in association with dysfunctional hormesis and transduction of survival signals from GPCRs/PKC to mitochondrial effectors. Differential changes in proteins governing caveolar and mitochondrial function may contribute to signal dysfunction and stress-intolerance.

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 ϳ50%) and postischemic contractile dysfunction (eliminating contracture, doubling force development). Cardioprotection persisted for 5 to 7 days after treatment. SLP was induced specifically by ␦-receptor and not -or -opioid receptor agonism, was eliminated by ␦-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 ␦-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.

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 A1AR agonism or IPC. Pretreatment of mouse hearts with 100 nM of A1AR agonist 2-chloro- N6-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/d t). 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 A1AR agonism, with responses blocked by AG1478, GM6001, and CRM197. Studies in HL-1 myocytes confirm A1AR-dependent Erk1/2 phosphorylation is negated by AG1478 or GM6001, and reduced with CRM197 (as was Akt activation). These data collectively reveal that A1AR- 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 A1AR agonism is similarly MMP/HB-EGF/EGFR dependent. Thus MMP-mediated EGFR activation appears essential to cardiac protection and signaling via A1ARs and preconditioning.

See Hoe LE, Schilling JM, Tarbit E, Kiessling CJ, Busija AR, Niesman IR, Du Toit E, Ashton KJ, Roth DM, Headrick JP, Patel HH, Peart JN. Sarcolemmal cholesterol and caveolin-3 dependence of cardiac function, ischemic tolerance, and opioidergic cardioprotection. Am J Physiol Heart Circ Physiol 307: H895-H903, 2014. First published July 25, 2014; doi:10.1152/ajpheart.00081.2014.-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 ischemiareperfusion (I/R) and cardioprotective stimuli are unclear. We assessed the effects of graded cholesterol depletion with methyl-␤-cyclodextrin (M␤CD) 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 M␤CD 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. M␤CD 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 Ն20 M M␤CD, whereas SLP was more robust and only inhibited with Ն200 M M␤CD. 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.cardioprotection; caveolae; caveolin-3; cholesterol; contractility; ischemia-reperfusion; membrane microdomains; opioid receptors SARCOLEMMAL MICRODOMAINS are critical regulators of cellular function and signaling (34) and may be important in sex-, age-, and disease-dependent differences in cardiac and vascular function (3,10,11,14,24,49,62). Caveolae are lipid rich (i.e., cholesterol and glycosphingolipid) microdomains containing scaffolding proteins, caveolins, that present as distinct molecular platforms for the regulation of cytoprotective and other signaling (52). However, the importance of membrane cholesterol and microdomains to the maintenance of myocardial and coronary function as well as intrinsic responses to insult/stress a...

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 build-up of acidic metabolites results in decreased intracellular and extracellular pH that can reach as low as 6.0-6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly impacts cardiac function. Methods: We used genetic and pharmacological 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 (hiPSC-CMs) as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and post-conditioning therapeutic agents. Results: Analysis of human complex trait genetics indicate that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using hiPSC-CMs 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 pharmacological inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction (MI) and two 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 pre- or post-conditioning 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 impact on cardiac ion channels regulating baseline electromechanical coupling and physiological performance. Conclusions: Collectively, our data provide compelling evidence for a novel pharmacological strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.