Stress Signaling In Myocardial Ischemia

Complex cell signaling pathways exist in the cell involved in growth, cell survival or cell death. Intracellular signaling is considered as an important transducer of both adaptive and maladaptive responses of the cell to stress. Signaling pathways are activated by ligand-receptor interaction or receptor independent stimuli such as mechanical stress, osmotic and oxidative stress or haemodynamic loading. Activation of intracellular signaling includes phosphorylations by tyrosine kinases and/or serine/threonine kinases while phosphatases are physiological negative feedback regulators of these pathways. Protein phosphorylations change enzyme activities and protein conformations. The eventual outcome is changes in gene expression and in the function of the responding cells. The intracellular signaling system consists of cell surface signal transduction receptors, intracellular signaling pathways and end-effectors. Signal pathways are not clear cut in their sequences; not all the intermediates are known while unknown feedback loops and signaling cross-talk exist. They can converge or diverge and are more like a web than straight lines.

2.1. Membrane bound receptors

Signal transduction receptors are of different classes:

a) Receptors with intrinsic enzymatic activity. These receptors are capable of auto-phosphorylation as well as phosphorylation of other substrates. Receptors with intrinsic enzymatic activity include those that are tyrosine kinases (e.g PDGF, insulin, EGF, FGF receptors), tyrosine phosphatases (e.g CD45 protein of T cells and macrophages), guanyl cyclases (e.g natriuretic peptide receptors) and serine/threonine kinases (e.g TGF-fS). Receptors with intrinsic enzymatic activity can interact with intracellular proteins; insulin receptor is associated with a protein termed IRS-1 which in turn is associated with the PI3K signaling. This protein acts as a docking or adapter protein. Growth factor receptor binding protein 2 (Grb2) is another common adapter protein through which receptors with intrinsic enzymatic activity can interact with Ras signaling. Figure 7.

b) Receptors lacking intrinsic enzymatic activity. These receptors are coupled to intracellular non receptor tyrosine kinases, such as Src and Lck protein and Janus kinase (JAK). This class of receptors includes all of the cytokine receptors, e.g IL-2 receptor. Figure 8.

c) Receptors (GPCRs) which are coupled, inside the cell, to GTP-binding and hydrolyzing proteins, collectively termed G-proteins. GPCRs modulate adenyl cyclase activity and the production of c-AMP with a negative regulation to occur by binding of the receptor to Gi-protein, e.g P2-adrenergic receptor, figure 9 and positive regulation by binding to Gi-protein, e.g ^-adrenergic or P2-adrenergic receptor. Figure 10. Table 1. c-AMP diffuses to protein kinase A (PKA) which phosphorylates glycogen synthase, phosphorylase and the trancription factor CREB (transcribes genes for gluconeogen-esis). ERK or Akt intracellular signaling can be regulated through receptors bound to Gi. Figure 9. GPCRs which are bound to Gq activate phospholipase C (PLC) leading to hydrolysis of polyphosphoinositides (e.g PIP2) generating the second messengers dia-cylglycerol (DAG) and inositol-triphosphate (IP3). IP3 increases the release of stored calcium. Calcium together with DAG activate PKC dependent signaling. Figure 11. This class of receptors include the angiotensin (ATj), the c^-adrenergic and endothelin receptors. Table 1.

d) Receptors which are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription. Figure 12.

Ligand

Ligand

Myocardial Ischemic Preconditioning

Figure 7. Schematic of a receptor with intrinsic enzymatic activity. Ligand mediated activation results in phosphorylation of phospholipase C (y isoform) leading to hydrolysis of polyphosphoinositides (PIP2). Dia-cyl-glycerol (DAG) and inositol-triphosphate (IP3) are formed. IP3 increases the release of stored calcium. Calcium together with DAG can activate PKC signalling pathway. Association of this type of receptor with adapter proteins results in activation of Ras/Raf/ERK signalling or in activation of PI3K/Akt signalling.

Figure 7. Schematic of a receptor with intrinsic enzymatic activity. Ligand mediated activation results in phosphorylation of phospholipase C (y isoform) leading to hydrolysis of polyphosphoinositides (PIP2). Dia-cyl-glycerol (DAG) and inositol-triphosphate (IP3) are formed. IP3 increases the release of stored calcium. Calcium together with DAG can activate PKC signalling pathway. Association of this type of receptor with adapter proteins results in activation of Ras/Raf/ERK signalling or in activation of PI3K/Akt signalling.

Ligand

Extracellular Space

Ligand

m~RNA

Figure 8. Schematic of membrane receptors lacking intrinsic enzymatic activity. Intracellular signaling is initiated by coupling of the receptor to an intracellular kinase (e.g JAK kinase). These receptors mediate cytokine signaling (prosurvival protective and maladaptive pathways leading to apoptosis). JAK/STAT signalling is involved in the cellular response to ischemia. Activation of STAT3 reduces ischemia induced apoptosis, whereas activation of STAT 1 has the opposite effect.

m~RNA

Figure 8. Schematic of membrane receptors lacking intrinsic enzymatic activity. Intracellular signaling is initiated by coupling of the receptor to an intracellular kinase (e.g JAK kinase). These receptors mediate cytokine signaling (prosurvival protective and maladaptive pathways leading to apoptosis). JAK/STAT signalling is involved in the cellular response to ischemia. Activation of STAT3 reduces ischemia induced apoptosis, whereas activation of STAT 1 has the opposite effect.

Ligand

Ligand

I'lynre 9. Schematic ofGs-couplcd receptors. This type of receptor is coupled to adenyl cyclase (AC) via the activated stimulatory G s protein and leads to the formation of c- AM P. c- AMP in turn activates protein kinase A (PKA), PKA regulates calcium homeostasis and may induce apoptosis due to the elevation of intracellular calcium levels or prevent apoptosis by inactivating the proapoptotic Bad. 13, and R adrenergic signaling pathways are mediated tlirough this receptor. Jror PKA targeted proteins, see figure 17.

Extracellular Space

Adenyl Cyclase

Cascade adivailon

Adenyl Cyclase

I ERK Palhway

Cascade adivailon

Extracellular Space

Ahl Pathway :

Figure 10. Schematic of Gi-coupled receptors, Adctiyl cyclase (AC) activity is inhibited by the Gi protein. Ras signaling is activated by Gi-coupled rcccptors. Acetylcholine, adenosine, opioid or jyadrenergic signaling is mediated through this type of rcccptor. Gi-coupled rcccptors arc able to trigger the preconditioned state in the heart (sec chapter 3). For ERK and Akt signaling pathway, see figure 19.

Ligand

Extracellular Space

Protein Phosphorylation

Figure 11, Schematic of Gq-coupled rcceptors. Activation of phospholipase C (PLC isofomi (1) results in hydrolysis of polyphosphoinositides (P1PZ) generating the diacyl-glycerol (DAG) and inositol-triphosphate (IP3). IP3 increases the release of stored calcium. Calcium together with DAG activate PKC signaling. The angiotensin AT; receptor, a -adrenergic and en doth el in-1 signaling is mediated tlirough this type of receptor. For PKC targeted proteins, see figure 18.

Ligand

Extracellular Space

Protein Phosphorylation

Figure 11, Schematic of Gq-coupled rcceptors. Activation of phospholipase C (PLC isofomi (1) results in hydrolysis of polyphosphoinositides (P1PZ) generating the diacyl-glycerol (DAG) and inositol-triphosphate (IP3). IP3 increases the release of stored calcium. Calcium together with DAG activate PKC signaling. The angiotensin AT; receptor, a -adrenergic and en doth el in-1 signaling is mediated tlirough this type of receptor. For PKC targeted proteins, see figure 18.

Steroid Hormone

Extracellular Space

Signal Transdiiction Pathway

Element

Figure 12. General receptor pattern for steroid hormones action. Ligand binding dissociates receptor from heat shock proteins (e,g )lsp90) Receptor dinners are formed and translocate to nucleus. This results in gene transcription (genomic effects). Activation of intracellular signaling with immediate physiological effects can also occur (non genomic effects).

Signal Transdiiction Pathway

Steroid Hormone

Extracellular Space

Element

Figure 12. General receptor pattern for steroid hormones action. Ligand binding dissociates receptor from heat shock proteins (e,g )lsp90) Receptor dinners are formed and translocate to nucleus. This results in gene transcription (genomic effects). Activation of intracellular signaling with immediate physiological effects can also occur (non genomic effects).

2.2. Triggers of cell signaling

2.2.1. Receptor dependent endogenous triggers

Several endogenous mediators are released during ischemia and reperfusion that could potentially modulate intracellular signaling by acting on surface membrane receptors or on intracellular components of signaling pathways. See Table 1 and figures 9,10, 11. Depending on their site of origin the mediators are termed autocrine when they act on the cell that produces them and paracrine when they act on a neighboring cell. The potential cardioprotective role of autocrine and paracrine mediators in the context of ischemia and reperfusion has been intensively investigated.

An increase in presynaptic release of endogenous catecholamines with concomitant regulation of the adrenergic signaling at various levels occurs during ischemia and reperfusion. The density of a and p receptors and the activity of adenyl cyclase are increased early in the course of subacute ischemia. In prolonged ischemia, adenyl cyclase activity gradually declines resulting in reduced responsiveness of the adrenergic system. Furthermore, energy depletion and intracellular acidosis differentially regulate P-adrenergic receptors, adenyl cyclase and protein kinase C; energy depletion induces an increase in P-adrenergic receptors, yet fails to activate adenyl cyclase and protein kinase C. Acidosis leads to the activation of protein kinase C and sensitization of cardiac adenyl cyclase activity without affecting the density of P-adrenergic receptors.90

P-adrenergic receptor activation results in stimulation of most plasma membrane currents (inward and outward), gap junction channels, and SR Ca2+-ATPase. p, and P2-receptors exert opposite effects on apoptosis; in adult ventricular myocytes activation of P2 adrenergic receptor attenuates apoptosis while activation of P, adrenergic receptors induces apoptosis.91 A possible beneficial effect of beta adrenergic signaling has been suggested. Exogenous preischemic activation of P-receptor with isoproterenol increased tolerance of the perfused rat heart subjected to ischemia and reperfusion, an effect that was abolished by Pt blockade.92 However, administration of dobutamine at reperfusion, (an agent with dominant pt receptor action) had a detrimental effect on postischemic recovery of function in perfused rat hearts.93

Preischemic activation of a-adrenergic signaling also results in cardioprotection. Pretreatment with norepinephrine induces bimodal (early and delayed) myocardial functional adaptation to ischemia in rats. PKC appears to be involved in the early response. Delayed protection was shown to be associated with the expression of genes encoding fetal contractile proteins (increase in P-MHC mRNA).94 However, a-receptor agonists can trigger arrhythmias in the setting of ischemia and reperfusion.55

Acetylcholine is released during ischemia. Acetylcholine can induce cardioprotection by its action on the muscarinic surface receptors with subsequent activation of the PI3K signaling pathway and Src-kinases. Table 1. Figures 10, 11. This leads to the mitochondrial KATp channels opening, mitochondrial reactive oxygen species (ROS) production and induction of survival signaling.95 See also chapter 3 and figures 3,4.

Cardiac tissue angiotensin II is increased in myocardial ischemia and seems to serve an important physiological role. Figure 13. Angiotensin II can induce oxidative stress through the activation of NADH/NADPH oxidase system thereby generating reactive oxygen species (ROS) with subsequent induction of cardioprotective genes adapting the heart to the angiotensin detrimental effects.96 Angiotensin II activates Akt, an effect that is blocked by either wortmannin, a PI3K inhibitor or by the overexpression of cata-

lase.97 A beneficial role for angiotensin has been identified in the context of ischemia and reperfusion. Exogenous angiotensin II limited infarct size in a concentration-dependent manner in the perfused rat heart without having an effect on contractile stunning.98

Bradykinin, endothelin and prostacyclin are released in myocardial ischemia. Bradykinin acts through B2 and Bj receptor and is inactivated by ACE and NEP (cell surface zinc metalloprotease). Figure 13. Activation of B2 receptor can confer protection through activation of the nitric oxide/protein kinase C pathway or through the activation ofPI3K/Aktprosurvival pathway (see figures 10,11). Furthermore, activation of B( receptor mediates protection to endothelium, limits noradrenaline outflow and reduces the occurrence of arrhythmias induced by ischemia and reperfusion.99

Endothelin-1 is a potent vasoconstrictor peptide derived from endothelial cells.100 Its physiological function is mediated by two receptors the ET-A and ET-B. Table 1. Figure 11. ET-A and ET-B receptors are located in vascular smooth muscle and their activation causes vasoconstriction, whereas ET-B receptor is also located in the endothelium and its activation results in vasodilation by increasing nitric oxide or prostacyclin. Endothelin is released following myocardial ischemia and reperfusion. Endothelin reduces infarct size in a perfused rat heart model of ischemia and reperfusion through activation of protein kinase C and KATp channel.101 Furthermore, in neonatal rat ventricular myocytes, endothelin is shown to activate the calcineurin-NFAT (nuclear factor of activated cells) pathways and enhance the expression of Bcl-2.102 However, endogenous blockade of endothelin at the level of the ET-A receptor reduced infarct size in a pig model of coronary occlusion and reperfusion.103

Prostacyclin is increased in response to ischemia and reperfusion through activation of the cyclooxygenase-2 pathway. Inhibition of cyclooxygenase-2 by celecoxib or meloxicam resulted in a concentration dependent exacerbation of the myocardial dysfunction and damage in a perfused rabbit heart model of ischemia and reperfusion, indicating a cardioprotective role for prostacyclin.104

Endogenous opioid peptides are increased in myocardial ischemia. Their effect is mediated through presynaptic and postsynaptic mechanisms. Opioids limit the release of stimulating catecholamines by its presynaptic action while opioid receptor agonists act via Gi -linked pathways postsynaptically and alter myocardial channel activity and intracellular activities of protein kinases. Table 1. Figure 10. Blockade of 5 and x-opioid receptors reduced the tolerance of the isolated rabbit heart to ischemia and reperfusion.105 Furthermore, blockade of 8-opioid receptor abrogated the ischemic preconditioning mediated cardioprotective effect while activation of S-opioid receptor by morphine decreased infarct size and apoptosis in a rabbit model of coronary occlusion and reperfusion.106

Adenosine is released during ischemia and can exert several effects on the myocardium.107 It can interact with three receptors; the A: receptor (myocardium, mast cell), the A2 receptor (vascular) and the A3 receptor (myocardium). See table 1 and figures 9, 10, 11. Adenosine induces opening of the potassium channel (At receptor) with subsequent bradycardia (sinus node, AV node), induces vasodilation (vascular KATp, A2 receptor) and increases calcium channel closing time (negative inotropic effect, A, receptor). Its cardioprotective role has been identified in several studies.108 Adenosine is released during the brief episodes of ischemia and reperfusion of the preconditioning protocol and triggers cardioprotective signaling. This effect is mediated through the Aj and/or A} receptors.109,110 See also chapter 3.

Histamine is released in the setting of ischemia and reperfusion. The mast cell degranulates during ischemia and the wash out of mast cells releases histamine dur-

Table 1. List of important G-protein membrane-bound receptors involved in cardiovascular physiology

Membrane receptors coupling to G-proteins

Receptors

G-proteins

a, adrenergic receptor

Gq, Gs

a2 adrenergic receptor

Gi

ß, adrenergic receptor

Gs

ß2 adrenergic receptor

Gs, Gi

Dopamine receptor 1

Gs

Dopamine receptor 2

Gi

Adenosine receptor (A,)

Gi

Adenosine receptor (A2)

Gs

Adenosine receptor (A3)

Gi, Gq

Muscarinic receptor (M,)

Gi, Gq

Muscarinic receptor (M2)

Gi

Muscarinic receptor (M3)

Gi, Gq

Angiotensin receptor type 1 (AT,)

Gq

Angiotensin receptor type 2 (AT2)

Gi

Endothelin receptor

Gq

Histamine receptor type 2

Gq, Gs

Bradykinin receptors

Gq, Gi

Opioid receptors

Gi

PTH receptor

Gs, Gq

ing reperfusion. Alternatively, it has been suggested that reperfusion induces mast cell degranulation and histamine release during this period. Adenosine can induce mast cell degranulation and histamine release through the adenosine A, receptor. Histamine acts on the myocardium via the H , H2 and the presynaptic H3 receptors. Transgenic mice lacking H3, display a greater incidence of ischemia induced arrhythmias, indicating a possible cardioprotective role of histamine. This effect has been attributed to the histamine presynaptic inhibitory effect on catecholamine release.111

2.2.2. Non receptor triggers; reactive oxygen species and nitric oxide

Reactive oxygen species are generated during ischemia and particularly during reperfusion. They can act as triggers or intermediates of both cell survival or cell death. Reactive oxygen species (ROS) are molecules with one or more misdonated unpaired electrons. Singlet oxygen is an oxygen molecule with an unpaired electron moved to a higher orbital. The unpaired electron alters the chemical reactivity of an atom or mole-

Angiotensinogen

Angiotensin I

Angiotensin il

Gaq ROS TK

JAK/STAT

Inflammation Vasoconstriction GrowîhiProlife ration Apoptosis

ACE or NEP

Angiotensin il

Phosphatases

Degradation

Bradykinin

B,/B; Receptors

Vasodilation Growth Inhibition Apoptosis/Regeneration

Figure 13. Schcmatic of angiotensin and bradykinin cellular actions. Note that angiotensin and bradykinin actions can be differentiated at the receptor level; c.g activation of AT! causes vasoconstriction, while activation of AT, results in vasodilation. Both angiotensin and braykinin have been linked to cardioprotection against ischemia and reperfusion injury. Sec text for a more detailed explanation.

uule and makes il more reactive. To some extent, reactive oxygen species are normal products of oxidative metabolism of physiological importance. Superoxide, which is converted to hydrogen peroxide (H202) by superoxide dismutase (SOD), is produced in the respiratory chain or from other sources shown in figure 14. The copper-zinc SOD is loeatcd in cytosol while the manganese SOD (MnSOD) is located in mitochondria. Cells have a well adapted mechanism that can control excess of free radical formation by the reduction of H 02 to H,0 via the catalase or the glutathione system. Antioxidants exist as a network to remove oxidant stress; myocardial hydrophilic antioxidants such as ascorbate and glutathione are utilized first, while with more severe stress, lipophilic antioxidants are also involved.112 In addition, ROS production can be regulated by mitochondrial uncoupling. Figure 14.

Mitochondria are the site of ROS production during ischemia. Araehidonic acid, eNOS, NADPH oxidase and xanthine oxidase are sources of reactive oxygen species at rcperfusion. Cytochrome P450 mono oxygenases (CYPs) have also been implicated. CYP catalyzes araehidonic acid oxidation to a variety of biologically active eicosanoids and generates reactive oxygen species.113

NADPH, Xanthine

Oxidases Mitochondria Neutrophils Catecholamines

NADPH, Xanthine

Oxidases Mitochondria Neutrophils Catecholamines

Figure 14. Schematic of reactive oxygen species production and cellular defense mechanisms (antioxidant enzymes; catalase, SOD, GSH). Reactive oxygen species (ROS) production is defrimental for the cell. However, ROS, in small amounts and for short periods, can act as intermediates of cardioprotective signaling.

Figure 14. Schematic of reactive oxygen species production and cellular defense mechanisms (antioxidant enzymes; catalase, SOD, GSH). Reactive oxygen species (ROS) production is defrimental for the cell. However, ROS, in small amounts and for short periods, can act as intermediates of cardioprotective signaling.

ROS can cause direct cell membrane damage, lipid peroxidation and damage to proteins and sulfhydryl bonds. The detrimental role of reactive oxygen species in ischemia and reperfusion has been shown by studies using antioxidants or removing endogenous antioxidant defense. Antioxidant administration resulted in reduced infarct size.114 Furthermore, mice with reduced expression of the mitochondrial SOD had a potent deficit in postischemic myocardial function compared with wild-type or mice with diminished expression of cytosolic SOD.115 However, no protection is demonstrated with antioxidant administration in rabbit hearts.116 Patients treated with streptokinase and N-acetylcysteine (NAC), a precursor of GSH synthesis, showed improved functional recovery."7 Furthermore, inhibition of cytochrome P450 monooxygenases by chloramphenicol reduced infarct size in perfused rat and rabbit heart models of ischemia and reperfusion.118

Paradoxically, ROS are not only injurious but may also be protective. ROS are shown to upregulate survival signaling.119 Furthermore, the cardioprotective abilities of ischemic preconditioning are completely abolished with the administration of N-acetylcysteine indicating that ROS can act as important mediators of the preconditioning response.120 (See also chapter 3).

Nitric oxide is a molecule with pleiotropic effects on the cardiovascular system. Figure 15. Nitric oxide (NO) is produced when NO synthase (NOS) converts L-ar-ginine to citrulline. The major target of NO in the cardiovascular system is the NO sensitive soluble guanyl cyclase that converts guanosine triphosphate (GTP) to c-GMP.

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