Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

Lethal Caspase-1/4-Dependent Injury Occurs in the First Minutes of Coronary Reperfusion and Requires Calpain Activity.

To study the relationship between caspase-1/4 and reperfusion injury, we measured infarct size (IS) in isolated mouse hearts undergoing 50 min global ischemia/2 h reperfusion. Starting VRT-043198 (VRT) at reperfusion halved IS. The pan-caspase inhibitor emricasan duplicated VRT's protection. IS in caspase-1/4-knockout hearts was similarly reduced, supporting the hypothesis that caspase-1/4 was VRT's only protective target. NLRC4 inflammasomes activate caspase-1. NLRC4 knockout hearts were not protected, eliminating NLRC4 as caspase-1/4's activator. The amount of protection that could be achieved by only suppressing caspase-1/4 activity was limited. In wild-type (WT) hearts, ischemic preconditioning (IPC) was as protective as caspase-1/4 inhibitors. Combining IPC and emricasan in these hearts or preconditioning caspase-1/4-knockout hearts produced an additive IS reduction, indicating that more protection could be achieved by combining treatments. We determined when caspase-1/4 exerted its lethal injury. Starting VRT after 10 min of reperfusion in WT hearts was no longer protective, revealing that caspase-1/4 inflicted its injury within the first 10 min of reperfusion. Ca++ influx at reperfusion might activate caspase-1/4. We tested whether Ca++-dependent soluble adenylyl cyclase (AC10) could be responsible. However, IS in AC10-/- hearts was not different from that in WT control hearts. Ca++-activated calpain has been implicated in reperfusion injury. Calpain could be releasing actin-bound procaspase-1 in cardiomyocytes, which would explain why caspase-1/4-related injury is confined to early reperfusion. The calpain inhibitor calpeptin duplicated emricasan's protection. Unlike IPC, adding calpain to emricasan offered no additional protection, suggesting that caspase-1/4 and calpain may share the same protective target.

Ticagrelor Does Not Protect Isolated Rat Hearts, Thus Clouding Its Proposed Cardioprotective Role Through ENT 1 in Heart Tissue.

P2Y12 receptor-blocking drugs given at reperfusion offer protection against myocardial infarction in animal models of transient coronary occlusion. Two recent reports concluded that ticagrelor was more cardioprotective than clopidogrel and attributed this to ticagrelor's unique ability to raise tissue adenosine by blocking the equilibrative nucleoside transporter 1. Indeed, an adenosine receptor blocker attenuated ticagrelor's protection. The related P2Y12 inhibitor cangrelor, which does not block the transporter, protects hearts only when platelets are in the perfusate, while adenosine is known to protect equally in situ blood-perfused and crystalloid-perfused isolated hearts. We, therefore, tested whether ticagrelor liberates a sufficient amount of adenosine to protect a Krebs buffer-perfused isolated rat heart subjected to 40 minutes of global ischemia followed by 2 hours of reperfusion. In untreated hearts, 77.6% ± 4.0% of the ventricle was infarcted as measured by triphenyltetrazolium staining. Ischemically preconditioned hearts had only 32.7% ± 3.6% infarction ( P < .001 vs untreated), indicating that our model could be protected by preconditioning which is known to involve adenosine. Strikingly, hearts treated with 10 µmol/L ticagrelor in the buffer throughout the reperfusion period had 77.5% ± 2.4% infarction comparable to unprotected controls ( P = NS vs untreated). These data strongly suggest that ticagrelor was unable to release sufficient adenosine from the crystalloid-perfused rat heart to protect it against infarction. Our previous studies have found no difference in the anti-infarct potency among clopidogrel, cangrelor, and ticagrelor in open-chest rats and rabbits, and surprisingly adenosine receptor antagonists block protection from all 3 drugs. We have no explanation why ticagrelor is more protective in the pig than clopidogrel but suspect a species or perhaps a treatment schedule difference that may or may not involve adenosine.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

Feasibility of using thermal response to Ka band millimeter wave heating to assess skin blood flow.

OBJECTIVE: Implementation of clinical guidelines for diagnosing peripheral artery disease will demand screening many millions of patients who are considered at-risk. This will require faster, easier screening technologies to identify patients with compromised blood flow to the extremities. APPROACH: The feasibility of using surface temperature response to Ka band (26.5-40 GHz) near-field irradiation to assess skin blood flow was explored using an animal model. Ears of domestic rabbits were subjected to low-power continuous wave radio frequency heating from an open-ended waveguide (WR-28) at f = 35 GHz. Three flow conditions were evaluated: (1) a baseline flow condition, (2) occluded flow and (3) reactive hyperemia. Surface temperatures were monitored continuously by means of an infrared thermography camera during each 2 min exposure. MAIN RESULTS: Ensemble average results showed significant differences (p < .05) at exposure times 30, 60, 90 and 120 s between baseline and occluded conditions, and between baseline and reactive hyperemia conditions. The occluded condition (N = 12) resulted in an average temperature increase of 21.4 °C ± 3.9 after 2 min, compared with an average increase of 12.1 °C ± 1.6 for baseline conditions (N = 9) and 4.7 °C ± 3.6 for post-occlusion/hyperemic conditions (N = 8). SIGNIFICANCE: Results are compared with the results of a simple two parameter mathematical model. These results suggest a method for non-invasive skin blood flow assessment to screen for peripheral artery disease and associated risk of cardiovascular events.

Epidemiology and antifungal susceptibilities of yeast isolates causing invasive infections across urban Beijing, China.

AIM: To investigate the species distribution and antifungal susceptibility profiles of yeast isolates causing invasive infections across Beijing. MATERIALS & METHODS: A total of 1201 yeast isolates recovered from blood and other sterile body fluids were correctly identified by matrix-assisted laser desorption/ionization TOF MS supplemented by DNA sequencing. Antifungal susceptibility testing was performed according to the Clinical and Laboratory Standards Institute broth microdilution method. RESULTS: Candida (95.5%) remained the most common yeast species isolated; Candida albicans (38.8%) and Candida parapsilosis (22.6%) were the leading species of candidemia. Azole resistances were mainly observed in Candida glabrata and Candida tropicalis isolates. CONCLUSION: This study outlined the epidemiologic data of invasive yeast infections and highlighted the need for continuous monitoring of azole resistances among C. glabrata and C. tropicalis isolates in Beijing.

The Highly Selective Caspase-1 Inhibitor VX-765 Provides Additive Protection Against Myocardial Infarction in Rat Hearts When Combined With a Platelet Inhibitor.

Use of ischemic postconditioning and other related cardioprotective interventions to treat patients with acute myocardial infarction (AMI) has failed to improve outcomes in clinical trials. Because P2Y12 inhibitors are themselves postconditioning mimetics, it has been postulated that the loading dose of platelet inhibitors routinely given to patients treated for AMI masks the anti-infarct effect of other intended cardioprotective interventions. To further improve outcomes of patients with AMI, an intervention must be able to provide additive protection in the presence of a P2Y12 platelet inhibitor. Previous studies reported an anti-infarct effect using a peptide inhibitor of the pro-inflammatory caspase-1 in animal models of AMI. Herein we tested whether a pharmacologic caspase-1 inhibitor can further limit infarct size in open-chest, anesthetized rats treated with a P2Y12 inhibitor. One hour occlusion of a coronary branch followed by 2 hours of reperfusion was used to simulate clinical AMI and reflow. One group of rats received an intravenous bolus of 16 mg/kg of the highly selective caspase-1 inhibitor VX-765 30 minutes prior to onset of ischemia. A second group received a 60 µg/kg intravenous bolus of the P2Y12 inhibitor cangrelor 10 minutes prior to reperfusion followed by 6 µg/kg/min continuous infusion. A third group received treatment with both inhibitors as above. Control animals received no treatment. Infarct size was measured by tetrazolium stain and volume of muscle at risk by fluorescent microspheres. In untreated hearts, 73.7% ± 4.1% of the ischemic zone infarcted. Treatment with either cangrelor or VX-765 alone reduced infarct size to 43.8% ± 2.4% and 39.6% ± 3.6% of the ischemic zone, respectively. Combining cangrelor and VX-765 was highly protective, resulting in only 14.0% ± 2.9% infarction. The ability of VX-765 to provide protection beyond that of a platelet inhibitor alone positions it as an attractive candidate therapy to further improve outcomes in today's patients with AMI.

Cangrelor-Mediated Cardioprotection Requires Platelets and Sphingosine Phosphorylation.

In animal models platelet P2Y12 receptor antagonists put the heart into a protected state, not as a result of suppressed thrombosis but rather through protective signaling, similar to that for ischemic postconditioning. While both ischemic postconditioning and the P2Y12 blocker cangrelor protect blood-perfused hearts, only the former protects buffer-perfused hearts indicating that the blocker requires a blood-borne constituent or factor to protect. We used an anti-platelet antibody to make thrombocytopenic rats to test if that factor resides within the platelet. Infarct size was measured in open-chest rats subjected to 30-min ischemia/2-h reperfusion. Infarct size was not different in thrombocytopenic rats showing that preventing aggregation alone is not protective. While ischemic preconditioning could reduce infarct size in thrombocytopenic rats, the P2Y12 inhibitor cangrelor could not, indicating that it protects by interacting with some factor in the platelet. Ischemic preconditioning is known to require phosphorylation of sphingosine. In rats treated with dimethylsphingosine to block sphingosine kinase, cangrelor was no longer protective. Thus cangrelor's protective mechanism appears to also involve sphingosine kinase revealing yet another similarity to conditioning's mechanism.

Mitochondrially targeted Endonuclease III has a powerful anti-infarct effect in an in vivo rat model of myocardial ischemia/reperfusion.

Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers marked cytoprotection in cultured cells and a variety of injury models. In this study, we measured the effect of EndoIII, a fusion protein construct that traffics Endonuclease III, a DNA glycosylase/AP lyase, to the mitochondria, on infarct size in a rat model of myocardial ischemia/reperfusion. Open-chest, anesthetized rats were subjected to 30 min of occlusion of a coronary artery followed by 2 h of reperfusion. An intravenous bolus of EndoIII, 8 mg/kg, just prior to reperfusion reduced infarct size from 43.8 ± 1.4% of the risk zone in control animals to 24.0 ± 1.3% with no detectable hemodynamic effect. Neither EndoIII's vehicle nor an enzymatically inactive EndoIII mutant (K120Q) offered any protection. The magnitude of EndoIII's protection was comparable to that seen with the platelet aggregation inhibitor cangrelor (25.0 ± 1.8% infarction of risk zone). Because loading with a P2Y12 receptor blocker to inhibit platelets is currently the standard of care for treatment of acute myocardial infarction, we tested whether EndoIII could further reduce infarct size in rats treated with a maximally protective dose of cangrelor. The combination reduced infarct size to 15.1 ± 0.9% which was significantly smaller than that seen with either cangrelor or EndoIII alone. Protection from cangrelor but not EndoIII was abrogated by pharmacologic blockade of phosphatidylinositol-3 kinase or adenosine receptors indicating differing cellular mechanisms. We hypothesized that EndoIII protected the heart from spreading necrosis by preventing the release of proinflammatory fragments of mitochondrial DNA (mtDNA) into the heart tissue. In support of this hypothesis, an intravenous bolus at reperfusion of deoxyribonuclease I (DNase I) which should degrade any DNA fragments escaping into the extracellular space was as protective as EndoIII. Furthermore, the combination of EndoIII and DNase I produced additive protection. While EndoIII would maintain mitochondrial integrity in many of the ischemic cardiomyocytes, DNase I would further prevent mtDNA released from those cells that EndoIII could not save from propagating further necrosis. Thus, our mtDNA hypothesis would predict additive protection. Finally to demonstrate the toxicity of mtDNA, isolated hearts were subjected to 15 min of global ischemia. Infarct size doubled when the coronary vasculature was filled with mtDNA fragments during the period of global ischemia. To our knowledge, EndoIII and DNase are the first agents that can both be given at reperfusion and add to the protection of a P2Y12 blocker, and thus should be effective in today's patient with acute myocardial infarction.

Triple therapy greatly increases myocardial salvage during ischemia/reperfusion in the in situ rat heart.

BACKGROUND: Cangrelor, a P2Y12 receptor blocker, administered just prior to reperfusion reduced but did not eliminate myocardial infarction in rabbits. Combining cangrelor with ischemic postconditioning offered no additional protection suggesting they protected by a similar mechanism. To determine if cangrelor's protection might be additive to other cardioprotective interventions we tested cangrelor in combination with ischemic preconditioning, cariporide, a sodium-hydrogen exchange blocker, and mild hypothermia. METHODS: Open-chest rats underwent 30-min coronary occlusion/2-h reperfusion. RESULTS: Cangrelor, administered as a bolus (60 µg/kg) 10 min before reperfusion and continued as an infusion (6 µg/kg/min) for the duration of the experiment, decreased infarction from 45.3 % of risk zone in control hearts to 25.0 %. Combining cangrelor and ischemic preconditioning offered no additional protection. Mild hypothermia (32-33 °C) instituted by peritoneal lavage with cold saline just prior to coronary occlusion resulted in 25.2 % infarction, and combining cangrelor and hypothermia nearly halved infarction to 14.1 % of risk zone. Cariporide (0.5 mg/kg) just prior to occlusion resulted in 27.2 % infarction and 15.8 % when combined with cangrelor. Combining cangrelor, hypothermia and cariporide further halved infarction to only 6.3 %. We also tested another P2Y12 inhibitor ticagrelor which is chemically similar to cangrelor. Ticagrelor (20 mg/kg) fed 1 h prior to surgery reduced infarct size by an amount similar to that obtained with cangrelor (25.6 % infarction), and this protective effect was abolished by chelerythrine and wortmannin, thus implicating participation of PKC and PI3-kinase, resp., in signaling. CONCLUSIONS: Cardioprotection from a P2Y12 receptor antagonist can be combined with at least 2 other strategies to magnify the protection. Combining multiple interventions that use different cardioprotective mechanisms could provide powerful protection against infarction in patients with acute coronary thrombosis.

Two classes of anti-platelet drugs reduce anatomical infarct size in monkey hearts.

BACKGROUND: Recent studies in rabbits have demonstrated that platelet P2Y12 receptor antagonists are cardioprotective, and that the mechanism is surprisingly not related to blockade of platelet aggregation but rather to triggering of the same signal transduction pathway seen in pre- and postconditioning. We wanted to determine whether this same cardioprotection could be documented in a primate model and whether the protection was limited to P2Y12 receptor antagonists or was a class effect. METHODS: Thirty-one macaque monkeys underwent 90-min LAD occlusion/4-h reperfusion. RESULTS: The platelet P2Y12 receptor blocker cangrelor started just prior to reperfusion significantly decreased infarction by an amount equivalent to that seen with ischemic postconditioning (p < 0.001). For any size of risk zone, infarct size in treated hearts was significantly smaller than that in control hearts. OM2, an investigational murine antibody against the primate collagen receptor glycoprotein (GP) VI, produced similar protection (p < 0.01) suggesting a class effect. Both cangrelor and OM2 were quite effective at blocking platelet aggregation (94 % and 97 %, respectively). CONCLUSIONS: Thus in a primate model in which infarct size could be determined directly platelet anti-aggregatory agents are cardioprotective. The important implication of these investigations is that patients with acute myocardial infarction who are treated with platelet anti-aggregatory agents prior to revascularization may already be in a postconditioned state. This hypothesis may explain why in recent clinical trials postconditioning-mimetic interventions which were so protective in animal models had at best only a modest effect.

Platelet P2Y12 blockers confer direct postconditioning-like protection in reperfused rabbit hearts.

BACKGROUND: Blockade of platelet activation during primary percutaneous intervention for acute myocardial infarction is standard care to minimize stent thrombosis. To determine whether antiplatelet agents offer any direct cardioprotective effect, we tested whether they could modify infarction in a rabbit model of ischemia/reperfusion caused by reversible ligation of a coronary artery. METHODS AND RESULTS: The P2Y12 (adenosine diphosphate) receptor blocker cangrelor administered shortly before reperfusion in rabbits undergoing 30-minute regional ischemia/3-hour reperfusion reduced infarction from 38% of ischemic zone in control hearts to only 19%. Protection was dose dependent and correlated with the degree of inhibition of platelet aggregation. Protection was comparable to that seen with ischemic postconditioning (IPOC). Cangrelor protection, but not its inhibition of platelet aggregation, was abolished by the same signaling inhibitors that block protection from IPOC suggesting protection resulted from protective signaling rather than anticoagulation. As with IPOC, protection was lost when cangrelor administration was delayed until 10 minutes after reperfusion and no added protection was seen when cangrelor and IPOC were combined. These findings suggest both IPOC and cangrelor may protect by the same mechanism. No protection was seen when cangrelor was used in crystalloid-perfused isolated hearts indicating some component in whole blood is required for protection. Clopidogrel had a very slow onset of action requiring 2 days of treatment before platelets were inhibited, and only then the hearts were protected. Signaling inhibitors given just prior to reperfusion blocked clopidogrel's protection. Neither aspirin nor heparin was protective. CONCLUSIONS: Clopidogrel and cangrelor protected rabbit hearts against infarction. The mechanism appears to involve signal transduction during reperfusion rather than inhibition of intravascular coagulation. We hypothesize that both drugs protect by activating IPOC's protective signaling to prevent reperfusion injury. If true, patients receiving P2Y12 inhibitors before percutaneous intervention may already be postconditioned thus explaining failure of recent clinical trials of postconditioning drugs.

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin.

BACKGROUND AND PURPOSE: A(2B) adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined. EXPERIMENTAL APPROACH: HEK 293 cells stably transfected with human A(2B) adenosine receptors (HEK-A(2B) ) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye. KEY RESULTS: In HEK-A(2B) cells, the selective A(2B) receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G(i) or G(o) proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK. CONCLUSION AND IMPLICATIONS: Activation of A(2B) adenosine receptors reduced superoxide generation from mitochondrial complex I through G(i/o) , ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.

Cardioprotection by mild hypothermia during ischemia involves preservation of ERK activity.

Cooling the ischemic heart by just a few degrees protects it from infarction without affecting its mechanical function, but the mechanism of this protection is unknown. We investigated whether signal transduction pathways might be involved in the anti-infarct effect of mild hypothermia (35°C). Isolated rabbit hearts underwent 30 min of coronary artery occlusion/2 h of reperfusion. They were either maintained at 38.5°C or cooled to 35°C just before and only during ischemia. Infarct size was measured. The effects of the protein kinase C inhibitor chelerythrine, the nitric oxide synthase inhibitor N (ω)-nitro-L: -arginine methyl ester (L: -NAME), the phosphatidylinositol 3-kinase antagonist wortmannin, or either of the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors PD98059 or U0126 on cooling's protection were examined. Myocardial ATP assays were performed and the level of phosphorylation of extracellular signal-regulated kinase (ERK) and MEK was examined by western blotting. To investigate an effect of cooling on protein phosphatase (PPase), a PPase inhibitor cantharidin was tested in the infarct model and the effect of mild hypothermia on PP2A activity in vitro was measured. Infarct size was 34.4 ± 2.2% of the ischemic zone in normothermic (38.5°C) hearts, but only 15.6 ± 8.7% in hearts cooled to 35°C during ischemia. Mechanical function was unaffected. Neither chelerythrine, L: -NAME, nor wortmannin had any effect, but both PD98059 and U0126 completely eliminated protection. Ischemia rather than reperfusion was the critical time when ERK had to be active to realize protection. Phosphorylation of ERK and MEK fell during normothermic ischemia, but during hypothermic ischemia phosphorylation of ERK remained high while that of MEK was increased. Cooling only slightly delayed the rate at which ATP fell during ischemia, and ERK inhibition did not affect that attenuation suggesting ATP preservation was unrelated to protection. Cantharidin, like cooling, also protected during ischemia but not at reperfusion, and its protection was dependent on ERK phosphorylation. However, mild hypothermia had a negligible effect on PP2A activity in an in vitro assay. Hence, mild hypothermia preserves ERK and MEK activity during ischemia which somehow protects the heart. While a PPase inhibitor mimicked cooling's protection, a direct effect of cooling on PP2A could not be demonstrated.

Cardioprotective PKG-independent NO signaling at reperfusion.

Cell models of ischemic preconditioning (IPC) indicate nitric oxide (NO) is involved in protection accruing during reoxygenation but disagree whether it acts through PKG. Using a more relevant intact heart model, we studied isolated rabbit hearts subjected to 30-min coronary artery occlusion/120-min reperfusion. We previously found protection from PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT-cGMP) at reperfusion was blocked by A(2b) adenosine receptor (A(2b)AR), ERK, or phosphatidylinositol 3-kinase (PI3-kinase) blockers. In this investigation A(2b)AR agonist BAY 60-6583 or CPT-cGMP at reperfusion reduced infarction comparably to IPC. Their protection was abrogated by N(ω)-nitro-l-arginine methyl ester (l-NAME), suggesting a PKG-independent NO synthase in IPC's mediator pathway downstream of PKG and A(2b)AR. NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) at reperfusion also protected. This protection was not blocked by PI3-kinase inhibitor wortmannin or ERK antagonist PD-98059, suggesting NO acted downstream of these kinases. Protection from SNAP was not affected by mitochondrial ATP-sensitive K(+) channel closer 5-hydroxydecanoate, PKC antagonist chelerythrine, reactive oxygen species scavenger N-2-mercaptopropionylglycine, or soluble guanylyl cyclase antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Absence of ODQ effect indicated NO was acting independently of PKG. BAY 58-2667, a soluble guanylyl cyclase activator, was protective, and l-NAME blocked its infarct-sparing effect, indicating a second signaling event dependent on NO generation but independent of PKG. SB216763, a blocker of glycogen synthase kinase-3ß (GSK-3ß), decreased infarct size, and its infarct-sparing effect was not affected by l-NAME, suggesting GSK-3ß acted downstream or independently of NO. Hence, NO signaling occurs in IPC's mediator pathway downstream of Akt and ERK, and its protection is independent of PKG.

AMP579 is revealed to be a potent A2b-adenosine receptor agonist in human 293 cells and rabbit hearts.

The mixed A1/A2a-adenosine agonist AMP579 given at reperfusion is protective in animal models of myocardial infarction. Receptor-blocking studies have indicated that the protection came from an adenosine receptor (AR), but neither A1- nor A2a-selective agonists could duplicate its protection. We recently found that A2b-selective agonists given at reperfusion are protective, and, therefore, tested whether AMP579 might also be an A2b agonist. We used human embryonic kidney cells overexpressing human A2b receptors as an assay system. In these cells, A2b receptor occupancy causes phosphorylation of ERK. AMP579 induced ERK phosphorylation with an EC50 of 250 nM and this phosphorylation could be blocked by MRS1754 or PSB1115, two highly selective blockers of human A2b receptors. We attempted to confirm our A2b hypothesis in a rabbit heart model of ischemia-reperfusion. AMP579 (500 nM) for 1 h starting at reperfusion reduced infarct size in isolated rabbit hearts exposed to 30 min of regional ischemia and 2 h of reperfusion (12.9 +/- 2.2% infarction of risk zone vs. 32.0 +/- 1.9% in untreated hearts). PSB1115 (500 nM) given for the first 15 min of reperfusion blocked AMP579's protection (32.2 +/- 3.1% infarction) which is consistent with an A2b mechanism. We conclude that AMP579 is a non-selective, but potent A2b-AR agonist, and that its protection against infarction is through that receptor.

Attenuation of infarction in cynomolgus monkeys: preconditioning and postconditioning.

Ischemic pre- (IPC) and post- (IPOC) conditioning are very protective in laboratory animals, but it has not been possible to measure their anti-infarct potency in human hearts. Non-human primates are genetically closer to humans than other laboratory animals, but until now there have been no studies of IPC or IPOC in any primate species. Accordingly the left anterior descending coronary artery of cynomolgus monkeys was occluded for 90 min and reperfused for 4 h. In control animals, only 44% of the risk zone infarcted indicating cynomolgus myocardium is much more resistant to infarction than that of rabbits or rats. The regression line for the infarct-risk zone plot was very linear (r = 0.99), and intersected the risk zone axis at 0.82 cm3. Even small changes in infarct size could be detected as a shift in this line. Collateral flow in 12 monkeys was 6.6% of flow to normal myocardium and not a covariate of infarct size. IPC with two cycles of 10-min coronary occlusion/10-min reperfusion reduced infarction to near zero indicating that the innate resistance to infarction was not caused by constitutive preconditioning. Wortmannin, an antagonist of phosphatidylinositol 3-kinase (PI3-K), administered just before release of the 90-min coronary occlusion attenuated IPC's infarct-sparing effect by approximately 50% suggesting that PI3-K was involved in preconditioning's protection. IPOC with six cycles of 30-s reperfusion/30-s coronary reocclusion, a very protective protocol in most species, was much less protective than IPC. We conclude that ischemic preconditioning is extremely protective in cynomolgus hearts despite their sparse collateralization but, surprisingly, the protocol of IPOC used in this study offers less protection.

BAY 58-2667, a nitric oxide-independent guanylyl cyclase activator, pharmacologically post-conditions rabbit and rat hearts.

AIMS: BAY 58-2667 (BAY-58) directly activates soluble guanylyl cyclase without tolerance in a nitric oxide (NO)-independent manner, and its haemodynamic effect is similar to that of nitroglycerin. We tested whether BAY-58 could make both rabbit and rat hearts resistant to infarction when given at the end of an ischaemic insult. METHODS AND RESULTS: All hearts were exposed to 30 min regional ischaemia followed by 120-(isolated hearts) or 180-(in situ hearts) min reperfusion. BAY-58 (1-50 nM) infused for 60 min starting 5 min before reperfusion significantly reduced infarction from 33.0 +/- 3.2% in control isolated rabbit hearts to 9.5-12.7% (P < 0.05). In a more clinically relevant in situ rabbit model, infarct size was similarly reduced with a loading dose of 53.6 microg/kg followed by a 60 min infusion of 1.25 microg/kg/min (41.1 +/- 3.1% infarction in control hearts to 16.0 +/- 4.4% in treated hearts, P < 0.05). BAY-58 similarly decreased infarction in the isolated rat heart, and protection was abolished by co-treatment with a protein kinase G (PKG) antagonist, or a mitochondrial K(ATP) channel antagonist. Conversely, N(omega)-nitro-L-arginine-methyl-ester-hydrochloride, a NO-synthase inhibitor, failed to block BAY-58's ability to decrease infarction, consistent with the latter's putative NO-independent activation of PKG. Finally, BAY-58 increased myocardial cGMP content in reperfused hearts while cAMP was unchanged. CONCLUSION: When applied at reperfusion, BAY-58 is an effective cardioprotective agent with a mechanism similar to that of ischaemic pre-conditioning and, hence, should be a candidate for treatment of acute myocardial infarction in man.

Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C.

PKG activator 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT) at reperfusion protects ischemic hearts, but the mechanism is unknown. We recently proposed that in preconditioned hearts PKC lowers the threshold for adenosine to initiate signaling from low-affinity A2b receptors during early reperfusion thus allowing endogenous adenosine to activate survival kinases phosphatidylinositol 3-kinase (PI3K) and ERK. We tested whether CPT might also sensitize A2b receptors to adenosine. CPT (10 microM) during the first minutes of reperfusion markedly reduced infarction in isolated rabbit hearts undergoing 30-min regional ischemia/2-h reperfusion, and salvage was blocked by MRS 1754, an A2b-selective antagonist. Coadministration of wortmannin (PI3K inhibitor) or PD-98059 (MEK1/2 and therefore ERK1/2 inhibitor) also blocked protection. In nonischemic hearts, 10-min infusion of CPT did not change phosphorylation of Akt or ERK1/2. Neither did a subthreshold dose (2.5 nM) of the nonselective but A2b-potent receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA). However, when 2.5 nM NECA was combined with 10 microM CPT, both phospho-Akt and phospho-ERK1/2 significantly increased, indicating CPT had lowered the threshold for A2b-dependent signaling. The PKC antagonist chelerythrine blocked this phosphorylation induced by CPT + NECA. Chelerythrine also blocked the anti-infarct effect of CPT as did nonselective (glibenclamide) and mitochondrial-selective (5-hydroxydecanoate) K(ATP) channel blockers. A free radical scavenger, N-(2-mercaptopropionyl)glycine, also blocked CPT protection. We propose CPT targets PKG, which activates PKC through mitochondrial K(ATP) channel (mitoKATP)-dependent redox signaling, a sequence mimicking that already documented in preconditioning. Activated PKC then augments sensitivity of normally low-affinity cardiac adenosine A2b receptors so endogenous adenosine can protect by activating Akt and ERK.

Acidosis, oxygen, and interference with mitochondrial permeability transition pore formation in the early minutes of reperfusion are critical to postconditioning's success.

Repetitive cycles of reflow/reocclusion in the initial 2 min following release of a prolonged coronary occlusion, i.e., ischemic postconditioning (IPoC), salvages ischemic myocardium. We have proposed that the intermittent ischemia prevents formation of mitochondrial permeability transition pores (MPTP) by maintaining an acidic myocardial pH for several minutes until survival kinases can be activated. To determine other requisites of IPoC, isolated rabbit hearts were subjected to 30 min of regional myocardial ischemia and 120 min of reperfusion. Infarct size was determined by staining with triphenyltetrazolium chloride. During the first 2 min of reperfusion the perfusate was either at pH 7.4 following equilibration with 95% O(2)/5% CO(2), pH 6.9 following equilibration with 80% N(2)/20% CO(2), or pH 7.8 following equilibration with 100% O(2). Whereas acidic, oxygenated perfusate for the first 2 min of reperfusion was cardioprotective, protection was lost when acidic perfusate was hypoxic. However, the acidic, hypoxic hearts could be rescued by addition of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, to the perfusate. Therefore, both low pH and restoration of oxygenation are necessary for protection, and the signaling step requiring combined oxygen and H(+) must be upstream of PKC. To gain further insight into the mechanism of IPoC, the latter was effected with 6 cycles of 10-s reperfusion/10-s reocclusion. Its protective effect was abrogated by either making the oxygenated perfusate alkaline during the reperfusion phases or making the reperfusion buffer hypoxic. Presumably the repeated coronary occlusions during IPoC keep myocardial pH low while the resupply of oxygen during the intermittent reperfusion provides fuel for the redox signaling that acts to prevent MPTP formation even after restoration of normal myocardial pH. Hearts treated simultaneously with IPoC and alkaline perfusate could not be rescued by addition to the perfusate of either PMA or SB216763 which inhibits GSK-3beta, the putative last cytoplasmic signaling step in the signal transduction cascade leading to MPTP inhibition. Yet cyclosporin A which also inhibits MPTP formation does rescue hearts made alkaline during IPoC. In view of prior studies in which the ROS scavenger N-2-mercaptopropionyl glycine aborts IPoC's protection, our data reveal that IPoC's reperfusion periods are needed to support redox signaling rather than improve metabolism. The low pH, on the other hand, is equally necessary and seems to suppress MPTP directly rather than through upstream signaling.