Initial Despair and Current Hope of Identifying a Clinically Useful Treatment of Myocardial Reperfusion Injury: Insights Derived from Studies of Platelet P2Y12 Antagonists and Interference with Inflammation and NLRP3 Assembly.

Myocardial necrosis following the successful reperfusion of a coronary artery occluded by thrombus in a patient presenting with ST-elevation myocardial infarction (STEMI) continues to be a serious problem, despite the multiple attempts to attenuate the necrosis with agents that have shown promise in pre-clinical investigations. Possible reasons include confounding clinical risk factors, the delayed application of protective agents, poorly designed pre-clinical investigations, the possible effects of routinely administered agents that might unknowingly already have protected the myocardium or that might have blocked protection, and the biological differences of the myocardium in humans and experimental animals. A better understanding of the pathobiology of myocardial infarction is needed to stem this reperfusion injury. P2Y12 receptor antagonists minimize platelet aggregation and are currently part of the standard treatment to prevent thrombus formation and propagation in STEMI protocols. Serendipitously, these P2Y12 antagonists also dramatically attenuate reperfusion injury in experimental animals and are presumed to provide a similar protection in STEMI patients. However, additional protective agents are needed to further diminish reperfusion injury. It is possible to achieve additive protection if the added intervention protects by a mechanism different from that of P2Y12 antagonists. Inflammation is now recognized to be a critical factor in the complex intracellular response to ischemia and reperfusion that leads to tissue necrosis. Interference with cardiomyocyte inflammasome assembly and activation has shown great promise in attenuating reperfusion injury in pre-clinical animal models. And the blockade of the executioner protease caspase-1, indeed, supplements the protection already seen after the administration of P2Y12 antagonists. Importantly, protective interventions must be applied in the first minutes of reperfusion, if protection is to be achieved. The promise of such a combination of protective strategies provides hope that the successful attenuation of reperfusion injury is attainable.

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.

The Infarct-Limiting Effect of Remote Ischemic Conditioning in Rats Is Not Affected by Aspirin.

PURPOSE: Remote ischemic conditioning (RIC) has been shown to be a powerful cardioprotective therapy in animal models. However, a protective effect in patients presenting with acute myocardial infarction has failed to be confirmed. A recent pre-clinical study reported that aspirin which is routinely given to patients undergoing reperfusion therapy blocked the infarct-limiting effect of ischemic postconditioning. The present study was designed to test whether aspirin could also be blocking the infarct-limiting effect of RIC. METHODS: This was investigated in vivo using male Sprague Dawley rats (n = 5 to 6 per group) subjected to either 30 min of regional myocardial ischemia, followed by 120-min reperfusion, or additionally to a RIC protocol initiated after 20-min myocardial ischemia. The RIC protocol included four cycles of 5-min hind limb ischemia interspersed with 5-min reperfusion. Intravenous aspirin (30 mg/kg) or vehicle (saline) was administered after 15-min myocardial ischemia. RESULTS: RIC significantly reduced infarct size (IS) normalized to the area at risk, by 47%. Aspirin administration did not affect IS nor did it attenuate the infarct-limiting effect of RIC. CONCLUSION: Aspirin administration in the setting of myocardial infarction is not likely to interfere with the cardioprotective effect of RIC.

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.

Guidelines for evaluating myocardial cell death.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

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.

The impact of irreproducibility and competing protection from P2Y12 antagonists on the discovery of cardioprotective interventions.

Scientists and clinicians have been concerned by the lack of a clinically suitable strategy for cardioprotection in patients with acute myocardial infarction despite decades of intensive pre-clinical investigations and a surprising number of clinical trials based on those observations which have uniformly been disappointing. However, it would be a mistake to abandon this search. Rather it would be useful to examine these past efforts and determine reasons for the multiple failures. It appears that earlier clinical trials were often based on results from a single experimental laboratory, thus minimizing the importance of establishing reproducibility in multiple laboratories by multiple scientists and in multiple models. Clinical trials should be discouraged unless robust protection is demonstrated in pre-clinical testing. After approximately 2005 a loading dose of a platelet P2Y12 receptor antagonist became increasingly widespread in patients with acute myocardial infarction prior to revascularization and quickly became standard-of-care. These agents are now thought to be a cause of failure of recent clinical trials since these pleiotropic drugs also happen to be potent postconditioning mimetics. Thus, introduction of an additional cardioprotective strategy such as ischemic postconditioning which uses the same signaling pathway as these P2Y12 antagonists would be redundant and doomed to failure. Additive cardioprotection could be achieved only if the second intervention had a different mechanism of cardioprotection. This concept has been demonstrated in experimental animals. So lack of reproducibility of earlier studies and failure to examine interventions in experimental animals also treated with anti-platelet agents could well explain past failures. These realizations should clear the way for development of interventions which can be translated into successful clinical treatments.

Prospects for Creation of Cardioprotective and Antiarrhythmic Drugs Based on Opioid Receptor Agonists.

It has now been demonstrated that the µ, δ1 , δ2 , and κ1 opioid receptor (OR) agonists represent the most promising group of opioids for the creation of drugs enhancing cardiac tolerance to the detrimental effects of ischemia/reperfusion (I/R). Opioids are able to prevent necrosis and apoptosis of cardiomyocytes during I/R and improve cardiac contractility in the reperfusion period. The OR agonists exert an infarct-reducing effect with prophylactic administration and prevent reperfusion-induced cardiomyocyte death when ischemic injury of heart has already occurred; that is, opioids can mimic preconditioning and postconditioning phenomena. Furthermore, opioids are also effective in preventing ischemia-induced arrhythmias.

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.

Does mild hypothermia protect against reperfusion injury? The debate continues.

Mild hypothermia (32-35°C) salvages ischemic myocardium and reduces infarct size in hearts undergoing ischemia/reperfusion. It is clear that a cardioprotective effect is evident when the heart is cooled during ischemia, and the protection is greater as the duration of normothermic ischemia is increasingly limited. The effect of cooling just before and at reperfusion is more controversial. Multiple experimental studies have revealed no effect of mild hypothermia on myocardial infarction when cooling was initiated in the waning minutes of ischemia. But Götberg et al. have demonstrated a small effect in pigs cooled with cold intravenous saline and a venous thermode, although the effect of cooling during ischemia continued to be more prominent. Clinical studies have been disappointing, and possible explanations are offered. Götberg's new data are encouraging, but it is questioned whether this is the correct time to conduct a new large-scale clinical trial.

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.

Evidence for an intracellular localization of the adenosine A2B receptor in rat cardiomyocytes.

Protection achieved by ischemic preconditioning is dependent on A(2B) adenosine receptors (A(2B)AR) in rabbit and mouse hearts and, predictably, an A(2B)AR agonist protects them. But it is controversial whether cardiomyocytes themselves actually express A(2B)AR. The present study tested whether A(2B)AR could be demonstrated on rat cardiomyocytes. Isolated rat hearts experienced 30 min of ischemia and 120 min of reperfusion. The highly selective, cell-permeant A(2B)AR agonist BAY60-6583 (500 nM) infused at reperfusion reduced infarct size from 40.4 ± 2.0% of the risk zone in control hearts to 19.9 ± 2.8% indicating that A(2B)AR are protective in rat heart as well. Furthermore, BAY60-6583 reduced calcium-induced mitochondrial permeability transition in isolated rat cardiomyocytes. A(2B)AR protein could be demonstrated in isolated cardiomyocytes by western blotting. In addition, message for A(2B)AR was found in individual cardiomyocytes using quantitative RT-PCR. Surprisingly, immunofluorescence microscopy did not show A(2B)AR on the cardiomyocyte's sarcolemma but rather at intracellular sites. Co-staining with MitoTracker Red in isolated cardiomyocytes revealed A(2B)AR are localized to mitochondria. Western blot analysis of a mitochondrial fraction from either rat heart biopsies or isolated cardiomyocytes revealed a strong A(2B)AR band. Thus, the present study demonstrates that activation of A(2B)AR is strongly cardioprotective in rat heart and suppresses transition pores in isolated cardiomyocytes, and A(2B)AR are expressed in individual cardiomyocytes. However, surprisingly, A(2B)AR are present in or near mitochondria rather than on the sarcolemma as are other adenosine receptors. Because A(2B)AR signaling is thought to result in inhibition of mitochondrial transition pores, this convenient location may be important.

The Role of Pyroptosis in Ischemic and Reperfusion Injury of the Heart.

While ischemia itself can kill heart muscle, much of the infarction after a transient period of coronary artery occlusion has been found to result from injury during reperfusion. Here we review the role of inflammation and possible pyroptosis in myocardial reperfusion injury. Current evidence suggests pyroptosis's contribution to infarction may be considerable. Pyroptosis occurs when inflammasomes activate caspases that in turn cleave off an N-terminal fragment of gasdermin D. This active fragment makes large pores in the cell membrane thus killing the cell. Inhibition of inflammation enhances cardiac tolerance to ischemia and reperfusion injury. Stimulation of the purinergic P2X7 receptor and the ß-adrenergic receptor and activation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) by toll-like receptor (TLR) agonists are all known to contribute to ischemia/reperfusion (I/R) cardiac injury through inflammation, potentially by pyroptosis. In contrast, stimulation of the cannabinoid CB2 receptor reduces I/R cardiac injury and inhibits this pathway. MicroRNAs, Akt, the phosphate and tension homology deleted on chromosome 10 protein (PTEN), pyruvate dehydrogenase and sirtuin-1 reportedly modulate inflammation in cardiomyocytes during I/R. Cryopyrin and caspase-1/4 inhibitors are reported to increase cardiac tolerance to ischemic and reperfusion cardiac injury, presumably by suppressing inflammasome-dependent inflammation. The ambiguity surrounding the role of pyroptosis in reperfusion injury arises because caspase-1 also activates cytotoxic interleukins and proteolytically degrades a surprisingly large number of cytosolic enzymes in addition to activating gasdermin D.

Both A2a and A2b adenosine receptors at reperfusion are necessary to reduce infarct size in mouse hearts.

Pre- and postconditioning depend on the activation of adenosine receptors (ARs) at the end of the index ischemia. The aim of this study was to determine which receptor subtypes must be activated. In situ mouse hearts underwent 30 min of regional ischemia, followed by 2 h of reperfusion. As expected, either ischemic postconditioning (6 cycles of 10 s of reperfusion and 10 s of coronary occlusion) or infusion of the selective A(2b) adenosine receptor (A(2b)AR) agonist BAY60-6583 (BAY60) for 60 min, starting 5 min before reperfusion reduced infarct size in wild-type C57Bl/6N mice. Protection from either was abolished by the selective A(2b)AR antagonist MRS-1754, confirming a role for A(2b)AR. Additionally, the coadministration of ischemic postconditioning and a selective A(2a)AR antagonist led to the loss of protection as well. 5'-Ectonucleotidase (CD73) is thought to be necessary for the production of adenosine during ischemia. As predicted, ischemic postconditioning did not protect CD73 knockout mice. Selective agonists of either A(2b)AR (BAY60) or A(2a)AR (CGS-21680), as well as the coadministration of ischemic postconditioning and BAY60, also failed to protect hearts of the CD73 knockout mice. But the nonselective A(1)/A(2)AR agonist 5'-(N-ethylcarboxamido)adenosine (NECA) was protective, suggesting that the activation of multiple AR subtypes might be required. The coadministration of CGS-21680 and BAY60 also elicited profound protection, indicating that two AR subtypes, A(2a) and A(2b), must be simultaneously activated for protection to occur.

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.