Amanda J. Zatta

Remote postconditioning. Brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infarct size via endogenous activation of adenosine receptors.

AbstractObjectivesA series of brief coronary artery reperfusions and reocclusionsnapplied during the early minutes of coronary artery reflow (“postconditioning”)nattenuates reperfusion injury. However, it is not knownnwhether brief ischemia–reperfusion applied to a distant organ at the onset ofnmyocardial reperfusion (i.e. “remote postconditioning”, remote PostC)nreduces infarct size in the reperfused myocardium. In an in vivo anesthetizednrat model of myocardial infarction induced by coronary artery occlusion andnreperfusion, this study tested the hypothesis that remote postC induced by ansingle 5 minute episode of renal artery (RA) occlusion and reperfusionnapplied immediately before the onset of coronary artery reperfusion protectsnthe myocardium from reperfusion injury by mechanisms involving endogenousnadenosine receptor activation.MethodsAll rats were subjected to antotal of 30 minutes of left coronary artery occlusion (LCAO) and 3 hours ofnreperfusion. The rats were randomized to one of six groups: 1) Control: LCAOnand reperfusion only with no other intervention; 2) Remote PostC: after 24nminutes of LCAO the RA was occluded for 5 minutes and released 1 minnbefore coronary artery reperfusion; 3) Permanent RA occlusion: the RA wasnpermanently occluded after 24 minutes LCAO continuing to the end ofnreperfusion; 4) Delayed Remote PostC: after 26 minutes LCAO the RA wasnoccluded for 5 minutes, and its release was delayed until 1 min after coronarynartery reperfusion; 5) CON + SPT: rats with LCAO and reperfusion receivedn10 mg/kg IV of the non–selective adenosine receptor antagonist 8–sulfophenylntheophylline [SPT] administered 5 minutes before coronary artery reperfusion;nand 6) Remote PostC + SPT: after 24 minutes of LCAO the RA wasnoccluded for 5 minutes and released 1 minute before coronary artery reperfusionnin the presence of 10 mg/kg SPT given 5 min before coronary arterynreperfusion.ResultsMyocardial infarct size (percentage necrosis/area atnrisk, mean ± SEM) was reduced by 50% in Remote PostC (25 ± 4%) comparednto Control (49 ± 4%, p = 0.003), consistent with a reduction in plasma CKnactivity (44 ± 5 vs. 67 ± 6 U/ml, p = 0.023). In contrast, permanent RA occlusionnbefore LCAO and reperfusion failed to reduce myocardial infarct size (47n± 5%) vs Control. Delaying the release of the RA occlusion (delayed RemotenPostC) abrogated the myocardial infarct reduction observed with RemotenPostC (48 ± 6%). SPT alone had no effect on infarct size (47 ± 4% in CON +nSPT vs. 49 ± 4% in CON); however, Remote PostC+SPT abrogated thenmyocardial infarct size reduction in Remote PostC (50 ± 3% in RemotenPostC + SPT vs. 25 ± 4% in Remote PostC).ConclusionsRemote renal postconditioningnapplied immediately before the onset of coronary artery reperfusionnprovides potent myocardial infarct size reduction likely exerted during the first minutes of coronary artery reperfusion. This inter–organ remotenpostconditioning phenomenon is likely mediated in part by release ofnadenosine by the ischemic–reperfused kidney and subsequent activation ofnadenosine receptors.

Postconditioning--A new link in nature's armor against myocardial ischemia-reperfusion injury.

AbstractReperfusion injury is a complex process involving several cellntypes (endothelial cells, neutrophils, and cardiomyocytes), soluble proinflammatory mediators, oxidants, ionic and metabolic dyshomeostasis, andncellular and molecular signals. These participants in the pathobiology ofnreperfusion injury are not mutually exclusive. Some of these events take placenduring the very early moments of reperfusion, while others, seeminglyntriggered in part by the early events, are activated within a later timeframe.nPostconditioning is a series of brief mechanical interruptions of reperfusionnfollowing a specific prescribed algorithm applied at the very onset of reperfusion.nThis algorithm lasts only from 1 to 3 minutes depending on species.nAlthough associated with re–occlusion of the coronary artery or re–impositionnof hypoxia in cell culture, the reference to ischemia has been dropped.nPostconditioning has been observed to reduce infarct size and apoptosis asnthe “end games” in myocardial therapeutics; salvage of infarct size was similarnto that achieved by the gold standard of protection, ischemic preconditioning.nThe cardioprotection was also associated with a reduction in:nendothelial cell activation and dysfunction, tissue superoxide anion generation,nneutrophil activation and accumulation in reperfused myocardium,nmicrovascular injury, tissue edema, intracellular and mitochondrial calciumnaccumulation. Postconditioning sets in motion triggers and signals that arenfunctionally related to reduced cell death. Adenosine has been implicated innthe cardioprotection of postconditioning, as has e–NOS, nitric oxide andnguanylyl cyclase, opening of KATP channels and closing of the mitochondrialnpermeability transition pore. Cardioprotection by postconditioning has alsonbeen associated with the activation of intracellular survival pathways such asnERK1/2 and PI3 kinase – Akt pathways. Other pathways have yet to be identified. Although many of the pathways involved in postconditioning have alsonbeen identified in ischemic preconditioning, some may not be involved innpreconditioning (ERK1/2). The timing of action of these pathways and othernmediators of protection in postconditioning differs from that of preconditioning.nIn contrast to preconditioning, which requires a foreknowledge of thenischemic event, postconditioning can be applied at the onset of reperfusionnat the point of clinical service, i.e. angioplasty, cardiac surgery, transplantation.

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.

Inhibition of myocardial apoptosis by postconditioning is associated with attenuation of oxidative stress-mediated nuclear factor-κB translocation and TNFα release

ABSTRACT Oxidative stress-stimulated nuclear factor-κB (NF-κB) activation has been associated with rapid transcription of TNF-α and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-κB and release of TNF-α secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 ± 0.08 vs. 0.8 ± 0.06* μM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-κB to nuclei (167% ± 21% vs. 142% ± 18%*), decreased the level of plasma TNF-α (1,994 ± 447 vs. 667 ± 130* pg/mL), and inhibited caspase-3 activity (0.57% ± 0.1% vs. 0.21% ± 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% ± 1% vs. 11% ± 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-κB expression (42% ± 8%*) and reduction of release of TNF-α (231 ± 72* pg/mL). Caspase-3 activity (0.33% ± 0.1%*) and apoptotic cells (12% ± 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-κB-TNF-α signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K ATP channels during reperfusion

This study tested the hypothesis that inhibition of myocardial injury and modulation of mitochondrial dysfunction by postconditioning (Postcon) after 24xa0h of reperfusion is associated with activation of KATP channels. Thirty dogs undergoing 60xa0min of ischemia and 24xa0h of reperfusion (R) were randomly divided into four groups: Control: no intervention at R; Postcon: three cycles of 30xa0s R alternating with 30xa0s re-occlusion were applied at R; 5-hydroxydecanoate (5-HD): the mitochondrial KATP channel blocker was infused 5xa0min before Postcon; HMR1098: the sarcolemmal KATP channel blocker was administered 5xa0min before Postcon. After 24xa0h of R, infarct size was smaller in Postcon relative to Control (27xa0±xa04%* Vs. 39xa0±xa02% of area at risk), consistent with a reduction in CK activity (66xa0±xa07* Vs. 105xa0±xa07xa0IU/g). The infarct-sparing effect of Postcon was blocked by 5-HD (48xa0±xa05%†), but was not altered by HMR1098 (29xa0±xa03%*), consistent with the change in CK activity (102xa0±xa08† in 5-HD and 71xa0±xa06*xa0IU/g in HMR1098). In H9c2 cells exposed to 8xa0h hypoxia and 3xa0h of reoxygenation, Postcon up-regulated expression of mito-KATP channel Kir6.1 protein, maintained mitochondrial membrane potential and inhibited mitochondrial permeability transition pore (mPTP) opening evidenced by preserved fluorescent TMRE and calcein staining. The protective effects were blocked by 5-HD, but not by HMR1098. These data suggest that in a clinically relevant model of ischemia-reperfusion (1) Postcon reduces infarct size and decreases CK activity after prolonged reperfusion; (2) protection by Postcon is achieved by opening mitochondrial KATP channels and inhibiting mPTP opening. *Pxa0<xa00.05 Vs. Control; †Pxa0<xa00.05 Vs. Postcon.

Long-term inhibition of myocardial infarction by postconditioning during reperfusion.

AbstractCardioprotection with postconditioning has been well demonstratednafter a short period of reperfusion. This study tested the hypothesisnthat postconditioning reduces infarct size, vascular dysfunction, and neutrophilnaccumulation after a long-term reperfusion. Canines undergoing 60nmin left anterior descending artery (LAD) occlusion were divided into twoncontrol groups of either 3 h or 24 h of full reperfusion and two postconditioningngroups with three 30 s cycles of reperfusion and re-occlusion appliednat the onset of either 3 h or 24 h of reperfusion. Size of the area at risk (AAR)nand collateral blood flow during ischemia were similar among groups. Inncontrols, infarct size as percentage of the AAR (30 ± 3 vs. 39 ± 2* %) by TTCnstaining, superoxide anion generation from the post-ischemic coronary arteriesnby lucigenin-enhanced chemiluminescence [(89 ± 5 vs. 236 ± 27* relativenlight units (RLU/mg)], and neutrophil (PMN) accumulation by immunohistochemicalnstaining in the AAR (52 ± 11 vs. 84 ± 14* cells/mm2nmyocardium) significantly increased between 3 and 24 h of reperfusion.nPostconditioning reduced infarct size (15 ± 4† and 27 ± 3.6† %), superoxidenanion generation (24 ± 4† and 43 ± 11† RLU/mg), and PMN accumulationn(19 ± 6† and 45 ± 8† cells/mm2 myocardium) in the 3 and 24 h reperfusionngroups relative to time-matched controls. These data suggest that myocardialninjury increases with duration of reperfusion; reduction in infarct sizenand attenuation in inflammatory responses with postconditioning persist afterna prolonged reperfusion. * p < 0.05 24 vs. 3 h control; † p < 0.05 postconditioningnvs. time-matched control.

Myocardial protection in reperfusion with postconditioning

Reperfusion is the definitive treatment for coronary occlusive disease. However, reperfusion carries the potential to exacerbate lethal injury, termed ‘reperfusion injury’. Studies have suggested that reperfusion injury events are triggered during the early moments of reflow, and determine, in part, the severity of downstream manifestations of postischemic injury, including endothelial dysfunction, infarction and apoptosis. The application of brief iterative episodes of reflow (reoxygenation) and reocclusion (ischemia, hypoxia) at the immediate onset of reperfusion, which has been termed ‘postconditioning’ by the authors, reduces many manifestations of postischemic injury, notably infarct size, apoptosis, coronary vascular endothelial injury and reperfusion arrhythmias. Cardioprotection with postconditioning has been reported to be comparable with that observed using the gold standard maneuver ischemic preconditioning. In contrast to preconditioning, which exerts its effects primarily during the index ischemia, postconditioning appears to exert its effects during reperfusion alone. Postconditioning modifies the early phase of reperfusion in ways that are just beginning to be understood. It appears to first: reduce the oxidant burden and consequent oxidant-induced injury; secondly, attenuate the local inflammatory response to reperfusion; and thirdly, engage end effectors and signaling pathways implicated in other cardioprotective maneuvers, such as ischemic and pharmacologic preconditioning. Postconditioning seems to trigger the upregulation of survival kinases principally known to attenuate the pathogenesis of apoptosis and possibly necrosis. The postconditioning phenomenon has been reproduced by a number of independent laboratories and has been observed in both large and small animal in vivo models, as well as in ex vivo and cell culture models. In contrast to preconditioning, postconditioning may have widespread clinical application because it can be applied during reperfusion at the point of service for angioplasty, stenting, cardiac surgery and organ transplantation.

Mediators of coronary reactive hyperaemia in isolated mouse heart

1 Mechanisms regulating coronary tone under basal conditions and during reactive hyperaemia following transient ischaemia were assessed in isolated mouse hearts. 2 Blockade of NO‐synthase (50u2003μM L‐NAME), KATP channels (5u2003μM glibenclamide), A2A adenosine receptors (A2AARs; 100u2003nM SCH58261), prostanoid synthesis (100u2003μM indomethacin), and EDHF (100u2003nM apamin+100u2003nM charybdotoxin) all reduced basal flow ∼40%. Effects of L‐NAME, glibenclamide, and apamin+charybdotoxin were additive, whereas coadministration of SCH58261 and indomethacin with these inhibitors failed to further limit flow. 3 Substantial hyperaemia was observed after 5–40u2003s occlusions, with flow increasing to a peak of 48±1u2003mlu2003min−1u2003g−1. Glibenclamide most effectively inhibited peak flows (up to 50%) while L‐NAME was ineffective. 4 With longer occlusions (20–40u2003s), glibenclamide alone was increasingly ineffective, reducing peak flows by ∼15% after 20u2003s occlusion, and not altering peak flow after 40u2003s occlusion. However, cotreatment with L‐NAME+glibenclamide inhibited peak hyperaemia by 70 and 25% following 20 and 40u2003s occlusions, respectively. 5 In contrast to peak flow changes, sustained dilation and flow repayment over 60u2003s was almost entirely KATP channel and NO dependent (each contributing equally) with all occlusion durations. 6 Antagonism of A2AARs with SCH58261 reduced hyperaemia 20–30% whereas inhibition of prostanoid synthesis was ineffective. Effects of A2AAR antagonism were absent in hearts treated with L‐NAME and glibenclamide, supporting NO and KATP‐channel‐dependent effects of A2AARs. 7 EDHF inhibition alone exerted minor effects on hyperaemia and only with longer occlusions. However, residual hyperaemia after 40u2003s occlusion in hearts treated with L‐NAME+glibenclamide+SCH58261+indomethacin was abrogated by cotreatment with apamin+charybdotoxin. 8 Data support a primary role for KATP channels and NO in mediating sustained dilation after coronary occlusion. While KATP channels (and not NO) are also important in mediating initial peak flow adjustments after brief 5–10u2003s occlusions, their contribution declines with longer 20–40u2003s occlusions. Intrinsic activation of A2AARs is important in triggering KATP channel/NO‐dependent hyperaemia. Synergistic effects of combined inhibitors implicate interplay between mediators, with compensatory changes occurring in KATP channel, NO, and/or EDHF responses when one is individually blocked.