Protection Of Postconditioning Research Articles

Redox signaling in ischemic postconditioning protection involves PKCε and Erk1/2 pathways and converges indirectly in Nrf2 activation

Ischemic-postconditioning (iPostC) exerts cardioprotection by preserving redox homeostasis in the reperfused heart. This protective effect has been associated with the activation of endogenous antioxidant response driven by transcription factor Nrf2 and with the activation of ‘reperfusion injury salvage kinases’ (RISK) as PI3K, PKC and Erk1/2. Redox homeostasis is essential for normal cell physiology since reactive oxygen species (ROS) are crucial for processes that involve protein signaling. Thus, it has become clear that not only the perturbation of redox balance to oxidative state is deleterious but also towards a reductive state contributing to pathogenesis of diseases. However, there is still a scarce knowledge about the role of ROS in the cardioprotective signals mediated by RISK in postconditioned hearts. Therefore, we studied the role of ROS as initiator of RISK signaling molecules in iPostC-conferred cardioprotection. With the aim to study the relationship between redox-dependent RISK activation and the downstream activation of the transcription factor Nrf2, we evaluated the effect of redox signaling disruption by the effect of ascorbic acid in iPostC hearts. Our results showed that PKCε and Erk1/2 activation is redox-dependent and that concurs downstream with Nrf2 deficient activation. Besides, using inhibitors we found that neither PI3K nor Erk1/2 are directly related with Nrf2 activation, indicating that these kinases have other targets. We conclude that redox signaling participates in cardioprotection triggered by iPostC through the action of kinase-dependent and -independent mechanisms and concurred with the downstream regulation of Nrf2-mediated antioxidant response to prolonged redox balance during long reperfusion.

Nesfatin-1 and the Cardiovascular System: Central and Pheripheral Actions and Cardioprotection.

Recently, the hypothalamic 82-aa peptide Nesfatin-1 received notable attention for its anorexigenic and anti-hyperglycemic properties. In mammalian hypothalamus, Nesfatin-1 is expressed, together with the precursor Nucleobindin 2 (NUCB2), in regions controlling water-food intake, body weight, and glucose homeostasis. The peptide is also peripherally expressed, as shown in the rat heart, in which it is present together with NUCB2. In addition to a central modulation of nutrition and energy balance, and of the nervous circuits responsible for blood pressure and heart rate control, Nesfatin-1 also acts peripherally on several districts, including the cardiovascular (CV) system. Accordingly, the peptide is regarded with interest as a multifunctional hormone not only linked to alimentary homeostasis. This review aims to analyze the literature on Nesfatin-1, with focus on its emerging CV activity. Few available studies show that the peptide affects energy metabolism of murine and human cardiomyocytes, by eliciting insulin-like effects. On the ex vivo rat heart, it directly depresses contractility and relaxation via cGMP, PKG and ERK1/2, and limits ischemia/reperfusion (I/R) damage, acting in post-conditioning protection. Nesfatin-1 actions are proposed to involve an unknown G-protein coupled receptor. However, in the rat heart, functional studies, co-immunoprecipitation and local sequence alignment analyses suggest an interaction with the Natriuretic Peptide Receptor-type A (NPR-A). These data open up novel perspectives to clarify not only the biological significance of the peptide, but also its putative biomedical potential in the presence of nutrition-dependent cardiovascular diseases.

Abstract 21: Remote Limb Ischemic Postconditioning Improves Postresuscitation Cerebrovascular Circulation and Survival/Neurological Prognoses via In Situ and Remote Activation of Akt-eNOS-NO Signaling Pathway

Introduction: Cerebrovascular circulation is usually compromised after cardiac arrest and CPR. Remote limb ischemic post-conditioning (RLIP) is clinically feasible and can potentially mitigate post-resuscitation neurological deficits. Since nitric oxide (NO) is implicated in post-conditioning protection, we aim to investigate if RLIP impacts post-resuscitation cerebral perfusion and prognosis via NO-related mechanism. Hypothesis: RLIP improves post-CPR cerebral perfusion and prognosis through in situ and remote activation of Akt-eNOS-NO signaling. Methods: Using an established rat model of asphyxia cardiac arrest and CPR, we randomized the rats to the following groups: (1) sham, (2) standard CPR, (3) RLIP 5 min after return of spontaneous circulation (ROSC). RLIP was done by 3 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion. Arterial blood was sampled for colorimetric determination of nitrate/nitrite. The cerebral perfusion was continuously recorded by OxyFLO probe. Two hours after ROSC, the brain and left femoral artery were harvested for measuring phosphorylated endothelial NO synthase (p-eNOS at Ser1177) and protein kinase B (p-Akt at Ser473). In a subgroup the survival and neurological outcomes were monitored up to 3 days. Results: The cerebral perfusion was significantly decreased (0.6-0.8 folds that of baseline) after ROSC in standard CPR group. If RLIP was employed, the cerebral perfusion was significantly augmented (up to 1.6 folds, P < 0.001) in the post-resuscitation phase. This was associated with improved survival (log-rank P < 0.05) and neurological scores at 6, 24, 48 and 72 h (all P < 0.05). Plasma NO as indicated by nitrate/nitrite was significantly increased in the RLIP group ( P < 0.05). Most of all, p-eNOS and p-Akt were significantly increased not only in left femoral artery but also in brain. If NOS inhibitor N ω -nitro-L-arginine methyl ester (10 mg/kg) was used, not only the NO increase was reversed, the improvement in survival and neurological outcomes were also abrogated. Conclusions: RLIP enhances post-resuscitation cerebral perfusion and improves survival and neurological prognoses not only via in situ limb artery derived NO but remote activation of Akt-eNOS signaling in the brain.

Infarct size reduction by cyclosporine A at reperfusion involves inhibition of the mitochondrial permeability transition pore but does not improve mitochondrial respiration

IntroductionIschemic postconditioning (PoCo) and cyclosporine A (CysA) given prior to reperfusion reduce myocardial infarct size after ischemia/reperfusion. Ischemic postconditioning's protection is characterized by better preservation of mitochondrial respiration and calcium retention capacity. Protection by CysA is not entirely clear. Cyclosporine A inhibits not only mitochondrial permeability transition pore (mPTP) opening but also the phosphatase calcineurin. We have investigated whether CysA mediates protection not only by mPTP inhibition but also through a more upstream inhibition of calcineurin with subsequently better preserved mitochondrial respiration.Material and methodsAnesthetized pigs were subjected to 90 min ischemia and 10 min reperfusion initiated with either PoCo (6 × 20 s reperfusion/re-occlusion; n = 9), CysA infusion (5 mg/kg i.v.; 5 min before reperfusion; n = 4), or immediate full reperfusion (IFR; n = 8). Mitochondria were isolated from myocardial tissue for measurement of respiration and calcium retention capacity.ResultsIn mitochondria from ischemic/reperfused myocardium, ADP-stimulated complex I respiration was similar between CysA (116 ±11 nmol O2/min/mg protein) and IFR (117 ±8), but better preserved with PoCo (160 ±9; p < 0.05). Calcium retention capacity was greater with both PoCo and CysA (1096 ±45 and 1287 ±128 nmol Ca2+/mg protein) than with IFR (756 ±103; p < 0.05).ConclusionsCyclosporine A's protection is not associated with improved mitochondrial respiration. Protection is unlikely related to an upstream calcineurin inhibition, but is indeed secondary to mPTP inhibition.

THE EFFECTS OF ISCHAEMIC POSTCONDITIONING ON THE EXPRESSION OF BCL-2 AND BAX PROTEIN IN RATS FOLLOWING ISCHAEMIA/REPERFUSION INJURY

ObjectivesRecently, many researchers have discovered that ischaemic postconditioning (IP) was a neuroprotective factor on cerebral ischaemia/reperfusion injury, which could have many mechanisms. Our researches purpose is going to observe the...

Editorial Comment: Myocardial protection with postconditioning in cardiac surgery: the importance of the model

In this issue of the European Journal of Cardio-Thoracic Surgery, Maruyama and Chambers [1] pose the question of whether postconditioning (POC) enhances the protection provided by normothermic St Thomas’ number 2 (ST2) cardioplegia solution. This is an excellent question, posed by a highly productive group, and whether POC provides protection in addition to that exerted by cardioplegia is a question that has been asked by others [2]. Maruyama and Chambers found that a POC algorithm applied after increasing durations of arrest induced by a single infusion of warm St Thomas’ number 2 (ST2) cardioplegia did not enhance the primary end points of recovery of contractile function or reduce infarct size relative to that observed after arrest and reperfusion alone. It is important that the intervention targets the end points or relevant mechanisms. POC has been shown to target mechanisms impacting these two end points; the improvement in global contractile function is likely through the reduction in infarct size since no effect has been demonstrated on contractile function per se. POC did not enhance the postcardioplegic outcomes at any duration of ischaemia, but was only effective when the magnesium concentration of ST2 cardioplegia solution was reduced to zero. Maruyama and Chambers conclude that POC has limited efficacy after arrest with ST2 cardioplegia solution, and there is likely a magnesium-sensitive component of POC protection. POC is defined as reperfusion interrupted by brief periods of ischaemia applied during the opening minutes of reflow [3]. In cardiac surgery, POC can be applied by directly clamping and declamping the aorta, as performed by Luo et al. [4], or by iteratively infusing and halting the cardioplegia solution using a dedicated delivery system (Quest Medical Inc.’ MPS, Terumo Cardiovascular Systems System 1 Master/Follower, etc). In contrast to cardioplegia solutions and adjunct strategies such as hypothermia that are designed to reduce the severity of ischaemia that, in part, also determines the severity of reperfusion injury, POC exclusively targets reperfusion injury. Conceptually, both cardioplegia and POC could reduce post-ischaemic injury. If the cardioplegia solution and related strategies successfully limit ischaemic injury, then there is little subsequent reperfusion injury on which POC may exert protection. If, on the other hand, the majority of injury occurs during ischaemia with or without cardioplegia, there may be little room for protection from reperfusion injury, and again POC may not play a role as in the 45 and 60 min ischaemia-only group (Figure 2). It was important for the authors to show that the POC algorithm was effective in improving functional recovery and reducing infarct size, at least after 30 min of normothermic ischaemia alone (no cardioplegia). However, the observation that POC did not improve functional recovery or reduce infarct size does not necessarily represent a failure or loss of cardioprotection, but rather represents a limitation of that algorithm to stimulate the triggers and cardioprotective pathways [5] that impact the effectors of POC; this phenomenon has been called the ‘threshold’ of protection [6]. When a threshold is encountered, whereby protection is no longer observed after longer durations of ischaemia, increasing the number of postconditioning cycles has been shown to re-establish cardioprotection. The authors may have encountered such a threshold at the 45 and 60 min of index ischaemia in the present study. This should be determined in further studies. The model often profoundly affects the outcomes of a study. In the Langendorff isolated perfused, non-working (not ejecting and performing external work) heart preparation, there may be fundamental species differences in biology, metabolic differences that effect the time course of ischaemic injury and efficacy of interventions. Whether the injury occurs primarily during ischaemia or during reperfusion is a fundamental question in this study. ST2 cardioplegia solution was used at 37°C, which differs from its use clinically, and is not consistent with clinical studies testing the efficacy of POC after hypothermic cardioplegia. Tables 2 and 3 show left ventricular end-diastolic pressures >30 mmHg at the end of ischaemia (0 min reperfusion), suggesting that contracture has already occurred to some extent during ischaemia with or without cardioplegia; contracture is associated with the onset of irreversible injury at the end of ischaemia that would not be amenable to reperfusion therapy such as POC. In addition, the isolated perfused heart preparation inherently lacks components of the inflammatory response, i.e. plasma-borne inflammatory cells, proteases and cytokines that contribute to reperfusion injury. POC has been shown to inhibit neutrophil accumulation, activation of the coronary vascular endothelium,

CGMP/PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion

Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague–Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by 31P-NMR spectroscopy (3.6 ± 0.2 min and 3.5 ± 0.2 min respectively vs. 1.4 ± 0.2 min in control group, P < 0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P < 0.01), but only postconditioning increased myocardial cGMP levels (P = 0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na +/H +-exchange mediated pHi normalization after acidification induced by a NH 4Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na +/H +-exchanger.

E0152 Endogenous κ opioid peptide mediates the cardioprotection induced by ischaemic postconditioning

AimPostconditioning is brief cycles of reperfusion and ischaemia during the early phase of reperfusion following a prolonged ischaemic insult. Opioids are well-known endogenous triggers of preconditioning. Because postconditioning shares the...

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.

The pH Hypothesis of Postconditioning

It is unclear how reperfusion of infarcting hearts with alternating cycles of coronary reperfusion/occlusion attenuates infarction, but prevention of mitochondrial permeability transition pore (MPTP) formation is crucial. Acidosis also suppresses MPTP formation. We tested whether postconditioning protects by maintaining acidosis during early reoxygenation. After 30-minute regional ischemia in isolated rabbit hearts, reperfusion with buffer (pH 7.4) caused 34.4+/-2.2% of the risk zone to infarct, whereas 2 minutes of postconditioning (6 cycles of 10-second reperfusion/10-second occlusion) at reperfusion resulted in 10.7+/-2.9% infarction. One minute (3 cycles) of postconditioning was not protective. Hypercapnic buffer (pH 6.9) for the first 2 minutes of reperfusion in lieu of postconditioning caused equivalent cardioprotection (15.0+/-2.6% infarction), whereas 1 minute of acidosis did not protect. Delaying postconditioning (6 cycles) or 2 minutes of acidosis for 1 minute aborted protection. Reperfusion with buffer (pH 7.7) blocked postconditioning protection, but addition of the MPTP closer cyclosporin A restored protection. Reactive oxygen species scavenger N-2-mercaptopropionyl glycine, protein kinase C antagonist chelerythrine, and mitochondrial K(ATP) channel closer 5-hydroxydecanoate each blocked protection from 2 minutes of acidosis as they did for postconditioning. Thus, postconditioning prevents MPTP formation by maintaining acidosis during the first minutes of reperfusion as reoxygenated myocardium produces reactive oxygen species that activate protective signaling to inhibit MPTP formation after pH normalization.

No loss of cardioprotection by postconditioning in connexin 43-deficient mice

In situ hearts and isolated cardiomyocytes from heterozygous connexin 43-deficient (Cx43+/-) mice cannot be protected by ischemic preconditioning or diazoxide. We have now addressed the role of connexin 43 in ischemic postconditioning (PC). Wild type (WT) and Cx43+/- mice were subjected to 30 min coronary occlusion and 120 min reperfusion, with and without a PC protocol of three cycles of 10 s coronary occlusion/10 s reperfusion. Infarct size (TTC staining) was reduced by PC from 54+/-5 to 37+/-3% of area at risk in WT. Likewise, infarct size was reduced by PC from 53+/-4 to 34+/-3% of area at risk in Cx43+/-. We conclude that connexin 43 is no prerequisite for PC's protection. To this end, the signal transduction of ischemic preconditioning and postconditioning differs.