Second Window Of Protection Research Articles

#6635 ARNT AND ALK3 MEDIATE RENO PROTECTION AFTER ISCHEMIC PRECONDITIONING

Abstract Background and Aims Ischemia and following hypoxia are a common cause of acute kidney injury (AKI). Strategies for prevention and treatment of AKI are limited. In several animal models, ischemic preconditioning (IPC) has been established as an effective intervention to prevent ischemic organ injury. An IPC protocol of ischemia and reperfusion mediates organ protection for a few hours. This first phase of organ protection is followed by a second, protective phase occurring after 24–48 h. The second phase, named second window of protection (SWOP), persists for 3–4 d. Mechanisms and transcriptional mediators during SWOP are mostly unknown. The aim of the underlying study was to investigate effectors and target genes during SWOP and to identify a potential pharmacological therapeutic target. Method 6–8-week-old C57BL/6 mice were undergoing an IPC intervention; five minutes of renal sinistra artery ischemia was followed by five minutes of reperfusion. This cycle was performed five times. Mice were sacrificed 2, 4, 8, 12, 25, 48, 72 h after IPC. Two additional groups of mice were pharmacologically preconditioned for 48 h with low dose FK506 and small molecule GPI1046. Analysis of murine kidneys were performed by SDS-Page and Western blot. Results The first phase after IPC (2–4 h) was characterized by an increasing expression of hypoxia inducible factor-1α (HIF1α); 48 h after intervention a significant increase of expression of aryl hydrocarbon receptor nuclear translocator (ARNT) and consecutive activin receptor-like kinase 3 (ALK3) was observed. Comparable induction of ARNT and ALK3 expression was obtained by pharmacological preconditioning with FK506 or GPI1046. Conclusion Affected by hypoxia, HIF1α activates transcriptional upregulation of ARNT 48 h after IPC. The increasing expression of ARNT, follows an increased ARNT homodimerization. ARNT homodimer binds to the palindromic E-box 5′-CACGTG-3′ of the proximal ALK3 promoter. By this it induces the expression of the renoprotective ALK3 during SWOP. The organ-protective effects of constitutively expressed ARNT during SWOP can be mimicked pharmacologically by administration of FK506 or GPI1046 and may be used as a future therapeutic target.

Angiotensin 1-7: A Second Window of Protection in Hypertensive Patients

The effects of the renin-angiotensin-aldosterone system on the human body are so diverse and our knowledge about them is ever growing. Angiotensin 1-7 has been proven to play protective roles in patients with cardiovascular disorders including but not limited to hypertension.
 As is the case with Africa, the prevalence of hypertension in Sudan is rising, and its complications could be delayed by pharmacologically manipulating the levels of renin-angiotensin system metabolites.
 The aim of this review is to compare the advantageous and deleterious effects of Angiotensin 2 in contrast to those of Angiotensin 1-7 and to assert the well-established protective effects of Angiotensin 1-7 (systemically and locally) in hypertensive patients

The effect of exogenous HSP70 on expression kinetics of HSP70, TLR2, and TLR4 in peripheral blood mononuclear cells and possible crosstalk between them in Black Bengal goat

ABSTRACT The present study aims to investigate the modulatory effect of eHSP70 on major heat stressed genes in in-vitro cell culture system. HSP70 (eHSP70) on mRNA expression profile of heat-shock protein 70 (HSP70), toll-like receptor 2 (TLR2) and TLR4 in peripheral blood mononuclear cells (PBMCs) in cell culture model of Black Bengal goat. After thermal challenge of cultured PBMCs were treated with a specific dose of eHSP70 (50 ng/ml) at 37°C for 0, 1, 2, 4, 8, 12, 16, 20, and 24 h. The mRNA expressions of the above-mentioned genes were determined by quantitative real-time PCR (qRT-PCR). The study showed that on administration of eHSP70 to the PBMCs cell culture system, the relative mRNA expression of iHSP70 and HSP90 increased significantly (p < 0.05) at 4, 20 and 24 h compared with control, and and TLR2 and TLR4 showed significantly higher expression (p < 0.05) at 2, 4, 8 and 24 h compared with control. Immunoreactivity revealed that all genes under study were localized in cytoplasm as well as in nucleus. The present findings specify that eHSP70 is probably involved in heat stress adaptive response in Black Bengal goat and the biphasic expression pattern may be providing a second window of protection during heat stress. Abbreviations: ANOVA: analysis of variance; GAPDH: gleceraldehyde 3-Phosphate dehydrogenase; bp: base pairs; cDNA: complementary DNA; Ct value: cycle threshold values; DEPC: diethyl pyrocarbonate; dNTP’s: deoxynucleoside triphosphate; DW: distilled water; DPBS: dulbecco’s phosphate buffer saline; FBS: fetal bovine serum; HSP: heat-shock protein; HSF: Heat shock factors; IG: immunoglobulin; KDa: kilo dalton; LN2: liquid nitrogen; NF-κB: nuclear factor kappa beta; TLR: toll-like receptor; PBMC: peripheral blood mononuclear cell; eHSP70: exogenous heat-shock protein 70.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle

Six male Tharparkar cattle aged 2-3years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15days and then exposed to 42°C temperature up to 23days followed by 12days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P<0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P<0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P<0.05) after 10days of heat exposure (corresponds to chronic heat stress) as compared to the first 5days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Intestinal ischemic preconditioning ameliorates hepatic ischemia/reperfusion injury in rats: role of heme oxygenase 1 in the second window of protection.

Preconditioning by brief ischemia protects not only the concerned organ but also other distant organs against subsequent lethal damage; this is called remote ischemic preconditioning (RIPC). This study was designed to investigate the impact of intestinal RIPC on hepatic ischemia/reperfusion injury (IRI) with a special interest in heme oxygenase 1 (HO-1) induction in the second window of protection (SWOP). Male Wistar rats were randomly assigned to 1 of 2 groups: an RIPC group or a sham group. Before hepatic IRI, either intestinal RIPC, consisting of 2 cycles of 4-minute superior mesenteric artery clamping separated by 11 minutes of declamping (RIPC group), or a sham procedure (sham group) was performed. After 48 hours of recovery, the rats were exposed to 30 minutes of total hepatic IRI. Transaminase releases and proinflammatory cytokines were determined at several time points after reperfusion. Histopathological analysis and animal survival were also investigated. Intestinal RIPC significantly lowered transaminase release (alanine aminotransferase at 2 hours: 873.3 ± 176.4 IU/L for the RIPC group versus 3378.7 ± 871.1 IU/L for the sham group, P < .001) as well as proinflammatory cytokine production (tumor necrosis factor α at 2 hours: 930 ± 42 versus 387 ± 17 pg/μL, P < .001). The morphological integrity of the liver and the ileum was maintained significantly better with intestinal RIPC; this reached statistical significance not only in Suzuki's liver injury score (3.5 ± 0.2 versus 0.7 ± 0.5, P = .007) but also in Park's score for intestinal damage (4.0 ± 0.4 versus 2.0 ± 0.2, P = .007). Animal survival was also markedly improved (83.1% versus 15.4%, P < .001). As a mechanism underlying this protection, HO-1 was substantially induced in liver tissue, especially in hepatocytes, with remarkable up-regulation of bradykinin in the portal blood, whereas HO-1 protein induction in enterocytes was not significant. In conclusion, intestinal RIPC remarkably attenuates hepatic IRI in the SWOP, presumably by HO-1 induction in hepatocytes.

Expression of HSPs: an adaptive mechanism during long-term heat stress in goats (Capra hircus).

Menacing global rise in surface temperature compelled more focus of research over understanding heat stress response mechanism of animals and mitigation of heat stress. Twenty-four goats divided into four groups (n = 6) such as NHS (non-heat-stressed), HS (heat-stressed), HS + VC (heat-stressed administered with vitamin C), and HS + VE + Se (heat-stressed administered with vitamin E and selenium). Except NHS group, other groups were exposed to repeated heat stress (42 °C) for 6 h on 16 consecutive days. Blood samples were collected at the end of heat exposure on days 1, 6, 11, and 16. When groups compared between days, expression of all heat shock proteins (HSPs) showed a similar pattern as first peak on day 1, reached to basal level on the sixth day, and followed by second peak on day 16. The relative messenger RNA (mRNA) and protein expression of HSP 60, HSP70, and HSP90 was observed highest (P < 0.05) in HS group, followed by antioxidant-administered group on days 1 and 16, which signifies that antioxidants have dampening effect on HSP expression. HSP105/110 expression was highest (P < 0.05) on day 16. We conclude that HSP expression pattern is at least two-peak phenomenon, i.e., primary window of HSP protection on the first day followed by second window of protection on day 16. HSP60, HSP70, and HSP90 play an important role during the initial phase of heat stress acclimation whereas HSP105/110 joins this cascade at later phase. Antioxidants may possibly attenuate the HSP expression by reducing the oxidative stress.

Reduced expression of mitochondrial electron transport chain proteins from hibernating hearts relative to ischemic preconditioned hearts in the second window of protection

Although protection against necrosis has been observed in both hibernating (HIB) and ischemic preconditioned hearts in the second window of protection (SWOP), a comparison of the mitochondrial proteome between the two entities has not been previously performed. Anesthetized swine underwent instrumentation with a fixed constrictor around the LAD artery and were followed for 12weeks (HIB; N=7). A second group of anesthetized swine underwent ischemic preconditioning by inflating a balloon within the LAD artery 10 times for 2min, each separated by 2min reperfusion and were sacrificed 24h later (SWOP; N=7). Myocardial blood flow and high-energy nucleotides were obtained in the LAD region and normalized to remote regions. Post-sacrifice, protein content as measured with iTRAQ was compared in isolated mitochondria from the LAD area of a Sham heart. Basal regional blood flow in the LAD region when normalized to the remote region was 0.86±0.04 in HIB and 1.02±0.02 in SWOP tissue (P<0.05). Despite reduced regional blood flows in HIB hearts, ATP content in the LAD region, when normalized to the remote region was similar in HIB versus SWOP (1.06±0.06 and 1.02±0.05 respectively; NS) as was the transmural phosphocreatine (PCr) to ATP ratio (2.1±0.2 and 2.2±0.2 respectively; NS). Using iTRAQ, 64 common proteins were identified in HIB and SWOP hearts. Compared with SWOP, the relative abundance of mitochondrial proteins involved with electron transport chain (ETC) were reduced in HIB including NADH dehydrogenase, Cytochrome c reductase and oxidase, ATP synthase, and nicotinamide nucleotide transhydrogenase. Within chronically HIB heart tissue with reduced blood flow, the relative abundance of mitochondrial ETC proteins is decreased when compared with SWOP tissue. These data support the concept that HIB heart tissue subjected to chronically reduced blood flow is associated with a down-regulation in the expression of key mitochondrial proteins involved in electron transport.

Delayed anesthetic preconditioning protects against myocardial infarction via activation of nuclear factor-κB and upregulation of autophagy

Delayed volatile anesthetic preconditioning (APC) can protect against myocardial ischemia/reperfusion (I/R) injury; the delayed phase is called the second window of protection (SWOP), but the underlying mechanism is unclear. Nuclear factor-κB (NF-κB) is involved in the myocardial protection conferred by APC in the acute phase; autophagy has been reported to confer apoptosis inhibition and infarction reduction. We hypothesized that APC initiates delayed cardioprotection against I/R injury via the activation of NF-kB and upregulation of autophagy, thus attenuating the inflammatory response and apoptosis After a rat I/R model was set up, left ventricular samples were obtained before I/R to assess NF-κB-DNA binding activity and microtubule-associated protein 1 light chain 3 (LC3) and cathepsin B protein expression, and to examine autophagosomes with a transmission electron microscope. Infarct size and the expressions of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and caspase-3 were measured at the end of 2-h reperfusion. The infarct size was significantly reduced in the SWOP group (30±3%) when compared with that in the I/R group (47±7%, P<0.05), and this finding was associated with increased NF-κB-DNA binding activity and autophagosomes. In addition, the expressions of LC3-II and cathepsin B were also up-regulated, and the expressions of TNF-α, IL-1β, and caspase-3 were attenuated in the SWOP group when compared with the findings in the I/R group. However, this protection was abolished by the administration of parthenolide (PTN) before sevoflurane inhalation, which resulted in an infarct size that was significantly increased (47±5%, P<0.05 PTN+SWOP vs. SWOP group). Delayed APC protected the rat heart from I/R injury. The underlying mechanisms may include NF-κB activation, upregulation of autophagy, and the attenuation of TNF-α, IL-1β, and caspase-3 expressions.

A novel in vitro model of ischaemic preconditioning: its effect on cell viability in human skeletal muscle and a possible explanation for the ‘second window of protection’

BackgroundThe ischaemia–reperfusion (IR) injury causes significant morbidity. Ischaemic preconditioning (IPC) is a technique for limiting the effects of the IR injury. Its potential has not yet been harnessed in orthopaedics. AimsTo establish a novel in vitro IR model using a human skeletal muscle cell line. Secondly, to introduce simulated IPC to the model and examine the effect of this on cell viability. MethodsA human skeletal muscle cell line was cultured in vitro. Placing the cells in a hypoxic buffer and a closed hypoxic environment simulated ischaemia. Reversing this process simulated reperfusion. IPC was simulated by alternate cycles of ischaemia and reperfusion. Cell viability comparisons were made between control and experimental groups of cells. ResultsA reproducible in vitro IR model was established. The addition of simulated IPC is associated with increased cell death at 12 and 24h of reperfusion. Significantly greater cell survival is seen in the IPC group when measured at 72h reperfusion. ConclusionsWe hypothesise that IPC initially decreases cell number. The remaining cells are more robust. This selected cell line then expands over the course of 72h and displays greater resistance to the IR injury. This theory can help explain delayed preconditioning.

Preconditioning with periodic acceleration (pGz) provides second window of cardioprotection

AimsWhole body periodic acceleration (pGz) is achieved with a motorized platform that moves the supine body with repetitive, sinusoidal head to foot motion. This increases pulsatile shear stress to the endothelium thereby activating endothelial nitric oxide synthase (eNOS) to produce increased release of nitric oxide (eNO). The current investigation was undertaken to determine whether or not pGz preconditioning can induce second window of protection (SWOP) cardioprotective affects in an in-vivo rat model of myocardial infarction. Main methodsRats received 1, 3 or 7 daily pGz treatments of 60minutes, or no pGz (CONT). Twenty-four hours after the last pGz treatment, ischemia reperfusion injury was produced by ligation of the left coronary artery (LCA) for 30minutes followed by 90minutes of reperfusion. Key findingsAll pGz preconditioned animals survived 90minutes. Three and 7days of pGz preconditioning reduced myocardial infarct size by 41 and 38% respectively, with better left ventricular function. Additionally, pGz preconditioning increased eNOS, p-eNOS, Akt and p-Akt, HSP70 and nNOS proteins. L-NAME (NOS inhibitor) or Wortmannin (PI3/Akt inhibitor) 15minutes prior to LCA ligation abolished pGz's cardioprotective effects. SignificancepGz preconditioning provides SWOP, with increased survival, infarct size reduction, and improved contractility, in part due to up regulation of HSP70 and the PI3/Akt/eNOS , which last at least 72hours.

Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ(®) and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG(0)), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation.

The fascinating and elusive life of cardiac fatty acids

This editorial refers to ‘Second window of preconditioning normalizes palmitate use for oxidation and improves function during low-flow ischaemia’ by R.K. Kudej et al ., pp. 394–400, this issue. Kudej et al. 1 attempted to elucidate the role of fatty acids (FAs) in the second window of protection of the chronically underperfused myocardium. Using the in vivo conscious swine heart model, they elegantly showed that ischaemic preconditioning resulted in less severe thinning of the left ventricular (LV) wall 24h after the onset of ischaemia. The improved mechanical function of the ischaemic region was not due to changes in residual blood flow and, hence, increased supply of oxygen. The contribution of FAs to cardiac oxidative ATP production was estimated by infusion of 13C-palmitate and subsequent in vitro NMR analysis of palmitate-derived acetyl-CoA. The contribution of FA to oxidative energy production declined in the ischaemic LV wall accompanied by decreased malonyl-CoA levels in the ischaemic subendocardium. Interestingly, preconditioning substantially enhanced the relative contribution of FA oxidation to oxidative energy production in association with a three-fold increase in malonyl-CoA concentration. The study of Kudej et al. 1 deserves special attention especially for its iconoclastic message. Two important paradigms concerning cardiac FA utilization read that first, myocardial FA oxidation is detrimental under challenging conditions2,3 and, secondly, cardiac FA oxidation is controlled by malonyl-CoA, in that elevated malonyl-CoA levels are inhibitory.4,5 The present study, …

Clinically Useful Cardioprotection: Ischemic Preconditioning Then and Now

Ischemic preconditioning (IP) is the most effective, reproducible form of protection against myocardial cell death yet described. The mechanism of classic IP has not been identified, but recent investigations have focused on the mitochondrial permeability transition pore (mPTP). Similarly, the mechanism of the "second window of protection" (SWOP) is not known but thought to involve increased expression of important gene products. Currently, IP in the clinical arena is limited to cardiac surgery, planned angioplasty, and organ preservation protocols. To move preconditioning into a broader clinical arena will require resolution of important fundamental yet stubborn problems involving both basic and clinical science. Important unresolved issues include the mechanisms involved in the transition from reversible to irreversible injury, the amount of potential salvageable myocardium present at the onset of reperfusion, the identity and signaling of the mPTP, the optimization of protocols, the identity of end effectors (SWOP), and the identification of the best experimental model systems. From a clinical standpoint, important issues include the influence of comorbidities on cardioprotection, identification of appropriate animal models, the lack of a biologic marker of the cardioprotective state, the influence of coexistent therapeutic drugs, potential toxicity of pharmacologic mimics, and the window of opportunity for significant protection. Ischemic preconditioning has yielded promising results in other organs including the brain as well as tissue preservation for certain surgical procedures that will require definition of the underlying mechanism(s) to be fully exploited clinically. Over the past 25 years, the scientific community has learned much regarding the biology and potential mechanisms of IP and the concept has been expanded to many other organ systems in many other clinically relevant scenarios. To realize the full clinical potential will require continued investigation into the mechanism.

Coordinated Post-transcriptional Regulation of Hsp70.3 Gene Expression by MicroRNA and Alternative Polyadenylation

Heat shock protein 70 (Hsp70) is well documented to possess general cytoprotective properties in protecting the cell against stressful and noxious stimuli. We have recently shown that expression of the stress-inducible Hsp70.3 gene in the myocardium in response to ischemic preconditioning is NF-κB-dependent and necessary for the resulting late phase cardioprotection against a subsequent ischemia/reperfusion injury. Here we show that the Hsp70.3 gene product is subject to post-transcriptional regulation through parallel regulatory processes involving microRNAs and alternative polyadenylation of the mRNA transcript. First, we show that cardiac ischemic preconditioning of the in vivo mouse heart results in decreased levels of two Hsp70.3-targeting microRNAs: miR-378* and miR-711. Furthermore, an ischemic or heat shock stimulus induces alternative polyadenylation of the expressed Hsp70.3 transcript that results in the accumulation of transcripts with a shortened 3'-UTR. This shortening of the 3'-UTR results in the loss of the binding site for the suppressive miR-378* and thus renders the alternatively polyadenylated transcript insusceptible to miR-378*-mediated suppression. Results also suggest that the alternative polyadenylation-mediated shortening of the Hsp70.3 3'-UTR relieves translational suppression observed in the long 3'-UTR variant, allowing for a more robust increase in protein expression. These results demonstrate alternative polyadenylation of Hsp70.3 in parallel with ischemic or heat shock-induced up-regulation of mRNA levels and implicate the importance of this process in post-transcriptional control of Hsp70.3 expression.

The Second Window of Preconditioning (SWOP) Where Are We Now?

A standard ischemic preconditioning (IPC) stimulus of one or more brief episodes of non-lethal myocardial ischemia and reperfusion elicits a bi-phasic pattern of cardioprotection. The first phase manifests almost immediately following the IPC stimulus and lasts for 1-2 h, after which its effect disappears (termed classical or early IPC). The second phase of cardioprotection appears 12-24 h later and lasts for 48-72 h (termed the Second Window of Protection [SWOP] or delayed or late IPC). The cardioprotection conferred by delayed IPC is robust and ubiquitous but is not as powerful as early IPC. Although there are some similarities in the mechanisms underlying early and delayed IPC, one of the major distinctions between the two is the latter's requirement for de novo protein synthesis of distal mediators such as iNOS and COX-2 which mediate the cardioprotection 24 h after the IPC stimulus. The phenomenon of delayed IPC has been demonstrated in man using a variety of experimental models. However, its clinical application has been limited by the same factors which affect early IPC- i.e. the need to intervene before the onset of myocardial ischemia, thereby restricting its potential clinical utility to planned settings of acute myocardial ischemia-reperfusion injury such as coronary artery bypass graft surgery, cardiac transplantation and percutaneous coronary intervention. In this article, the focus will be on the origins of delayed IPC, the mechanisms underlying its delayed cardioprotective effect, and the potential areas for its clinical application.

A Favorable Energetic State Is Observed During Low Flow Myocardial Ischemia in the Second Window of Preconditioning (SWOP) in Swine Despite an Increased Expression of Uncoupling Protein-2

Background: Mitochondrial expression of uncoupling protein (UCP)-2 is increased following ischemic preconditioning and is protective in the Second Window of Protection (SWOP). Although UCP-2 expression protects against mitochondrial sources of oxidant damage, it dissipates the electrochemical gradient by inducing a hydrogen leak across the inner membrane and could create an unfavorable energetic state during myocardial ischemia. Methods: Fourteen anesthetized pigs underwent a SWOP protocol, with ten 2-minute balloon inflations in the LAD artery, separated by 2minutes reperfusion.

Mechanism of volatile anesthetic preconditioning-induced "second window" protection against myocardial ischemia/reperfusion injury

Volatile anesthetic preconditioning can elicit acute and delayed (also called as second window) myocardial protection,and the latter may be more clinicaUy significant in decreasing the risk of myocardial ischemia/reperfusion injury for patientswith cardiovascular disease.The second protection might be achieved by stimulating myocardium to trigger adenosine and nitric oxide release,passing through protein kinase C and nuclear factor-κB signal transduction pathways,activating the ATP-sensirive potassium channel and reactive oxygen species as the final effectors,presenting a delayed myocardial protective effect.This review summarizes the latest studies about volatile anesthetic preconditioning-induced second protection. Key words: Anesthetics; inhalation; Ischemic preconditioning; Myocardial reperfusion injury; Second window of protection

Isoflurane causes powerful second window of protection (SWOP) against myocardial stunning in the dog

Despite intensive investigation, the ability of isoflurane (ISO) to cause SWOP in the myocardium remains a matter of debate. We tested the hypothesis that inhalation of ISO can reduce extent of myocardial stunning produced by ischemia 24 hours later. The findings with ISO were compared to those with ischemic preconditioning (IPC) produced by a 10 min coronary artery occlusion (CAO). 8 mongrel dogs were chronically instrumented to measure wall thickening (WT) in the left ventricle. In group 1 (n=4), dogs inhaled 1 MAC (1.4 %) ISO for 60 min, after which time they were allowed to recover for 24 hrs and studied in the conscious state. A 10 min CAO was induced and the recovery of WT was monitored for 24 hrs. In group 2 (n=4), dogs underwent IPC, induced by a 10 min CAO; 24 hrs later they were subjected to another 10 min CAO. The recovery of WT following the initial occlusion served as control (CON) for the effects of both ISO and IPC. CAO caused akinesis in all groups. Three hours of reperfusion were required for complete recovery of WT in the ISO and IPC. At this time WT remained significantly depressed (−25±4%) in CON; between 12 and 24 hrs of reperfusion was required for complete recovery. We conclude that isoflurane has powerful SWOP effects against myocardial stunning and that this effect is comparable to ischemic preconditioning in the dog.

Cardiac remodelling hinders activation of cyclooxygenase-2, diminishing protection by delayed pharmacological preconditioning: role of HIF1α and CREB

We tested whether delayed pharmacologic preconditioning elicited by isoflurane is protective in infarct-remodelled hearts. Male Wistar rats were treated with the preconditioning drug isoflurane 6 weeks after permanent ligation of the left anterior descending coronary artery. Twenty-four and 48 h later, hearts were perfused on the Langendorff system and treated with cyclooxygenase-2 or 12-lipoxygenase inhibitors before exposure to 40 min of ischaemia followed by 90 min of reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining and lactate dehydrogenase release. Cyclooxygenase-2 expression and activity were measured by Western blotting and colorimetric assay. Nuclear translocation of cyclooxygenase-2-inducing transcription factors HIF1alpha, CREB, STAT3, and NFkappaB was determined. Post-infarct, remodelled hearts exhibit alterations in cellular signalling, time course and extent of isoflurane-induced late protection. While remodelled, preconditioned hearts exhibited protection exclusively at 24 h, healthy hearts showed sustained protection for up to 48 h, which correlated with cyclooxygenase-2 protein expression and enzymatic activity. The cyclooxygenase-2 inhibitors celecoxib and NS-398, but not the 12-lipoxygenase inhibitor cinnamyl-3,4-dihydroxycyanocinnamate, abolished delayed protection in both healthy and remodelled hearts, identifying cyclooxygenase-2 as a key mediator of late protection in both models. Isoflurane induced nuclear translocation of HIF1alpha in all hearts, but CREB was exclusively activated in healthy but not remodelled myocardium, which expressed higher levels of the CREB antagonist ICER. Delayed protection by isoflurane in remodelled hearts was more vulnerable to inhibition by celecoxib. Isoflurane failed to mobilize cyclooxygenase-2-inducing CREB in ICER-overexpressing, remodelled hearts, which was associated with a shortening of the second window of protection.

Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes

Current cellular models of ischemic preconditioning (IPC) rely on inducing preconditioning in vitro and may not accurately represent complex pathways triggered by IPC in the intact heart. Here, we show that it is possible to precondition the intact heart and to subsequently isolate individual ventricular myocytes that retain the protection triggered by IPC. Myocytes isolated from Langendorff-perfused hearts preconditioned with three cycles of ischemia-reperfusion were exposed to metabolic inhibition and reenergization. Injury was assessed from induction of hypercontracture and loss of Ca(2+) homeostasis and contractile function. IPC induced an immediate window of protection in isolated myocytes, with 64.3 +/- 7.6% of IPC myocytes recovering Ca(2+) homeostasis compared with 16.9 +/- 2.4% of control myocytes (P < 0.01). Similarly, 64.1 +/- 5.9% of IPC myocytes recovered contractile function compared with 15.3 +/- 2.2% of control myocytes (P < 0.01). Protection was prevented by the presence of 0.5 mM 5-hydroxydecanoate during the preconditioning stimulus. This early protection disappeared after 6 h, but a second window of protection developed 24 h after preconditioning, with 54.9 +/- 4.7% of preconditioned myocytes recovering Ca(2+) homeostasis compared with 12.6 +/- 2.9% of control myocytes (P < 0.01). These data show that "true" IPC of the heart confers both windows of protection in the isolated myocytes, with a similar temporal relationship to in vivo preconditioning of the whole heart. The model should allow future studies in isolated cells of the protective mechanisms induced by true ischemia.