Window Of Ischemic Preconditioning Research Articles

Secreted frizzled-related protein 2, a novel mechanism to induce myocardial ischemic protection through angiogenesis.

Our hypothesis is that Secreted Frizzled-Related Protein 2 (sFPR2) is an important mechanism mediating ischemic cardioprotection, since it is the most upregulated gene in the third window of ischemic preconditioning. One week after permanent coronary artery occlusion (CAO), sFRP2 TG mice exhibited a 49% higher LV ejection fraction and a 36% reduction in infarct size, p < 0.05, and reduced fibrosis in both adjacent and remote zones, along with an increase in collagen type III and a decrease in the collagen type I/III ratio compared with WTL. The ischemic cardioprotection was associated with increased angiogenesis and arteriogenesis, reflected by increased capillary and arteriolar proliferation in the ischemic zone, thereby preserving blood flow after CAO. The angiogenesis and arteriogenesis were mediated by cross talk between myocytes and endothelial cells. The mechanism for cardioprotection and angiogenesis/arteriogenesis did not involve a traditional vascular growth hormone, e.g., VEGF or FGF, but rather cTGF, and ATF6 through the stress signaling pathway. The ATF6 inhibitor, AEBSF, blocked the upregulation of cTGF and both the angiogenesis and arteriogenesis, resulting in abolition of the reduced infarct size and protection of cardiac function in the sFRP2 TG mouse following permanent CAO. sFRP2 is a novel mechanism to induce angiogenesis/arteriogenesis, mediated through the endoplasmatic reticulum (ER) stress signaling pathway, ATF6 and cTGF, which protects the heart from myocardial ischemia.

The 4th Window: A Novel, Clinically Relevant Model of Delayed Ischemic Preconditioning in Conscious, Chronically Instrumented Pigs

Most studies on cardiac ischemic preconditioning (IPC) focused on the 1st or 2nd window, where the preconditioning ischemic stimulus was induced briefly and the cardiac protection all occurred within a few hours or days. More recently 3rd window models have been described with IPC induced over several days. The goal of this investigation was to determine if IPC could be extended for a longer time, making it more clinically relevant. In this new IPC model, which we named the 4th window, pigs, chronically instrumented with coronary blood flow transducers, hydraulic occluders, and ultrasonic wall thickness crystals implanted in potentially ischemic and non‐ischemic zones, underwent IPC induced by 6 episodes of 90 min coronary stenosis (40% reduction in blood flow but no necrosis) every 12 hours over 3 days. 60 min coronary artery occlusion (CAO) followed by 3 days of coronary reperfusion, induced one week after IPC, resulted in infarct size, as a fraction of the area at risk, of 11±2.5%, less (p&lt;0.05) than in the absence of IPC (42±3.9%). Most genes expressed in this model were novel, compared to prior IPC models, included genes mediating angiogenesis. To confirm angiogenesis, we found increased capillary density adjacent to the infarct in the 4th window by 154% (p&lt;0.05), and 38% increased, p&lt;0.05, blood flow (measured with microspheres) during the 60min CAO, compared in the same samples with brief CAO before IPC. In summary, this novel model of 4th window IPC induces angiogenesis and markedly different gene regulation than previous IPC models.

The Effect of Ischemic Preconditioning in the Experimental Model of Renal Ischemia Reperfusion Injury Using Single Nephrectomized Porcine.

Purpose: We evaluate organ protective function of remote ischemic preconditioning (IPC) in renal ischemia reperfusion injury using a single-kidney porcine model. Methods: A total of 15 Yorkshire pigs, weighting 35-38kg and 20 week-old, were used. One week (as an adaption period) later from left nephrectomy, we performed IPC and right renal artery clamping (warm ischemia). We divided the animals into 3 experimental groups of 5 animals each; control group: 90 min warm ischemia without IPC, group 1: 2 cycles of 10 min IPC followed by 90 min warm ischemia, group 2: 2 cycles of 10 min IPC, and 24 hours as a window period followed by 90 min warm ischemia. Urinary NGAL, KIM-1, IL-18, cystatin C and microalbumin were measured as renal injury makers before renal ischemia at POD 1 and 3. Also gene microarray was investigated using kidney tissue after euthanasia. Results: There were no differences in serum creatinine changes between groups (figure 1). However, Group 3 showed lower level of KIM-1 and NGAL than control and group 1 at 72 hours. Microalbumin also showed a tendency of lower level in group 2. In the microarray, group 1 had 198 gene expression differences compared to the control group (figure 2). However group 2 had 1027 gene expression differences. Ischemic precondition groups (group 1 & 2) had 179 altered gene expressions in common that are mostly related with apoptosis, inflammation, and oxidation. When we compare group 1 and 2, group 2 had more increases in inflammation regulatory gene expressions (C3, C1QA, and SAA2).Figure: No Caption available.Table: No Caption available.Conclusion: Remote IPC can mitigate renal damage from ischemia reperfusion injury. Late window IPC is especially more efficient than early window.

Metabolomic analysis of two different models of delayed preconditioning

Recently we described an ischemic preconditioning induced by repetitive coronary stenosis, which is induced by 6 episodes of non-lethal ischemia over 3days, and which also resembles the hibernating myocardium phenotype. When compared with traditional second window of ischemic preconditioning using cDNA microarrays, many genes which differed in the repetitive coronary stenosis appeared targeted to metabolism. Accordingly, the goal of this study was to provide a more in depth analysis of changes in metabolism in the different models of delayed preconditioning, i.e., second window and repetitive coronary stenosis. This was accomplished using a metabolomic approach based on liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS) techniques. Myocardial samples from the ischemic section of porcine hearts subjected to both models of late preconditioning were compared against sham controls. Interestingly, although both models involve delayed preconditioning, their metabolic signatures were radically different; of the total number of metabolites that changed in both models (135 metabolites) only 7 changed in both models, and significantly more, p<0.01, were altered in the repetitive coronary stenosis (40%) than in the second window (8.1%). The most significant changes observed were in energy metabolism, e.g., phosphocreatine was increased 4 fold and creatine kinase activity increased by 27.2%, a pattern opposite from heart failure, suggesting that the repetitive coronary stenosis and potentially hibernating myocardium have enhanced stress resistance capabilities. The improved energy metabolism could also be a key mechanism contributing to the cardioprotection observed in the repetitive coronary stenosis and in hibernating myocardium. This article is part of a Special Issue entitled “Focus on Cardiac Metabolism”.

Metabolic Profiles of the second and third windows of ischemic preconditioning

There have been numerous studies on the differences between the first window of ischemic preconditioning (IPC) and late IPC, i.e. the second window of IPC.Recently our group has shown the presence of a third window of IPC, which is induced by 6 episodes of non lethal ischemia over 3 days. This model differs from classical second window IPC, induced by 1 or 2 episodes of non lethal ischemia, followed 24 hr. later by a longer period of ischemia. The underlying molecular mechanisms mediating the second and third windows of IPC are very different, but both paradigms of IPC appear to affect myocardial metabolism. The goal of this study was to provide a global analysis of the metabolic profile of these two late IPC models, using a metabolomic approach based on liquid chromatography‐mass spectrometry (LC‐MS) and gas chromatography‐mass spectrometry (GC‐MS) techniques. Myocardial samples from the ischemic section of porcine hearts subjected to second and third window IPC were compared against sham controls. Out of 287 assessed metabolites, there were more changes in metabolic pathways noted in third window (119 metabolites) than second (24 metabolites), but surprisingly only 7 metabolites were common to both models. The results of this study confirm the importance of metabolic pathways mediating late IPC, and again illustrate major differences between the third and second windows of IPC.

Late Preconditioning Induced by Transgenic Cardiac Overexpression of α1A Adrenergic Receptors in Rats

We determined the extent to which cardiac overexpression of the α1A‐adrenergic receptor (AR) in transgenic (TG) rats affords cardioprotection, and if it is involved in first or second window ischemic preconditioning (IPC). Without IPC, infarct size, induced by coronary artery occlusion (CAO) for 30min and 3hrs reperfusion (CAR; expressed as % area at risk), was reduced in TG vs. non‐transgenic littermates (NTL) (35±5% vs 50±2%, P&lt;0.05). After 1st window IPC, induced by 3 episodes of 5min CAO/5min CAR followed by 30min CAO/3hr CAR, infarct size of α1A‐AR TG was reduced further (12±1%) and was similar to NTL (10±1.1%). In contrast, 2nd window IPC, induced by 3 episodes of 5min CAO/5min CAR followed by 30min CAO/3hr CAR at 24 hours after IPC, reduced infarct size only in NTL (29±3%), but not in TG (30±1%), suggesting that TG already exhibited 2nd window IPC. Furthermore, blocking key molecules involved in 2nd window IPC, (iNOS and the MEK/ERK pathway) abrogated cardioprotection in TG rats. Cardiac myocyte microarrays revealed that only 4.6% of 3172 upregulated and 8.8% of 3498 downregulated genes were directionally similar in α1A‐TGs without IPC compared to NTL with 2nd window IPC. Thus, TG rats with cardiac α1A‐adrenergic receptor overexpression exhibit ischemic cardioprotection that resembles 2nd window IPC, with only a minor fraction of overlapping genes.

SIRT1-mediated acute cardioprotection

Overexpression studies have revealed a role for silent information regulator of transcription 1 (SIRT1) lysine deacetylase in cardioprotection against ischemia-reperfusion injury via long-term transcriptional effects. However, short-term SIRT1-mediated lysine deacetylation, within the context of acute cardioprotection, is poorly understood. In this study, the role of SIRT1 in the acute cardioprotective paradigm of first window ischemic preconditioning (IPC) was studied using SIRT1-deficient (SIRT1(+/-)) and SIRT1-overexpressing (SIRT1(+++)) mice. In wild-type hearts, cytosolic lysine deacetylation was observed during IPC, and overacetylation was observed upon pharmacological SIRT1 inhibition. Consistent with a role for SIRT1 in IPC, SIRT1(+/-) hearts could not be preconditioned and exhibited increased cytosolic lysine acetylation. Furthermore, SIRT1(+++) hearts were endogenously protected against ischemia-reperfusion injury and exhibited decreased cytosolic acetylation. Both of these effects in SIRT1(+++) mice were reversed by pharmacological SIRT1 inhibition on an acute timescale. Several downstream targets of SIRT1 were examined, with data suggesting possible roles for endothelial nitric oxide synthase phosphorylation, NF-κB, and stimulation of autophagy. In conclusion, these data suggest that SIRT1, acting on nontranscriptional targets, is required for cardioprotection by acute IPC and that SIRT1-dependent lysine deacetylation occurs during IPC and may play a role in cardioprotective signaling.

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.

Preemptive conditioning of the swine heart by H11 kinase/Hsp22 provides cardiac protection through inducible nitric oxide synthase

The second window of ischemic preconditioning (SWOP) provides maximal protection against ischemia through regulation of the inducible nitric oxide synthase (iNOS), yet its application is limited by the inconvenience of the preliminary ischemic stimulus required for prophylaxis. Overexpression of H11 kinase/Hsp22 (Hsp22) in a transgenic mouse model provides cardioprotection against ischemia that is equivalent to that conferred by SWOP. We hypothesized that short-term, prophylactic overexpression of Hsp22 would offer an alternative to SWOP in reducing ischemic damage through a nitric oxide (NO)-dependent mechanism. Adeno-mediated overexpression of Hsp22 was achieved in the area at risk of the left circumflex (Cx) coronary artery in chronically instrumented swine and compared with LacZ controls (n = 5/group). Hsp22-injected myocardium showed an average fourfold increase in Hsp22 protein expression compared with controls and a doubling in iNOS expression (both P < 0.05). Four days after ischemia-reperfusion, regional wall thickening was reduced by 58 ± 2% in the Hsp22 group vs. 82 ± 7% in the LacZ group, and Hsp22 reduced infarct size by 40% (both P < 0.05 vs. LacZ). Treatment with the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) before ischemia suppressed the protection induced by Hsp22. In isolated cardiomyocytes, Hsp22 increased iNOS expression through the transcription factors NF-κB and STAT, the same effectors activated by SWOP, and reduced by 60% H(2)O(2)-mediated apoptosis, which was also abolished by NOS inhibitors. Therefore, short-term, prophylactic conditioning by Hsp22 provides NO-dependent cardioprotection that reproduces the signaling of SWOP, placing Hsp22 as a potential alternative for preemptive treatment of myocardial ischemia.

Abstract 1610: Repetitive Ischemia Elicits a Third Window of Ischemic Preconditioning

Ischemic preconditioning (IPC), following 1 or 2 episodes of coronary occlusion (CO) is the most powerful mechanism of cardioprotection, but it remains unclear whether IPC occurs clinically. Since patients with coronary disease experience episodes of repetitive ischemia (RI), we hypothesized that a pattern of RI would provide more clinically relevant cardioprotection. Conscious, chronically instrumented pigs (6/group) were submitted to RI induced by 6 episodes, 12 hrs apart, of coronary stenosis for 90 min (RCS) and compared to pigs submitted to second window IPC (SWIPC) induced by 2 episodes of 10 min CO / 24 hr reperfusion (CR) and to shams. Lethal ischemia, induced by 60 min CO, resulted in an infarct size/area at risk (IS/AAR) of 42±4% in shams, and significantly less, p&lt;0.05, after RCS (6±3%) and after SWIPC (16±4%). SWIPC showed 2–3-fold increases, p&lt;0.05, in translocation of PKCepsilon and the inducible isoform of nitric oxide synthase (NOS), which were not found in RCS. Pretreatment with the NOS inhibitor L-NNA (35 mg/kg) prevented the protection by SWIPC but not by RCS. Microarrays were performed to further elucidate the differences in molecular mechanisms between the SWIPC and RCS models. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394). Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Since another difference between the two models is that one involves CS and the other complete CO, we examined another group of RI pigs (n=5) with 6 episodes, 12 hrs apart, of 2–10 min periods of CO, which showed reduced IS/AAR with 60 min CO after RI (14±4%); similar to RCS the cardioprotection was still observed after L-NNA. Both RI models, in further contrast to SWIPC, demonstrated downregulation of genes involved in mitochondrial function, and upregulation of genes involving autophagy (such as cathepsins B and D), endoplasmic reticulum stress (Grp78) and cell cycle (E2F1). These results were confirmed at the protein level by western blotting. Therefore, RI, over a time course of several days, provides a NOS-independent “third window” of protection, which is as effective as traditional SWIPC, but is mechanistically distinct. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Repetitive Ischemia by Coronary Stenosis Induces a Novel Window of Ischemic Preconditioning

The hypothesis of the present study was that molecular mechanisms differ markedly when mediating ischemic preconditioning induced by repetitive episodes of ischemia versus classic first- or second-window preconditioning. To test this, chronically instrumented conscious pigs were subjected to either repetitive coronary stenosis (RCS) or a traditional protocol of second-window ischemic preconditioning (SWIPC). Lethal ischemia, induced by 60 minutes of coronary artery occlusion followed by reperfusion, resulted in an infarct size/area at risk of 6+/-3% after RCS and 16+/-3% after SWIPC (both groups P<0.05, less than shams 42+/-4%). Two molecular signatures of SWIPC, the increased expression of the inducible isoform of nitric oxide synthase and the translocation of protein kinase Cepsilon to the plasma membrane, were observed with SWIPC but not with RCS. Confirming this, pretreatment with a nitric oxide synthase inhibitor prevented the protection conferred by SWIPC but not by RCS. Microarray analysis revealed a qualitatively different genomic profile of cardioprotection between ischemic preconditioning induced by RCS and that induced by SWIPC. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394) animals. Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Broad categories of genes induced by RCS but not SWIPC included those involved in autophagy, endoplasmic reticulum stress, and mitochondrial oxidative metabolism. The upregulation of these pathways was confirmed by Western blotting. RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional ischemic preconditioning but is mediated through radically different mechanisms.

Abstract 602: Repetitive Coronary Stenosis Induces a Third Window of Ischemic Preconditioning

The mechanisms responsible for the 1 st and 2 nd window of protection following ischemic preconditioning (IPC) have been studied in detail. We tested the hypothesis that chronic myocardial ischemia (repetitive bouts of coronary stenosis, CS) may induce IPC mediated by different mechanisms. To study this, 6 episodes of 90 min chronic CS (reduction in coronary blood flow, &gt;35% from baseline), followed by reperfusion every 12 hrs were examined in conscious pigs chronically instrumented to measure left ventricular (LV) pressure and regional wall thickness. Infarct size/area at risk (IF/AAR) was assessed in all groups after 60 min of coronary artery occlusion (CAO) followed by 4 days of reperfusion. This resulted in IF/AAR of 42±5% in control pigs without IPC (n=5). Following repetitive CS, IF/AAR was markedly decreased (p&lt;0.05) (4±6%, n=4). The 2 nd window of IPC was induced in 5 pigs by two 10 min periods of CAO. 24 hrs later, the 60 min of CAO also resulted in reduced IF/AAR of 16±4%. In 6 pigs, 24 hrs after IPC without 60 min of CAO, the IPC myocardium was found to have significant, p&lt;0.05, 2– 4-fold increases in iNOS, Cox-2, phosphorylated PKC and AMPK. None of these proteins was elevated in myocardium subjected to repetitive CS. Pretreatment with an NO synthase inhibitor, L-NNA, prevented the 2 nd window of IPC as expected, since iNOS was elevated, but failed to prevent the ischemic protection following repetitive CS, where iNOS was not elevated. Following repetitive CS, genomic microarray analysis revealed upregulation of cytoprotective genes, e.g., IAP, H11 kinase, αB-crystallin, Akt 1, Bcl-2. These cytoprotective genes were also found to be upregulated in samples from patients with chronic coronary artery disease and hibernating myocardium. Thus, repetitive CS induces powerful protection against lethal myocardial ischemia, with mechanisms radically different from that observed in the 2 nd window of IPC, suggesting a novel “third window” of IPC, which could also be involved in the protection inherent to hibernating myocardium.

Obligatory Role of Cardiac Nerves and α 1 -Adrenergic Receptors for the Second Window of Ischemic Preconditioning in Conscious Pigs

We tested the hypothesis that cardiac nerves may mediate ischemic preconditioning. Pigs were chronically instrumented to measure aortic, left atrial and left ventricular pressures, and regional myocardial function (wall thickening). Hemodynamic variables, area at risk, and tissue blood flows (radioactive microspheres) were similar among groups. Myocardial infarct size following 60 minutes coronary artery occlusion and 4 days reperfusion, expressed as a fraction of the area at risk, was 42+/-4.0%, in innervated pigs and similar in pigs with regional cardiac denervation (CD, 41+/-2.5%). Infarct size in innervated pigs during the first window of preconditioning (first window) was markedly reduced (6+/-1.8%, P<0.01), as it was in the second window of preconditioning (second window) (16+/-3.3%, P<0.01). Although infarct size was still reduced in pigs with CD and first window preconditioning (9+/-1.8%, P<0.01), the protective effects of second window were abrogated in pigs with CD resulting in an infarct size of 38+/-5.6%. In another group of innervated pigs during pharmacological alpha(1)-adrenergic receptor (AR) blockade, infarct size was also not reduced during the second window (48+/-3.2%). Additionally, Western blot analysis of inducible nitric oxide synthase and cyclooxygenase-2 proteins demonstrated significant (P<0.05) upregulation following the second window in innervated pigs, but not in pigs with CD or alpha(1)-AR blockade. Thus, the mechanism of protection during the second window, but not the first window, appears to be dependent on cardiac nerves and alpha(1)-AR stimulation.

Induction of 72-kDa heat shock protein does not produce second window of ischemic preconditioning in rat heart.

Ischemic preconditioning (PC) induces delayed phase of protection, known as the second window of protection (SWOP). We investigated this phenomenon in rat and correlated it with the expression of 72-kDa heat shock protein (HSP 72). Rats were preconditioned with 1, 2, and 3 cycles of 5-min left anterior descending artery occlusions, each separated by a 10-min reperfusion (PC x 1, PC x 2 and PC x 3, respectively). Another group of rats was preconditioned with heat shock (HS) by raising temperature to 42 degreesC for 15 min. Twenty-four hours later, rats were given sustained ischemia for 30 min and 90 min of reperfusion. Infarct sizes (%risk area) were 40.0 +/- 7.5, 37.6 +/- 5.6, and 47.6 +/- 2.4 (mean +/- SE) for PC x 1, PC x 2, and PC x 3 hearts, respectively, which were not different from the sham (49.9 +/- 3.9, P > 0.05). In contrast, infarct size was reduced from 47.5 +/- 3.8% in sham to 4.7 +/- 2.3% (P < 0.01) 24 h after HS. Additionally, early PC significantly reduced infarct size from 47.5 +/- 3.8% in controls to 6.0 +/- 1.2 and 5.0 +/- 1.1% with PC x 1 and PC x 3. Repeated PC cycles induced over a threefold increase in HSP 70 mRNA after 2 h compared with sham (P < 0.05). HSP 72, which increased 24 h after PC or HS, was not significantly different between the two PC stimuli. We conclude that PC does not induce SWOP in rat heart despite enhanced expression of HSP 72. In contrast, HS-induced delayed protection was associated with enhanced accumulation of HSP 72. It is possible that SWOP and HS have distinct mechanisms of protection that may not be exclusively related to HSP 72 expression.

Monophosphoryl lipid A attenuates myocardial stunning in dogs: role of ATP-sensitive potassium channels.

Results of previous studies indicate that monophosphoryl lipid A (MLA) reduces myocardial infarct size when administered 24 but not 1 h before a prolonged period of regional ischemia in dogs and rabbits. This cardioprotective effect of MLA could be reversed by the administration of the adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) blockers, glibenclamide, or 5-hydroxydecanoate. MLA also was shown to attenuate myocardial stunning in dogs; however, its mechanism in this model remains unknown. Therefore the major aim of our study was to determine the dose-related effect of MLA to enhance contractile function in stunned myocardium and to determine the role of the K(ATP) channel in mediating its cardioprotective effect. To produce myocardial stunning, barbital-anesthetized dogs were subjected to five cycles of 5 min of left anterior descending (LAD) coronary artery occlusion interspersed with 10 min of reperfusion and finally followed by 2 h of reperfusion. Regional segment shortening (%SS) was determined by sonomicrometers implanted in the subendocardium of the ischemic region. Single intravenous doses of MLA in the range of 10-35 microg/kg given 24 h before ischemia resulted in an improvement in %SS over a 2-h reperfusion period. Similar to results obtained in the canine and rabbit infarct models, cardioprotection against stunning with MLA appears to require activation of K(ATP) channels during ischemia, because glibenclamide (50 microg/kg, 15 min before ischemia) completely blocked the effect of MLA to improve regional %SS during reperfusion. Cardioprotective doses of MLA were without effect on systemic hemodynamics, blood gases, and pH throughout the experiment. No treatment-related effects on regional myocardial blood flow were observed during ischemia or reperfusion. These results suggest that MLA improves %SS at doses of 10-35 microg/kg by an ATP-sensitive potassium channel-dependent process, and that MLA may mimic the antistunning effects observed during the second window of ischemic preconditioning.

ATP-sensitive potassium (KATP) channel is involved in the second window of ischemic preconditioning in rabbit

ATP-sensitive potassium (KATP) channel is involved in the second window of ischemic preconditioning in rabbit

Role of Inducible Nitric Oxide Synthase in Pharmacological “Preconditioning” with Monophosphoryl Lipid A

Pretreatment with monophosphoryl lipid A (MLA) can pharmacologically mimic the second window of ischemic preconditioning (SWOP) to protect the heart from prolonged ischemia and reperfusion injury. Based on the delayed time course for development of MLA associated cardioprotection, this study was designed to test if MLA's cardioprotective effect is mediated by signalling through production of inducible nitric oxide synthase (iNOS), a proposed effector of SWOP. Rabbits were assigned to one of four groups: (1) vehicle control; (2) MLA; (3) vehicle+aminoguanidine (AMG) control; or (4) MLA+AMG. Monophosphoryl lipid A (35 μg/kg) or vehicle was given intravenously 24 h before ischemia. The selective iNOS inhibitor AMG (300 mg/kg) was injected subcutaneously 1 h before ischemia. All rabbits experienced 30 min coronary artery occlusion followed by 3 h of reperfusion. Infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Myeloperoxidase activity, an index of neutrophil infiltration, was also quantified in heart tissue collected from the post-ischemic viable border zone surrounding the infarct area. MLA pretreatment significantly reduced infarct size and neutrophil infiltration in rabbit hearts compared to control (P<0.05). Inhibition of iNOS activity by AMG abolished the infarct size reductive effect of MLA. Aminoguanidine also blocked the ability of MLA to significantly reduce neutrophil infiltration. Although measurement of iNOS activity did not show induction of the enzyme in normal myocardial tissue 24 h after MLA pretreatment, an increase in iNOS activity in ischemic tissue relative to non-ischemic tissue was found after either 15 or 30 min of coronary occlusion in MLA treated rabbits. These results suggest that MLA pretreatment may enhance iNOS enzyme activity by MLA during ischemia which may be responsible for the observed cardioprotection.

Monophosphoryl Lipid A: A Novel Agent for Inducing Pharmacologic Myocardial Preconditioning.

Myocardial tissue appears to possess an endogenous protective mechanism whereby brief ischemic periods precondition cells to better withstand both reversible and irreversible injury associated with prolonged subsequent ischemic events. Protection develops within minutes of transient ischemia, dissipates within 1-2 hours, and then reappears 12-24 hours following the preconditioning ischemic event. This phenomena, known as ischemic preconditioning (IP), is associated with limitation of infarct size, contractile stunning, and ventricular arrhythmias in postischemic/reperfused hearts. Preconditioned myocardium displays reduced anerobic metabolism. ATPase function, and, hence, improved ATP preservation during ischemia, reduced cytosolic calcium concentrations during reperfusion, and preservation of ultrastructural and myofilament integrity. Efforts to dissect the intracellular signal transduction pathway operative in IP have met with some success. Ischemic preconditioning is associated w ith activation of myocyte Gi protein-coupled receptors such as adenosine and acetylcholine, activation of PKC, production of nitric oxide, and, eventually, opening of ATP-sensitive potassium (KATP) channels. Preconditioning can also be elicited by pharmacologic means using adenosine receptor agonists, activators of PKC, nitric oxide inducers, and KATP openers, among other strategies. Monophosphoryl lipid A (MLA), a nontoxic derivative of the endotoxin pharmacophore lipid A, has been evaluated for cardioprotective activity in numerous preclinical models of cardiac ischemia/reperfusion injury. MLA, when given as a single dose pretreatment in various canine and rabbit models 12-24 hours prior to ischemia, limits infarct size and reduces regional and global contractile dysfunction. Cardioprotection in various models is associated with preservation of ATP during ischemia, enhanced 5'-nucleotidase and adenosine kinase function during reperfusion, and in these aspects mimics ischemic pr econditioning. Priming of KATP channel for enhanced opening during ischemia may be a prerequisite for the cardioprotective activity of MLA and is another feature establishing a similarity between MLA and ischemia induced preconditioning. Efforts continue to further our understanding regarding how MLA may regulate KATP channel and thereby precondition myocardium. Ongoing studies include evaluation of a possible direct effect on the KATP channel, investigation of the ability of MLA to induce a secondary mediator of potassium channel modulation, and evaluation of MLA's ability to phosphorylate the KATP channel as a consequence of kinase activation. Pretreatment with MLA represents a novel method of pharmacologically preconditioning myocardium, displaying a time course for development similar to that of the second window of ischemic preconditioning. Prior clinical experience with MLA indicates that intravenous doses of up to at least 20 µg/kg may be given safely to humans. The drug is c urrently being evaluated in patients undergoing coronary artery bypass engraftment surgery and may prove to be a useful way to protect myocardium from anticipated ischemic events.