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Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I–forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell–derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT1) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell–deficient mice than in control hearts. Thus, mast cell–derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell–derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

Enhanced production of angiotensin II and excessive release of norepinephrine in the ischemic heart are major causes of arrhythmias and sudden cardiac death. Mast cell-dependent mechanisms are pivotal in the local formation of angiotensin II and modulation of norepinephrine release in cardiac pathophysiology. Cardiac mast cells increase in number in myocardial ischemia and are located in close proximity to sympathetic neurons expressing angiotensin AT1- and histamine H3-receptors. Once activated, cardiac mast cells release a host of potent pro-inflammatory and pro-fibrotic cytokines, chemokines, preformed mediators (e.g., histamine) and proteases (e.g., renin). In myocardial ischemia, angiotensin II (formed locally from mast cell-derived renin) and histamine (also released from local mast cells) respectively activate AT1- and H3-receptors on sympathetic nerve endings. Stimulation of angiotensin AT1-receptors is arrhythmogenic whereas H3-receptor activation is cardioprotective. It is likely that in ischemia/reperfusion the balance may be tipped toward the deleterious effects of mast cell renin, as demonstrated in mast cell-deficient mice, lacking mast cell renin and histamine in the heart. In these mice, no ventricular fibrillation occurs at reperfusion following ischemia, as opposed to wild-type hearts which all fibrillate. Preventing mast cell degranulation in the heart and inhibiting the activation of a local reninangiotensin system, hence abolishing its detrimental effects on cardiac rhythmicity, appears to be more significant than the loss of histamine-induced cardioprotection. This suggests that therapeutic targets in the treatment of myocardial ischemia, and potentially congestive heart failure and hypertension, should include prevention of mast cell degranulation, mast cell renin inhibition, local ACE inhibition, ANG II antagonism and H3-receptor activation.

Although protein kinase C (PKC) plays a key role in ischemic preconditioning (IPC), the actual mechanism of that protection is unknown. We recently found that protection from IPC requires activation of adenosine receptors during early reperfusion. We, therefore, hypothesized PKC might act to increase the heart’s sensitivity to adenosine. IPC limited infarct size in isolated rabbit hearts subjected to 30-min regional ischemia/2-h reperfusion and IPC’s protection was blocked by the PKC inhibitor chelerythrine given during early reperfusion revealing involvement of PKC at reperfusion. Similarly chelerythrine infused in the early reperfusion period blocked the increased phosphorylation of the protective kinases Akt and ERK1/2 observed after IPC. Infusing phorbol 12-myristate 13-acetate (PMA), a PKC activator, during early reperfusion mimicked IPC’s protection. As expected, the protection triggered by PMA at reperfusion was blocked by chelerythrine, but surprisingly it was also blocked by MRS1754, an adenosine A2b receptor–selective antagonist, suggesting that PKC was somehow facilitating signaling from the A2b receptors. NECA [5′-(N-ethylcarboxamido) adenosine], a potent but not selective A2b receptor agonist, increased phosphorylation of Akt and ERK1/2 in a dose-dependent manner. Pretreating hearts with PMA or brief preconditioning ischemia had no effect on phosphorylation of Akt or ERK1/2 per se, but markedly lowered the threshold for NECA to induce their phosphorylation. BAY 60-6583, a highly selective A2b agonist, also caused phosphorylation of ERK 1/2 and Akt. MRS1754 prevented phosphorylation induced by BAY 60-6583. BAY 60-6583 limited infarct size when given to ischemic hearts at reperfusion. These results suggest that activation of cardiac A2b receptors at reperfusion is protective, but because of the very low affinity of the receptors endogenous cardiac adenosine is unable to trigger their signaling. We propose that the key protective event in IPC occurs when PKC increases the heart’s sensitivity to adenosine so that endogenous adenosine can activate A2b-dependent signaling.

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient ‘iron-signal’ that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this ‘iron signal’ and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min (‘delay’) prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the ‘delay’. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the ‘iron-signal’ by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the ‘delay’. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Cardiac function is compromised by oxidative stress which occurs upon exposing the heart to ischemia reperfusion (I/R) for a prolonged period. The reactive oxygen species (ROS) that are generated during I/R incur extensive damage to the myocardium and result in subcellular organelle remodeling. The cardiac nucleus, glycocalyx, myofilaments, sarcoplasmic reticulum, sarcolemma, and mitochondria are affected by ROS during I/R injury. On the other hand, brief periods of ischemia followed by reperfusion, or ischemic preconditioning (IPC), have been shown to be cardioprotective against oxidative stress by attenuating the cellular damage and alterations of subcellular organelles caused by subsequent I/R injury. Endogenous defense mechanisms, such as antioxidant enzymes and heat shock proteins, are activated by IPC and thus prevent damage caused by oxidative stress. Although these cardioprotective effects of IPC against I/R injury are considered to be a consequence of changes in the redox state of cardiomyocytes, IPC is considered to promote the production of NO which may protect subcellular organelles from the deleterious actions of oxidative stress. The article is intended to focus on the I/R-induced oxidative damage to subcellular organelles and to highlight the cardioprotective effects of IPC. In addition, the actions of various endogenous cardioprotective interventions are discussed to illustrate that changes in the redox state due to IPC are cardioprotective against I/R injury to the heart.

Background

Nitric oxide (NO) has been noted to produce ischemic preconditioning (IPC)-mediated cardioprotection. Caveolin is a negative regulator of NO, which inhibits endothelial nitric oxide synthase (eNOS) by making caveolin-eNOS complex. The expression of caveolin is increased during diabetes mellitus (DM). The present study was designed to investigate the involvement of caveolin in attenuation of the cardioprotective effect of IPC during DM in rat.

Methods

Experimental DM was induced by single dose of streptozotocin (50 mg/Kg, i.p,) and animals were used for experiments four weeks later. Isolated heart was mounted on Langendorff's apparatus, and was subjected to 30 min of global ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Kreb's-Henseleit solution (K-H). Extent of injury was measured in terms of infarct size by triphenyltetrazolium chloride (TTC) staining, and release of lactate dehydrogenase (LDH) and creatin kinase-MB (CK-MB) in coronary effluent. The cardiac release of NO was noted by measuring the level of nitrite in coronary effluent.

Results

IPC- induced cardioprotection and release of NO was significantly decreased in diabetic rat heart. Pre-treatment of diabetic rat with daidzein (DDZ) a caveolin inhibitor (0.2 mg/Kg/s.c), for one week, significantly increased the release of NO and restored the attenuated cardioprotective effect of IPC. Also perfusion of sodium nitrite (10 μM/L), a precursor of NO, significantly restored the lost effect of IPC, similar to daidzein in diabetic rat. Administration of 5-hydroxy deaconate (5-HD), a mito KATP channel blocker, significantly abolished the observed IPC-induced cardioprotection in normal rat or daidzein and sodium nitrite perfused diabetic rat heart alone or in combination.

Conclusions

Thus, it is suggested that attenuation of the cardioprotection in diabetic heart may be due to decrease the IPC mediated release of NO in the diabetic myocardium, which may be due to up -regulation of caveolin and subsequently decreased activity of eNOS.

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning.

Background

During renal transplantation, the kidney remains without blood flow for a period of time. The following reperfusion of this ischemic kidney causes functional and structural injury. Formation of oxygen-derived free radicals (OFR) and subsequent lipid peroxidation (LP) has been implicated as the causative factors of these injuries. Vitamin E is known to be the main endogenous antioxidant that stabilizes cell membranes by interfering with LP. The present study was designed to examine the role of ischemic-preconditioning (repeated brief periods of ischemia, IPC) in prevention of renal injury caused by ischemia-reperfusion (IR) in rats.

Methods

IPC included sequential clamping of the right renal artery for 5 min and release of the clamp for another 5 min for a 3 cycles. IR was induced by 30 min ischemia followed by 10 min reperfusion. Four groups of male rats were used: Control, IPC, IR and IPC-IR. Vitamin E, an endogenous antioxidant and as an index of LP, was measured by HPLC and UV detection in renal venous plasma and tissue. Renal function was assessed by serum creatinine and BUN levels. Renal damage was assessed in sections stained with Haematoxylin and Eosin.

Results

In the IR group, there was a significant decrease in vitamin E in plasma and tissue compared to a control group (p,0.05). In the IPC-IR group, vitamin E concentration was significantly higher than in the IR group (p,0.01). The results showed that 30 min ischemia in the IR group significantly (p,0.05) reduced renal function demonstrated by an increase in serum creatinine levels as compared with the control group. These results in the IPC group also showed a significant difference with the IR group but no significant difference in serum BUN and creatinine between IR and IPC-IR group were detected. Histological evaluation showed no structural damage in the IPC group and an improvement in the IPC-IR group compared to IR alone.

Conclusions

In this study, IPC preserved vitamin E levels, but it could not markedly improve renal function in the early phase (1–2 h) of reperfusion. IPC may be a useful method for antioxidant preservation in organ transplantation.

In ischemic preconditioning (IPC) brief ischemia/reperfusion renders the heart resistant to infarction from any subsequent ischemic insult. Protection results from binding of surface receptors by ligands released during the preconditioning ischemia. The downstream pathway involves redox signaling as IPC will not protect in the presence of a free radical scavenger. To determine when the redox signaling occurs, five groups of isolated rabbit hearts were studied. All hearts underwent 30 min of coronary branch occlusion and 2 h of reperfusion. IPC groups were subjected to 5 min of regional ischemia followed by 10 min of reperfusion prior to the 30-min coronary occlusion. The Control group had only the 30-min occlusion and 2-h reperfusion. The second group had IPC alone. The third group was also preconditioned, but the free radical scavenger N-2-mercaptopropionyl glycine (MPG, 300 µM) was infused during the 10-min reperfusion and therefore was present in the myocardium in the distribution of the snared coronary artery during the entire reperfusion phase and also during the subsequent 30-min ischemia. In another preconditioned group MPG was added to the perfusate before the preconditioning ischemia and therefore was present in the tissue only during the preconditioning ischemia and then was washed out during reperfusion. In the fifth group MPG was added to the perfusate for only the last 5 min of the preconditioning reperfusion and therefore was present in the tissue during the last minutes of the reperfusion phase and the 30 min of ischemia. Infarct size and risk size were measured by triphenyltetrazolium staining and fluorescent microspheres, resp. IPC reduced infarct size from 31.3±2.7% of the ischemic zone in control hearts to only 8.4±1.9%. MPG completely blocked IPC’s protection in the 3rd group (39.4±2.8%) but did not affect its protection in groups 4 (8.1±1.5%) or 5 (7.8±1.1%). Hence redox signaling occurs during the reperfusion phase of IPC.

We and others have reported that Rho-kinase plays an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. Studies have also demonstrated that the activation of Rho-kinase is reversed in ischemic preconditioning (IPC). However, the mechanisms by which Rho-kinase is increased in I/R and reversed in IPC are not thoroughly understood. In female Wistar rats, we created I/R by ligating the left anterior–descending branch of the coronary artery (LAD) for 30 min and releasing the ligature for 180 min. IPC rats underwent IPC (two cycles of 5-min ligation of the LAD and 5-min reflow) before I/R. IPC caused a significant increase in extracellular signal–regulated kinase (ERK)1/2 activity and reduced Rho-kinase activity and cardiomyocyte apoptosis (P < 0.05 versus I/R). Administration of PD98059, an inhibitor of ERK–mitogen-activated protein kinase (MAPK), increased cardiomyocyte apoptosis, Caspase-3 activity and myocardial infarction size (P < 0.05 versus IPC). Western-blot analysis showed that administration of PD98059 increased Rho-kinase activity. Treatment with fasudil, an inhibitor of Rho-kinase, reversed cell apoptosis caused by treatment with PD98059 in IPC. In addition, ROCK1 (Rho-kinase 1) may be the major Rho-kinase isoform that is opposed by ERK-MAPK signaling in IPC. These results indicate that ERK-MAPK signaling is required in IPC to oppose Rho-kinase activity in cardiomyocyte apoptosis in vivo.

A series of brief ischemia/reperfusion cycles (termed ischemic preconditioning, IPC) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Over the last 2 decades our understanding of IPC’s mechanism has increased exponentially. Hearts exposed to IPC have a better metabolic and ionic status during prolonged ischemia compared to naïve hearts. However, this difference is not thought to be the main mechanism by which IPC protects against infarction. Signaling pathways that are activated by IPC distinguish IPC hearts from naïve hearts. During the trigger phase of IPC, adenosine, bradykinin and opioid receptors are occupied. Although these three receptors trigger signaling through divergent pathways, the signaling converges on protein kinase C. We have proposed that at the end of the index ischemia the activated PKC sensitizes the low-affinity A2b adenosine receptor (A2bAR) through phosphorylation of either the receptor or its coupling proteins so that A2bAR can be activated by endogenous adenosine released by the previously ischemic cardiomyocytes. The sensitized A2bAR would then be responsible for activation of the survival kinases including PI3 kinase, Akt and ERK which then act to inhibit lethal mitochondrial permeability transition pore formation which normally uncouples mitochondria and destroys many myocytes in the first minutes of reperfusion. Herein we review the evidence for the above mechanisms and their functional details.

Acute administration of ethanol can reduce cardiac ischemia/reperfusion injury. Previous studies demonstrated that the acute cytoprotective effect of ethanol on the myocardium is mediated by protein kinase C epsilon (PKCε). We recently identified aldehyde dehydrogenase 2 (ALDH2) as an PKCε substrate, whose activation is necessary and sufficient to confer cardioprotection in vivo. ALDH2 metabolizes cytotoxic reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), which accumulate during cardiac ischemia/reperfusion. Here, we used a combination of PKCε knockout mice and a direct activator of ALDH2, Alda-44, to further investigate the interplay between PKCε and ALDH2 in cardioprotection. We report that ethanol preconditioning requires PKCε, whereas direct activation of ALDH2 reduces infarct size in both wild type and PKCε knockout hearts. Our data suggest that ALDH2 is downstream of PKCε in ethanol preconditioning and that direct activation of ALDH2 can circumvent the requirement of PKCε to induce cytoprotection. We also report that in addition to ALDH2 activation, Alda-44 prevents 4-HNE induced inactivation of ALDH2 by reducing the formation of 4-HNE-ALDH2 protein adducts. Thus, Alda-44 promotes metabolism of cytotoxic reactive aldehydes that accumulate in ischemic myocardium. Taken together, our findings suggest that direct activation of ALDH2 may represent a method of harnessing the cardioprotective effect of ethanol without the side effects associated with alcohol consumption.

Ischemia-reperfusion is a major cause of acute kidney injury and inflammation has been well-established as a detrimental process in the pathogenesis of kidney ischemia reperfusion injury (IRI). The kidney has the ability to be preconditioned by a non-lethal period of ischemia, rendering the kidney refractory to further ischemia-induced dysfunction (ischemic preconditioning: IPC). Regulatory T (Treg) cells are lymphocytes that suppress immune responses. We hypothesized that IPC is partially mediated by Treg cells. To test this, a model of delayed IPC was used by subjecting mice to 24 min of bilateral renal IRI or sham surgery on day 0, then 28 min IRI on day 7 (IPC = IRI/IRI; non-IPC = Sham/IRI). IPC significantly inhibited the accumulation of neutrophils and macrophages, tubular necrosis and loss of kidney function induced by IRI. The initial 24 min IRI caused a significant increase in kidney CD4+CD25+FoxP3+ and CD4+CD25+IL-10+ Treg cells at 7 days of reperfusion. Use of a Treg cell-depleting antibody (PC61) in preconditioned mice reversed the effect of IPC on kidney neutrophil accumulation and partially inhibited the functional and histological protection of IPC. Adoptive transfer of Treg cells, prior to IR, in naïve mice, mimicked the protective and anti-inflammatory effects of IPC on the kidney. These results demonstrate that suppression of inflammation and a significant fraction of kidney protection, imparted by delayed IPC, is mediated by Treg cells.

In previous studies, inhibition of mitogen activated protein kinase (MAP) p38 significantly improved recovery and attenuated apoptosis after retinal ischemia in rats. Yet, ischemic preconditioning (IPC) attenuated the ischemia-induced increase in p38 expression. We hypothesized that p38 was required for induction of ischemic tolerance by IPC. We examined the mechanisms of involvement of p38 in IPC neuroprotection. IPC or ischemia was induced in rat retina in vivo. Recovery after ischemia performed 24 h after IPC was assessed functionally (electroretinography) and histologically at 7 days after ischemia in the presence or absence of inhibition of p38. We examined the role of p38α in the mimicking of IPC produced by opening mitochondrial KATP channels using diazoxide, or stimulation of p38 activation by anisomycin. The importance of adenosine receptors in p38 activation after IPC was assessed using specific blockers of adenosine A1 and A2a receptors. Interfering RNA (siRNA) or SB203580 were used to block p38α. Phosphorylated p38 levels were measured. Phosphorylated p38 protein increased with IPC. Interfering RNA (siRNA) to p38α prior to IPC, or inhibiting p38 activation with SB203580, with ischemia following 24 h later, significantly attenuated the neuroprotective effect of IPC. Anisomycin administered to increase p38 mimicked IPC, an effect blocked by SB203580. IPC mimicking with diazoxide, an opener of mitochondrial KATP channels, was diminished with p38α siRNA. Adenosine receptor blockade did not decrease the elevated levels of phosphorylated p38 after IPC. Specific inhibition of p38α suggests that this MAPK is involved in the protective effects of IPC, and that p38 is downstream of mitochondrial KATP channels, but not adenosine receptors, in this neuroprotection.

Ischemic preconditioning (IPC) affords cardioprotection against ischemia-reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP sensitive K+ channel (mKATP) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mKATP and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mKATP channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mKATP channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mKATP channels during IPC.

Mitochondrial dysfunction is a key pathologic event in cardiac ischemia-reperfusion (IR) injury, and protection of mitochondrial function is a potential mechanism underlying ischemic preconditioning (IPC). Acknowledging the role of nitric oxide (NO•) in IPC, it was hypothesized that mitochondrial protein S-nitrosation may be a cardioprotective mechanism. The reagent S-nitroso-2-mercaptopropionyl-glycine (SNO-MPG) was therefore developed to enhance mitochondrial S-nitrosation and elicit cardioprotection. Within cardiomyocytes, mitochondrial proteins were effectively S-nitrosated by SNO-MPG. Consistent with the recent discovery of mitochondrial complex I as an S-nitrosation target, SNO-MPG inhibited complex I activity and cardiomyocyte respiration. The latter effect was insensitive to the NO• scavenger c-PTIO, indicating no role for NO•-mediated complex IV inhibition. A cardioprotective role for reversible complex I inhibition has been proposed, and consistent with this SNO-MPG protected cardiomyocytes from simulated IR injury. Further supporting a cardioprotective role for endogenous mitochondrial S-nitrosothiols, patterns of protein S-nitrosation were similar in mitochondria isolated from Langendorff perfused hearts subjected to IPC, and mitochondria or cells treated with SNO-MPG. The functional recovery of perfused hearts from IR injury was also improved under conditions which stabilized endogenous S-nitrosothiols (i.e. dark), or by pre-ischemic administration of SNO-MPG. Mitochondria isolated from SNO-MPG-treated hearts at the end of ischemia exhibited improved Ca2+ handling and lower ROS generation. Overall these data suggest that mitochondrial S-nitrosation and complex I inhibition constitute a protective signaling pathway that is amenable to pharmacologic augmentation.

Objective

To examine the protective role of ischaemic preconditioning (IPC) in rat hearts using 99mTc-glucarate (GLA) and a stationary SPECT imager, FastSPECT.

Methods

Twenty-four rats with 30 min myocardial ischaemia and 150 min reperfusion (IR) were studied as follows. The IPC group (n = 6) underwent IPC (five cycles of 4 min ligation of the left coronary artery and reflow) before IR. The control group (n = 7) was treated by IR without IPC. The SPT group (n = 6) was subjected to IPC and an adenosine antagonist, 8-(p-sulfophenyl)-theophylline (SPT). The vehicle group (n = 5) received IPC and SPT carrier vehicle. GLA was delivered intravenously 30 min post-reperfusion, and 2-h dynamic cardiac images were acquired by FastSPECT.

Results

GLA showed ‘hot-spot’ accumulation in the ischaemic area-at-risk (IAR) and exhibited lower retention (% 5 min peak) in the IPC and vehicle groups (33.8 ± 2.6 vs. 35.7 ± 9.2, P>0.05) than in the control and SPT groups (63.1 ± 5.3 vs. 54.8 ± 4.8, P>0.05). The infarct size (% IAR) was larger in the control and SPT groups (48.2 ± 6.3 vs. 41.7 ± 6.3, P>0.05) than that in the IPC and vehicle groups (21.0 ±1.9 vs. 19.1 ± 4.6, P>0.05). In terms of the ex-vivo IAR-to-normal radioactivity ratio, there was a statistical difference between the control and IPC groups (7.4 ± 0.9 vs. 3.0 ± 0.4), as well as the SPT and vehicle groups (7.4 ± 1.0 vs. 3.4 ± 0.5).

Conclusion

IPC offers cardioprotection and relates to the activation of adenosine receptors in rat hearts. FastSPECT GLA imaging is not only useful in detecting early ischaemia–reperfusion injury, but also valuable in evaluating cardioprotection.

Objective

Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex.

Methods and Results

In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 minutes of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP).

Conclusion

Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε.

Introduction

The function of the repolarization reserve in the prevention of ventricular arrhythmias during cardiac ischemia/reperfusion and the impact of ischemia on slowly activated delayed rectifier potassium current (IKs) channel subunit expression are not well understood.

Methods and Results

The responses of monophasic action potential duration (MAPD) prolongation and triangulation were investigated following an L-768,673-induced blockade of IKs with or without ischemia/reperfusion in a rabbit model of left circumflex coronary artery occlusion/reperfusion. Ischemia/reperfusion and IKs blockade were found to significantly induce MAPD90 prolongation and increase triangulation at the epicardial zone at 45 min, 60 min, and 75 min after reperfusion, accompanied with an increase in premature ventricular beats (PVBs) during the same period. Additionally, IKs channel subunit expression was examined following transient ischemia or permanent infarction and changes in monophasic action potential (MAP) waveforms challenged by β-adrenergic stimulation were evaluated using a rabbit model of transient or chronic cardiac ischemia. The epicardial MAP in the peri-infarct zone of hearts subjected to infarction for 2 days exhibited increased triangulation under adrenergic stimulation. KCNQ1 protein, the α subunit of the IKs channel, was downregulated in the same group. Both findings were consistent with an increased incidence of PVBs.

Conclusion

Blockade of IKs caused MAP triangulation, which precipitated ventricular arrhythmias. Chronic ischemia increased the incidence of ventricular arrhythmias under adrenergic stimulation and was associated with increased MAP triangulation of the peri-infarct zone. Downregulation of KCNQ1 protein may be the underlying cause of these changes.

The reversible S-nitrosation and inhibition of mitochondrial complex I is a potential mechanism of cardioprotection, recruited by ischemic preconditioning (IPC), S-nitrosothiols, and nitrite. Previously, to exploit this mechanism, the mitochondrial S-nitrosating agent S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) was developed, and protected perfused hearts and isolated cardiomyocytes against ischemia-reperfusion (IR) injury. In the present study, the murine left anterior descending coronary artery (LAD) occlusion model of IR injury was employed, to determine the protective efficacy of SNO-MPG in vivo. Intraperitoneal administration of 1 mg/kg SNO-MPG, 30 min. prior to occlusion, significantly reduced myocardial infarction and improved EKG parameters, following 30 min. occlusion plus 2 or 24 hr. reperfusion. SNO-MPG protected to the same degree as IPC, and notably was also protective when administered at reperfusion. Cardioprotection was accompanied by increased mitochondrial protein S-nitrosothiol content, and inhibition of complex I, both of which were reversed after 2 hr. reperfusion. Finally, hearts from mice harboring a heterozygous mutation in the complex I NDUSF4 subunit were refractory to protection by either SNO-MPG or IPC, suggesting that a fully functional complex I, capable of reversible inhibition is critical for cardioprotection. Overall, these results are consistent with a role for mitochondrial S-nitrosation and complex I inhibition in the cardioprotective mechanism of IPC and SNO-MPG in vivo.

The cardioprotective effects of ischemic preconditioning (IPC) can be mimicked or blocked by pharmacologic agents which modulate the mitochondrial ATP-sensitive potassium (mKATP) channel, thereby implicating this channel in the mechanism of IPC. Cardioprotection can also be achieved via inhibition of mitochondrial respiratory complex II, and significant pharmacologic overlap exists between complex II inhibitors and mKATP channel agonists. However, the relationship between complex II and the mKATP channel remains unclear. Atpenin A5 (AA5) is a potent and specific complex II inhibitor, and herein we report that AA5 (1 nM) also activates the mKATP channel and protects against simulated ischemia-reperfusion (IR) injury in isolated cardiomyocytes. Similar to known mKATP agonists, AA5-mediated protection was sensitive to the mKATP antagonists 5-hydroxydecanoate (5HD) and glyburide. Notably, the optimal mKATP opening and protective concentration of AA5 had no effect on complex II enzymatic activity, suggesting an interaction of AA5 with complex II, but not inhibition of the complex per se, is necessary for protection. A cardioprotective effect of AA5 was also observed in isolated perfused hearts, wherein AA5 increased post-IR contractile function and decreased infarct size, in a 5HD-sensitive manner. In conclusion, the specific complex II inhibitor AA5 is the most potent mKATP activator discovered to date, and provides a novel method of activating mKATP channels and protecting the heart from IR injury.

Background

Although recent studies indicate that renal ischemic preconditioning (IPC) protects the kidney from ischemia-reperfusion (I/R) injury, the precise protective mechanism remains unclear. In the current study, we investigated whether early IPC could upregulate hypoxia inducible transcription factor-1α (HIF-1α) expression and could reduce endoplasmic reticulum (ER) stress after renal I/R and whether pharmacological inhibition of nitric oxide (NO) production would abolish these protective effects.

Methods

Kidneys of Wistar rats were subjected to 60 min of warm ischemia followed by 120 min of reperfusion (I/R group), or to 2 preceding cycles of 5 min ischemia and 5 min reperfusion (IPC group), or to intravenously injection of NG-nitro-L-arginine methylester (L-NAME, 5 mg/kg) 5 min before IPC (L-NAME+IPC group). The results of these experimental groups were compared to those of a sham-operated group. Sodium reabsorption rate, creatinine clearance, plasma lactate dehydrogenase (LDH) activity, tissues concentrations of malonedialdehyde (MDA), HIF-1α and nitrite/nitrate were determined. In addition, Western blot analyses were performed to identify the amounts of Akt, endothelial nitric oxide synthase (eNOS) and ER stress parameters.

Results

IPC decreased cytolysis, lipid peroxidation and improved renal function. Parallely, IPC enhanced Akt phosphorylation, eNOS, nitrite/nitrate and HIF-1α levels as compared to I/R group. Moreover, our results showed that IPC increased the relative amounts of glucose-regulated protein 78 (GRP78) and decreased those of RNA activated protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 4 (ATF4) and TNF-receptor-associated factor 2 (TRAF2) as judged to I/R group. However, pre treatment with L-NAME abolished these beneficial effects of IPC against renal I/R insults.

Conclusion

These findings suggest that early IPC protects kidney against renal I/R injury via reducing oxidative and ER stresses. These effects are associated with phosphorylation of Akt, eNOS activation and NO production contributing thus to HIF-1α stabilization. The beneficial impact of IPC was abolished when NO production is inhibited before IPC application.

Objective:

The cardioprotective potential of human recombinant erythropoietin (alpha) (Epo) against ischemia-reperfusion-induced injury is well known. But, the underlying mechanisms are not well elucidated. The aim of this study was to characterize the mechanism involved in the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.

Materials and Methods:

The heart was mounted on a Langendorff apparatus. After 10 min of stabilization, four cycles of ischemic preconditioning (IPC) were given followed by 30 min of global ischemia and 120 min of reperfusion. Epo preconditioning was induced by four cycles of 5-min perfusion of K-H solution containing Epo (1.0 U/ml) followed by 5 min perfusion with K-H solution. Myocardial infarct size was estimated macroscopically using the triphenyltetrazolium chloride staining technique. The extent of myocardial injury was measured by release of lactate dehydrogenase and creatine kinase-MB in the coronary effluent.

Results:

The present study demonstrates that Epo preconditioning was almost as effective as IPC. Administration of Wortmannin (100 nM), a PI-3K inhibitor, or Chelerythrine (1 µM), a protein kinase-C (PKC) inhibitor, or AG490 (5 µM), a JAK-2 inhibitor, significantly attenuated the cardioprotective effects of Epo-induced preconditioning.

Conclusion:

Our result suggest that the cardioprotective potential of Epo-induced preconditioning in isolated rat heart was due to an interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of the three pathways was sufficient to block the cardioprotective effect of Epo-induced preconditioning in isolated rat heart.

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A2BAR gene knock-out (A2BKO)/β-galactosidase reporter gene knock-in mice and the A2BAR antagonist ATL-801 to investigate the potential involvement of the A2BAR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A2BKO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A2BKO hearts by ~30–40%, and equivalently decreased the release of cardiac tropinin I during the reperfusion period (from 5,969±925 to 1,595±674 ng/g and 4,376±739 to 2,278±462 ng/g using WT and A2BKO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifest in experiments using A2BKO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A2BAR agonist BAY 60-658 (~25% reduction at a dose of 1.0 mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A2BAR. We present the idea that the A2BAR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.