Cardioprotection Of Ischemic Preconditioning Research Articles

Endothelial Derived Neuregulin-1 may be Important for Cardioprotection Induced by Ischemic Preconditioning

Aim of review: The underlying mechanisms of ischemic preconditioning (IPC) have been studied for many years, but have not been elucidated completely. The available literatures indicate that endothelial derived neuregulin-1 (NRG-1) is involved in myocardial ischemia/reperfusion injury (IRI) and protects cardiomyocytes against H2O2-induced apoptosis by regulating endoplasmic reticulum stress (ERS). The aim of this review is to provide the evidence that endothelial derived NRG-1 maybe a crucial biomolecule mediating powerful cardioprotection by IPC.Methods: According to the available literatures, this review will first discuss the factors attributable to cardioprotection of IPC and then provide an overview of the cellular and molecular mechanisms of endothelial derived NRG-1 maybe involved in IPC induced cardioprotection.Recent findings: Multiple factors are attributable to cardioprotection induced by IPC. It has been shown that anoxia preconditioning in vitro can not provide cardioprotection as much as the IPC in vivo. During myocardial ischemic conditioning, endothelial cells may play several roles: a “receptor” for blood-borne conditioning moieties, a sensor for hypoxic stress and a paracrine organ. The NRG-1 produced by endothelial cells has been proved to protect against myocardial IRI through a PI3K/Akt pathway. Furthermore, unbalanced endoplasmic reticulum stress is one of the important mechanisms of IRI, and IPC has been demonstrated to protect myocardial IRI by regulating endoplasmic reticulum stress. In addition, NRG-1 may attenuate IRI by regulating cold inducible RNA-binding protein with its downstream endoplasmic reticulum stress related signaling pathways.Summary: Endothelial derived NRG-1 and its downstream signaling pathways are involved in multiple aspects of cardiac physiology and function, and can provide a significant protection against myocardial injury. The available evidence indicates that selective deletion of endothelial derived NRG-1 in vivo decreases the tolerance to IRI, as demonstrated by impaired recovery of post-ischemic myocardial contraction function. Thus, endothelial derived NRG-1 maybe a crucial biomolecule mediating powerful cardioprotection by IPC. If this new view is proved by basic and clinical experiments, a crucial biomolecule mediated cardioprotection induced by IPC would be revealed. Citation: Gui-Zhen Yang, Fu-Shan Xue, Chao Sun, Xu Liao, Jian-Hua Liu. Endothelial derived neuregulin-1 may be important for cardioprotection induced by ischemic preconditioning. J Anesth Perioper Med 2017; 4: 225-30. doi: 10.24015/JAPM.2017.0008This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Angiotensin (1-7) facilitates cardioprotection of ischemic preconditioning on ischemia-reperfusion-challenged rat heart.

Ischemic preconditioning (IPC) is one of the most promising strategies used to protect the myocardium from ischemia-reperfusion injury. Ang (1-7) exhibits cardioprotective activity; however, its therapeutic potential on IPC-induced cardioprotection has not been reported in ischemia-reperfusion injury till date. Therefore, the first set of experiment was designed to evaluate the direct effect of Ang (1-7), in perfusion medium, on cardioprotective activity of IPC in rat heart challenged to ischemia-reperfusion injury. In addition, the acute and chronic effects of pretreated Ang (1-7) were investigated on cardioprotection of IPC in ischemia-reperfusion-challenged hearts in subsequent sets of experiments. The results showed that Ang (1-7) potentiated the IPC-induced increase in coronary flow and heart rate, decrease in lactate dehydrogenase and creatine kinase activity, ventricular fibrillation, and infarct size in ischemia-reperfusion-challenged animals in direct and chronic sets of experiments. Further, Ang (1-7) enhanced the IPC-induced attenuation in mitochondrial dysfunction, oxidative stress, and apoptosis in ischemia-reperfusion-challenged hearts in both sets of experiments. A-779, Mas receptor antagonist, abrogated potentiation effects of Ang (1-7) on IPC-induced cardioprotection in ischemia-reperfusion-challenged rats in the above set of experiments. These observations indicate that Ang (1-7)/Mas receptor activation could be a potential adjuvant to IPC during ischemia-reperfusion-induced cardiac injury.

Cardioprotection of ischemic preconditioning in rats involves upregulating adiponectin

It has been reported that ischemic preconditioning (IPC) and adiponectin (APN) are cardioprotective in many cardiovascular disorders. However, whether APN mediates the effect of IPC on myocardial injury has not been elucidated. This study was conducted to investigate whether IPC affects myocardial ischemic injury by increasing APN expression. Male adult rats with cardiac knockdowns of APN and its receptors via intramyocardial small-interfering RNA injection were subjected to IPC and then myocardial infarction (MI) at 24 h after IPC. Globular APN (gAd) was injected at 10 min before MI. APN mRNA and protein levels in myocardium as well as the plasma APN concentration were markedly high at 6 and 12 h after IPC. IPC ameliorated myocardial injury as evidenced by improved cardiac functions and a reduced infarct size. Compared with the control MI group, rats in the IPC + MI group had elevated levels of left ventricular ejection fraction and fractional shortening and a smaller MI size (P < 0.05). However, the aforementioned protective effects were ameliorated in the absence of APN and APN receptors, followed by the inhibition of AMP-activated protein kinase (AMPK) phosphorylation, but reversed by gAd treatment in wild-type rats, and AMPK phosphorylation increased (P < 0.05). Overall, our results suggest that the cardioprotective effects of IPC are partially due to upregulation of APN and provide a further insight into IPC-mediated signaling effects.

Ischemic preconditioning in pigs: a causal role for signal transducer and activator of transcription 3

Ischemic preconditioning (IPC), i.e., brief episodes of nonlethal myocardial ischemia-reperfusion (I/R) before sustained ischemia with subsequent reperfusion, reduces infarct size in all species tested so far, including humans. In rodents, the cardioprotective signal transduction causally involves an activation of Akt, ERK1/2, and STAT3. However, there are apparent species differences in the signal transduction between rodents and larger mammals such as pigs, where data on IPC's signal transduction are inconsistent for Akt and ERK1/2. The role of STAT3 has not yet been analyzed. Pigs were subjected to 60 min of left anterior descending coronary artery occlusion and 180 min of reperfusion without or with IPC (2 cycles of 3-min occlusion separated by 2 min of reperfusion 15 min before sustained I/R). Infarct size was analyzed by triphenyl tetrazolium chloride staining, and Akt, ERK1/2, and STAT3 phosphorylation was quantified in myocardial biopsies taken at baseline and early reperfusion. AG490 was used to block the STAT3 signaling pathway. IPC reduced infarct size (%area at risk; mean ± SE, I/R, 45 ± 3 vs. IPC, 18 ± 3, P < 0.05). Akt and ERK1/2 phosphorylation was increased at early reperfusion without and with IPC. In contrast, STAT3 phosphorylation at early reperfusion was only increased with IPC (%baseline; mean ± SE, I/R, 126 ± 29 vs. IPC, 408 ± 147, P < 0.05). AG490 prevented the IPC-related increase of STAT3 phosphorylation at reperfusion (%baseline; mean ± SE, 82 ± 12) and abolished IPC's cardioprotection (%area at risk; mean ± SE, 35 ± 4). In pigs, increased phosphorylation of STAT3 is causally involved, whereas Akt and ERK1/2 seem to play no role in IPC's cardioprotection.NEW & NOTEWORTHY In pig hearts in situ, ischemic preconditioning (IPC) causally involves increased phosphorylation of STAT3, whereas Akt and ERK1/2 play no role for cardioprotection. The cardioprotective signal transduction of IPC is similar to that of ischemic postconditioning and remote IPC in pigs.

Regulation of protein kinase C-epsilon and its age-dependence

Protein kinase C (PKC) is an important mediator in the cardioprotection of ischemic preconditioning and has been shown to translocate to mitochondria upon activation. However, little is known about the cellular signaling underlying the translocation of PKC isoforms to mitochondria and its age-dependence. The present study aimed to explore whether adenosine-induced translocation of PKCε to mitochondria is mediated by caveolin-3 and/or adenosine A2B receptor/PI3 kinase mediated signaling, and whether the mitochondrial targeting of PKCε is age-related. Immunofluorescence imaging of isolated mitochondria from cardiomyocytes and H9c2 cells showed that while adenosine-induced increase in mitochondrial PKCε was inhibited by adenosine A1 receptor blocker, pretreatment with adenosine A2B receptor specific inhibitor MRS 1754 or PI3K inhibitor Wortmannin did not significantly reduce adenosine-mediated increase in mitochondrial PKCε. Interestingly, adenosine-induced increase in mitochondrial translocation of PKCε was significantly blocked by suppressing caveolin-3 expression with specific siRNA. When compared to that in young adult rat hearts, the level of mitochondrial PKCε in middle-aged rat hearts was significantly lower at the basal condition and in response to adenosine treatment, along with largely decreased mitochondrial HSP90 and TOM70 protein expression. We demonstrate that adenosine-induced translocation of PKCε to mitochondria is mediated by a caveolin-3-dependent mechanism and this process is age-related, possibly in part, through regulation of HSP90 and TOM70 expression. These results point out a novel mechanism in regulating PKC function in mitochondria.

Remote Ischemic Preconditioning Cardio-protection via Enhanced Cell Volume Regulation Requires Activation of Swelling-Activated Chloride (Icl- Swell) Channels

Introduction: We have previously shown that remote ischemic preconditioning induced in cardiomyocytes by a brief direct incubation in rabbit blood dialysate, originated from other rabbits initially subjected to brief periods of limb ischemia/ reperfusion, given prior exposure to a prolonged ischemia/reperfusion protects cardiomyocytes against necrosis. In this study, we examined the hypothesis that sarcolemmal protein kinase C epsilon interacts with the swellingactivated Cl- channel to both enhance cardiomyocyte volume regulation and protect against cardiomyocyte necrosis in limb ischemia/reperfusion remote ischemic preconditioning. Methods: Cultured (forty-eight hours) rabbit cardiomyocytes (control and remote ischemic preconditioned dialysate treated) were, after stabilization, subjected to either thirty-minute hypo-osmotic stress or to seventy-five minutes of simulated ischemia (severe hypoxia plus metabolic inhibition) followed by sixty minutes of simulated reperfusion (in oxygenated media), with or without a specific swelling-activated chloride channel inhibitor or its vehicle given ten minutes prior and during the hypo-osmotic stress or the simulate ischemia, to measure peak cell swelling (between eight to twelve minutes of hypo-osmotic stress), regulatory volume decrease and cell necrosis (by trypan blue staining). Results: Specific inhibition of swell-activated chloride channels not only substantially inhibited remote ischemic preconditioned dialysate induced protection against cardiomyocyte necrosis but it also significantly impaired cardiomyocyte volume regulation. PKCe was found to co-immunoprecipitate with ClC-3, consistent with this kinase influencing swell-activated chloride channel activity. Conclusion: These findings indicate that swelling-activated chloride channels are essential for the cardioprotection by remote ischemic preconditioning.

Molecular mechanism underlying adenosine receptor-mediated mitochondrial targeting of protein kinase C

Activation of protein kinase C (PKC) via adenosine receptors is known to be involved in the cardioprotection of ischemic preconditioning (IPC). Specifically, activation of PKCε is critical for cardioprotection. There is ample evidence that PKCε resides in cardiac mitochondria. However, the signals that promote translocation of PKCε are largely unknown. The present study was designed to determine whether and how adenosine receptor activation induces translocation of PKCε to mitochondria. Freshly isolated adult rat cardiac myocytes and rat heart-derived H9c2 were used in the study. Immunofluorescence imaging of isolated mitochondria showed that PKCε but not PKCδ was localized in mitochondria and this mitochondrial localization of PKCε was significantly increased by adenosine treatment. The adenosine-induced increase in PKCε-positive mitochondria was largely prevented not only by PKC inhibitor chelerythrine, but also by the HSP90 inhibitor geldanamycin and by siRNA targeting HSP90. Immunoblot analysis from percoll-purified mitochondria further demonstrated that adenosine mediated a significant increase in mitochondrial PKCε but not PKCδ. This effect was blocked by inhibiting PKC activity with chelerythrine and bisindolylmaleimide. Furthermore, co-immunoprecipitation data showed that PKCε but not PKCδ was associated with TOM70 and HSP90, and this association was enhanced by adenosine treatment. Moreover, adenosine-induced association of PKCε with TOM70 was reduced by suppressing HSP90 expression with siRNA. In conclusion, we demonstrate that adenosine induces HSP90-dependent translocation of PKCε to mitochondria, possibly through mitochondrial import machinery TOM70. These results point out a novel mechanism in regulating PKC in mitochondria and suggest an important implication in ischemic preconditioning or postconditioning.

Abstract 270: A Novel Mechanism of Preconditioning: Attenuating Reperfusion Injury through Enhanced Myocardial Substrate Uptake via Insulin-Stimulated Akt and AMPK Activation

Introduction: Ischemic preconditioning (IPC) is an intrinsic protective mechanism which markedly enhanced the ability of the heart to withstand a subsequent ischemic injury. We previously found that IPC cardioprotection was markedly attenuated in diabetic rats. Therefore, we hypothesized that insulin-regulated myocardial substrate metabolism may play a key role in IPC-afforded cardioprotection. Methods: Adult male rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion (MI/R). IPC was achieved by two cycles of 5 min ischemia and 5 min reperfusion. Myocardial glucose and fatty acid (FA) uptake were assessed at the end of 1 h reperfusion by determining 18 F-2-deoxy-2-fluoro-D-glucose uptake and fatty acid translocase (FAT)/CD36 translocation, respectively. Results: IPC significantly improved cardiac function (n=8, P &lt;0.05) with reduced myocardial infarction, apoptotic cell death and blood creatine kinase/lactate dehydrogenase levels following MI/R (all P &lt;0.05). Myocardial glucose uptake was markedly elevated after IPC treatment (17.0±1.5 vs. 12.4±1.0 in MI/R, n=10-12, P &lt;0.05), as well as translocation of glucose transporter 4 (GLUT4) to plasma membrane (PM) ( P &lt;0.01). Meanwhile, myocardial PI3K expression and Akt phosphorylation were significantly enhanced in IPC group ( P &lt;0.05). Interestingly, IPC also increased CD36 translocation to PM and AMPK phosphorylation ( P &lt;0.05). Wortmannin not only abrogated the cardioprotective effect of IPC, but also inhibited IPC-induced Akt/AMPK phosphorylation and subsequent GLUT4/CD36 translocation. Furthermore, the cardioprotection of IPC was markedly blunted in STZ-induced insulin-deficient diabetic hearts with failure of increase in glucose/FA uptake and impaired IPC-stimulated PI3K-Akt and AMPK signaling (n=6, P &lt;0.05). Conclusions: These results suggest that IPC increases both glucose and FA uptake during early reperfusion to resist myocardial injury via insulin-stimulated, and PI3K-dependent Akt and AMPK activation. Therefore, augmenting insulin signaling may be a potential therapy to improve myocardial substrate uptake and restore the cardioprotection of IPC in diabetic hearts.

Genetically determined angiotensin converting enzyme level and myocardial tolerance to ischemia

Angiotensin I-converting enzyme (ACE; kininase II) levels in humans are genetically determined. ACE levels have been linked to risk of myocardial infarction, but the association has been inconsistent, and the causality underlying it remains undocumented. We tested the hypothesis that genetic variation in ACE levels influences myocardial tolerance to ischemia. We studied ischemia-reperfusion injury in mice bearing 1 (ACE1c), 2 (ACE2c, wild type), or 3 (ACE3c) functional copies of the ACE gene and displaying an ACE level range similar to humans. Infarct size in ACE1c was 29% lower than in ACE2c (P<0.05). Pretreatment with a kinin B2 receptor antagonist suppressed this reduction. In ACE3c, infarct size was the same as in ACE2c. But ischemic preconditioning, which reduced infarct size in ACE2c (-63%, P<0.001) and ACE1c (-52%, P<0.05), was not efficient in ACE3c (-2%, NS, P<0.01 vs. ACE2c). In ACE3c, ischemic preconditioning did not decrease myocardial inflammation or cardiomyocyte apoptosis. Pretreatment with a renin inhibitor had no cardioprotective effect in ACE2c, but in ACE3c partially restored (38%) the cardioprotection of ischemic preconditioning. Thus, a modest genetic increase in ACE impairs myocardial tolerance to ischemia. ACE level plays a critical role in cardiac ischemia, through both kinin and angiotensin mediated mechanisms.

Autophagy induced by ischemic preconditioning is essential for cardioprotection.

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.

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 microg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 microg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- 6.4%, n = 7) and MnSOD (79.9 +/- 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP.

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

Signalosomes: delivering cardioprotective signals from GPCRs to mitochondria

Non‐viral delivery of therapeutic nucleic acids to investigate the role of transcriptional networks in the ischemic heart

Nuclear factor‐kappaB (NF‐κB) is a key player in the physiological outcomes of myocardial ischemic events including ischemia‐reperfusion (I/R), permanent occlusion (PO), and the cardioprotection of ischemic preconditioning (IPC). Our lab has demonstrated that NF‐κB plays a protective role in late IPC and after PO, yet has an injurious role after I/R. Such results suggest the transcriptional activation of differential gene sets by each ischemic event. This was confirmed in our lab by a gene microarray analysis comparing I/R, PO, and IPC. Our microarray results also suggested potential cross‐talk of NF‐κB with other transcriptional networks, such as those regulated by HIF‐1α, AP‐1, and HSF‐1. Our aim is to deliver transcription factor decoys and interfering RNAs to the heart in vivo and begin to elucidate the relative role of these gene networks in myocardial ischemia. We have demonstrated nucleic acid delivery to the heart via novel polyglycoamidoamine (PGAA) glycopolymers that act through formation of nanoscale “polyplexes” and enhance cellular uptake via endocytosis resulting in nearly transmural transfection of the myocardium without eliciting cytotoxicity. Our work provides a high‐throughput in vivo approach to the study of the regulation of interlaced gene networks during myocardial ischemic events and will augment the identification of new clinical targets for the treatment of myocardial ischemia. This work was supported by NIH grant R01 HL63034 (WKJ) and NSF award number 0333377.

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.

An obligatory role of STAT1 in the upregulation of cardioprotective proteins and delayed cardioprotection in ischemic preconditioning

An obligatory role of STAT1 in the upregulation of cardioprotective proteins and delayed cardioprotection in ischemic preconditioning

Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43

Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C epsilon and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 +/- 3.3 (n = 17) to 34.2 +/- 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 +/- 4.0, n = 10) and preconditioned hearts (65.4 +/- 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.

Connexin 43 and ischemic preconditioning: effects of age and disease

Connexin 43 (Cx43) is the constitutive protein for the formation of cardiac gap junctions and therefore essential for cell–cell coupling and normal cardiac function. Apart from its localization at the sarcolemma, Cx43 is present in the inner mitochondrial membrane. Cx43 is involved in cardiomyocyte free oxygen radical formation and in the cardioprotection by ischemic preconditioning (IP). This review will discuss Cx43 deficiency in aged and diseased myocardium and the potential impact of age and disease on IP's cardioprotection.

Heat shock proteins, end effectors of myocardium ischemic preconditioning?

The purpose of this study was to investigate (1) whether ischemia-reperfusion increased the content of heat shock protein 72 (Hsp72) transcripts and (2) whether myocardial content of Hsp72 is increased by ischemic preconditioning so that they can be considered as end effectors of preconditioning. Twelve male minipigs (8 protocol, 4 sham) were used, with the following ischemic preconditioning protocol: 3 ischemia and reperfusion 5-minute alternative cycles and last reperfusion cycle of 3 hours. Initial and final transmural biopsies (both in healthy and ischemic areas) were taken in all animals. Heat shock protein 72 messenger ribonucleic acid (mRNA) expression was measured by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method using complementary DNA normalized against the housekeeping gene cyclophilin. The identification of heat shock protein 72 was performed by immunoblot. In our "classic" preconditioning model, we found no changes in mRNA hsp72 levels or heat shock protein 72 content in the myocardium after 3 hours of reperfusion. Our experimental model is valid and the experimental techniques are appropriate, but the induction of heat shock proteins 72 as end effectors of cardioprotection in ischemic preconditioning does not occur in the first hours after ischemia, but probably at least 24 hours after it, in the so-called "second protection window."

G protein-coupled receptor internalization signaling is required for cardioprotection in ischemic preconditioning.

The present study is designed to explore the role of G protein-coupled receptors (GPCRs) in the protection afforded by ischemic preconditioning (PC). We used TG mice with cardiac-specific overexpression of a Gbetagamma-sequestering peptide, betaARKct (TG betaARKct mice), to test whether the protection of PC is Gbetagamma-dependent. To test the role of G(i) protein, we used wild-type mice pretreated with the G(i) inhibitor pertussis toxin. Recovery of left ventricular developed pressure and infarct size were measured as indices of protection. PC induced protection in wild-type mice, but this protection was blocked by pertussis toxin treatment and was also blocked in TG betaARKct mice. To determine the mechanism of Gbetagamma-induced protection in PC, we investigated one of the downstream targets of Gbetagamma, the PI3K/p70S6K pathway. PC-induced phosphorylation of p70S6K was not blocked in TG betaARKct hearts; therefore, we investigated other targets of Gbetagamma. Recent studies suggest a role for Gbetagamma in GPCR internalization. We found that betaARKct, a specific PI3K inhibitor wortmannin, and bafilomycin A1, which all block receptor recycling, all blocked the protective effect of PC. To additionally test whether PI3K is involved in PC-activated receptor internalization and endosomal signaling, we used TG mice with cardiac-specific overexpression of a catalytically inactive mutant PI3Kgamma, which disrupts the recruitment of functional PI3K to agonist-activated GPCRs in vivo. We found that the catalytically inactive mutant of PI3Kgamma blocks the protection of PC. In summary, these data suggest the novel finding that the cardioprotective effect of PC requires receptor internalization.

Kappa- but not delta-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats.

Two series of experiments were performed in the isolated perfused rat heart to determine the role of kappa- and delta-opioid receptors (OR) in cardioprotection of ischemic preconditioning (IP). In the first series of experiments, it was found that IP with two cycles of 5-min regional ischemia followed by 5-min reperfusion each reduced infarct size induced by 30-min ischemia, and the ameliorating effect of IP on infarct was attenuated with blockade of either 5 x 10(-6) mol/l nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, or 5 x 10(-6) mol/l naltrindole (NTD), a selective delta-OR antagonist. The second series showed that U50,488H, a selective kappa-OR agonist, or D-Ala(2)-D-leu(5)-enkephalin (DADLE), a selective delta-OR agonist, dose dependently reduced the infarct size induced by ischemia, which mimicked the effects of IP. The effect of 10(-5) mol/l U50,488H on infarct was significantly attenuated by blockade of protein kinase C (PKC) with specific PKC inhibitors, 5 x 10(-6) mol/l chelerythrine or 8 x 10(-7) mol/l calphostin C, as well as by blockade of ATP-sensitive K(+) (K(ATP)) channels with blockers of the channel, 10(-5) mol/l glibenclamide or 10(-4) mol/l 5-hydroxydecanoate. IP also reduced arrhythmia induced by ischemia. Nor-BNI, but not NTD, attenuated, while U50,488H, but not DADLE, mimicked the antiarrhythmic action of IP. In conclusion, the present study has provided first evidence that kappa-OR mediates the ameliorating effects of IP on infarct and arrhythmia induced by ischemia, whereas delta-OR mediates the effects only on infarct. Both PKC and K(ATP) channels mediate the effect of activation of kappa-OR on infarct.