Late Ischemic Preconditioning Research Articles

Ischemic preconditioning delays the time of exhaustion in cycling performance during the early but not in the late phase

Abstract Aims: To investigate the early and late ischemic preconditioning (IPC) effect on the trained cyclists’ performance during incremental cycling test until exhaustion. Methods: Twenty-one male cyclists allocated to an IPC (2 x 5-min of blood flow occlusion at 50 mm Hg above systolic pressure followed + 5-min of deflation), SHAM (2 x 5-min at 20 mm Hg) or control (CON; no occlusion) interventions, performed three incremental cycling test (ICT) until exhaustion on separate days. The ICT were conducted pre interventions (baseline), 5-min and 24-h after interventions. The heart rate (HR) and power output (PO) were recorded during all ICT. Results: The IPC group increased ICT performance (4.4 ± 4.0 %; effect size (ES) = 0.27) 5-min post intervention, accompanied by HR mean reduction, compared to baseline (p 0.05) and IPC group decreased the performance (-4.6 ± 3.6 %; ES = 0.16) compared to 5-min post intervention (p 0.05). There were no difference (p > 0.05) among groups for PO peak, HR and ICT performance in all moments (baseline, 5-min and 24-h post intervention). Conclusion: The IPC increases early but not late incremental cycling test performance.

A review on Late phase ischemic preconditioning.

A review on Late phase ischemic preconditioning.

From the S.P. Botkin’s idea of “preexposure” to preconditioning phenomenon. Perspectives for use of phenomena of ischemic and pharmacological preconditioning

The phenomenon of ischemic preconditioning based on the S.P. Botkin’s idea about defense effect of disturbing factors acting in small intensities is observed in the review. The modern literature data about main types of preconditioning exposure, triggers and mechanisms of ischemic preconditioning are reviewed. This phenomenon was supported in many experiments in vivo and in vitro on animals of different spices as well as in humans in clinical conditions. Ischemic preconditioning is qualified as transient positive changes in the organs and tissues produced by activation of rapid endogenous adoptive processes in them during the short period of subletal ischemia and reperfusion and which defend them from subsequent ischemic episodes. There are early and late ischemic preconditioning (the second window of defense). The first type of ischemic preconditioning belongs to classic type of preconditioning and is produced by the short ischemic episodes (3-5 min) and similar intervals of reperfusion. Ischemic preconditioning observed in a day or more after preconditioning stimuli is named as late preconditioning with genes expression, synthesis of heat shock proteins (HSP 72 in particular) and NO synthase as the basis mechanisms underlying of it. Administration of triggers like adenosine, forbol ether, bradykinine or glycerol derivatives into the blood or ischemic tissues produces defense action similar to ischemic preconditioning and qualified as pharmacological preconditioning. Preconditioning induced by pharmacological agents are more preference than short ischemic episodes. Antihypoxic effects of benzimidazol derivatives in both an acute hypoxia and hypoxic preconditioning are described in the article. Other perspectives of pharmacological preconditioning in practical use are also discussed.

The second window of desflurane-induced preconditioning is mediated by STAT3: role of Pim-1 kinase.

Late ischemic preconditioning is mediated via nuclear transcription factor signal transducer and activator of transcription 3 (STAT3). Pim-1 kinase reduces infarct size in cardiomyocytes and is regulated by STAT3. We tested the hypothesis that late desflurane-induced preconditioning (DES-SWOP) is mediated via STAT3 and Pim-1. After institutional approval, pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min coronary artery occlusion (CAO) and 3 h reperfusion. Control animals received no additional intervention. Desflurane was administered 48 h before CAO either alone or in combination with the janus kinase/STAT3 inhibitor AG-490 (40 μg/g i.p., 20 min before desflurane administration) or the Pim-1 kinase inhibitor II (PIM-Inh.II, 10 μg/g i.p., 15 min before CAO). Infarct size (IS) and area at risk were determined with triphenyltetrazolium chloride and Evans blue, respectively. Additionally, cytosolic and nuclear fractions were separated at two different time points and expression of STAT3, phospho-STAT3(Ser727) , phospho-STAT3(Tyr705) , Pim-1, Bad and phospho-Bad(Ser112) were determined by Western Blot analysis. Data were analyzed with one-way or two-way ANOVA and post hoc Duncan test and are presented as mean ± SEM. IS was 47 ± 2% (n = 7-8 per group) in control animals (CON). DES-SWOP reduced myocardial infarct size to 23 ± 4%* (*P < 0.05 vs. CON). AG-490 alone did not affect myocardial infarct size (44 ± 7%), but abolished DES-SWOP (44 ± 4%). Blockade of Pim-1 did not affect the protection by DES-SWOP (34 ± 4%*). Desflurane reduced cytosolic content and enhanced nuclear content of phospho-STAT(S) (er727) . After 48 h, desflurane enhanced Pim-1 activity, whereas Pim-1 expression remained unchanged. These data suggest that DES-SWOP is mediated by activation and nuclear translocation of STAT3. The impact of Pim-1 in DES-SWOP signaling remains unclear.

Targeting MicroRNA to Upregulate Cardioprotective Heat Shock Proteins

Ischemic preconditioning (IPC) is a protective stimulus whereby the heart undergoes a series of brief, non-lethal bouts of ischemia and reperfusion that protect it from subsequent prolonged ischemia-reperfusion injury. The late phase of IPC relies on expression of protective genes, including Hsp70, Hsp90, and possibly heat shock proteins of the Hsp40 (Dnaj) family. This study investigates the hypothesis that IPC causes downregulation of microRNAs, allowing enhanced expression of their protective gene targets. Previous work has shown that modulation by microRNA is key to regulating the levels of Hsp70.3 after IPC. Because it is known that each microRNA can have multiple mRNA targets, and conversely, each mRNA can be regulated by multiple microRNAs, we took a network-based approach. Overexpression and knockdown of microRNAs was achieved by transfecting synthetic microRNAs or antisense inhibitors, respectively. Luciferase reporter assays and Western blots were used to determine the effects on expression of the predicted target genes. Results: microRNAs work together to regulate expression of the heat shock proteins studied. Specifically, miR-148a and miR-148b regulate Hsp70.1 and Hsp70.3, as well as Dnaja1. MiR-30b regulates Hsp90aa1 and Dnajb4, which is also a target of let-7a*. Knockdown of the microRNAs revealed that protein levels of the target genes increased, indicating that these microRNAs negatively regulate their expression. Because microRNA downregulation is sufficient to enhance the expression of these genes, it is likely that the decrease in these microRNAs observed in preconditioned cells and heart tissue constitutes an important mechanism in late IPC.

Effects of ischemic preconditioning protocols on skeletal muscle ischemia–reperfusion injury

BackgroundIschemic preconditioning (IPC) is described as brief ischemia–reperfusion (I/R) cycles to induce tolerance to subsequent in response to longer I/R insults. Various IPC protocols can be performed in four combinations as follows: at early or late phases and on local or distant organs. Although many experimental studies have been performed on IPC, no consensus has been established on which IPC protocol is most effective. The aims of the present study were as follows: (1) to compare the variables of preconditioning in different combinations (in early versus late phases; local versus remote organ implementations) and (2) to determine the most therapeutic IPC protocol(s). Materials and methodsA subtotal hind limb amputation model with clamping an intact femoral pedicle was used for I/R injury. IPC was induced using hind limb tourniquet with 3 × 10 min I/R cycles before longer I/R insult. Forty-nine rats were divided into seven groups (n = 7), sham, IsO (ischemia only), I/R, early ischemic preconditioning (e-IPC), late ischemic preconditioning (l-IPC), early remote ischemic preconditioning (e-RIPC), and l-RIPC (late-remote) groups, respectively. In the sham group, pedicle occlusion was not performed. Six hours ischemia was challenged in the IsO group. Three hours ischemia followed by 3 h reperfusion was performed in the I/R group. The e-IPC group was immediately preconditioned, whereas the l-IPC group was preconditioned 24 h before I/R injury on the same hind limb. In the e-RIPC and l-RIPC groups, the same protocols were performed on the contralateral hind limb. At the end of the experiments, skeletal muscle tissue samples were obtained for biochemical analysis (Malondialdehyde [MDA], catalase, myeloperoxidase [MPO], and nitric oxide end products [NOx]), light microscopy, and caspase-3 immunohistochemistry for determination of apoptosis. ResultsTissue biochemical markers were improved in nearly all the IPC groups compared with IsO and I/R groups (P < 0.05). Similarly, the histologic damage scores were decreased in all the IPC groups (P < 0.05). The lowest damage score was in the e-RIPC group followed by the l-RIPC, e-IPC, and l-IPC groups, respectively. The apoptosis scores were significantly high in the I/R group compared with the e-RIPC and l-RIPC groups (P < 0.05). Although apoptosis scores of the e-IPC and l-IPC groups were lower than the I/R group, this finding was not statistically significant (P > 0.05). ConclusionsAll IPC protocols were effective in reducing I/R injury. Among these protocols, e-RIPC achieved most protection.

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.

Effects of Early and Late Ischemic Preconditioning of the Brain on the Severity of Hippocampal Neuron Injury and the Degree of Neurological Deficit in Rats

The aim of the present work was to investigate the neuroprotective effects of early and late ischemic preconditioning (IPreC) in the acute phase of ischemic brain damage in rats. Single 5-min ischemic episodes of early IPreC were found not to produce significant neuroprotective effects as compared with controls, while three 5-min episodes of early IPreC led to significant increases in neurological deficit and increases in the level of neuron injury in hippocampal field CA1. Conversely, late IPreC, consisting of a single 5-min episode 24 h before modeling test ischemia, produced a significant neuroprotective effect, with decreased neurological deficit and retention of hippocampal field CA1 neuron viability.

Nox1 NADPH oxidase is necessary for late but not early myocardial ischaemic preconditioning

Ischaemic preconditioning (IPC) is an adaptive mechanism that renders the myocardium resistant to injury from subsequent hypoxia. Although reactive oxygen species (ROS) contribute to both the early and late phases of IPC, their enzymatic source and associated signalling events have not yet been understood completely. Our objective was to investigate the role of the Nox1 NADPH oxidase in cardioprotection provided by IPC. Wild-type (WT) and Nox1-deficient mice were treated with three cycles of brief coronary occlusion and reperfusion, followed by prolonged occlusion either immediately (early IPC) or after 24 h (late IPC). Nox1 deficiency had no impact on the cardioprotection afforded by early IPC. In contrast, deficiency of Nox1 during late IPC resulted in a larger infarct size, cardiac remodelling, and increased myocardial apoptosis compared with WT hearts. Furthermore, expression of Nox1 in WT hearts increased in response to late IPC. Deficiency of Nox1 abrogated late IPC-mediated activation of cardiac nuclear factor-κB (NF-κB) and induction of tumour necrosis factor-α (TNF-α) in the heart and circulation. Finally, knockdown of Nox1 in cultured cardiomyocytes prevented TNF-α induction of NF-κB and the protective effect of IPC on hypoxia-induced apoptosis. Our data identify a critical role for Nox1 in late IPC and define a previously unrecognized link between TNF-α and NF-κB in mediating tolerance to myocardial injury. These findings have clinical significance considering the emergence of Nox1 inhibitors for the treatment of cardiovascular disease.

Spinal Cord Early Ischemic Preconditioning Activates the Stabilized Fraction of β-Catenin After Thoracoabdominal Aortic Occlusion in Pigs

Paraplegia after thoracoabdominal aortic surgery is a devastating complication attributed to motor neurons loss and dysfunction, due to spinal cord ischemia. β-Catenin is a protein that has been associated with cell survival and healing and many studies have correlated this protein with late ischemic preconditioning (IPC). Herein we investigate the potential contribution of β-catenin in an early IPC animal model, and its relationship with heat shock protein 70 (Hsp70), suggesting a possible role of this protein as a first window of protection. A total of 42 pigs were used in an experimental thoracoabdominal aortic occlusion model. Twelve animals were used for neurologic evaluation and were randomly assigned to 2 groups (A and B). The remaining 30 animals were used in experiments for biologic measurements and innunohistochemical studies, and were randomly assigned to 5 groups (1-5). Western blotting analysis and immunoprecipitations were performed to study the levels of β-catenin and its binding relationship with Hsp70. The cellular distribution of β-catenin at various time-points was investigated by immunohistochemical studies. According to neurologic evaluation, the animals in the IPC+ischemia group had significantly better neurologic scores compared with those in the ischemia group, indicating a protective role for IPC. The biologic measurements demonstrated a significant (P=0.03) increase in β-catenin levels and translocation of the protein in the nucleus at the end of ischemic preconditioning. Our results suggest a significant role of β-catenin in early IPC protection of spinal cord after thoracoabdominal occlusion, as IPC seems to trigger the activation of the β-catenin stabilized fraction and, thus, its survival pathway.

REGULATORY T CELLS PARTIALLY MEDIATE CARDIO-PROTECTION OF LATE ISCHAEMIC PRECONDITIONING IN RATS

ObjectivesMyocardial ischaemia-reperfusion (IR) injury (IRI) is associated with activation of the innate immune system and the resultant inflammatory response. Myocardial ischaemic preconditioning (IPC) is the most powerful endogenous protective mechanism...

Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation

The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC. Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection. Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.

Expression of MicroRNA-1 and MicroRNA-21 in Different Protocols of Ischemic Conditioning in an Isolated Rat Heart Model

Background: ‘Conditioning’ [ischemic preconditioning (IPC), ischemic postconditioning (IPO) and remote ischemic preconditioning (RIPC)] the heart to render it more resistant to an episode of acute myocardial ischemia-reperfusion (I/R) injury is an endogenous cardioprotective strategy. There are several mechanisms proposed for ‘conditioning’, such as endogenous mediators or cytoprotective proteins. In recent reports, microRNAs (miRNAs) were involved in controlling the expression of myocardial ischemia-related genes. Some studies have demonstrated that cardiac miRNA-1 and miRNA-21 were significantly increased by late IPC with an increase in their target proteins [endothelial nitric oxide synthase and heat shock protein 70 (HSP70)], but their expression levels in ‘conditioning’ strategies are currently unknown. Methods: In the current study, Langendorff-perfused Sprague-Dawley rat hearts were randomly assigned to one of four groups [control group (CON group, n = 12), IPC group (n = 12), IPO group (n = 12) and RIPC group (n = 12)]. Cardiac function was digitalized and analyzed. The expression of miRNA-1 and miRNA-21 was detected by real-time reverse transcription polymerase chain reaction. The expression of HSP70, programmed cell death protein 4 (PDCD4), B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax) was detected by Western blot. Cardiac infarct size and myocardial apoptosis were determined using the 2,3,5-triphenyltetrazolium chloride assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, respectively. Results: The results revealed that miRNA-1 (233 ± 45%) and miRNA-21 (356 ± 33%) expression was up-regulated in the IPC group, but the expression of miRNA-1 was down-regulated in the RIPC (61 ± 16%) group and IPO group (61 ± 13%). The expression of PDCD4 [IPC (74 ± 11%), RIPC (81 ± 16%), IPO (83 ± 12%)], HSP70 [IPC (74 ± 5%), RIPC (81 ± 6%), IPO (67 ± 11%)] and Bax [IPC (27 ± 6%), RIPC (21 ± 3%), IPO (27 ± 4%)] was down-regulated in the conditioning groups compared with the CON group [PDCD4 (130 ± 11%), HSP70 (121 ± 11%) and Bax (63 ± 8%)]. In the conditioning hearts, infarct size [IPC (31.7 ± 4.1%), RIPC (29.6 ± 6.19%) and IPO (32.8 ± 4.71%)] and myocardial apoptosis [IPC (15.2 ± 4.21%), RIPC (17.2 ± 1.92%) and IPO (15.6 ± 4.04%)] were significantly decreased compared with the CON group (infarct size: 51.77 ± 4.3%, myocardial apoptosis: 32.8 ± 3.96%). Conclusion: We concluded that miRNA-1 and miRNA-21 expression differed in IPC, RIPC and IPO groups, and their target proteins were not inversely correlated with the miRNAs in all the conditioning groups, which revealed that the miRNAs were regulated but complicated by the different conditioning protocols.

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.

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

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

Differential consequences of protein kinase C activation during early and late hepatic ischemic preconditioning

Activation of protein kinase C (PKC) has been implicated in the protection of ischemic preconditioning (IPC), but the exact role of PKC in early and late hepatic IPC is still unclear. The present study was conducted in order to investigate the differential role of PKC during early and late hepatic IPC. Rats were subjected to 90 min of partial hepatic ischemia followed by 3 (early IPC) and 24 h (late IPC) of reperfusion. IPC was induced by 10 min of ischemia following 10 min of reperfusion prior to sustained ischemia, and chelerythrine, a PKC inhibitor, was injected 10 min before IPC (5 mg/kg, i.v.). Chelerythrine abrogated the protection of early IPC, as indicated by increased serum aminotransferase activities and decreased hepatic glutathione content. While the IPC-treated group showed a few apoptotic cell deaths during both phases, chelerythrine attenuated these changes only at late IPC and limited IPC-induced inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) overexpression. Membrane translocation of PKC-δ and -ε during IPC was blocked by chelerythrine. Our results suggest that PKC might play a differential role in early and late IPC; activation of PKC-δ and -ε prevents necrosis in early IPC through preservation of redox state and prevents apoptosis in late IPC with iNOS and HO-1 induction. Therefore, PKC represents a promising target for hepatocyte tolerance to ischemic injury, and understanding the differential role of PKC in early and late IPC is important for clinical application of IPC.

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.

Characterization of a critical role for CFTR chloride channels in cardioprotection against ischemia/reperfusion injury

To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury. CFTR knockout (CFTR(-/-)) mice and age- and gender-matched wild-type (CFTR(+/+)) and heterozygous (CFTR(+/-)) mice were used. In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined. In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR(+/+)) (from 40.4% ± 5.3% to 10.4% ± 2.0%, n=8, P<0.001) and heterozygous (CFTR(+/-)) littermates (from 39.4% ± 2.4% to 15.4% ± 5.1%, n=6, P<0.001) but failed to protect CFTR knockout (CFTR(-/-)) mice from I/R induced myocardial infarction (46.9% ± 6.2% vs 55.5% ± 7.8%, n=6, P>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTR gene abolished the protective effects of IPC against I/R-induced apoptosis. These results provide compelling evidence for a critical role for CFTR Cl(-) channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.

Identification of a NF-κB cardioprotective gene program: NF-κB regulation of Hsp70.1 contributes to cardioprotection after permanent coronary occlusion

The transcription factor Nuclear Factor Kappa B (NF-κB) has been shown to be cardioprotective after permanent coronary occlusion (PO) and late ischemic preconditioning (IPC), and yet it is cell injurious after ischemia/reperfusion (I/R) in the heart. There is limited information regarding NF-κB-dependent cardioprotection, and the NF-κB-dependent genes that contribute to the cardioprotection after PO are completely unknown. The objective of the study was to identify NF-κB-dependent genes that contribute to cardioprotection after PO. Microarray analysis was used to delineate genes that potentially contribute to the NF-κB-dependent cardioprotection by determining the overlap between the set of PO regulated genes and genes regulated by NF-κB, using mice with genetic abrogation of NF-κB activation in the heart. This analysis identified 16 genes as candidates for NF-κB-dependent effects after PO. This set of genes overlaps with, but is significantly different from the set of genes we previously identified as regulated by NF-κB after IPC. The genes encoding heat shock protein 70.3 ( hspa1a) and heat shock protein 70.1 ( hspa1b) were the most significantly regulated genes after PO and were up-regulated by NF-κB. Results using knockout mice show that Hsp70.1 contributes to NF-κB-dependent cardioprotection after PO and likely underlies, at least in part, the NF-κΒ-dependent cardioprotective effect. Our previous results show that Hsp70.1 is injurious after I/R injury. This demonstrates that, like NF-κB itself, Hsp70.1 has antithetical effects on myocardial survival and suggests that this may underlie the similar antithetical effects of NF-κB after different ischemic stimuli. The significance of the research is that understanding the gene network regulated by NF-κB after ischemic insult may lead to identification of therapeutic targets more appropriate for clinical development.

NF-κB driven cardioprotective gene programs; Hsp70.3 and cardioprotection after late ischemic preconditioning

It has been shown that the transcription factor NF-κB is necessary for late phase cardioprotection after ischemic preconditioning (IPC) in the heart, and yet is injurious after ischemia/reperfusion (I/R). However the downstream gene expression programs that underlie the contribution of NF-κB to cardioprotection after late IPC are incompletely understood. The objective of this study was to delineate the specific genes that are regulated by NF-κB immediately after a late IPC stimulus and validate the methodology for the identification of NF-κB-dependent genes that contribute to cardioprotection. A directed microarray analysis identified 238 genes as up or downregulated in an NF-κB-dependent manner 3.5 h after late IPC. Among these are several genes previously implicated in late IPC. Gene ontological analysis showed that the most significant group of NF-κB-dependent genes are heat shock response genes, including the genes encoding Hsp70.1 and Hsp70.3. Though an Hsp70.1/70.3 double knockout failed to exhibit cardioprotection, late IPC was intact in the Hsp70.1 single knockout. After I/R, the Hsp70.1/70.3 double knockout and the Hsp70.1 single knockout had significantly increased and reduced infarct size, respectively. These results delineate the immediate NF-κB-dependent transcriptome after late IPC. One of the major categories of NF-κB-dependent genes induced by late IPC is the heat shock response. The results of infarct studies confirm that Hsp70.3 is protective after IPC. However, though Hsp70.1 and Hsp70.3 are coordinately regulated, their functions are opposing after I/R injury.