Ischemic Preconditioning Stimulus Research Articles

Ergogenic effect of ischemic preconditioning is not directly conferred to isolated skeletal muscle via blood.

Ischemic preconditioning (IPC) in humans has been demonstrated to confer ergogenic benefit to aerobic exercise performance, with an improvement in the response rate when the IPC stimulus is combined with concurrent exercise. Despite potential performance improvements, the nature of the neuronal and humoral mechanisms of conferral and their respective contributions to ergogenic benefit remain unclear. We sought to examine the effects of the humoral component of ischemic preconditioning on skeletal muscle tissue using preconditioned human serum and isolated mouse soleus. Isolated mouse soleus was electrically stimulated to contract while in human serum preconditioned with either traditional (IPC) or augmented (AUG) ischemic preconditioning compared to control (CON) and exercise (ERG) preconditioning. Force frequency (FF) curves, twitch responses, and a fatigue-recovery protocol were performed on muscles before and after the addition of serum. After preconditioning, human participants performed a 4km cycling time trial in order to identify responders and non-responders to IPC. No differences in indices of contractile function, fatiguability, nor recovery were observed between conditions in mouse soleus muscles. Further, no human participants improved performance in a 4-km cycling time trial in response to traditional nor augmented ischemic preconditioning compared to control or exercise conditions (CON 407.7 ± 41.1s, IPC 411.6 ± 41.9s, ERG 408.8 ± 41.4s, AUG 414.1 ± 41.9s). Our findings do not support the conferral of ergogenic benefit via a humoral component of IPC at the intracellular level. Ischemic preconditioning may not manifest prominently at submaximal exercise intensities, and augmented ischemic preconditioning may have a hormetic relationship with performance improvements.

Plasma exosomes generated by ischaemic preconditioning are cardioprotective in a rat heart failure model

BackgroundExosomes released into the plasma after brief cardiac ischaemia mediate subsequent cardioprotection. Whether donor exosomes can provide cardioprotection to recipients with chronic heart failure, which confers the highest perioperative risk, is unknown. We examined whether ischaemic preconditioning (IPC)-induced plasma exosomes exerted cardioprotection after their transfer from normal donors to post-infarcted failing hearts. MethodsPlasma exosomes were obtained from adult rats after IPC or sham. An exosome inhibitor GW4869 was administrated before IPC in an in vivo model of ischaemia/reperfusion (I/R) injury in normal rats. The IPC exosomes or control exosomes from normal donor rats were perfused to the normal or post-infarcted failing rat hearts before ischaemia in Langendorff perfusion experiments. Infarct size, cardiac enzymes, cardiac function, and pro-survival kinases were quantified. ResultsThe IPC stimulus increased the release of exosomes, whereas GW4869 inhibited the rise of plasma exosomes. Pre-treatment with GW4869 reversed IPC-mediated cardioprotection against in vivo I/R injury. In the Langendorff perfusion experiments, IPC exosomes from normal donor rats reduced mean infarct size from 41.05 (1.87)% to 31.43 (1.81)% and decreased lactate dehydrogenase activity in the post-infarcted failing rat hearts. IPC exosomes but not control exosomes activated pro-survival kinases in the heart tissues. ConclusionsIschaemic preconditioning-induced exosomes from normal rats can restore cardioprotection in heart failure after myocardial infarction, which is associated with activation of pro-survival protein kinases. These results suggest a potential perioperative therapeutic role for ischaemic preconditioning-induced exosomes.

Impact of hyperglycemia on myocardial ischemia\u2013reperfusion susceptibility and ischemic preconditioning in hearts from rats with type 2 diabetes

BackgroundThe mechanisms underlying increased mortality in patients with diabetes and admission hyperglycemia after an acute coronary syndrome may involve reduced capacity for cardioprotection. We investigated the impact of hyperglycemia on exogenously activated cardioprotection by ischemic preconditioning (IPC) in hearts from rats with type 2 diabetes mellitus (T2DM) that were endogenously cardioprotected by an inherent mechanism, and the involvement of myocardial glucose uptake (MGU) and myocardial O-linked β-N-acetylglucosamine (O-GlcNAc).Methods and resultsIn isolated, perfused rat hearts subjected to ischemia–reperfusion, infarct size (IS) was overall larger during hyper- ([Glucose] = 22 mmol/L]) than normoglycemia ([Glucose] = 11 mmol/L]) (p < 0.001). IS was smaller in 12-week old Zucker diabetic fatty rats with recent onset T2DM (fa/fa) than in rats without T2DM (fa/+) (n = 8 in each group) both during hyperglycemia (p < 0.05) and normoglycemia (p < 0.05). IPC (2 × 5 min cycles) reduced IS during normo- (p < 0.01 for both groups) but not during hyperglycemia independently of the presence of T2DM. During hyperglycemia, an intensified IPC stimulus (4 × 5 min cycles) reduced IS only in hearts from animals with T2DM (p < 0.05). IPC increased MGU and O-GlcNAc levels during reperfusion in animals with and without T2DM at normoglycemia (MGU: p < 0.05, O-GlcNAc: p < 0.01 for both groups) but not during hyperglycemia. Intensified IPC at hyperglycemia increased MGU (p < 0.05) and O-GlcNAc levels (p < 0.05) only in hearts from animals with T2DM.ConclusionWhile the effect of IPC is reduced during hyperglycemia in rats without T2DM, endogenous cardioprotection in animals with T2DM is not influenced by hyperglycemia and the capacity for exogenous cardioprotection by IPC is preserved. MGU and O-GlcNAc levels are increased by exogenously induced cardioprotection by IPC but not by endogenous cardioprotection in animals with T2DM reflecting different underlying mechanisms by exogenous and endogenous cardioprotection.

Enhanced Metabolic Stress Augments Ischemic Preconditioning for Exercise Performance.

Purpose: To identify the combined effect of increasing tissue level oxygen consumption and metabolite accumulation on the ergogenic efficacy of ischemic preconditioning (IPC) during both maximal aerobic and maximal anaerobic exercise.Methods: Twelve healthy males (22 ± 2 years, 179 ± 2 cm, 80 ± 10 kg, 48 ± 4 ml.kg−1.min−1) underwent four experimental conditions: (i) no IPC control, (ii) traditional IPC, (iii) IPC with EMS, and (iv) IPC with treadmill walking. IPC involved bilateral leg occlusion at 220 mmHg for 5 min, repeated three times, separated by 5 min of reperfusion. Within 10 min following the IPC procedures, a 30 s Wingate test and subsequent (after 25 min rest) incremental maximal aerobic test were performed on a cycle ergometer.Results: There was no statistical difference in anaerobic peak power between the no IPC control (1211 ± 290 W), traditional IPC (1209 ± 300 W), IPC + EMS (1206 ± 311 W), and IPC + Walk (1220 ± 288 W; P = 0.7); nor did VO2max change between no IPC control (48 ± 2 ml.kg−1.min−1), traditional IPC (48 ± 6 ml.kg−1.min−1), IPC + EMS (49 ± 4 ml.kg−1.min−1) and IPC + Walk (48 ± 6 ml.kg−1.min−1; P = 0.3). However, the maximal watts during the VO2max increased when IPC was combined with both EMS (304 ± 38 W) and walking (308 ± 40 W) compared to traditional IPC (296 ± 39 W) and no IPC control (293 ± 48 W; P = 0.02).Conclusion: This study shows that in a group of participants for whom a traditional IPC stimulus was not effective, the magnification of the IPC stress through muscle contractions while under occlusion led to a subsequent exercise performance response. These findings support that amplification of the ischemic preconditioning stimulus augments the effect for exercise capacity.

Influence of diabetes mellitus duration on the efficacy of ischemic preconditioning in a Zucker diabetic fatty rat model.

Augmented mortality and morbidity following an acute myocardial infarction in patients with diabetes mellitus Type 2 (T2DM) may be caused by increased sensitivity to ischemia reperfusion (IR) injury or altered activation of endogenous cardioprotective pathways modified by T2DM per se or ischemic preconditioning (IPC). We aimed to investigate, whether the duration of T2DM influences sensitivity against IR injury and the efficacy of IPC, and how myocardial glucose oxidation rate was involved. Male Zucker diabetic fatty rats (homozygote (fa/fa)) at ages 6-(prediabetic), 12- (onset diabetes) and 24-weeks of age (late diabetes) and their age-matched non-diabetic controls (heterozygote (fa/+) were subjected to IR injury in the Langendorff model and randomised to IPC stimulus or control. T2DM rats were endogenously protected at onset of diabetes, as infarct size was lower in 12-weeks T2DM animals than in 6- (35±2% vs 53±4%; P = 0.006) and 24-weeks animals (35±2% vs 72±4%; P<0.0001). IPC reduced infarct size in all groups irrespective of the presence of T2DM and its duration (32±3%; 20±2%; 36±4% respectively; (ANOVA P<0.0001). Compared to prediabetic rats, myocardial glucose oxidation rates were reduced during stabilisation and early reperfusion at onset of T2DM, but these animals retained the ability to increase oxidation rate in late reperfusion. Late diabetic rats had low glucose oxidation rates throughout stabilisation and reperfusion. Despite inherent differences in sensitivity to IR injury, the cardioprotective effect of IPC was preserved in our animal model of pre-, early and late stage T2DM and associated with adaptations to myocardial glucose oxidation capacity.

Ischemic preconditioning enhances critical power during a 3 minute all-out cycling test

ABSTRACTThis study tested the hypothesis that ischemic preconditioning (IPC) would increase critical power (CP) during a 3 minute all-out cycling test. Twelve males completed two 3 minute all-out cycling tests, in a crossover design, separated by 7 days. These tests were preceded by IPC (4 x 5 minute intervals at 220 mmHg bilateral leg occlusion) or SHAM treatment (4 x 5 minute intervals at 20 mmHg bilateral leg occlusion). CP was calculated as the mean power output during the final 30 s of the 3 minute test with W′ taken as the total work done above CP. Muscle oxygenation was measured throughout the exercise period. There was a 15.3 ± 0.3% decrease in muscle oxygenation (TSI; [Tissue saturation index]) during the IPC stimulus, relative to SHAM. CP was significantly increased (241 ± 65 W vs. 234 ± 67 W), whereas W′ (18.4 ± 3.8 vs 17.9 ± 3.7 kJ) and total work done (TWD) were not different (61.1 ± 12.7 vs 60.8 ± 12.7 kJ), between the IPC and SHAM trials. IPC enhanced CP during a 3 minute all-out cycling test without impacting W′ or TWD. The improved CP after IPC might contribute towards the effect of IPC on endurance performance.

Is There an Optimal Ischemic-Preconditioning Dose to Improve Cycling Performance?

Ischemic preconditioning (IPC) may enhance endurance performance. No previous study has directly compared distinct IPC protocols for optimal benefit. To determine whether a specific IPC protocol (ie, number of cycles, amount of muscle tissue, and local vs remote occlusion) elicits greater performance outcomes. Twelve cyclists performed 5 different IPC protocols 30min before a blinded 375-kJ cycling time trial (TT) in a laboratory. Responses to traditional IPC (4 × 5-min legs) were compared with those to 8 × 5-min legs and sham (dose cycles), 4 × 5-min unilateral legs (dose tissue), and 4 × 5-min arms (remote). Rating of perceived exertion and blood lactate were recorded at each 25% TT completion. Power (W), heart rate (beats/min), and oxygen uptake ([Formula: see text]) (mL · kg-1 · min-1) were measured continuously throughout TTs. Magnitude-based-inference statistics were employed to compare variable differences to the minimal practically important difference. Traditional IPC was associated with a 17-s (0, 34) faster TT time than sham. Applying more dose cycles (8 × 5min) had no impact on performance. Traditional IPC was associated with likely trivial higher blood lactate and possibly beneficial lower [Formula: see text] responses vs sham. Unilateral IPC was associated with 18-s (-11, 48) slower performance than bilateral (dose tissue). TT times after remote and local IPC were not different (0 [-16, 16] s). The traditional 4 × 5-min (local or remote) IPC stimulus resulted in the fastest TT time compared with sham; there was no benefit of applying a greater number of cycles or employing unilateral IPC.

Ischemic preconditioning, retinal neuroprotection and histone deacetylase activities

Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 min of acute ischemia, and retinal neuroprotection induced by 5 min of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.

Extracellular Adenosine Formation by Ecto-5'-Nucleotidase (CD73) Is No Essential Trigger for Early Phase Ischemic Preconditioning.

BackgroundAdenosine is a powerful trigger for ischemic preconditioning (IPC). Myocardial ischemia induces intracellular and extracellular ATP degradation to adenosine, which then activates adenosine receptors and elicits cardioprotection. Conventionally extracellular adenosine formation by ecto-5’-nucleotidase (CD73) during ischemia was thought to be negligible compared to the massive intracellular production, but controversial reports in the past demand further evaluation. In this study we evaluated the relevance of ecto-5’-nucleotidase (CD73) for infarct size reduction by ischemic preconditioning in in vitro and in vivo mouse models of myocardial infarction, comparing CD73-/- and wild type (WT) mice.Methods and Results3x5 minutes of IPC induced equal cardioprotection in isolated saline perfused hearts of wild type (WT) and CD73-/- mice, reducing control infarct sizes after 20 minutes of ischemia and 90 minutes of reperfusion from 46 ± 6.3% (WT) and 56.1 ± 7.6% (CD73-/-) to 26.8 ± 4.7% (WT) and 25.6 ± 4.7% (CD73-/-). Coronary venous adenosine levels measured after IPC stimuli by high-pressure liquid chromatography showed no differences between WT and CD73-/- hearts. Pharmacological preconditioning of WT hearts with adenosine, given at the measured venous concentration, was evenly cardioprotective as conventional IPC. In vivo, 4x5 minutes of IPC reduced control infarct sizes of 45.3 ± 8.9% (WT) and 40.5 ± 8% (CD73-/-) to 26.3 ± 8% (WT) and 22.6 ± 6.6% (CD73-/-) respectively, eliciting again equal cardioprotection. The extent of IPC-induced cardioprotection in male and female mice was identical.ConclusionThe infarct size limiting effects of IPC in the mouse heart in vitro and in vivo are not significantly affected by genetic inactivation of CD73. The ecto-5’-nucleotidase derived extracellular formation of adenosine does not contribute substantially to adenosine’s well known cardioprotective effect in early phase ischemic preconditioning.

229 THE IMPAIRMENT OF INNATE CARDIOPROTECTIVE SIGNALING IN THE AGED, DIABETIC RAT HEART

<h3>Introduction</h3> Aging and type 2 diabetes represent two interacting risk factors for ischaemic heart disease (IHD). Some contradictory clinical data suggest the diabetic heart to be less vulnerable to infarction; however patients still experienced worse clinical outcomes. This led us to hypothesize that the diabetic heart, may develop smaller infarcts but could be more susceptible to ischaemic damage i.e. the threshold at which infarct size translates into worse clinical outcomes is lower for diabetic patients. To verify this, we employed a type 2 diabetic rat model at different ages and investigated the susceptibility of the heart to varied durations of ischaemia- reperfusion injury(IRI) and the ability to condition against this lethal event using ischaemic preconditioning (IPC). Impaired phosphorylation of pro-survival Akt, a known mediator in the cardioprotective effect of preconditioning, has been suggested to be involved in insulin resistance. Therefore, we assessed whether Akt activation was impaired in these aging diabetic animals and if the changes we found had any detrimental effects on some downstream cardioprotective targets. <h3>Methods</h3> Hearts from diabetic Goto-Kakizaki (GK) rats, aged 3–18 months were assigned to one of the following experimental groups: a) Langendorff isolated heart perfusions, subjected to 20min (sublethal ischaemia) or 35 min (lethal ischaemia) followed by 60 min reperfusion; some hearts were pretreated prior to lethal ischemia with 3 cycles of 5 min ischaemia, (ischaemic preconditioning, IPC, a well-known cardioprotective maneuver); the end point was to compare the size of the infarct amongst groups; b) Western blot analysis (for prosurvival Akt, PGC-1α, a regulator of mitochondrial biogenesis, and catalase, an endogenous antioxidant); and c) measurements of the intracellular ROS/H₂O₂ content, at baseline. <h3>Results</h3> The aged GK heart demonstrated an increased susceptibility to sub-lethal ischaemia with infarct sizes of 38.6%±3.1 (3 months) and 55.5%±3.3 (18 months) and to lethal ischaemia with infarct sizes of 45.5%±3.1 (3 months) and 62.1%±6.0 (18 months). With IPC there was a 63% decrease in infarct size at 3months and no protection seen at 18 months. The 18 month GK heart also demonstrated a decreased rate pressure product output compared to the 3month group (34623±4312.4 <i>vs.</i> 54437.4±6937.4, mmHg/sec). Surprisingly, an age-related <b>increase</b> in phosphorylated Akt was seen, with no subsequent increase in activation following an IPC stimulus. Furthermore, a decrease in basal PGC-1α and catalase expression and increased H₂O₂ and ROS levels were also observed in the 18 month GK heart. <h3>Conclusions</h3> Overall our data seem to indicate that chronic activation of Akt occurs in aging diabetic hearts which could lead to alterations in downstream signaling, rendering the heart more susceptible to IRI and less amenable to IPC. This may occur via a down regulation of factors essential for maintaining mitochondrial function and antioxidant defense.

Endothelial nitric oxide synthase S‐glutathionylation and Ser1177 phosphorylation modulate myocardial protection

Endothelial nitric oxide synthase (eNOS) plays an important role in cardiovascular function, myocardial injury and protection. The role of eNOS is well recognized in ischemic preconditioning (IPC); however, questions remain regarding how eNOS is regulated during ischemia‐reperfusion (I/R) and IPC. eNOS phosphorylation at Ser1177 enhances enzyme activation and nitric oxide (NO) production; however, S‐glutathionylation of eNOS uncouples the enzyme leading to loss of NO production and vascular dysfunction. We measure myocardial eNOS S‐glutathionylation and phosphorylation in the process of I/R injury and investigate the role of these modifications in cardioprotection. Isolated rat hearts were subjected to 30‐min of global ischemia and/or reperfusion with or without IPC stimuli. Ischemia induced a 4–5 fold increase in eNOS S‐glutathionylation that was completely reversed by â‐mercaptoethanol. Importantly, IPC reduced I/R‐induced eNOS S‐glutathionylation by 50%, decreased myocardial superoxide generation by &gt;;80% and attenuated myocardial infarction by 75%. Levels of eNOS phosphorylation at Ser1177 decreased with I/R but were increased by IPC. Thus, IPC stimuli inhibit I/R‐induced eNOS S‐glutathionylation but increase Ser1177 phosphorylation to maintain eNOS coupling, activation and NO production that protect myocardium against I/R injury. (Support: NIH grants HL63744, HL65608 and HL38324).

Reciprocal Endothelial NO Synthase (eNOS) Ser1177 Phosphorylation and Thr495 Dephosphorylation is Key for Robust in vivo Cardioprotection: Therapeutic Implication of a Novel Ischemic Preconditioning Stimuli

Ischemic preconditioning (IPC) induces powerful cardioprotection, but its clinical application has been limited. The cardioprotective effect of IPC may be limited by the design of the IPC protocol and downstream mechanisms. While IPC‐induced eNOS phosphorylation is reported for in vitro cardioprotection; the importance of eNOS phosphorylation at Ser1177 (stimulation) and Thr495 (inhibition) is unknown for in vivo cardioprotection. Therefore, we investigate whether a novel rapid cycles IPC (RIPC) protocol induces in vivo cardioprotection; and if so, what is its cardioprotective efficacy and mechanism(s) relative to classical IPC (C‐IPC). Rats were subjected to in vivo ischemia/reperfusion (I/R) preceded by either C‐IPC (3 × 5‐min I &amp; 5‐min R) or R‐IPC (6 × 1‐min I &amp; 1‐min R). After 24‐hrs R, cardiac function, infarction, reactive oxygen species (ROS) generation, prosurvival kinase and eNOS proteins were characterized. R‐IPC conferred robust cardioprotection and that was superior to C‐IPC. The R‐IPC effect was manifested by reduced MI, improved function, reduced ROS, activation of Akt and ERK1/2, and reciprocal phosphorylation of eNOS at Ser1177 and dephosphorylation at Thr495. Thus, reciprocal eNOS phosphorylation plays an important role in acute cardioprotection, and the novel R‐IPC is highly effective in activating cardioprotective signaling pathways. (NIH grants HL63744, HL65608 and HL38324).

Proteomic analysis of mouse brain cortex identifies metabolic down‐regulation as a general feature of ischemic pre‐conditioning

The molecular mechanisms that lead to ischemic pre-conditioning are not completely understood, and proteins are important players. We compared the mouse brain cortex proteome from different ischemia sets: transient (7 min) middle cerebral artery occlusion (7'MCAo, pre-conditioning stimulus), permanent MCAo (pMCAo, severe ischemia), and pMCAo 4 days after 7'MCAo (7'MCAo/pMCAo, pre-conditioned model). Proteins were analyzed by two-dimensional electrophoresis coupled to liquid chromatography-tandem mass spectrometry. Overall, 28 proteins were expressed differentially from sham controls, and identified. The ischemic pre-conditioning stimulus alone up-regulated the stress protein heat-shock protein 70 (HSP70), possibly activated by the androgen receptor. Western blotting confirmed the increased expression of HSP70 and showed that androgen receptor expression paralleled that of HSP70. In the ischemic-tolerant group (7'MCAo/pMCAo), a number of proteins over-expressed after pMCAo returned to sham levels, seven proteins remained up-regulated as in pMCAo, and five proteins mainly involved in energy metabolism and mitochondrial electron transport and unchanged in pMCAo were down-regulated only in ischemic tolerance, suggesting a role in brain pre-conditioning. Astrocytes participated in ischemic-tolerance induction, as shown by the down-regulation of glutamine synthetase in the 7'MCAo/pMCAo group. The results suggest that metabolic down-regulation was a general feature of ischemic pre-conditioning, playing a pivotal role in neuroprotection.

The Akt1 isoform is an essential mediator of ischaemic preconditioning

Phosphatidyl-inositol-3-kinase (PI3K)-Akt pathway is essential for conferring cardioprotection in response to ischaemic preconditioning (IPC) stimulus. However, the role of the individual Akt isoforms expressed in the heart in mediating the protective response to IPC is unknown. In this study, we investigated the specific contribution of Akt1 and Akt2 in cardioprotection against ischaemia-reperfusion (I-R) injury. Mice deficient in Akt1 or Akt2 were subjected to in vivo regional myocardial ischaemia for 30 min. followed by reperfusion for 2 hrs with or without a prior IPC stimulus. Our results show that mice deficient in Akt1 were resistant to protection with either one or three cycles of IPC stimulus (42.7 ± 6.5% control versus 38.5 ± 1.9% 1 χ IPC, N = 6, NS; 41.4 ± 6.3% control versus 32.4 ± 3.2% 3 χ IPC, N = 10, NS). Western blot analysis, performed on heart samples taken from Akt1−/− mice subjected to IPC, revealed an impaired phosphorylation of GSK-3β, a downstream effector of Akt, as well as Erk1/2, the parallel component of the reperfusion injury salvage kinase pathway. Akt2−/− mice, which exhibit a diabetic phenotype, however, were amenable to protection with three but not one cycle of IPC (46.4 ± 5.6% control versus 35.9 ± 5.0% in 1 χ IPC, N = 6, NS; 47.0 ± 6.0% control versus 30.8 ± 3.3% in 3 χ IPC, N = 6; *P = 0.039). Akt1 but not Akt2 is essential for mediating a protective response to an IPC stimulus. Impaired activation of GSK-3β and Erk1/2 might be responsible for the lack of protective response to IPC in Akt1−/− mice. The rise in threshold for protection in Akt2−/− mice might be due to their diabetic phenotype.

Hypoxia-inducible factor 1 transcriptional activity in endothelial cells is required for acute phase cardioprotection induced by ischemic preconditioning

Infarction occurs when myocardial perfusion is interrupted for prolonged periods of time. Short episodes of ischemia and reperfusion protect against tissue injury when the heart is subjected to a subsequent prolonged ischemic episode, a phenomenon known as ischemic preconditioning (IPC). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates adaptive responses to hypoxia/ischemia and is required for IPC. In this study, we performed a cellular and molecular characterization of the role of HIF-1 in IPC. We analyzed mice with knockout of HIF-1α or HIF-1β in Tie2(+) lineage cells, which include bone marrow (BM) and vascular endothelial cells, compared with control littermates. Hearts were subjected to 30 min of ischemia and 120 min of reperfusion, either as ex vivo Langendorff preparations or by in situ occlusion of the left anterior descending artery. The IPC stimulus consisted of two cycles of 5-min ischemia and 5-min reperfusion. Mice lacking HIF-1α or HIF-1β in Tie2(+) lineage cells showed complete absence of protection induced by IPC, whereas significant protection was induced by adenosine infusion. Treatment of mice with a HIF-1 inhibitor (digoxin or acriflavine) 4 h before Langendorff perfusion resulted in loss of IPC, as did administration of acriflavine directly into the perfusate immediately before IPC. We conclude that HIF-1 activity in endothelial cells is required for acute IPC. Expression and dimerization of the HIF-1α and HIF-1β subunits is required, suggesting that the heterodimer is functioning as a transcriptional activator, despite the acute nature of the response.

Ischemic Preconditioning Attenuates Brain Edema After Experimental Intracerebral Hemorrhage

Ischemic preconditioning (IPC) provides protection against subsequent severe ischemic injury. A recent study found that cerebral IPC prolongs bleeding time. In this study, we examined whether IPC protects against intra-cerebral hemorrhage (ICH)-induced brain edema formation and whether IPC affects blood coagulation. There were three sets of experiments in this study. In the first set, male Sprague-Dawley rats were preconditioned with either 15 min of left middle cerebral artery occlusion, an IPC stimulus, or a sham operation. Three days later, rats received an infusion of autologous whole blood in the ipsilateral or contralateral caudate. Rats were killed 24 h later for brain water content measurement. In the second set, rats underwent 15 min of IPC or a sham operation. Three days later, rats were used for bleeding and thrombin clotting time tests. In the third set, the levels of p44/42 mitogen-activated protein kinases (MAPKs), heme oxygenase-1 (HO-1), transferrin (Tf), and transferrin receptor (TfR) in the brain 24 or 72 h after IPC were examined. We found that IPC reduced ICH-induced brain edema when blood was injected into the ipsilateral caudate but it did not when blood was injected into the contralateral caudate. IPC resulted in prolongation of bleeding time and thrombin clotting time. IPC also induced the activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR levels in the ipsilateral caudate. These results suggest that IPC protects against ICH-induced brain edema formation and decreases blood coagulation. The protection of IPC against ICH is mainly due to local factors in the brain and may be related to activation of p44/42 MAPKs and upregulation of HO-1, Tf, and TfR.

Novel Rapid‐Multiple‐Short‐Cycle Preconditioning Stimuli Induces Robust Cardioprotective Signaling Mechanisms and Protects the Heart against in vivo Ischemia Reperfusion Injury: An Effective Approach with Clinical Justification

Despite the large body of basic research, the well‐proved phenomenon of ischemic preconditioning (IPC) still lacks its clinical benefits. In clinics, balloon inflation/deflation cycles during coronary interventions are restricted to &lt;1‐min in duration; and multiple balloon inflations/deflations are required for complex lesions. Recently, we have shown that rapid brief 60‐sec cycle of ischemia/reperfusion (I/R) induces small burst of reactive oxygen species (ROS) formation that could trigger IPC like protection. Therefore, to investigate whether a novel rapid‐multiple‐short‐cycle preconditioning (RPC) stimuli induces in vivo cardioprotection, rats were subjected to in vivo regional I/R (30‐min I and 24‐hr R) preceded by RPC (6 cycles of 60‐sec I and 60‐ sec R) or classical IPC (C‐IPC; 3 cycles of 5‐min I and 5‐min R). Cardiac function, infarction, ROS generation, survival kinase and nitric oxide synthase (NOS) pathways were characterized. RPC conferred robust cardioprotection compared to C‐IPC. This was manifested by reduced myocardial infarction, improved postischemic cardiac function, reduced ROS generation, effective activation of Akt and ERK1/2, as well as enhanced NOS expression and activation. Thus, this novel IPC stimuli RPC is highly effective in preserving myocardial viability and function in the setting of acute myocardial I/R. (Funding: NIH grants HL63744, HL65608, and HL38324 to JLZ)

Ischemic Conditioning Protects the Uremic Heart in a Rodent Model of Myocardial Infarction

Outcomes after acute myocardial infarction in patients with chronic kidney disease are extremely poor. Ischemic conditioning techniques are among the most powerful cytoprotective strategies discovered to date. However, experimental data suggest that comorbidity may attenuate the protective effects of ischemic conditioning. We conducted investigations into the effects of chronic uremia on myocardial infarct size and the protective effects of ischemic preconditioning (IPC), remote ischemic preconditioning, and ischemic postconditioning in 2 rodent models of chronic uremia. In addition, a limited investigation into the signaling mechanisms involved in cardioprotection after IPC was performed in both uremic and nonuremic animals. Myocardial infarct size was increased in uremic animals, but all 3 conditioning strategies (IPC, remote IPC, ischemic postconditioning) proved highly efficacious in reducing myocardial infarct size (relative reduction, 86%, 39%, and 65% [P<0.005, P<0.05, and P<0.05], respectively). Moreover, some protocols (IPC and ischemic postconditioning) appeared to be more effective in uremic than in sham (nonuremic) animals. Analysis of the signaling mechanisms revealed that components of both the reperfusion injury salvage kinase and survivor activating factor enhancement pathways were similarly upregulated in both uremic and nonuremic animals after an IPC stimulus. Conditioning strategies may present the best opportunity to improve outcomes for patients with chronic kidney disease after an acute coronary syndrome.

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.

Glial Cells Drive Preconditioning-Induced Blood-Brain Barrier Protection

The cerebrovascular contribution to ischemic preconditioning (IPC) has been scarcely explored. Using in vivo and in vitro approaches, we investigated the involvement of the blood-brain barrier and the role of its cellular components. Seven-minute occlusion of the right middle cerebral artery, used as in vivo IPC stimulus 4 days before permanent occlusion of the right middle cerebral artery, significantly reduced brain infarct size (8.45±0.7 versus 13.61±0.08 mm3 measured 7 days after injury) and preserved blood-brain barrier function (Evans blue leakage, 0.54±0.1 versus 0.89±0.1 ng/mg). Assessment of neuronal, endothelial, and glial gene expression revealed that IPC specifically increased glial fibrillary acidic protein mRNA, thus showing selective astrocyte activation in IPC-protected mice. The blood-brain barrier was modeled by coculturing murine primary brain microvessel endothelial and astroglial cells. One-hour oxygen-glucose deprivation (OGD), delivered 24 hours before a 5-hour OGD, acted as an IPC stimulus, significantly attenuating the reduction in transendothelial electric resistance (199.17±11.7 versus 97.72±3.4 Ωcm2) and the increase in permeability coefficients for sodium fluorescein (0.98±0.11×10(-3) versus 1.8±0.36×10(-3) cm/min) and albumin (0.12±0.01×10(-3) versus 0.29±0.07×10(-3) cm/min) induced by severe OGD. IPC also prevented the 5-hour OGD-induced disorganization of the tight junction proteins ZO-1 and claudin-5. IPC on glial (but not endothelial) cells alone preserved transendothelial electric resistance, permeability coefficients, and ZO-1 localization after 5 hours of OGD. Astrocyte metabolic inhibition by fluorocitrate abolished IPC protection, confirming the critical role of astrocytes. IPC significantly increased glial fibrillary acidic protein, interleukin-6, vascular endothelial growth factor-a, and ciliary neurotrophic factor gene expression after OGD in glial cells, indicating that multiple pathways mediate the glial contribution to IPC. Our data show that the blood-brain barrier can be directly preconditioned and that astrocytes are major mediators of IPC protection.