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Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.
Hibernating mammals, like the arctic ground squirrel (AGS), exhibit robust resistance to myocardial ischemia/reperfusion (IR) injury. Regulated preference for lipid over glucose to fuel metabolism may play an important role. We tested whether providing lipid in an emulsion protects hearts from summer-active AGS better than hearts from Brown Norway (BN) rats against normothermic IR injury. Langendorff-prepared AGS and BN rat hearts were perfused with Krebs solution containing 7.5 mM glucose with or without 1% Intralipid™. After stabilization and cardioplegia, hearts underwent 45-min global ischemia and 60-min reperfusion. Coronary flow, isovolumetric left ventricular pressure, and mitochondrial redox state were measured continuously; infarct size was measured at the end of the experiment. Glucose-only AGS hearts functioned significantly better on reperfusion than BN rat hearts. Intralipid™ administration resulted in additional functional improvement in AGS compared to glucose-only and BN rat hearts. Infarct size was not different among groups. Even under non-hibernating conditions, AGS hearts performed better after IR than the best-protected rat strain. This, however, appears to strongly depend on metabolic fuel: Intralipid™ led to a significant improvement in return of function in AGS, but not in BN rat hearts, suggesting that year-round endogenous mechanisms are involved in myocardial lipid utilization that contributes to improved cardiac performance, independent of the metabolic rate decrease during hibernation. Comparative lipid analysis revealed four candidates as possible cardioprotective lipid groups. The improved function in Intralipid™-perfused AGS hearts also challenges the current paradigm that increased glucose and decreased lipid metabolism are favorable during myocardial IR.
BACKGROUND - Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM.
METHODS AND RESULTS - RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in expression with ICM. encodes the Kir6.2 subunit of the cardioprotective K channel. Using wild-type mice and -deficient (-null) mice, we examined the effect of expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in -null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in -null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in -null mice. Despite impaired relaxation, -null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca, thereby increasing sarcoendoplasmic reticulum Ca-mediated calcium reuptake. However, -null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca-ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca function, thereby contributing to diastolic dysfunction.
CONCLUSIONS - expression is decreased in human ICM. Lack of expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca-ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.
© 2017 American Heart Association, Inc.
Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.
Copyright © 2016 Elsevier Inc. All rights reserved.
OBJECTIVE - β(2)-adrenoreceptor activation has been shown to protect cardiac myocytes from cell death. We hypothesized that acute β(2)-adrenoreceptor stimulation, using arformoterol (ARF), would attenuate myocardial ischemia/reperfusion (R) injury via NO synthase activation and cause a subsequent increase in NO bioavailability.
METHODS AND RESULTS - Male C57BL/6J and endothelial NO synthase (eNOS) knockout mice were subjected to 45 minutes of myocardial ischemia and 24 hours of R. ARF or vehicle was administered 5 minutes before R. Serum troponin-I was measured, and infarct size per area-at-risk was evaluated at 24 hours of R. Echocardiography was performed at baseline and 2 weeks after R. Myocardial cAMP, protein kinase A, eNOS/Akt phosphorylation status, and NO metabolite levels were assayed. ARF (1 µg/kg) reduced infarct size per area-at-risk by 53.1% (P<0.001 versus vehicle) and significantly reduced troponin-I levels (P<0.001 versus vehicle). Ejection fraction was significantly preserved in ARF-treated hearts compared with vehicle hearts at 2 weeks of R. Serum cAMP and nuclear protein kinase A C-α increased 5 and 15 minutes after ARF injection, respectively (P<0.01). ARF increased Akt phosphorylation at Thr(308) (P<0.001) and Ser(473) (P<0.01), and eNOS phosphorylation at Ser(1177) (P<0.01). ARF treatment increased heart nitrosothiol levels (P<0.001) at 15 min after injection. ARF failed to reduce infarct size in eNOS(-/-) mice.
CONCLUSIONS - Our results indicate that β(2)-adrenoreceptor stimulation activates cAMP, protein kinase A, Akt, and eNOS and augments NO bioavailability. Activation of this prosurvival signaling pathway attenuates myocardial cell death and preserves cardiac function after ischemia/reperfusion.
UNLABELLED - CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs.
CONTROL - 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Modulation of purinergic signaling is critical to myocardial homeostasis. Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD-1; CD39) which converts the proinflammatory molecules ATP or ADP to AMP is a key regulator of purinergic modulation. However, the salutary effects of transgenic over expression of ENTPD-1 on myocardial response to ischemic injury have not been tested to date. Therefore we hypothesized that ENTPD-1 over expression affords myocardial protection from ischemia-reperfusion injury via specific cell signaling pathways. ENTPD-1 transgenic mice, which over express human ENTPDase-1, and wild-type (WT) littermates were subjected to either ex vivo or in vivo ischemia-reperfusion injury. Infarct size, inflammatory cell infiltrate and intracellular signaling molecule activation were evaluated. Infarct size was significantly reduced in ENTPD-1 versus WT hearts in both ex vivo and in vivo studies. Following ischemia-reperfusion injury, ENTPD-1 cardiac tissues demonstrated an increase in the phosphorylation of the cellular signaling molecule extracellular signal-regulated kinases 1/2 (ERK 1/2) and glycogen synthase kinase-3β (GSK-3β). Resistance to myocardial injury was abrogated by treatment with a non-selective adenosine receptor antagonist, 8-SPT or the more selective A(2B) adenosine receptor antagonist, MRS 1754, but not the A(1) selective antagonists, DPCPX. Additionally, treatment with the ERK 1/2 inhibitor PD98059 or the mitochondrial permeability transition pore opener, atractyloside, abrogated the cardiac protection provided by ENTPDase-1 expression. These results suggest that transgenic ENTPDase-1 expression preferentially conveys myocardial protection from ischemic injury via adenosine A(2B) receptor engagement and associated phosphorylation of the cellular protective signaling molecules, Akt, ERK 1/2 and GSK-3β that prevents detrimental opening of the mitochondrial permeability transition pore.
2011 Elsevier Ltd. All rights reserved.
Antioxidant enzymatic pathways form a critical network that detoxifies ROS in response to myocardial stress or injury. Genetic alteration of the expression levels of individual enzymes has yielded mixed results with regard to attenuating in vivo myocardial ischemia-reperfusion injury, an extreme oxidative stress. We hypothesized that overexpression of an antioxidant network (AON) composed of SOD1, SOD3, and glutathione peroxidase (GSHPx)-1 would reduce myocardial ischemia-reperfusion injury by limiting ROS-mediated lipid peroxidation and oxidative posttranslational modification (OPTM) of proteins. Both ex vivo and in vivo myocardial ischemia models were used to evaluate the effect of AON expression. After ischemia-reperfusion injury, infarct size was significantly reduced both ex vivo and in vivo, ROS formation, measured by dihydroethidium staining, was markedly decreased, ROS-mediated lipid peroxidation, measured by malondialdehyde production, was significantly limited, and OPTM of total myocardial proteins, including fatty acid-binding protein and sarco(endo)plasmic reticulum Ca(²+)-ATPase (SERCA)2a, was markedly reduced in AON mice, which overexpress SOD1, SOD3, and GSHPx-1, compared with wild-type mice. These data demonstrate that concomitant SOD1, SOD3, and GSHPX-1 expression confers marked protection against myocardial ischemia-reperfusion injury, reducing ROS, ROS-mediated lipid peroxidation, and OPTM of critical cardiac proteins, including cardiac fatty acid-binding protein and SERCA2a.
Oxidative stress is increased in the myocardium following infarction and plays a significant role in death of cardiac myocytes, leading to cardiac dysfunction. Levels of the endogenous antioxidant Cu/Zn-superoxide dismutase (SOD1) decrease following myocardial infarction. While SOD1 gene therapy studies show promise, trials with SOD1 protein have had little success due to poor pharmacokinetics and thus new delivery vehicles are needed. In this work, polyketal particles, a recently developed delivery vehicle, were used to make SOD1-encapsulated-microparticles (PKSOD). Our studies with cultured macrophages demonstrated that PKSOD treatment scavenges both intracellular and extracellular superoxide, suggesting efficient delivery of SOD1 protein to the inside of cells. In a rat model of ischemia/reperfusion (IR) injury, injection of PKSOD, and not free SOD1 or empty particles was able to scavenge IR-induced excess superoxide 3 days following infarction. In addition, only PKSOD treatment significantly reduced myocyte apoptosis. Further, PKSOD treatment was able to improve cardiac function as measured by acute changes in fractional shortening from baseline echocardiography, suggesting that sustained delivery of SOD1 is critical during the early phase of cardiac repair. These data demonstrate that delivery of SOD1 with polyketals is superior to free SOD1 protein therapy and may have potential clinical implications.
(c) 2009 Elsevier Ltd. All rights reserved.
The female sex has been associated with increased resistance to acute myocardial ischemia-reperfusion (I/R) injury. While enhanced antioxidant capacity has been implicated in female cardioprotection, there is little evidence to support this assumption. Previous studies have shown an important role of cellular glutathione peroxidase (GPx1) in protection of the myocardium from I/R injury. Whether GPx1 is mechanistic in the protection of female myocardium, post-I/R, has not been examined. We utilized a murine model with homozygous deletion of GPx1 and examined its impact on postischemic myocardial recovery in male and female mice. Following I/R, male GPx1(-/-) hearts were more susceptible to contractile and diastolic dysfunction, and this was associated with increased protein carbonyls, a marker of oxidative stress. In contrast, GPx1 deficiency in female hearts did not exacerbate dysfunction or oxidative stress post-I/R. Both wild-type and GPx1(-/-) female hearts exhibited reduced creatine kinase leakage and a more favorable ascorbate redox status compared with males. Following I/R, female GPx1(-/-) hearts showed a comparable decrease in glutathione redox status as their male counterparts; however, they exhibited a greater decrease in nitrate-to-nitrite ratio, suggesting a higher consumption of nitrate in female GPx1(-/-) hearts. Our findings demonstrate that GPx1 is critical for cardioprotection during I/R in male, but not female, mice. The maintenance of cardioprotection in female mice lacking GPx1 post-I/R may be due to an improved ascorbate redox homeostasis and enhanced nitrate-to-nitrite conversion, which would predictably be accompanied by enhanced production of cardioprotective nitric oxide.