ag-490 and Myocardial-Infarction

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.

Topics: Anesthetics, Inhalation; Animals; bcl-Associated Death Protein; Blood Pressure; Desflurane; Heart Rate; Ischemic Preconditioning, Myocardial; Isoflurane; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion; Proto-Oncogene Proteins c-pim-1; STAT3 Transcription Factor; Tyrphostins

Interleukin-17A, a pro-inflammatory cytokine, has a direct proapoptotic effect on cardiomyocytes. However, the specific mechanism has not been clarified. In the present study, an in-vitro model of cardiomyocyte apoptosis induced by IL-17A stimulation was employed and the roles of iNOS and Stat3 involved were investigated. Our data showed that the neonatal mouse cardiomyocytes express IL-17 receptors: IL-17RA and IL-17RC, but did not express IL-17A. Exogenous IL-17A significantly induces iNOS expression and hence the cardiomyocyte apoptosis. Moreover, IL-17A-induced cardiomyocyte apoptosis can be achieved directly via iNOS activation. We further showed that exogenous IL-17A simultaneously triggers Stat3 activation, which in turn inhibits IL-17A-induced iNOS expression in cardiomyocytes. And both ChIP and dual-luciferase results confirmed that Stat3 directly inhibits transcriptional activities of iNOS via binding to its specific promoter region. Consistent with these data, silencing of Stat3 in fact can aggravate IL-17A-triggered cardiomyocyte apoptosis. Finally, using an in vivo myocardial ischemia/reperfusion injury model, we verified that Stat3 inhibition increased iNOS expression and exacerbated cardiomyocyte apoptosis. Thus, our data strongly support the notion that Stat3 plays a compensatory anti-apoptotic role in IL-17A/iNOS-mediated cardiomyocyte apoptosis via inhibiting iNOS transcription, providing a novel molecular mechanism of apoptosis regulation and complicated interactions between IL-17A/iNOS and IL-17A/Stat3 signalings.

Topics: Animals; Apoptosis; Binding Sites; Cells, Cultured; Disease Models, Animal; Interleukin-17; Male; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide Synthase Type II; Phosphorylation; Promoter Regions, Genetic; Receptors, Interleukin-17; Signal Transduction; STAT3 Transcription Factor; Time Factors; Transfection; Tyrphostins

We have recently discovered that melatonin, given acutely and directly to the isolated heart at the concentration found in wine, confers cardioprotection against ischemia-reperfusion (I/R). However, whether the presence of melatonin in wine contributes to the cardioprotective effect of chronic and moderate consumption of wine and its signalling mechanisms of protection are unknown. We therefore used both in vivo and in vitro models of I/R to investigate whether the presence of melatonin in red wine may contribute to the cardioprotective effect of chronic and moderate consumption of red wine. Wistar rats and C57black6 mice (WT) received drinking water supplemented daily with a moderate amount of red wine or melatonin given at the concentration found in the red wine. Rats were also pretreated with luzindole, a specific inhibitor of melatonin receptors 1 and 2 (2.3 mg/kg/day, intraperitoneally) or prazosin, a specific inhibitor of melatonin receptor type 3 (2.5 mg/kg/day, intraperitoneally). After 14 days, hearts were subjected to I/R in vivo or ex vivo. Red wine reduced the infarct size in both rats and WT mice (p < 0.001). Luzindole did not affect wine-induced cardioprotection, while prazosin reduced the infarct sparing effect of red wine (p < 0.05). Furthermore, red wine or melatonin failed to protect tumor necrosis factor alpha (TNF) receptor 2 knockout or cardiomyocyte specific signal transducer and activator of transcription 3 (STAT3) deficient mice (n.s. vs. control). Our novel findings suggest that the presence of melatonin in red wine contributes to the cardioprotective effect of chronic and moderate consumption of red wine against lethal I/R injuries. This effect is most likely mediated, at least in part, via melatonin receptor 3 and the activation of TNF and STAT3, both key players of the prosurvival and well described SAFE pathway.

Topics: Animals; Cardiotonic Agents; Male; Melatonin; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Prazosin; Rats; Rats, Wistar; Receptor, Melatonin, MT1; Receptor, Melatonin, MT2; Receptors, Melatonin; Receptors, Tumor Necrosis Factor, Type II; STAT3 Transcription Factor; Tryptamines; Tyrphostins; Wine

αMUPA transgenic mice spontaneously consume less food compared with their wild type (WT) ancestors due to endogenously increased levels of the satiety hormone leptin. αMUPA mice share many benefits with mice under caloric restriction (CR) including an extended life span. To understand mechanisms linked to cardiac aging, we explored the response of αMUPA hearts to ischemic conditions at the age of 6, 18, or 24 months. Mice were subjected to myocardial infarction (MI) in vivo and to ischemia/reperfusion ex vivo. Compared to WT mice, αMUPA showed functional and histological advantages under all experimental conditions. At 24 months, none of the WT mice survived the first ischemic day while αMUPA mice demonstrated 50% survival after 7 ischemic days. Leptin, an adipokine decreasing under CR, was consistently ~60% higher in αMUPA sera at baseline. Leptin levels gradually increased in both genotypes 24h post MI but were doubled in αMUPA. Pretreatment with leptin neutralizing antibodies or with inhibitors of leptin signaling (AG-490 and Wortmannin) abrogated the αMUPA benefits. The antibodies also reduced phosphorylation of the leptin signaling components STAT3 and AKT specifically in the αMUPA myocardium. αMUPA mice did not show elevation in adiponectin, an adipokine previously implicated in CR-induced cardioprotection. WT mice treated for short-term CR exhibited cardioprotection similar to that of αMUPA, however, along with increased adiponectin at baseline. Collectively, the results demonstrate a life-long increased ischemic tolerance in αMUPA mice, indicating the attenuation of cardiac aging. αMUPA cardioprotection is mediated through endogenous leptin, suggesting a protective pathway distinct from that elicited under CR.

Topics: Adipokines; Animals; Biomarkers; Cellular Senescence; Disease Models, Animal; Echocardiography; Heart Ventricles; Kaplan-Meier Estimate; Leptin; Mice; Mice, Transgenic; Myocardial Infarction; Myocardium; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Ventricular Function

Acute myocardial infarction is a leading cause of mortality and morbidity worldwide. Although essential for successful recovery, myocardium reperfusion is associated with reperfusion injury. Two major cell survival signaling cascades are known to be protective against ischemia-reperfusion (I/R) injury: the reperfusion injury salvage kinase, including Akt, extracellular signal-regulated kinase 1/2, and the downstream target GSK-3β, and the survivor activating factor enhancement, which involves STAT-3. Pharmacologic inhibition of factor Xa has been shown to attenuate I/R injury, but the cellular mechanism is poorly understood. Our aim was to determine the role of whole blood in fondaparinux (FDX)-induced cardioprotection and the involvement of reperfusion injury salvage kinase and survivor activating factor enhancement pathways. We investigated FDX ability to prevent in vivo I/R injury using a transient coronary ligation rat model and ex vivo using a model of crystalloid-perfused isolated rat heart. In both models, infarct size was assessed after 120 min of reperfusion. Myocardial tissues were collected after 15 and 30 min of reperfusion for Western blot analysis. In vivo, FDX decreased infarct size by 29% and induced significant STAT-3 and GSK-3β phosphorylation in comparison to controls. Adding AG490, an inhibitor of JAK/STAT pathway, before I/R, prevented STAT-3 phosphorylation and abolished FDX-induced cardioprotection. On the contrary, FDX did not have an effect on infarct size or hemodynamic parameters in the isolated-heart model. Fondaparinux decreased I/R injury in vivo, but not in a crystalloid-perfused isolated heart. Under our experimental conditions, FDX required whole blood to be protective, and this beneficial effect was mediated through STAT-3 phosphorylation.

Topics: Animals; Cardiotonic Agents; Fondaparinux; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; In Vitro Techniques; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Polysaccharides; Rats; STAT3 Transcription Factor; Tyrphostins

In this study, we evaluated the effect of curcumin (Cur) post-treatment on isolated perfused rat hearts that had been subjected to a protocol of ischemia and reperfusion injury. We also examined whether the Janus kinase 2 and signal transducer and activator 3 of transcription (JAK2/STAT3) signaling pathway plays a role in the cardioprotective effects of Cur post-treatment. Isolated perfused rat hearts were subjected to 60 min of ischemia, followed by 60 min of reperfusion. The hearts were exposed to 1-μM Cur during the first 10 min of reperfusion in the absence or presence of the JAK kinase-specific inhibitor AG490 (AG, 1 μM). The Cur treatment conferred a cardioprotective effect, and the treated hearts demonstrated an improved post-ischemic cardiac functional recovery, a decreased myocardial infarct size and decreased lactate dehydrogenase release in the coronary flow, a reduced number of apoptotic cardiomyocytes, up-regulation of the anti-apoptotic protein Bcl2 and down-regulation of the pro-apoptotic protein Caspase3. AG blocked the Cur-mediated cardioprotection by inhibiting the JAK2/STAT3 signaling pathway, as reflected by the abrogation of the Cur-induced up-regulation of Bcl2 and down-regulation of Caspase3. The results suggest that Cur post-treatment can attenuate IR injury through the activation of the JAK2/STAT3 signaling pathway, which transmits a survival signal to the myocardium.

Topics: Animals; Apoptosis; Cardiotonic Agents; Caspase 3; Curcumin; In Vitro Techniques; Janus Kinase 2; L-Lactate Dehydrogenase; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Perfusion; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; STAT3 Transcription Factor; Time Factors; Tyrphostins; Ventricular Function, Left; Ventricular Pressure

Widespread death of implanted cells hampers the development of stem cell therapy for acute myocardial infarction (AMI). Our previous studies indicated that statins can protect implanted mesenchymal stem cells (MSCs) against the post-infarct microenvironment, thus increasing the therapeutic effect. However, the underlying mechanisms are unclear. The JAK-STAT pathway participates in regulation of stress responses of the myocardium to various insults. This study aimed to detect whether rosuvastatin (ROSU) facilitates the survival, engraftment, and differentiation of allogeneic bone marrow-derived MSCs in the post-infarct heart via the JAK-STAT signaling pathway.. Female Sprague-Dawley rats were randomized into 5 groups: AMI (control), ROSU gavage (group R), MSCs transplantation (group M), MSCs and ROSU (group M+R), or MSCs, ROSU and a JAK2 inhibitor AG-490 (group M+R+AG). MSCs from male rats were injected into the myocardium 1 week after AMI. Cardiac function and histology, as well as the expression of Y-chromosomal genes and JAK-STAT signaling proteins, were examined at 4 weeks after transplantation. Better functional recovery, increased survival and differentiation of MSCs occurred in group M+R. Furthermore, phosphorylation of JAK2 and STAT3 was higher in group M+R. The effects of ROSU, as well as of activated JAK-STAT proteins, could be attenuated by AG-490.. ROSU treatment improves the efficacy of stem cell transplantation in infarcted hearts by activation of the JAK2-STAT3 signaling pathway.

Topics: Animals; Cell Differentiation; Cell Survival; Cell Tracking; Cells, Cultured; Disease Models, Animal; Enzyme Activation; Female; Fluorobenzenes; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Janus Kinase 2; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myocardial Contraction; Myocardial Infarction; Myocardium; Phosphorylation; Protein Kinase Inhibitors; Pyrimidines; Rats; Rats, Sprague-Dawley; Recovery of Function; Rosuvastatin Calcium; Signal Transduction; STAT3 Transcription Factor; Stroke Volume; Sulfonamides; Time Factors; Transplantation, Homologous; Tyrphostins; Ventricular Function, Left; Y Chromosome

Ethanolamine is a biogenic amine found naturally in the body as part of membrane lipids and as a metabolite of the cardioprotective substances, sphingosine-1-phosphate (S1P) and anandamide. In the brain, ethanolamine, formed from the breakdown of anandamide protects against ischaemic apoptosis. However, the effects of ethanolamine in the heart are unknown. Signal transducer and activator of transcription 3 (STAT-3) is a critical prosurvival factor in ischaemia/reperfusion (I/R) injury. Therefore, we investigated whether ethanolamine protects the heart via activation of STAT-3. Isolated hearts from wildtype or cardiomyocyte specific STAT-3 knockout (K/O) mice were pre-treated with ethanolamine (Etn) (0.3 mmol/L) before I/R insult. In vivo rat hearts were subjected to 30 min ischaemia/2 h reperfusion in the presence or absence of 5 mg/kg S1P and/or the FAAH inhibitor, URB597. Infarct size was measured at the end of each protocol by triphenyltetrazolium chloride staining. Pre-treatment with ethanolamine decreased infarct size in isolated mouse or rat hearts subjected to I/R but this infarct sparing effect was lost in cardiomyocyte specific STAT-3 deficient mice. Pre-treatment with ethanolamine increased nuclear phosphorylated STAT-3 [control 0.75 ± 0.08 vs. Etn 1.50 ± 0.09 arbitrary units; P < 0.05]. Our findings suggest a novel cardioprotective role for ethanolamine against I/R injury via activation of STAT-3.

Topics: Amidohydrolases; Animals; Benzamides; Carbamates; Cardiovascular Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethanolamine; Janus Kinases; Lysophospholipids; Male; Mice; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Rats; Rats, Wistar; Sphingosine; STAT3 Transcription Factor; Tyrphostins

Ischaemic postconditioning (IPostC) is a powerful protective phenomenon that activates prosurvival intrinsic signalling cascades to limit reperfusion injury. We propose that IPostC confers its infarct-sparing effect via activation of the newly described prosurvival Survivor Activating Factor Enhancement (SAFE) pathway, which involves the activation of the cytokine tumour necrosis factor alpha (TNFalpha) and signal transducer and activator of transcription-3 (STAT-3).. Isolated ischaemic/reperfused hearts from TNF knockout, TNF receptor-1 knockout, TNF receptor-2 knockout, cardiomyocyte-specific STAT-3-deficient mice or their respective wild-type, (TNF-WT) or (STAT-3-WT), were postconditioned by ischaemic episodes (IPostC) or with exogenous TNFalpha (0.5 microg/L) (TNF-PostC) at the onset of reperfusion. IPostC reduced infarct size (IS) in TNF-WT and TNFR1(-/-) hearts (by 33 and 27%, respectively, P < 0.05), whereas hearts from TNF(-/-) or TNFR2(-/-) failed to be postconditioned. TNF-PostC reduced IS by 37% (P < 0.05) in STAT-3-WT hearts but failed to protect cardiac-specific STAT-3(-/-) hearts. Administration of wortmannin, an inhibitor of PI-3 kinase/Akt, or PD98059, an inhibitor of extracellular regulated kinase 1/2 (Erk1/2), during the postconditioning stimulus did not abolish the infarct-sparing effect of TNF-PostC. AG490, an inhibitor of STAT-3, abrogated the protective effect of TNFalpha. Western blot analysis did not demonstrate the involvement of Akt or Erk1/2 in TNF-PostC, whereas STAT-3 phosphorylation was increased in both IPostC and TNF-PostC.. The protective effect of the SAFE pathway is shown in IPostC, with the activation of TNFalpha, its receptor type 2, and STAT-3. This signalling cascade is activated independently of the well-known Reperfusion Injury Salvage Kinases (RISK) pathway, which involves the kinases Akt and Erk1/2.

Topics: Androstadienes; Animals; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Tumor Necrosis Factor, Type I; Receptors, Tumor Necrosis Factor, Type II; Signal Transduction; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha; Tyrphostins; Wortmannin

Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Atorvastatin; Cyclooxygenase 2; Disease Models, Animal; Electrophoretic Mobility Shift Assay; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunoblotting; Janus Kinases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardium; NF-kappa B; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Peptides; Phosphorylation; Protein Kinase Inhibitors; Pyrroles; Time Factors; Tyrphostins; Up-Regulation

Activation of the heart renin-angiotensin system (RAS) under pathophysiological conditions has been correlated with the development of ischemic injury. The binding of angiotensin II to its receptors triggers induction of several, perhaps multifunctional, intracellular signaling pathways, notable among them the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. In this study, we investigated whether the JAK/STAT signaling is involved in the ischemia/reperfusion injury in adult rat myocardium.. We report here that 2 components of the JAK/STAT signaling pathway, namely STAT 5A and STAT 6, are selectively activated in the rat heart subjected to ischemia/reperfusion. The activated STATs bind to a conserved nucleotide sequence (St domain) in the promoter of the angiotensinogen (ANG) gene and consequently upregulate the level of ANG mRNA. Treatment of the hearts with losartan (4.5 micromol/L), an AT(1) blocker, or with tyrphostin AG490 (5 micromol/L), an inhibitor of JAK 2 phosphorylation, results in loss of the STAT/ANG promoter binding activity and an upregulated level of ANG mRNA. Hearts treated with the JAK 2 inhibitor tyrphostin AG490 showed a reduction in myocardial infarct size and in number of cardiomyocytes undergoing apoptosis. The treated hearts also showed a recovery in functional hemodynamics of the myocardium.. These findings suggest that activation of the JAK/STAT signaling pathway is a significant contributing factor to the pathogenesis of myocardial ischemia and that interference in activation of the pathway potentiates recovery in cardiac function.

Topics: Angiotensinogen; Animals; DNA-Binding Proteins; Enzyme Inhibitors; In Vitro Techniques; Janus Kinase 2; Male; Milk Proteins; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; RNA, Messenger; Signal Transduction; STAT5 Transcription Factor; STAT6 Transcription Factor; Trans-Activators; Tyrphostins; Up-Regulation

The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 +/- 53%) and JAK2 (+238 +/- 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 +/- 61%) and pTyr-STAT3 (+253 +/- 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 gamma-IFN activation site DNA-binding activity (+606 +/- 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress.

Topics: Animals; Base Sequence; DNA Probes; Ischemic Preconditioning; Male; Mice; Mice, Inbred ICR; Myocardial Infarction; Nitric Oxide Synthase; Protein-Tyrosine Kinases; Trans-Activators; Tyrphostins

We recently demonstrated that ischemic preconditioning (IPC) induced by cyclic episodes of short durations of ischemia and reperfusion potentiates a signal transduction cascade involving protein tyrosine kinases and MAP kinases. A rapid activation of janus kinase (JAK) and several signal transducers and activators of the transcription (STATs) including STAT3, STAT5A and STAT6 has been shown to occur during myocardial ischemia and reperfusion. This study sought to examine if JAK/STAT signaling pathway play any role in classical early phase of IPC. Isolated working rat hearts were perfused for 15 min with KHB buffer in the absence or presence of a JAK kinase inhibitor tyrphostin AG490 (5 microm) followed by IPC, 30 min global ischemia and 2 h of reperfusion. The results demonstrated extensive phosphorylation of JAK2 and STAT3 in the IPC hearts which was almost completely abolished by an inhibitor of JAK2, AG490. IPC displayed cardioprotection as evidenced by improved post-ischemic contractile recovery, decreased myocardial infarct size and reduced number of apoptotic cardiomyocytes. AG490 blocked IPC-mediated cardioprotection by altering the IPC-mediated survival signal into death signal. Thus, IPC-induced upregulation of antiapoptotic gene bcl-2 and downregulation of pro-apoptotic gene bax are decreased and increased, respectively, in the AG490 treated hearts. The results suggest that early phase of IPC potentiates JAK/STAT signaling by activating STAT3 which transmits a survival signal to the myocardium.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Survival; DNA-Binding Proteins; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Heart; In Situ Nick-End Labeling; Ischemic Preconditioning, Myocardial; Myocardial Infarction; Myocardium; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Time Factors; Trans-Activators; Tyrphostins; Up-Regulation

We reported that the activation of gp130 transduced hypertrophic and cytoprotective signals via Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in cardiac myocytes. Recent in vivo experiments have demonstrated that the JAK/STAT pathway is activated in acute pressure overload hearts. The present study was designed to examine whether the JAK/STAT pathway is also activated in acute myocardial infarction (AMI) and to determine its pathophysiological roles in ischemic heart disease.. AMI model was generated by the ligation of proximal left anterior descending coronary artery of male Wistar rat. They were sacrificed at various time points ranging from 1 to 24 h after coronary ligation and their hearts were examined. Tyrosine phosphorylation of STAT3 was observed in the myocardium obtained from both the ischemic area and the healthy border area adjacent to the infarcted area. The AMI rats were next randomly assigned to two groups, one with coronary ligation only (group M), and the other with coronary ligation with AG-490 treatment (1 mg/kg i.v., every 4 h), a specific JAK2 inhibitor (group A). In group A, phosphorylation of STAT3 was significantly suppressed and caspase-3 activity and Bax expression were significantly increased in the myocardium after AMI. In group M, few apoptotic myocytes were identified in the border area by means of TUNEL assay. However, a significant increase in apoptotic cells was observed in group A.. Administration of JAK2 inhibitor resulted in deterioration of myocardial viability in AMI hearts. The JAK/STAT pathway is activated in AMI myocardium and plays a pivotal role in cytoprotective signaling.

Topics: Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspases; Cells, Cultured; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; In Situ Nick-End Labeling; Janus Kinase 1; Janus Kinase 2; Male; Mitogen-Activated Protein Kinases; Myocardial Infarction; Myocardium; Phosphatidylinositol 3-Kinases; Phosphorylation; Precipitin Tests; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Trans-Activators; Tyrphostins