Signal transducer and activator of transcription 3 (STAT3) activation is key for ischemic postconditioning (IPo) to attenuate myocardial ischemia-reperfusion injury (MIRI), but IPo loses cardioprotection in diabetes in which cardiac STAT3 activation is impaired and adiponectin (APN) reduced. We found that IPo increased postischemic cardiomyocyte-derived APN, activated mitochondrial STAT3 (mitoSTAT3), improved mitochondrial function, and attenuated MIRI in wild-type but not in APN knockout (Adipo(-/-)) mice subjected to 30 min coronary occlusion, followed by 2 or 24 h of reperfusion. Hypoxic postconditioning-induced protection against hypoxia/reoxygenation injury was lost in Adipo(-/-) cardiomyocytes but restored by recombinant APN, but this APN beneficial effect was abolished by specific STAT3 or APN receptor 1 (AdipoR1) gene knockdown, or caveolin-3 (Cav3) disruption. APN activated cardiac STAT3 and restored IPo cardioprotection in 4-week diabetic rats where AdipoR1 and Cav3 were functionally interactive but not in 8-week diabetic rats whose cardiac Cav3 was severely reduced and AdipoR1/Cav3 signaling impaired. We concluded that IPo activates mitoSTAT3 through APN/AdipoR1/Cav3 pathway to confer cardioprotection, whereas in diabetes, IPo loses cardioprotection due to impaired APN/AdipoR1/Cav3 signaling. Therefore, effective means that may concomitantly activate APN and repair APN signaling (i.e., AdipoR1/Cav3) in diabetes may represent promising avenues in the treatment of MIRI in diabetes.
Bupivacaine, a commonly used local anesthetic, has potential neurotoxicity through diverse signaling pathways. However, the key mechanism of bupivacaine-induced neurotoxicity remains unclear. Cultured human SH-SY5Y neuroblastoma cells were treated (bupivacaine) or untreated (control) with bupivacaine for 24 h. Compared to the control group, bupivacaine significantly increased cyto-inhibition, cellular reactive oxygen species, DNA damage, mitochondrial injury, apoptosis (increased TUNEL-positive cells, cleaved caspase 3, and Bcl-2/Bax), and activated autophagy (enhanced LC3II/LC3I ratio). To explore changes in protein expression and intercommunication among the pathways involved in bupivacaine-induced neurotoxicity, an 8-plex iTRAQ proteomic technique and bioinformatics analysis were performed. Compared to the control group, 241 differentially expressed proteins were identified, of which, 145 were up-regulated and 96 were down-regulated. Bioinformatics analysis of the cross-talk between the significant proteins with altered expression in bupivacaine-induced neurotoxicity indicated that phosphatidyl-3-kinase (PI3K) was the most frequently targeted protein in each of the interactions. We further confirmed these results by determining the downstream targets of the identified signaling pathways (PI3K, Akt, FoxO1, Erk, and JNK). In conclusion, our study demonstrated that PI3K may play a central role in contacting and regulating the signaling pathways that contribute to bupivacaine-induced neurotoxicity.
Oxidative stress plays a critical role in the pathogenesis of intestinal ischemia reperfusion (IIR) injury. Enhancement in endogenous Lipoxin A4 (LXA4), a potent antioxidant and mediator, is associated with attenuation of IIR. However, the effects of LXA4 on IIR injury and the potential mechanisms are unknown. In a rat IIR (ischemia 45 minutes and subsequent reperfusion 6 hours) model, IIR caused intestinal injury, evidenced by increased serum diamine oxidase, D-lactic acid, intestinal-type fatty acid-binding protein, and the oxidative stress marker 15-F2t-Isoprostane. LXA4 treatment significantly attenuated IIR injury by reducing mucosal 15-F2t-Isoprostane and elevating endogenous antioxidant superoxide dismutase activity, accompanied with Keap1/Nrf2 pathway activation. Meanwhile, LXA4 receptor antagonist Boc-2 reversed the protective effects of LXA4 on intestinal injury but failed to affect the oxidative stress and the related Nrf2 pathway. Furthermore, Nrf2 antagonist brusatol reversed the antioxidant effects conferred by LXA4 and led to exacerbation of intestinal epithelium cells oxidative stress and apoptosis, finally resulting in a decrease of survival rate of rat. Meanwhile, LXA4 pretreatment upregulated nuclear Nrf2 level and reduced hypoxia/reoxygenation-induced IEC-6 cell damage and Nrf2 siRNA reversed this protective effect of LXA4 in vitro. In conclusion, these findings suggest that LXA4 ameliorates IIR injury by activating Keap1/Nrf2 pathway in a LXA4 receptor independent manner.
The effect of sevoflurane postconditioning (sevo-postC) cardioprotection is compromised in diabetes which is associated with increased oxidative stress. We hypothesized that antioxidant N-Acetylcysteine may enhance or restore sevo-postC cardioprotection in diabetes. Control or streptozotocin-induced Type 1 diabetic rats were either untreated or treated with N-Acetylcysteine for four weeks starting at five weeks after streptozotocin injection and were subjected to myocardial ischemia-reperfusion injury (IRI), in the absence or presence of sevo-postC. Diabetes showed reduction of cardiac STAT3 activation (p-STAT3) and adiponectin with concomitantly increase of FoxO1 and CD36, which associated with reduced sevo-postC cardioprotection. N-Acetylcysteine and sevo-postC synergistically reduced the infarct size in diabetic groups. N-Acetylcysteine remarkably increased cardiac p-STAT3 which was further enhanced by sevo-postC. N-Acetylcysteine but not sevo-postC decreased myocardial FoxO1 while sevo-postC but not N-Acetylcysteine significantly increased myocardiac adiponectin in diabetic rats. It is concluded that late stage diabetic rats displayed reduction of cardiac p-STAT3, adiponectin deficiency, and increase of FoxO1 and CD36 expression, which may be responsible for the loss of myocardial responsiveness to sevo-postC cardioprotection. N-Acetylcysteine restored Sevo-postC cardioprotection in diabetes possibly through enhancing cardiac p-STAT3 and adiponectin and reducing Fox1 and CD36.
Necrosis amplifies inflammation and plays important roles in acute respiratory distress syndrome (ARDS). Necroptosis is a newly identified programmed necrosis that is mediated by receptor interacting protein 3 (RIP3). However, the potential involvement and impact of necroptosis in lipopolysaccharide (LPS)-induced ARDS remains unknown. We therefore explored the role and mechanism of RIP3-mediated necroptosis in LPS-induced ARDS. Mice were instilled with increasing doses of LPS intratracheally to induce different degrees of ARDS. Lung tissues were harvested for histological and TUNEL staining and western blot for RIP3, p-RIP3, X-linked inhibitor of apoptosis protein (XIAP), mixed lineage kinase domain-like protein (MLKL), total and cleaved caspases-3/8. Then, wild-type and RIP3 knock-out mice were induced ARDS with 30 mg/kg LPS. Pulmonary cellular necrosis was labeled by the propidium Iodide (PI) staining. Levels of TNF-a, Interleukin (IL)-1beta, IL-6, IL-1alpha, IL-10 and HMGB1, tissue myeloperoxidase (MPO) activity, neutrophil counts and total protein concentration were measured. Results showed that in high dose LPS (30mg/kg and 40mg/kg) -induced severe ARDS, RIP3 protein was increased significantly, accompanied by increases of p-RIP3 and MLKL, while in low dose LPS (10mg/kg and 20mg/kg) -induced mild ARDS, apoptosis was remarkably increased. In LPS-induced severe ARDS, RIP3 knock-out alleviated the hypothermia symptom, increased survival rate and ameliorated the lung tissue injury RIP3 depletion also attenuated LPS-induced increase in IL-1alpha/beta, IL-6 and HMGB1 release, decreased tissue MPO activity, and reduced neutrophil influx and total protein concentration in BALF in severe ARDS. Further, RIP3 depletion reduced the necrotic cells in the lung and decreased the expression of MLKL, but had no impact on cleaved caspase-3 in LPS-induced ARDS. It is concluded that RIP3-mediated necroptosis is a major mechanism of enhanced inflammation and lung tissue injury in high dose LPS- induced severe ARDS in mice.
BACKGROUND: Recent clinical and animal studies suggested that remote limb ischemic postconditioning (RIPostC) can invoke potent cardioprotection or neuroprotection. However, the effect and mechanism of RIPostC against renal ischemia/reperfusion injury (IRI) are poorly understood. T-LAK-cell-originated protein kinase (TOPK) is crucial for the proliferation and migration of tumor cells. However, the function of TOPK and the molecular mechanism underlying renal protection remain unknown. Therefore, this study aimed to evaluate the role of TOPK in renoprotection induced by RIPostC. MATERIALS AND METHODS: The renal IRI model was induced by left renal pedicle clamping for 45min followed by 24h reperfusion and right nephrectomy. All mice were intraperitoneally injected with vehicle, TOPK inhibitor HI-TOPK-032 or Akt inhibitor LY294002. After 24h reperfusion, renal histology, function, and inflammatory cytokines and oxidative stress were assessed. The proteins were measured by Western blotting. RESULTS: The results showed that RIPostC significantly protected the kidneys against IRI. The protective effects were accompanied by the attenuation of renal dysfunction, tubular damage, inflammation and oxidative stress. In addition, RIPostC increased the phosphorylation of TOPK, PTEN, Akt, GSK3beta and the nuclear translocation of Nrf2 and decreased the nuclear translocation of NF-kappaB. However, all of the above renoprotective effects of RIPostC were eliminated either by the inhibition of TOPK or Akt with HI-TOPK-032 or LY294002. CONCLUSIONS: The current data reveal that RIPostC protects against renal IRI via activation of TOPK/PTEN/Akt signaling pathway mediated anti-oxidation and anti-inflammation.
Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1x10(9) pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure-volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2'-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2mug/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10muM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.
BACKGROUND: Pretreatment with the angiotensin-converting inhibitor captopril or volatile anesthetic isoflurane has, respectively, been shown to attenuate myocardial ischemia reperfusion (MI/R) injury in rodents and in patients. It is unknown whether or not captopril pretreatment and isoflurane preconditioning (Iso) may additively or synergistically attenuate MI/R injury. METHODS AND RESULTS: Patients selected for heart valve replacement surgery were randomly assigned to five groups: untreated control (Control), captopril pretreatment for 3 days (Cap3d), or single dose captopril (Cap1hr, 1 hour) before surgery with or without Iso (Cap3d+Iso and Cap1hr+Iso). Rabbit MI/R model was induced by occluding coronary artery for 30 min followed by 2-hour reperfusion. Rabbits were randomized to receive sham operation (Sham), MI/R (I/R), captopril (Cap, 24 hours before MI/R), Iso, or the combination of captopril and Iso (Iso+Cap). In patients, Cap3d+Iso but not Cap1hr+Iso additively reduced postischemic myocardial injury and attenuated postischemic myocardial inflammation. In rabbits, Cap or Iso significantly reduced postischemic myocardial infarction. Iso+Cap additively reduced cellular injury that was associated with improved postischemic myocardial functional recovery and reduced myocardial apoptosis and attenuated oxidative stress. CONCLUSION: A joint use of 3-day captopril treatment and isoflurane preconditioning additively attenuated MI/R by reducing oxidative stress and inflammation.
Clinical evidence shows that circulating levels of adipocyte fatty-acid-binding protein (A-FABP) are elevated in patients with diabetes and closely associated with ischaemic heart disease. Patients with diabetes are more susceptible to myocardial ischaemia/reperfusion (MI/R) injury. The experiments in the present study investigated the role of A-FABP in MI/R injury with or without diabetes. Non-diabetic and diabetic (streptozotocin-induced) A-FABP knockout and wild-type mice were subjected to MI/R or sham intervention. After MI/R, A-FABP knockout mice exhibited reductions in myocardial infarct size, apoptotic index, oxidative and nitrative stress, and inflammation. These reductions were accompanied by an improved left ventricular function compared with the relative controls under non-diabetic or diabetic conditions. After diabetes induction, A-FABP knockout mice exhibited a preserved cardiac function compared with that in wild-type mice. Endothelial cells, but not cardiomyocytes, were identified as the most likely source of cardiac A-FABP. Cardiac and circulating A-FABP levels were significantly increased in mice with diabetes or MI/R. Diabetes-induced superoxide anion production was significantly elevated in wild-type mice, but diminished in A-FABP knockout mice, and this elevation contributed to the exaggeration of MI/R-induced cardiac injury. Phosphorylation of endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) were enhanced in both diabetic and non-diabetic A-FABP knockout mice after MI/R injury, but diminished in wild-type mice. The beneficial effects of A-FABP deficiency on MI/R injury were abolished by the NOS inhibitor N(G)-nitro-L-arginine methyl ester. Thus, A-FABP deficiency protects mice against MI/R-induced and/or diabetes-induced cardiac injury at least partially through activation of the eNOS/NO pathway and reduction in superoxide anion production.
Activation of PKCbeta (protein kinase Cbeta) plays a critical role in myocardial I/R (ischaemia/reperfusion) injury in non-diabetic rodents. In the myocardium of diabetes, PKCbeta2 overexpression is associated with increased vulnerability to post-ischaemic I/R injury with concomitantly impaired cardiomyocyte Cav (caveolin)-3 and Akt signalling compared with non-diabetic rats. We hypothesized that myocardial PKCbeta overexpression in diabetes exacerbates myocardial I/R injury through impairing Cav-3/Akt signalling. Streptozotocin-induced diabetic rats were treated with the selective PKCbeta inhibitor ruboxistaurin (RBX, 1 mg/kg per day) for 4 weeks, starting from 1 week after diabetes induction, before inducing myocardial I/R achieved by occluding the left descending coronary artery followed by reperfusion. Cardiac function was measured using a pressure-volume conductance system. In an in vitro study, cardiac H9C2 cells were exposed to high glucose (30 mmol/l) and subjected to hypoxia followed by reoxygenation (H/R) in the presence or absence of the selective PKCbeta2 inhibitor CGP53353 (1 mumol/l), siRNAs of PKCbeta2 or Cav-3 or Akt. Cell apoptosis and mitochondrial membrane potential were assessed by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) and JC-1 staining respectively. RBX significantly decreased post-ischaemic myocardial infarct size (35+/-5% compared with 49+/-3% in control, P<0.05) and attenuated cardiac dysfunction, and prevented the reduction in cardiac Cav-3 and enhanced phosphorylated/activated Akt (p-Akt) in diabetic rats (P<0.05). H/R increased cardiomyocyte injury under high glucose conditions as was evident by increased TUNEL-positive and increased JC-1 monomeric cells (P<0.05 compared with control), accompanied with increased PKCbeta2 phosphorylation/activation and decreased Cav-3 expression. Either CGP53353 or PKCbeta2 siRNA significantly attenuated all of these changes and enhanced p-Akt. Cav-3 gene knockdown significantly reduced p-Akt and increased post-hypoxic cellular and mitochondrial injury despite a concomitant reduction in PKCbeta2 phosphorylation. PKCbeta2 inhibition with RBX protects diabetic hearts from myocardial I/R injury through Cav-3-dependent activation of Akt.
Inhalation anesthetic isoflurane inhibits hypoxia pulmonary vasoconstriction (HPV), while dexmedetomidine (Dex) could reduce the dose of isoflurane inhalation and potentiate HPV, but the mechanism is unclear. Inhibition of reactive oxygen species (ROS) production can favor HPV during one-lung ventilation (OLV). Similarly, nitric oxide (NO), an important endothelium-derived vasodilator in lung circulation, can decrease the regional pulmonary vascular resistance of ventilated lung and reduce intrapulmonary shunting. We hypothesized that Dex may augment HPV and improve oxygenation during OLV through inhibiting oxidative stress and increasing NO release. Patients undergoing OLV during elective thoracic surgery were randomly allocated to either isoflurane + saline (NISO, n = 24) or isoflurane + dexmedetomidine (DISO, n = 25) group. Anesthesia was maintained with intravenous remifentanil and inhalational isoflurane (1.0-2.0%), with concomitant infusion of dexmedetomidine 0.7 mugkg(-1)h(-1) in DISO and saline 0.25 mL kg(-1)h(-1) in NISO group. Hemodynamic variables or depth of anesthesia did not significantly differ between groups. Administration of Dex significantly reduced Qs/Qt and increased PaO2 after OLV, accompanied with reduced lipid peroxidation product malondialdehyde and higher levels of SOD activity as well as serum NO (all P < 0.05 DISO versus NISO). In conclusion, reducing oxidative stress and increasing NO release during OLV may represent a mechanism whereby Dex potentiates HPV.
BACKGROUND: Postliver transplantation acute kidney injury (AKI) severely affects patient survival, whereas the mechanism is unclear and effective therapy is lacking. The authors postulated that reperfusion induced enhancement of connexin32 (Cx32) gap junction plays a critical role in mediating postliver transplantation AKI and that pretreatment/precondition with the anesthetic propofol, known to inhibit gap junction, can confer effective protection. METHODS: Male Sprague-Dawley rats underwent autologous orthotopic liver transplantation (AOLT) in the absence or presence of treatments with the selective Cx32 inhibitor, 2-aminoethoxydiphenyl borate or propofol (50 mg/kg) (n = 8 per group). Also, kidney tubular epithelial (NRK-52E) cells were subjected to hypoxia-reoxygenation and the function of Cx32 was manipulated by three distinct mechanisms: cell culture in different density; pretreatment with Cx32 inhibitors or enhancer; Cx32 gene knock-down (n = 4 to 5). RESULTS: AOLT resulted in significant increases of renal Cx32 protein expression and gap junction, which were coincident with increases in oxidative stress and impairment in renal function and tissue injury as compared to sham group. Similarly, hypoxia-reoxygenation resulted in significant cellular injury manifested as reduced cell growth and increased lactate dehydrogenase release, which was significantly attenuated by Cx32 gene knock-down but exacerbated by Cx32 enhancement. Propofol inhibited Cx32 function and attenuated post-AOLT AKI. In NRK-52E cells, propofol reduced posthypoxic reactive oxygen species production and attenuated cellular injury, and the cellular protective effects of propofol were reinforced by Cx32 inhibition but cancelled by Cx32 enhancement. CONCLUSION: Cx32 plays a critical role in AOLT-induced AKI and that inhibition of Cx32 function may represent a new and major mechanism whereby propofol reduces oxidative stress and subsequently attenuates post-AOLT AKI.
Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2O2) stimulation without or with MC degranulator compound 48/80 (CP). H2O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47(phox) and gp91(phox) protein expression, increases of the specific lipid peroxidation product 15-F2t-Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and beta-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation.
Endothelial dysfunction induced by oxidative stress and inflammation plays a critical role in the pathogenesis of cardiovascular diseases. The anesthetic sevoflurane confers cytoprotective effects through its anti-inflammatory properties in various pathologies such as systemic inflammatory response syndrome and ischemic-reperfusion injury but mechanism is unclear. We hypothesized that sevoflurane can protect against tumor necrosis factor (TNF)-alpha-induced endothelial dysfunction through promoting the production of endothelium-dependent nitric oxide (NO). Primary cultured human umbilical vein endothelial cells (HUVECs) were pretreated with different concentrations (0.5, 1.5 and 2.5 minimum alveolar concentration, MAC) of sevoflurane for 30 min before TNF-alpha (10 ng/mL) stimulation for 4 h. Sevoflurane pretreatment significantly reduced TNF-alpha-induced VCAM-1, ICAM-1, IkappaBalpha, and NF-kappaB activation, and blocked leukocytes adhesion to HUVECs. Meanwhile, sevoflurane (1.5 and 2.5 MAC) significantly induced endothelial nitric oxide synthase (eNOS) phosphorylation and enhanced NO levels both intracellularly and in the cell culture medium. All these cytoprotective effects of sevoflurane were abrogated by NG-nitro-l-arginine methyl ester (l-NAME), a non-specific nitric oxide synthase inhibitor. Collectively, these data indicate that sevoflurane protects against TNF-alpha -induced vascular endothelium dysfunction through activation of eNOS/NO pathway and inhibition of NF-kappaB.
OBJECTIVES: Heme oxygenase-1 is inducible in cardiomyocytes in response to stimuli such as oxidative stress and plays critical roles in combating cardiac hypertrophy and injury. Signal transducer and activator of transcription 3 plays a pivotal role in heme oxygenase-1-mediated protection against liver and lung injuries under oxidative stress. We hypothesized that propofol, an anesthetic with antioxidant capacity, may attenuate hyperglycemia-induced oxidative stress in cardiomyocytes via enhancing heme oxygenase-1 activation and ameliorate hyperglycemia-induced cardiac hypertrophy and apoptosis via heme oxygenase-1/signal transducer and activator of transcription 3 signaling and improve cardiac function in diabetes. DESIGN: Treatment study. SETTING: Research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: In vivo and in vitro treatments. MEASUREMENTS AND MAIN RESULTS: At 8 weeks of streptozotocin-induced type 1 diabetes in rats, myocardial 15-F2t-isoprostane was significantly increased, accompanied by cardiomyocyte hypertrophy and apoptosis and impaired left ventricular function that was coincident with reduced heme oxygenase-1 activity and signal transducer and activator of transcription 3 activation despite an increase in heme oxygenase-1 protein expression as compared to control. Propofol infusion (900 mug/kg/min) for 45 minutes significantly improved cardiac function with concomitantly enhanced heme oxygenase-1 activity and signal transducer and activator of transcription activation. Similar to the changes seen in diabetic rat hearts, high glucose (25 mmol/L) exposure for 48 hours led to cardiomyocyte hypertrophy and apoptosis, both in primary cultured neonatal rat cardiomyocytes and in H9c2 cells compared to normal glucose (5.5 mmol/L). Hypertrophy was accompanied by increased reactive oxygen species and malondialdehyde production and caspase-3 activity. Propofol, similar to the heme oxygenase-1 inducer cobalt protoporphyrin, significantly increased cardiomyocyte heme oxygenase-1 and p-signal transducer and activator of transcription protein expression and heme oxygenase-1 activity and attenuated high-glucose-mediated cardiomyocyte hypertrophy and apoptosis and reduced reactive oxygen species and malondialdehyde production (p < 0.05). These protective effects of propofol were abolished by heme oxygenase-1 inhibition with zinc protoporphyrin and by heme oxygenase-1 or signal transducer and activator of transcription 3 gene knockdown. CONCLUSIONS: Heme oxygenase-1/signal transducer and activator of transcription 3 signaling plays a critical role in propofol-mediated amelioration of hyperglycemia-induced cardiomyocyte hypertrophy and apoptosis, whereby propofol improves cardiac function in diabetic rats.
Protein kinase C (PKC)beta2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCbeta2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCbeta2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-beta-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCbeta2 phosphorylation. Inhibition of PKCbeta2 activation by compound CGP53353 or knockdown of PKCbeta2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCbeta2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2(-), nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCbeta2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCbeta2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.
N-Acetylcysteine (NAC) and allopurinol (ALP) synergistically reduce myocardial ischemia reperfusion (MI/R) injury in diabetes. However, the mechanism is unclear. We postulated that NAC and ALP attenuated diabetic MI/R injury by up-regulating phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase 2/signal transducer and activator of transcription-3 (JAK2/STAT3) pathways subsequent to adiponectin (APN) activation. Control (C) or streptozotocin-induced diabetic rats (D) were untreated or treated with NAC and ALP followed by MI/R. D rats displayed larger infarct size accompanied by decreased phosphorylation of Akt, STAT3 and decreased cardiac nitric oxide (NO) and APN levels. NAC and ALP decreased MI/R injury in D rats, enhanced phosphorylation of Akt and STAT3, and increased NO and APN. High glucose and hypoxia/reoxygenation exposure induced cell death and Akt and STAT3 inactivation in cultured cardiomyocytes, which were prevented by NAC and ALP. The PI3K inhibitor wortmannin and Jak2 inhibitor AG490 abolished the protection of NAC and ALP. Similarly, APN restored posthypoxic Akt and STAT3 activation and decreased cell death in cardiomyocytes. Gene silencing with AdipoR2 siRNA or STAT3 siRNA but not AdipoR1 siRNA abolished the protection of NAC and ALP. In conclusion, NAC and ALP prevented diabetic MI/R injury through PI3K/Akt and Jak2/STAT3 and cardiac APN may serve as a mediator via AdipoR2 in this process.
BACKGROUND: Large body of evidences accumulated in clinical and epidemiological studies indicate that hearts of diabetic subjects are more sensitive to ischemia reperfusion injury (IRI), which results in a higher rate of mortality at post-operation than that of non-diabetes. However, experimental results are equivocal and point to either increased or decreased susceptibility of the diabetic hearts to IRI, especially at the early stage of the disease. The present study was designed to test the hypothesis that the duration/severity of the indexed ischemia is a major determinant of the vulnerability to myocardial IRI at early stage of diabetes. METHODS: Four weeks streptozotocin (STZ)-induced diabetic (D) and non-diabetic (C) Sprague-Dawley rats were randomly assigned to receive 30 or 45 min of left anterior descending artery ligation followed by 2 or 3 hours of reperfusion, respectively. Cardiac function was recorded by using Pressure-Volume (PV) conduction system. Myocardial infarct size was determined with triphenyltetrazolium chloride staining. Plasma Creatine kinase-MB (CK-MB), Lactate dehydrogenase (LDH) release, myocardial nitric oxide(NO) content and nitrotyrosine formation, 15-F(2t)-Isoprostane and plasma superoxide dismutase (SOD) were measured with colorimetric assays. Cardiomyocyte apoptosis was assessed by TUNEL staining. Myocardial TNFalpha, Caspase-3, STAT3, Akt, and GSK-3beta were determined by Western blotting. RESULTS: Prolongation of ischemia but not reperfusion from 30 min to 45 min significantly increased infarct size in D compared to C rats (P < 0.05), accompanied with significantly increased plasma CK-MB (P < 0.05). Prolongation of the duration of either ischemia or reperfusion significantly increased plasma LDH release and myocardial 15-F(2t)-Isoprostane and reduced plasma SOD activity, with concomitant reduction of myocardial NO and increase of nitrotyrosine formation in D relative to C (P < 0.05). Prolongation of ischemia and reperfusion significantly reduced left ventricular ejection fraction and increased the peak rate of pressure, accompanied with increased end systolic pressure in D relative to C rats (P < 0.05) but reduced phosphorylations of myocardial STAT3 at site Ser727 and Akt at site Ser473 as well as GSK-3beta at Ser 9 (P < 0.05). CONCLUSIONS: Diabetic hearts, even at early stage of the disease are more sensitive to IRI, and this increased severity of post-ischemic myocardial injury depends more on the duration of ischemia than that of reperfusion.