Ischemia-reperfusion injury and cardioprotection: investigating PTEN,the phosphatase that negatively regulates PI3K,using a congenital model of PTEN haploinsufficiency

Activation of the PI3K/Akt pathway protects the heart from ischemia-reperfusion injury (IRI). The phosphatase PTEN is the main negative regulator of this pathway. We hypothesized that reduced PTEN levels could protect against IRI. Isolated perfused mouse hearts from PTEN^+/− and their littermates PTEN^+/+ (WT), were subjected to 35 min global ischemia and 30 min reperfusion, with and without 2, 4 or 6 cycles ischemic preconditioning (IPC). The end point was infarct size, expressed as a percentage of the myocardium at risk (I/R%). PTEN and Akt levels were determined using Western blot analysis. Unexpectedly, there were no significant differences in infarction between PTEN^+/− and WT (42.1 ± 5.0% Vs. 45.6 ± 3.3%). However, the preconditioning threshold was significantly reduced in the PTEN^+/− Vs. WT, with 4 cycles of IPC being sufficient to reduce I/R%, compared to 6 cycles in the WT (4 cycles IPC: 29.8. ± 3.69% in PTEN^+/− Vs. 45.5. ± 5.08% in WT, P < 0.01). In addition, the ratio between the phospho/total Akt (Ser473 and Thr308) was slightly but significantly increased in the PTEN^+/− indicating an upregulation of PI3K/Akt pathway. Interestingly, the levels of the other phosphatases that may negatively regulate the PI3K/Akt pathway (PP2A, SHIP2 and PHLPP) were not significantly different between littermates and PTEN^+/−. In conclusion, PTEN haploinsufficiency alone does not induce cardioprotection in this model; however, it reduces the threshold of protection induced by IPC. Returned for 1. Revision: 29 November 2007 1. Revision received: 5 May 2008 Returned for 2. Revision: 2 June 2008 2. Revision received: 5 June 2008     

TNFα protects cardiac mitochondria independently of its cell surface receptors

Our novel proposal is that TNFα exerts a direct effect on mitochondrial respiratory function in the heart, independently of its cell surface receptors. TNFα-induced cardioprotection is known to involve reactive oxygen species (ROS) and sphingolipids. We therefore further propose that this direct mitochondrial effect is mediated via ROS and sphingolipids. The protective concentration of TNFα (0.5 ng/ml) was added to isolated heart mitochondria from black 6 × 129 mice (WT) and double TNF receptor knockout mice (TNFR1&2^−/−). Respiratory parameters and inner mitochondrial membrane potential were analyzed in the presence/absence of two antioxidants, N-acetyl-l-cysteine or N-tert-butyl-α-(2-sulfophenyl)nitrone or two antagonists of the sphingolipid pathway, N-oleoylethanolamine (NOE) or imipramine. In WT, TNFα reduced State 3 respiration from 279.3 ± 3 to 119.3 ± 2 (nmol O₂/mg protein/min), increased proton leak from 15.7 ± 0.6% (control) to 36.6 ± 4.4%, and decreased membrane potential by 20.5 ± 3.1% compared to control groups. In TNFR1&2^−/− mice, TNFα reduced State 3 respiration from 205.2 ± 4 to 75.7 ± 1 (p < 0.05 vs. respective control). In WT mice, both antioxidants added with TNFα restored State 3 respiration to 269.2 ± 2 and 257.6 ± 2, respectively. Imipramine and NOE also restored State 3 respiration to 248.4 ± 2 and 249.0 ± 2, respectively (p < 0.01 vs. TNFα alone). Similarly, both antioxidant and inhibitors of the sphingolipid pathway restored the proton leak to pre-TNF values. TNFα-treated mitochondria or isolated cardiac muscle fibers showed an increase in respiration after anoxia–reoxygenation, but this effect was lost in the presence of an antioxidant or NOE. Similar data were obtained in TNFR1&2^−/− mice. TNFα exerts a protective effect on respiratory function in isolated mitochondria subjected to an anoxia–reoxygenation insult. This effect appears to be independent of its cell surface receptors, but is likely to be mediated by ROS and sphingolipids.     

Direct relationship between levels of TNF-α expression and endothelial dysfunction in reperfusion injury

We previously found that myocardial ischemia/reperfusion (I/R) initiates expression of tumor necrosis factor-α (TNF) leading to coronary endothelial dysfunction. However, it is not clear whether there is a direct relationship between levels of TNF expression and endothelial dysfunction in reperfusion injury. We studied levels of TNF expression by using different transgenic animals expressing varying amounts of TNF in I/R. We crossed TNF overexpression (TNF^++/++) with TNF knockout (TNF^−/−) mice; thus we have a heterozygote population of mice with the expression of TNF “in between” the TNF^−/− and TNF^++/++ mice. Mouse hearts were subjected to 30 min of global ischemia followed by 90 min of reperfusion and their vasoactivity before and after I/R was examined in wild type (WT), TNF^−/−, TNF^++/++ and TNF heterozygote (TNF^−/++, cross between TNF^−/− and TNF^++/++) mice. In heterozygote TNF^−/++ mice with intermediate cardiac-specific expression of TNF, acetylcholine-induced or flow-induced endothelial-dependent vasodilation following I/R was between TNF^++/++ and TNF^−/− following I/R. Neutralizing antibodies to TNF administered immediately before the onset of reperfusion-preserved endothelial-dependent dilation following I/R in WT, TNF^−/++ and TNF^++/++ mice. In WT, TNF^−/++ and TNF^++/++ mice, I/R-induced endothelial dysfunction was progressively lessened by administration of free-radical scavenger TEMPOL immediately before initiating reperfusion. During I/R, production of superoxide (O₂ ^·−) was greatest in TNF^++/++ mice as compared to WT, TNF^−/++ and TNF^−/− mice. Following I/R, arginase mRNA expression was elevated in the WT, substantially elevated in the TNF^−/++ and TNF^++/++ mice and not affected in the TNF^−/− mice. These results suggest that the level of TNF expression determines arginase expression in endothelial cells during myocardial I/R, which is one of the mechanisms by which TNF compromises coronary endothelial function in reperfusion injury.     

c-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets

Aims/hypothesis  Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. Methods  C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)^−/− and Jnk2 (also known as Mapk9)^−/− mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. Results   Jnk1 ^−/− islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 ^−/− islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 ^−/− but not Jnk1 ^−/− islets; VEGF blockade restored Jnk1 ^−/− islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ^−/− or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ^−/− recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 ^−/− recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. Conclusions/interpretation  We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.     

Antiapoptosis and Mitochondrial Effect of Pioglitazone Preconditioning in the Ischemic/reperfused Heart of Rat

Purpose  Pioglitazone, used clinically in the treatment of type 2 diabetes mellitus, has been implicated as a regulator of cellular inflammatory and ischemic responses. The present study examined whether pioglitazone could inhibit cadiocyte apoptosis and reduce mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of the rat. Furthermore, we investigated whether the protective effect of pioglitazone was related to opening of the mitochondrial_ATP-sensitive potassium channels. Methods  Adult male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 4 h of reperfusion. At 24 h before ischemia, rats were randomized to receive 0.9% saline, 5-hydroxydecanoate (5-HD, 10 mg kg⁻¹, i.v.) plus pioglitazone (3 mg kg⁻¹, i.v.) or pioglitazone (3 mg kg⁻¹, i.v.). One group served as sham control. We investigated mitochondrial structure, apoptosis rate and Bcl-2, Bax and Caspase-3 proteins by immunohistochemistry staining. RT-PCR was used to determine the expression of P38MAPKmRNA and JNKmRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65. A second group of rats were randomly divided into sham-operated, ischemia/reperfusion (I/R), pioglitazone treatment, 5-HD + pioglitazone and 5-HD groups and the size of myocardial infarction was determined. Primary cultured cardiomyocytes of neonatal Sprague–Dawley rats were divided into control, hypoxia reoxygenation, different concentrations of pioglitazone and 5-HD + pioglitazone groups. JC-1 staining flowcytometry was used to examine mitochondrial membrane potential (ΔΨm). Results  Pioglitazone decreased mitochondrial ultrastructural damage compared to I/R, and reduced infarct size from 34.93 ± 5.55% (I/R) to 20.24 ± 3.93% (P < 0.05). Compared with the I/R group, the apoptosis rate and positive cell index (PCI) of Bax and Caspase-3 proteins in the pioglitazone group were significantly decreased (P < 0.05), while the PCI of Bcl-2 protein was increased (P < 0.05). There was no significant difference between the I/R and 5-HD + pioglitazone groups. Compared with the sham-operated group, the expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in I/R was increased (P < 0.05). Pioglitazone did inhibit the increase in expressions vs I/R (P < 0.05). The rate of loss ΔΨm cells in the pioglitazone group was significantly lower than in the hypoxia reoxygenation group, while the addition of 5-HD inhibited the effect of pioglitazone. Conclusion  Pioglitazone inhibited cadiocyte apoptosis and reduced mitochondrial ultrastructure injury and membrane potential loss in the ischemic/reperfused heart of rat. These protective effects of pioglitazone may be related to opening mitochondrial_ATP-sensitive potassium channels.     

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase α (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 μg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 μg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 ± 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 ± 4%, n = 7), whereas the immunoreactivities of ATP synthase α (49.1 ± 6.4%, n = 7) and MnSOD (79.9 ± 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP’s cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP. K. Boengler and S. Stahlhofen contributed equally to this work.     

The Cardioprotective Effect of Necrostatin Requires the Cyclophilin-D Component of the Mitochondrial Permeability Transition Pore

Background Necrostatin (Nec-1) protects against ischemia–reperfusion (IR) injury in both brain and heart. We have previously reported in this journal that necrostatin can delay opening of the mitochondrial permeability transition pore (MPTP) in isolated cardiomyocytes. Aim The aim of the present study was to investigate in more detail the role played by the MPTP in necrostatin-mediated cardioprotection employing mice lacking a key component of the MPTP, namely cyclophilin-D. Method Anaesthetized wild type (WT) and cyclophilin-D knockout (Cyp-D−/−) mice underwent an open-chest procedure involving 30 min of myocardial ischemia and 2 h of reperfusion, with subsequent infarct size assessed by triphenyltetrazolium staining. Nec-1, given at reperfusion, significantly limited infarct size in WT mice (17.7 ± 3% vs. 54.3 ± 3%, P < 0.05) but not in Cyp-D−/− mice (28.3 ± 7% vs. 30.8 ± 6%, P > 0.05). Conclusion The data obtained in Cyp-D−/− mice provide further evidence that Nec-1 protects against myocardial IR injury by modulating MPTP opening at reperfusion.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle

Aims/hypothesis Insulin resistance and type 2 diabetes are associated with mitochondrial dysfunction. The aim of the present study was to test the hypothesis that oxidative phosphorylation and electron transport capacity are diminished in the skeletal muscle of type 2 diabetic subjects, as a result of a reduction in the mitochondrial content. Materials and methods The O₂ flux capacity of permeabilised muscle fibres from biopsies of the quadriceps in healthy subjects (n = 8; age 58 ± 2 years [mean±SEM]; BMI 28 ± 1 kg/m²; fasting plasma glucose 5.4 ± 0.2 mmol/l) and patients with type 2 diabetes (n = 11; age 62 ± 2 years; BMI 32 ± 2 kg/m²; fasting plasma glucose 9.0 ± 0.8 mmol/l) was measured by high-resolution respirometry. Results O₂ flux expressed per mg of muscle (fresh weight) during ADP-stimulated state 3 respiration was lower (p < 0.05) in patients with type 2 diabetes in the presence of complex I substrate (glutamate) (31 ± 2 vs 43 ± 3 pmol O₂ s⁻¹ mg⁻¹) and in response to glutamate + succinate (parallel electron input from complexes I and II) (63 ± 3 vs 85 ± 6 pmol s⁻¹ mg⁻¹). Further increases in O₂ flux capacity were observed in response to uncoupling by FCCP, but were again lower (p < 0.05) in type 2 diabetic patients than in healthy control subjects (86 ± 4 vs 109 ± 8 pmol s⁻¹ mg⁻¹). However, when O₂ flux was normalised for mitochondrial DNA content or citrate synthase activity, there were no differences in oxidative phosphorylation or electron transport capacity between patients with type 2 diabetes and healthy control subjects. Conclusions/interpretation Mitochondrial function is normal in type 2 diabetes. Blunting of coupled and uncoupled respiration in type 2 diabetic patients can be attributed to lower mitochondrial content.     

Glucagon-like peptide-1 receptor signalling selectively regulates murine lymphocyte proliferation and maintenance of peripheral regulatory T cells

Aims/hypothesis  

Glucagon-like peptide-1 receptor (GLP-1R) agonists improve glucose control in animals and humans with type 1 diabetes. However, there is little information on the role of the GLP-1R in the immune system. We studied the role of the GLP-1R in immune function in wild-type (WT) and non-obese diabetic (NOD) and Glp1r ^−/− mice.     

PKC-δ inhibition does not block preconditioning-induced preservation in mitochondrial ATP synthesis and infarct size reduction in rats

We have previously demonstrated that cardioprotection induced by the infusion of a selective δ₁-opioid agonist is mediated by the specific translocation of PKC-δ to the mitochondria in in vivo rat hearts and via opening of the mitochondrial K_ATP channel. Ischemic preconditioning (IPC) is also thought to involve the translocation of specific isoforms of PKC and K_ATP channel activation. Therefore, we utilized the PKC-δ selective antagonist, rottlerin, to assess the effect of inhibition of this isozyme on cardioprotection induced by one-cycle of IPC prior to 30 minutes of ischemia and 2 hours of reperfusion. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Non-preconditioned control animals had an IS/AAR of 59.7 ± 1.6. IPC significantly reduced the extent of myocardial infarction (6.3 ± 1.4). Rottlerin, 0.3 mg/kg, did not alter IS/AAR in control animals (55.0 ± 5.6), and had no significant effect on IS/AAR in preconditioned animals (14.4 ± 3.8). Additionally, we demonstrated, using a luciferase-based assay to determine the rate of ATP synthesis and state of mitochondrial bioenergetics, that IPC preserves ATP synthesis in the ischemic myocardium and that this preservation is attenuated by the isoform non-selective PKC inhibitor, chelerythrine, but not by the δ-selective antagonist, rottlerin. These data suggest that PKC-δ does not play an important role in IPC and that differences in isoform importance are evident during pharmacological versus ischemia-induced preconditioning. Received: 8 June 2001, Returned for revision: 2 July 2001, Revision received: 31 July 2001, Accepted: 2 August 2001     

Ginsenoside Rb1 Preconditioning Protects Against Myocardial Infarction After Regional Ischemia and Reperfusion by Activation of Phosphatidylinositol-3-kinase Signal Transduction

Background  Ginsenoside Rb1, a major bioactive component of Panax ginseng, bears various beneficial effects on the cardiovascular system. This study investigated whether ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia–reperfusion injury and its potential mechanism. Methods  Rats subjected to 45 min of myocardial ischemia followed by 120 min of reperfusion were assigned to the following groups: sham-operated, ischemia–reperfusion (I/R), ginsenoside Rb1+I/R, wortmannin(a specific PI3K inhibitor)+I/R, wortmannin drug vehicle (dimethyl sulfoxide, DMSO), wortmannin+sham, ginsenoside Rb1+ wortmannin +I/R. Infarct size was assessed by triphenyltetrazolium chloride staining. Plasma creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and troponin T levels were also measured. Akt phosphorylation expression was assessed by immunoblotting. Results  Ginsenoside Rb1 preconditioning reduced infarct size compared with that in the I/R group: 30 ± 2.6% versus 51 ± 2.7% (p < 0.01). Ginsenoside Rb1 preconditioning also markedly reduced the plasma CK, CK-MB, LDH and troponin T levels in blood. Akt phosphorylation expression increased after ginsenoside Rb1 preconditioning. These effects of ginsenoside Rb1 preconditoning were significantly inhibited by wortmannin. Conclusion  This is the first study to demonstrate that ginsenoside Rb1 preconditioning has protective effects on myocardial ischemia and reperfusion injury, partly by mediating the activation of the PI3K pathway and phosphorylation of Akt. Project supported by the National Basic Research Program of China (a.k.a. 973 Program) (No.2005CB523305).     

The Effect of the Phytoestrogen Genistein on Myocardial Protection, Preconditioning and Oxidative Stress

Introduction We have previously shown that estrogen administered in ovariectomized female rabbits significantly reduce myocardial infarct size. We now investigated whether the phytoestrogen genistein similarly protects ischemic myocardium and whether this is associated with its antioxidant properties. In addition, we examined whether genistein abolishes preconditioning, since at high doses, it inhibits tyrosine kinase. Materials and methods We studied five groups of New Zealand white female rabbits. Group A (n = 12) were normal controls, group B (n = 14) were ovariectomized 4 weeks prior to the experiment, group C (n = 10) were ovariectomized and treated with genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) for 4 weeks before the experiment, group D (n = 12) had intact gonads and were treated with genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) for 4 weeks before the experiment and group E (n = 8) were ovariectomized 4 weeks prior to the experiment and treated with a single dose of genistein (0.2 mg kg⁻¹ day⁻¹ subcutaneously) just prior to the experiment. All animals underwent 30 min of heart ischemia and 120 min of reperfusion, with (subgroup I) or without (subgroup II) preconditioning. Malondialdehyde (MDA) concentration just before the experiment was determined. Results We found significant differences between the groups—p < 0.0001 in factorial ANOVA. The groups with preconditioning had significant smaller infarcts compared to those without—AI vs AII (10.66 ± 1.42% vs 43.22 ± 2.67%), BI vs BII (18.53 ± 2.36% vs 43.05 ± 8.37%), CI vs CII (10.17 ± 2.07% vs 44.5 ± 5.47%), DI vs DII (14.98 ± 2.36% vs 37.79 ± 3.92%) and EI vs EII (17.11 ± 3.24% vs 42.08 ± 3.42%), p < 0.0005. Ovariectomy was not associated with larger myocardial infarctions—AII vs BII, p = NS. Genistein, for 4 weeks, did not protect ischemic myocardium in either ovariectomized or non-ovariectomized animals—BII vs CII and AII vs DII, p = NS. There was no significant difference between the preconditioned animals, with intact gonads or ovariectomized (AI vs BI, p = NS), ovariectomized with or without genistein (BI vs CI, p = NS) and non-ovariectomized whether treated with genistein or not (AI vs DI, p = NS). A single dose of genistein did not offer any protection (EII vs BII, p = NS), nor did it modify the preconditioning effect (EI vs BI, p = NS). We found no significant difference in MDA plasma levels between the groups. Conclusion Genistein, at this dose, does not reduce infarct size per se nor abolishes the protection induced by preconditioning, in both ovariectomized and non-ovariectomized animals. Preconditioning offers myocardial protection in animals with intact gonads as well as estrogen deprived; bilateral ovariectomy, at least during short-term, is not associated with larger myocardial infarcts compared to control animals. In addition estrogen deprivation, during short term, as well as genistein do not modify oxidative stress.     

Pattern Recognition Scavenger Receptors, SR-A and CD36, Have an Additive Role in the Development of Colitis in Mice

The multifunctional pattern recognition scavenger receptors, SR-A and CD36, are predominantly expressed by lamina propria macrophages and considered important in innate immunity. We examined the role of these receptors in the pathophysiology of inflammatory bowel disease. Colitis was induced in wild type (WT), SR-A^−/−, CD36^−/−, and SR-A/CD36 double deficient mice by administering DSS. DSS-induced moderately severe colitis in WT mice was manifested by weight loss, reduced hematocrit, and pathology. SR-A/CD36 double deficient mice developed significantly more severe colitis as indicated by anemia (P < 0.01), decreased colonic length due to inflammation (P < 0.01), and lesions when compared with WT and single deficient animals. Serum amyloid A was significantly more elevated in SR-A/CD36^−/− mice (P < 0.01) compared with WT and single deficient animals. However, the spleens of WT mice (P < 0.05) were significantly enlarged. Inflammatory cytokine levels were considerably increased in WT mice (IL-6 P < 0.001, TNFα P < 0.01). In contrast, SR-A deficient mice maintained more normal body and splenic weight and developed less severe colonic lesions compared to other groups. In conclusion, our data indicate that SR-A/CD36 double deficiency leads to more severe colonic lesions and dysregulated inflammatory response as compared with single SR-A or CD36 deficiency in colitis, suggesting additive effects between these two receptors in this model.     

Effect of sulfur dioxide preconditioning on rat myocardial ischemia/reperfusion injury by inducing endoplasmic reticulum stress

Sulfur dioxide has recently been found to be produced endogenously in the cardiovascular system and have important positive biological effects. However, it is unknown whether sulfur dioxide preconditioning has a protective effect on rat myocardial ischemia/reperfusion (I/R) injury and whether this process involves endoplasmic reticulum stress (ERS). In this study, we showed that preconditioning with sulfur dioxide 10 min before ischemia (with a low concentration of sulfur dioxide of 1–10 μmol/kg) could reduce myocardial infarct size and plasma activities of lactate dehydrogenase and creatine kinase in rats with I/R in vivo. Sulfur dioxide preconditioning also reduced myocardium apoptosis induced by I/R. In addition, sulfur dioxide preconditioning increased cardiac function in vitro. Sulfur dioxide preconditioning induced expression of myocardial glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation of the factor 2α-subunit (p-eIF2α) prior to myocardial I/R but suppressed expression of myocardial GRP78, C/EBP homologous protein, and p-eIF2α during myocardial I/R, in association with improved myocardial injury in vivo and in vitro. Pretreatment with dithiothreitol, an ERS stimulator mimicked the above cardioprotective effect. However, pretreatment with the ERS inhibitor 4-phenylbutyrate reversed the cardioprotection provided by sulfur dioxide preconditioning. These data indicated that sulfur dioxide preconditioning reduced I/R-induced myocardial injury in vivo and in vitro, and that augmenting ERS by sulfur dioxide preconditioning prior to I/R contributed to protection against myocardial I/R injury.     

Mitochondrial fragmentation and superoxide anion production in coronary endothelial cells from a mouse model of type 1 diabetes

Aims/hypothesis  

Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O₂ ⁻ ) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs.     

RISK and SAFE signaling pathway interactions in remote limb ischemic perconditioning in combination with local ischemic postconditioning

Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising methods to decrease ischemia–reperfusion (I/R) injury. We tested whether the use of the two procedures in combination led to an improvement in cardioprotection through a higher activation of survival signaling pathways. Rats exposed to myocardial I/R were allocated to one of the following four groups: Control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated 20 min after coronary artery occlusion; IPost+RIPer, IPost and RIPer in combination. Infarct size was significantly reduced in both IPost and RIPer (34.25 ± 3.36 and 24.69 ± 6.02%, respectively) groups compared to Control (54.93 ± 6.46%, both p < 0.05). IPost+RIPer (infarct size = 18.04 ± 4.86%) was significantly more cardioprotective than IPost alone (p < 0.05). RISK pathway (Akt, ERK1/2, and GSK-3β) activation was enhanced in IPost, RIPer, and IPost+RIPer groups compared to Control. IPost+RIPer did not enhance RISK pathway activation as compared to IPost alone, but instead increased phospho-STAT-3 levels, highlighting the crucial role of the SAFE pathway. In IPost+RIPer, a SAFE inhibitor (AG490) abolished cardioprotection and blocked both Akt and GSK-3β phosphorylations, whereas RISK inhibitors (wortmannin or U0126) abolished cardioprotection and blocked STAT-3 phosphorylation. In our experimental model, the combination of IPost and RIPer improved cardioprotection through the recruitment of the SAFE pathway. Our findings also indicate that cross talk exists between the RISK and SAFE pathways.     

Diabetes mellitus abrogates erythropoietin-induced cardioprotection against ischemic-reperfusion injury by alteration of the RISK/GSK-3β signaling

Recent studies reported cardioprotective effects of erythropoietin (EPO) against ischemia–reperfusion (I/R) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been reported to be impaired in diabetes and insulin resistance syndrome, we examined whether EPO-induced cardioprotection was maintained in rat models of type 1 diabetes and insulin resistance syndrome. Isolated hearts were obtained from three rat cohorts: healthy controls, streptozotocin (STZ)-induced diabetes, and high-fat diet (HFD)-induced insulin resistance syndrome. All hearts underwent 25 min ischemia and 30 min or 120 min reperfusion. They were assigned to receive either no intervention or a single dose of EPO at the onset of reperfusion. In hearts from healthy controls, EPO decreased infarct size (14.36 ± 0.60 and 36.22 ± 4.20% of left ventricle in EPO-treated and untreated hearts, respectively, p < 0.05) and increased phosphorylated forms of Akt, ERK1/2, and their downstream target GSK-3β. In hearts from STZ-induced diabetic rats, EPO did not decrease infarct size (32.05 ± 2.38 and 31.88 ± 1.87% in EPO-treated and untreated diabetic rat hearts, respectively, NS) nor did it increase phosphorylation of Akt, ERK1/2, and GSK-3β. In contrast, in hearts from HFD-induced insulin resistance rats, EPO decreased infarct size (18.66 ± 1.99 and 34.62 ± 3.41% in EPO-treated and untreated HFD rat hearts, respectively, p < 0.05) and increased phosphorylation of Akt, ERK1/2, and GSK-3β. Administration of GSK-3β inhibitor SB216763 was cardioprotective in healthy and diabetic hearts. STZ-induced diabetes abolished EPO-induced cardioprotection against I/R injury through a disruption of upstream signaling of GSK-3β. In conclusion, direct inhibition of GSK-3β may provide an alternative strategy to protect diabetic hearts against I/R injury.     

Nitric oxide/cGMP signalling mediates the cardioprotective action of adrenomedullin in reperfused myocardium

We demonstrated previously that adrenomedullin (AM), when given during early reperfusion, limited infarct size in rat heart. The present study was undertaken to provide direct evidence of the NO-dependency of AM’s cardioprotective action by assessing NO biosynthesis and involvement of the soluble guanylyl cyclase (sGC) pathway. Perfused hearts from male CD-1 mice were subjected to 30-min left coronary occlusion and 60-min reperfusion. Infarct size was determined by tetrazolium staining. AM 10 nM was administered from 20 min after coronary occlusion until 10 min after reperfusion. Coronary effluent was analysed for NO₂ ⁻ and NO₃ ⁻, and myocardial samples were analysed for NO₂ ⁻, NO₃ ⁻, nitroso-adducts and cGMP concentration. To examine the role of NO/sGC signalling in the infarct-limiting action of AM, further hearts received the sGC inhibitor ODQ 2 μM. AM treatment stimulated NO synthesis, indicated by increased NO₂ ⁻ efflux in coronary effluent throughout reperfusion (summarised as area under curve, AM 29.2 ± 3.9 vs. control 14.4 ± 2.8 μmol min² mL⁻¹, P < 0.05). AM limited infarct size (35.4 ± 2.7 vs. 12.2 ± 2.3%, P < 0.01), associated with a 2.45-fold increase (P < 0.05) in myocardial cGMP concentration at 10 min after reperfusion. ODQ abolished the infarct size-limiting effect of AM (28.9 ± 4.3%). These data provide the first evidence that AM increases NO bioavailability in intact murine myocardium and confirm that the NO/sGC/cGMP pathway is central to the cytoprotective action of AM against ischaemia–reperfusion injury.