Functional cardiac orexin receptors: role of orexin-B/orexin 2 receptor in myocardial protection

Orexins/hypocretins exert cardiovascular effects which are centrally mediated. In the present study, we tested whether orexins and their receptors may also act in an autocrine/paracrine manner in the heart exerting direct effects. Quantitative reverse transcription-PCR (RT-PCR), immunohistochemical and Western blot analyses revealed that the rat heart expresses orexins and orexin receptors (OXR). In isolated rat cardiomyocytes, only orexin-B (OR-B) caused an increase in contractile shortening, independent of diastolic or systolic calcium levels. A specific orexin receptor-2 (OX2R) agonist ([Ala11, d-Leu15]-Orexin B) exerted similar effects as OR-B, whereas a specific orexin receptor-1 (OX1R) antagonist (SB-408124) did not alter the responsiveness of OR-B. Treatment of the same model with OR-B resulted in a dose-dependent increase in myosin light chain and troponin-I (TnI) phosphorylation. Following ischaemia/reperfusion in the isolated Langendorff perfused rat heart model, OR-B, but not OR-A, exerts a cardioprotective effect; mirrored in an in vivo model as well. Unlike OR-A, OR-B was also able to induce extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2) and Akt phosphorylation in rat myocardial tissue and ERK1/2 phosphorylation in human heart samples. These findings were further corroborated in an in vivo rat model. In human subjects with heart failure, there is a significant negative correlation between the expression of OX2R and the severity of the disease clinical symptoms, as assessed by the New York Heart Association (NYHA) functional classification. Collectively, we provide evidence of a distinct orexin system in the heart that exerts a cardioprotective role via an OR-B/OX2R pathway.

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Effects of the SOD mimetic, M40403, on prostaglandin production in an in vivo model of ischemia and reperfusion in rat heart

Inflammation Research ◽

10.1007/s000110300037 ◽

2003 ◽

Vol 52 (0) ◽

pp. s23-s24 ◽

Cited By ~ 1

Author(s):

C. Marzocca ◽

A. Vannacci ◽

S. Cuzzocrea ◽

D. Salvemini ◽

P. F. Mannaioni ◽

...

Keyword(s):

Rat Heart ◽

In Vivo Model ◽

Ischemia And Reperfusion ◽

Prostaglandin Production

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Desensitization and Incomplete Recovery of Hepatic Target Genes After Chronic Thyroid Hormone Treatment and Withdrawal in Male Adult Mice

Endocrinology ◽

10.1210/en.2015-1848 ◽

2016 ◽

Vol 157 (4) ◽

pp. 1660-1672 ◽

Cited By ~ 16

Author(s):

Kenji Ohba ◽

Melvin Khee-Shing Leow ◽

Brijesh Kumar Singh ◽

Rohit Anthony Sinha ◽

Ronny Lesmana ◽

...

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Adult Male ◽

Target Genes ◽

Clinical Symptoms ◽

Monocarboxylate Transporter ◽

In Vivo Model ◽

Male Adult ◽

Tsh Levels

Abstract Clinical symptoms may vary and not necessarily reflect serum thyroid hormone (TH) levels during acute and chronic hyperthyroidism as well as recovery from hyperthyroidism. We thus examined changes in hepatic gene expression and serum TH/TSH levels in adult male mice treated either with a single T3 (20 μg per 100 g body weight) injection (acute T3) or daily injections for 14 days (chronic T3) followed by 10 days of withdrawal. Gene expression arrays from livers harvested at these time points showed that among positively-regulated target genes, 320 were stimulated acutely and 429 chronically by T3. Surprisingly, only 69 of 680 genes (10.1%) were induced during both periods, suggesting desensitization of the majority of acutely stimulated target genes. About 90% of positively regulated target genes returned to baseline expression levels after 10 days of withdrawal; however, 67 of 680 (9.9%) did not return to baseline despite normalization of serum TH/TSH levels. Similar findings also were observed for negatively regulated target genes. Chromatin immunoprecipitation analysis of representative positively regulated target genes suggested that acetylation of H3K9/K14 was associated with acute stimulation, whereas trimethylation of H3K4 was associated with chronic stimulation. In an in vivo model of chronic intrahepatic hyperthyroidism since birth, adult male monocarboxylate transporter-8 knockout mice also demonstrated desensitization of most acutely stimulated target genes that were examined. In summary, we have identified transcriptional desensitization and incomplete recovery of gene expression during chronic hyperthyroidism and recovery. Our findings may be a potential reason for discordance between clinical symptoms and serum TH levels observed in these conditions.

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MicroRNA-34a protects myocardial cells against ischemia–reperfusion injury through inhibiting autophagy via regulating TNFα expression

Biochemistry and Cell Biology ◽

10.1139/bcb-2016-0158 ◽

2018 ◽

Vol 96 (3) ◽

pp. 349-354 ◽

Cited By ~ 17

Author(s):

Haifeng Shao ◽

Lili Yang ◽

Li Wang ◽

Bozan Tang ◽

Jian Wang ◽

...

Keyword(s):

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Heart Tissue ◽

Neonatal Rat ◽

Cell Counting ◽

In Vivo Model ◽

Myocardial Cells ◽

Rat Cardiomyocytes ◽

Cardiac Tissues

Background: ischemia–reperfusion (I/R) is a consequence of restored blood supply after myocardial infarction. Myocardial I/R injury can be alleviated by reducing autophagy in heart tissue. MicroRNA-34a (miR-34a) has been shown to regulate autophagy in a renal model of I/R, but it is not known whether it can protect cardiac tissues from I/R injury. This study investigated how miR-34a protects myocardial cells from I/R injury by inhibiting autophagy via regulation of tumor necrosis factor α (TNFα). Methods: we constructed an I/R model in vivo using Langendorff perfusion, and we constructed an in vivo model by treating neonatal rat cardiomyocytes (NRCMs) with hypoxia–reoxygenation (H/R method). Transfected adenoviral-overexpressed miR-34a mimics and controlled NRCMs after H/R. We analyzed cell viability using the MTT assay and a cell counting kit-8 (CCK-8) assay. Changes in the rate of apoptosis were detected by flow cytometry. We investigated the effect mechanisms of miR-34a with Western blot and luciferase assays. Results: miR-34a expression decreased after in vivo reperfusion of the myocardial cells and heart tissues of neonatal rats. MiR-34a reduced apoptosis of the NRCMs and autophagy levels, simultaneously, after H/R injury. Further, miR-34a decreased the expression of Lc3-II and p62, indicating that miR-34a reduces myocardial I/R injury by decreasing TNFα expression. Conclusion: miR-34a can inhibit autophagy levels after I/R by targeting TNFα, thereby reducing myocardial injury.

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The Luciferase-Expressing Glioma-261 Murine Models Elicit an Immune-Mediated Antitumor Response

Neurosurgery ◽

10.1093/neuros/nyz310_812 ◽

2019 ◽

Vol 66 (Supplement_1) ◽

Author(s):

Victoria E Sanchez ◽

John Lynes ◽

Stuart Walbridge ◽

Xiang Wang ◽

Nancy A Edwards ◽

...

Keyword(s):

Inflammatory Response ◽

Tumor Growth ◽

Cell Line ◽

Median Survival ◽

Immune Cell ◽

Clinical Symptoms ◽

In Vivo Model ◽

Murine Models

Abstract INTRODUCTION Preclinical models that accurately recapitulate the immunosuppressive properties of human gliomas are essential to assess immune therapeutics. Glioma-261 (GL261) murine glioma cells are widely used as an in Vivo model of glioma. Our group has previously shown that the red-shifted luciferase-expressing cell line, GL261 Red-FLuc, creates an inflammatory response when implanted intracranially in C57BL/6 mice. However, it remains unclear if this is particular to GL261 Red-Fluc or any GL261 cell line transfected with luciferase-expressing genes. For this reason, we have additionally explored the inflammatory response of stably transfected, monoclonal GL261-luc2 cells. METHODS To evaluate the characteristics of these various cell lines, C57BL/6 mice (n = 10 in each group) underwent stereotaxic, intracranial implantation with GL261, GL261 Red-FLuc, or GL261-Luc2 cells at doses of 5 × 104 cells/5 μL or 3 × 105 cells/5 μL. Immunohistochemistry and flow cytometry of sacrificed mouse brains assess the frequency of immune cell populations. Magnetic resonance imaging (MRI) scans were also performed to monitor relative tumor growth. Finally, in Vitro cytokine profiles were evaluated by proteome microarray. RESULTS Kaplan-Meier survival analysis demonstrated that the median survival for mice implanted with GL261 cells at 5 × 104 cells/5 μL was 21 d. However, even at a higher tumor dose (3 × 105 cells/5 μL), the GL261-Red FLuc implanted mice did not reach median survival. Mice injected with the newly transfected GL261-Luc2 cells at 3 × 105 cells/5 μL reached median survival at 23 d, but median survival was not reached for GL261-Luc2 implanted mice at 5 × 104 cells/5 μL. MRI analysis reveals clear differences in tumor growth that correspond well with the onset of clinical symptoms and median survival. In addition, proteomic analysis reveals significantly elevated inflammatory cytokines such as IFNgamma, IL-7, and TNF-alpha in the supernatant of the GL261 Red-FLuc cells and upregulated IL-1alpha in GL261-Luc2 cells. Further immune characterization is ongoing. CONCLUSION Our data suggest that both GL261 Red-FLuc and GL261-luc2 murine models create an undesirable microenvironment for tumor growth by increasing proinflammatory modulators.

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Cardiac function and phosphorylation of contractile proteins

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1983.0040 ◽

1983 ◽

Vol 302 (1108) ◽

pp. 83-90 ◽

Cited By ~ 13

Keyword(s):

Light Chain ◽

Rat Heart ◽

Troponin I ◽

Contractile Proteins ◽

Perfused Heart ◽

Tension Development ◽

C Protein ◽

Perfused Hearts

The primary regulation of cardiac contractility is probably through changes in the level of cytoplasmic free Ca 2+ . In the stimulation of contraction by catecholamines, secondary controls may be present at the level of the contractile proteins. Troponin-I, a subunit of the troponin complex of the thin filament, and C-protein, a thick filament component, are both phosphorylated in perfused hearts in response to catecholamines over time courses similar to that for the increase in contraction. Both proteins are also phosphorylated rapidly in vitro by cyclic-AMP-dependent protein kinase. Phosphorylation of troponin-I causes a decrease in the sensitivity of both cardiac myofibrillar ATPase and tension development of skinned fibre preparations to Ca 2+ , and also an increase in the rate of dissociation of Ca 2+ from isolated troponin. These results support the hypothesis that the role of phosphorylation of cardiac troponin-I is to contribute to the increased rate of relaxation of the heart that is observed with catecholamines. C-protein is phosphorylated to a maximum of 4-5 mol phosphate per mole protein both in vivo and in vitro . At present, however, the functions of both C-protein itself and its phosphorylation are unknown. Dephosphorylation of these contractile proteins after catecholamine stimulation is slow in perfused heart, although the rate can be increased by cholinergic agents. Phosphorylase, in contrast, is rapidly dephosphorylated under these circumstances. Phosphoprotein phosphatases relatively specific for phosphorylase have been identified in rat heart, whereas troponin-I appears to be dephosphorylated by general phosphatases. These observations account for the different rates of dephosphorylation of phosphorylase and the contractile proteins, but do not explain the slow dephosphorylation of the latter. In control perfused hearts myosin P-light chain was 50 % phosphorylated, and this was not changed by perfusion with positive inotropic agents or by short-term ischaemia. It was also unchanged during long-term hormonal modifications. Perfusions with 32 P 1 in rat heart give a half-time for the turnover of phosphate bound to the P-light chain of 2-4 min, showing that the myosin light chain kinase and phosphatase are active in the heart. It is hypothesized that under control conditions the kinase is already fully active, and that an increase in cytoplasmic Ca 2+ cannot therefore cause further activation of the enzyme.

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Developing and Characterizing a Mouse Model of Hepatotoxicity Using Oral Pyrrolizidine Alkaloid (Monocrotaline) Administration, with Potentiation of the Liver Injury by Co-administration of LPS

Natural Product Communications ◽

10.1177/1934578x1000500922 ◽

2010 ◽

Vol 5 (9) ◽

pp. 1934578X1000500 ◽

Cited By ~ 1

Author(s):

Mohamed Sadek Abdel-Bakky ◽

Mohamed A. Hammad ◽

Larry A. Walker ◽

Mohammad K. Ashfaq

Keyword(s):

Liver Injury ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Pyrrolizidine Alkaloid ◽

Human Subjects ◽

Real Life ◽

Plasma Chemistry ◽

In Vivo Model ◽

In Vivo Models

Oral administration of xenobiotics is preferable for research in In Vivo models because it mimics the real life situation of human subjects. Therefore, oral (po) monocrotaline (MCT) (a common contaminant of dietary supplements)/intraperitoneal (ip) lipopolysaccharides (LPS)-induced liver injury possibly imitates idiosyncratic hepatotoxicity in humans. Cytokines, for example interleukin-1β (IL-1β) and transforming growth factor beta (TGF-β) are known to play a role in the development of toxicity and repair processes, respectively. The purpose of this study was to develop and characterize a model of po MCT/ip LPS hepatotoxicity which may elucidate the mechanisms of injury. ND4 male mice were given MCT (200 mg/kg) followed 4 h later by LPS (6 mg/kg). Blood samples were drawn for plasma chemistry and IL-1β. Animals were euthanized and livers were harvested at different time points. We have shown that MCT/LPS cotreatment results in significant elevation of plasma alanine aminotransferase (ALT), CRP, IL-1β and TGF-β. Histopathological evaluation revealed diffuse degenerative injury. In summary, we have established a reproducible in vivo model of hepatotoxicity by po MCT/ip LPS cotreatment that may closely mimic real life idiosyncratic hepatotoxicity.

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Ameliorating Effect of Klotho Protein on Rat Heart during I/R Injury

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/6427284 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Agnieszka Olejnik ◽

Anna Krzywonos-Zawadzka ◽

Marta Banaszkiewicz ◽

Iwona Bil-Lula

Keyword(s):

Oxidative Stress ◽

Rat Heart ◽

Troponin I ◽

Tissue Injury ◽

Heart Function ◽

Ion Homeostasis ◽

Contractile Dysfunction ◽

Rat Cardiomyocytes ◽

Cell Contractility ◽

Klotho Protein

An essential procedure for the treatment of myocardial infarction is restoration of blood flow in the obstructed infarct artery, which may cause ischaemia/reperfusion (I/R) injury. Heart I/R injury manifests in oxidative stress, metabolic and morphological disorders, or cardiac contractile dysfunction. Klotho protein was found to be produced in the heart tissue and participate in antioxidation or ion homeostasis. The aim of this study was to examine an influence of Klotho protein on the heart subjected to I/R injury. Wistar rats served as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischaemia, and isolated rat cardiomyocytes underwent chemical I/R in vitro, with or without recombinant Klotho protein administration. Haemodynamic parameters of heart function, cell contractility, markers of I/R injury and oxidative stress, and the level of contractile proteins such as myosin light chain 1 (MLC1) and troponin I (TnI) were measured. The treatment of hearts subjected to I/R injury with Klotho protein resulted in a recovery of heart mechanical function and ameliorated myocyte contractility. This improvement was associated with decreased tissue injury, enhanced antioxidant capacity, and reduced release of MLC1 and TnI. The present research showed the contribution of Klotho to cardioprevention during I/R. Thus, Klotho protein may support the protection from I/R injury and prevention of contractile dysfunction in the rat heart.

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GH receptor signaling in skeletal muscle and adipose tissue in human subjects following exposure to an intravenous GH bolus

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00024.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. E899-E905 ◽

Cited By ~ 52

Author(s):

Jens O. L. Jørgensen ◽

Niels Jessen ◽

Steen B. Pedersen ◽

Esben Vestergaard ◽

Lars Gormsen ◽

...

Keyword(s):

Dna Binding ◽

Kinase Activity ◽

Human Subjects ◽

Binding Activity ◽

In Vivo Model ◽

Gh Receptor ◽

Dna Binding Activity ◽

Igf I

Growth hormone (GH) regulates muscle and fat metabolism, which impacts on body composition and insulin sensitivty, but the underlying GH signaling pathways have not been studied in vivo in humans. We investigated GH signaling in biopsies from muscle and abdominal fat obtained 30 ( n = 3) or 60 ( n = 3) min after an intravenous bolus of GH (0.5 mg) vs. saline in conjunction with serum sampling in six healthy males after an overnight fast. Expression of the following signal proteins were assayed by Western blotting: STAT5/p-STAT5, MAPK, and Akt/PKB. IRS-1-associated PI 3-kinase activity was measured by in vitro phosphorylation of PI. STAT5 DNA binding activity was assessed with EMSA, and the expression of IGF-I and SOCS mRNA was measured by real-time RT-PCR. GH induced a 52% increase in circulating FFA levels with peak values after 155 min ( P = 0.03). Tyrosine-phosphorylated STAT5 was detected in muscle and fat of all subjects after GH. Activation of MAPK was observed in several lysates but without GH dependency. Neither PKB/Akt nor PI 3-kinase activity was affected by GH. GH-induced STAT5 DNA binding and expression of IGF-I mRNA were detected in fat, whereas expression of SOCS-1 and -3 tended to increase after GH in muscle and fat, respectively. We conclude that 1) STAT5 is acutely activated in human muscle and fat after a GH bolus, but additional downstream GH signaling was significant only in fat; 2) the direct GH effects in muscle need further characterization; and 3) this human in vivo model may be used to study the mechanisms subserving the actions of GH on substrate metabolism and insulin sensitivity in muscle and fat.

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