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MODULATION OF OXIDATIVE STRESS INDUCED APOPTOSIS AND AUTOPHAGY IN CARDIOMYOCYTES BY INTERMEDIN
  1. C McCarthy*,
  2. Y Zhao,
  3. P McKeag,
  4. D Treggiari,
  5. D McLaughlin,
  6. L Colhoun,
  7. D J Grieve,
  8. B J McDermott
  1. Queen's University Belfast, Centre for Vision and Vascular Science, School of Medicine, Dentistry and Biomedical Sciences, Grosvenor Road, Belfast BT12 6BA

    Abstract

    The heart is subjected to oxidative stress in conditions of increased reactive oxygen species (ROS) production, such as doxorubicin (DOX) chemotherapy. A major cause of associated ventricular dysfunction is cardiomyocyte loss through apoptosis, while autophagic processes can also be detrimental. Intermedin (IMD) has emerged as a major counter-regulatory peptide with cytoprotective properties. Here we examined its potential to be upregulated and attenuate indices of cardiomyocyte death by a mechanism involving NADPH oxidase-derived superoxide.

    In isolated adult C57BL/6J wild type (WT) mouse ventricular cardiomyocytes, DOX (5×10−7 M) increased preproIMD mRNA expression 3.5-fold, which was significantly decreased in NOX2-deficient cells. Similarly, IMD mRNA was upregulated by the direct pro-oxidant, H2O2 (10−7 M) in both WT and HL-1 cardiomyocytes, as was NOX2; DOX increased IMD protein (immuno-cytochemistry). Superoxide production stimulated by DOX or H2O2 (by ∼35%, lucigenin-enhanced chemiluminescence), was abolished by IMD (10−10 M–10−8 M) in both WT and HL-1 cardiomyocytes. Apoptosis in HL-1 cells, shown by DOX-induced increases in caspase 3/7 activity (3.5-fold), was decreased significantly by IMD (10−9 M), which also increased cell viability. Increased autophagosome formation after serum starvation or DOX treatment (by ∼50%, Cyto-ID fusion with LC3 protein), was decreased to control values by IMD (10−9 M). Confocal imaging showed numerous cytoplasmic punctate structures with DOX, whereas addition of IMD showed a diffuse LC-3 staining pattern similar to control.

    These findings indicate that a NOX2-mediated increase in IMD in cardiomyocytes could have a potential autocrine effect, acting at nM concentration to reduce levels of superoxide and so limit cell death by apoptotic and autophagic mechanisms.

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