FGF-2 Transcriptionally Down-Regulates the Expression of BNIP3L via PI3K/Akt/FoxO3a Signaling and Inhibits Necrosis and Mitochondrial Dysfunction Induced by High Concentrations of Hydrogen Peroxide in H9c2 Cells

Cell Physiol Biochem. 2016;40(6):1678-1691. doi: 10.1159/000453217. Epub 2016 Dec 23.

Abstract

Background/aims: Cardiovascular disease is a growing major global public health problem. Necrosis is one of the main forms of cardiomyocyte death in heart disease. Oxidative stress is regarded as one of the key regulators of cardiac necrosis, which eventually leads to cardiovascular disease. Many pharmacological and in vitro studies have suggested that FGF-2 can act directly on cardiomyocytes to maintain the integrity and function of the myocardium and prevent damage during oxidative stress. However, the mechanisms by which FGF-2 rescues the myocardium from oxidative stress damage in cardiovascular disease remain unclear. The present study explored the protective effects of FGF-2 in the H2O2-induced necrosis of H9C2 cardiomyocytes as well as the possible signaling pathways involved.

Methods: Necrosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using a Cell Counting Kit-8 (CCK8) assay and flow cytometry analysis. The cells were pretreated with the PI3K/Akt inhibitor Wortmannin to investigate the possible involvement of the PI3K/Akt pathway in the protection by FGF-2. The levels of Akt, p-Akt, FoxO3a, p-FoxO3a, and BNIP3L were detected by Western blot. Chromatin immuno-precipitation (ChIP) analysis was used to test whether FoxO3a binds directly to the BNIP3L promoter region. A luciferase assay was used to study the effects of FoxO3a on BNIP3L gene promoter activity. Mitochondrial ΔΨM was quantified using tetramethylrhodamine methyl ester perchlorate (TMRM). The mitochondrial oxygen consumption rate (OCR) was assessed with a Seahorse XF24 Analyzer.

Results: Treatment with H2O2 decreased the phosphorylation of Akt and FoxO3a, and it induced the nuclear localization of FoxO3a and the necrosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by the PI3K/Akt inhibitor Wortmannin. ChIP analyses indicated that FoxO3a binds directly to the BNIP3L promoter region. Using a luciferase assay, we further observed that FoxO3a increased BNIP3L gene promoter activity. As expected, overexpression of BNIP3L in H9C2 cardiomyoblast cells reduced the cardioprotection of FGF-2 in H2O2-induced necrosis and mitochondrial dysfunction.

Conclusions: The present data suggest that FGF-2 protects against H2O2-induced necrosis of H9C2 cardiomyocytes via the activation of the PI3K/Akt/FoxO3a pathway. Moreover, the present results demonstrate that FoxO3a is an important transcription factor that acts by binding to the promoter and promoting the transcription of BNIP3L, and it contributes to the necrosis and mitochondrial dysfunction induced by H2O2 in H9c2 cardiomyoblast cells.

MeSH terms

  • Animals
  • Base Sequence
  • Cardiotonic Agents / pharmacology
  • Cell Line
  • Cytoprotection / drug effects
  • Down-Regulation / drug effects*
  • Down-Regulation / genetics
  • Fibroblast Growth Factor 2 / pharmacology*
  • Forkhead Box Protein O3 / metabolism
  • Hydrogen Peroxide / toxicity*
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Models, Biological
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Necrosis
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphorylation / drug effects
  • Promoter Regions, Genetic / genetics
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Rats
  • Signal Transduction / drug effects
  • Transcription, Genetic / drug effects*

Substances

  • BNIP3L protein, rat
  • Cardiotonic Agents
  • FOXO3 protein, rat
  • Forkhead Box Protein O3
  • Membrane Proteins
  • Proto-Oncogene Proteins
  • Fibroblast Growth Factor 2
  • Hydrogen Peroxide
  • Proto-Oncogene Proteins c-akt