Pim1 Overexpression Prevents Apoptosis in Cardiomyocytes After Exposure to Hypoxia and Oxidative Stress via Upregulating Cell Autophagy

Cell Physiol Biochem. 2018;49(6):2138-2150. doi: 10.1159/000493817. Epub 2018 Sep 26.

Abstract

Background/aims: Microvascular obstruction (MVO), an undesirable complication of percutaneous coronary intervention, is independently associated with adverse left ventricle remodeling and poor prognosis after acute myocardial infarction. Hypoxia and oxidative stress major roles in the pathophysiology of MVO. Pim1 serves an important protective role in the ischemic myocardium, but the underlying mechanisms remain poorly defined. Autophagy in early hypoxia or during moderate oxidative stress has been demonstrated to protect the myocardium. In this study, we investigated the association between the protective effect of Pim1 and autophagy after hypoxia and oxidative stress.

Methods: Ventricular myocytes from neonatal rat heart (NRVMs) were isolated. NRVMs were exposed to hypoxia and H2O2. Rapamycin and 3-methyladenine (3-MA) were used as an activator and inhibitor of autophagy, respectively. pHBAd-Pim1 was transfected into NRVMs. We assessed cardiomyocyte apoptosis by Annexin V-FITC/PI flow cytometry. Autophagy was evaluated by mRFP-GFP-LC3 adenovirus infection by confocal microscopy. Western blotting was used to quantify apoptosis or autophagy protein (caspase-3, LC3, P62, AMPK, mTOR, ATG5) concentrations.

Results: Autophagy and apoptosis in NRVMs significantly increased and peaked at 3 h and 6 h, respectively, after exposure to hypoxia and H2O2. The mTOR inhibitor rapamycin induced autophagy and decreased cardiomyocyte apoptosis, but the autophagy inhibitor 3-MA decreased autophagy and increased apoptosis at 3 h after exposure to hypoxia and H2O2. Pim1 levels in NRVMs increased at 3 h and decreased gradually after exposure to hypoxia and H2O2. Pim1 overexpression enhanced autophagy and decreased apoptosis. Pim1-induced promotion of autophagy is partly the result of activation of the AMPK/mTOR/ATG5 pathway after exposure to hypoxia and H2O2.

Conclusion: Our results revealed that Pim1 overexpression prevented NRVMs from apoptosis via upregulating autophagy after exposure to hypoxia and oxidative stress, partly through activation of the AMPK/mTOR/ATG5 autophagy pathway.

Keywords: Apoptosis; Autophagy; Cardiomyocyte; Hypoxia; Oxidatve stress; Pim1.

MeSH terms

  • AMP-Activated Protein Kinases / metabolism
  • Animals
  • Apoptosis / drug effects
  • Autophagy* / drug effects
  • Autophagy-Related Protein 5 / metabolism
  • Cell Hypoxia*
  • Cells, Cultured
  • Hydrogen Peroxide / pharmacology
  • Microtubule-Associated Proteins / metabolism
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism
  • Oxidative Stress* / drug effects
  • Proto-Oncogene Proteins c-pim-1 / genetics
  • Proto-Oncogene Proteins c-pim-1 / metabolism*
  • Rats
  • Sequestosome-1 Protein / metabolism
  • Signal Transduction / drug effects
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / metabolism
  • Up-Regulation / drug effects

Substances

  • Autophagy-Related Protein 5
  • LC3 protein, rat
  • Microtubule-Associated Proteins
  • Sequestosome-1 Protein
  • Sqstm1 protein, rat
  • Hydrogen Peroxide
  • TOR Serine-Threonine Kinases
  • Pim1 protein, rat
  • Proto-Oncogene Proteins c-pim-1
  • AMP-Activated Protein Kinases
  • Sirolimus