Hypercholesterolemia downregulates autophagy in the rat heart

Lipids Health Dis. 2017 Mar 23;16(1):60. doi: 10.1186/s12944-017-0455-0.

Abstract

Background: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart.

Methods: Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot.

Results: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments.

Conclusions: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals.

Keywords: ATG8; Apoptosis; Autophagy; Caspase; Hypercholesterolemia; Necroptosis; Programmed necrosis; Receptor-interacting serine/threonine-protein kinase.

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Autophagy / drug effects*
  • Beclin-1 / genetics
  • Beclin-1 / metabolism
  • Biomarkers / metabolism
  • Caspase 3 / genetics
  • Caspase 3 / metabolism
  • Cholesterol / administration & dosage
  • Cholesterol / adverse effects*
  • Cholic Acid / administration & dosage
  • Cholic Acid / adverse effects*
  • Diet, High-Fat / adverse effects
  • Gene Expression Regulation / drug effects
  • Heart / drug effects
  • Hypercholesterolemia / etiology
  • Hypercholesterolemia / genetics
  • Hypercholesterolemia / metabolism*
  • Hypercholesterolemia / pathology
  • Male
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Necrosis / etiology
  • Necrosis / genetics
  • Necrosis / metabolism
  • Necrosis / pathology
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Rats
  • Rats, Wistar
  • Receptor-Interacting Protein Serine-Threonine Kinases / genetics
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
  • Ribosomal Protein S6 Kinases / genetics
  • Ribosomal Protein S6 Kinases / metabolism
  • Signal Transduction
  • TOR Serine-Threonine Kinases / genetics
  • TOR Serine-Threonine Kinases / metabolism
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Beclin-1
  • Becn1 protein, rat
  • Biomarkers
  • LC3 protein, rat
  • Microtubule-Associated Proteins
  • rab7 protein
  • Cholesterol
  • MLKL protein, rat
  • Protein Kinases
  • TOR Serine-Threonine Kinases
  • mTOR protein, rat
  • Protein-Serine-Threonine Kinases
  • RIPK1 protein, rat
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ribosomal Protein S6 Kinases
  • receptor-interacting protein 3, rat
  • Casp3 protein, rat
  • Caspase 3
  • rab GTP-Binding Proteins
  • Cholic Acid