Hypercholesterolemia downregulates autophagy in the rat heart.
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.
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary. giricz.zoltan@med.semmelweis-univ.hu.|Pharmahungary Group, Szeged, Hungary. giricz.zoltan@med.semmelweis-univ.hu.
Zoltán
http://orcid.org/0000-0003-2036-8665
Z
Giricz
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary.
Gábor
G
Koncsos
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojárov 10, 83232, Bratislava, Slovakia.
Tomáš
T
Rajtík
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary.|Department of Biochemistry, Faculty of Medicine, University of Szeged, Dóm tér 9, H-6720, Szeged, Hungary.
Zoltán V
ZV
Varga
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary.
Tamás
T
Baranyai
Department of Biochemistry, Faculty of Medicine, University of Szeged, Dóm tér 9, H-6720, Szeged, Hungary.
Csaba
C
Csonka
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojárov 10, 83232, Bratislava, Slovakia.
Adrián
A
Szobi
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Odbojárov 10, 83232, Bratislava, Slovakia.
Adriana
A
Adameová
Heart Institute, Cedars-Sinai Medical Center, 127 S. San Vicente Blvd., Los Angeles, CA, 90048, USA.
Roberta A
RA
Gottlieb
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary.|Pharmahungary Group, Szeged, Hungary.
Péter
P
Ferdinandy
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1 / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000008:administration & dosage / Q000009:adverse effects*; D019826:Cholic Acid / Q000008:administration & dosage / Q000009:adverse effects*; D059305:Diet, High-Fat / Q000009:adverse effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006321:Heart / Q000187:drug effects; D006937:Hypercholesterolemia / Q000209:etiology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009336:Necrosis / Q000209:etiology / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D000071186:Beclin-1; D015415:Biomarkers; D053148:Caspase 3; D002784:Cholesterol; D019826:Cholic Acid; D008869:Microtubule-Associated Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C512829:Becn1 protein, rat; C526109:Anxa3 protein, rat; C000716466:rab7 GTP-binding proteins, rat; C000610381:MLKL protein, rat; C546845:mTOR protein, rat; C586536:RIPK1 protein, rat; C582791:Ripk3 protein, rat; C505410:Casp3 protein, rat
ATG8; Apoptosis; Autophagy; Caspase; Hypercholesterolemia; Necroptosis; Programmed necrosis; Receptor-interacting serine/threonine-protein kinase