Role of Hypoxia-Inducible Factors (HIF) in Regulating Intracellular Trafficking of Na⁺/K⁺-ATPase Subunites in in-vitro Ischmic Heart Model


Gençay G., Kılınç E., Baloğlu E.

46. Ulusal Fizyoloji Kongresi, Samsun, Turkey, 8 - 10 October 2021, pp.21

  • Publication Type: Conference Paper / Summary Text
  • City: Samsun
  • Country: Turkey
  • Page Numbers: pp.21

Abstract

AIM: Regular activity of Na⁺/K⁺-ATPase pump is essential for optimal function of the heart. In ischemic heart disease, heart failure and experimental models pump activity and subunit expression decreases. Activity of hypoxia-induced transcription factors (HIF); critical for adaptation of cells to low oxygen tension, increases in ischemic heart and heart failure. Here we tested whether HIFs are involved in regulating expression and localization of Na⁺/K⁺-ATPase subunits in in-vitro ischemic heart.

METHODS: H9c2 cardiomyocytes were used for the experiments. Adenovirus mediated HIF-1α and HIF-2α silenced cells were kept in 1.5%O₂ for 24 hours. We investigated expression of Na⁺/K⁺-ATPase α1, α2, β1 subunits by Western Blot in cell membranes and cytoplasmic fractions after labeling cell surface with NHS-SS-biotin and immunoprecipitation. Confocal microscopy was used for intracellular localization experiments.

RESULTS: Hypoxia decreased membrane expression of α1 Na⁺/K⁺-ATPase (25%) (p=0.003) compared to normoxic cells (%19O₂) and increased internalization (p=0.004). HIF-1α silencing increased membrane translocation (p=0.004) and prevented internalization in hypoxic cells (p=0.029). There was no effect of HIF-2α silencing. Hypoxia increased membrane expression of α2 subunit (p=0.005) was prevented by HIF-2α silencing (p=0.006), intracellular expression was not affected. The decreased membrane expression of β1 subunit by hypoxia (p<0.001) was independent of HIFs. Confocal microscopy showed highly stained α1 subunit at cell-cell contact regions, co-localization with β1 subunit which seemed less pronounced in hypoxic cells, however unclear α1 and β1 localization in HIF-1α silenced cells. In hypoxia α2 subunit highly stained at the organels close to cell nucleus; HIF-2α silencing prevented these changes.

CONCLUSION: Results indicate that in hypoxic cardiomyocytes HIF-1α controls internalization and degradation of α1 subunit, α2 subunit is regulated transcriptionaly by HIF-2α and protein synthesis of β1 subunit is inhibited. Impact of HIFs on Na⁺/K⁺-ATPase activity will be investigated in future studies.

This study has been funded by TÜBİTAK project no: 119S162 Keywords: Cardiac Ischemia, Hypoxia, Na⁺/K⁺-ATPase, HIF.