HIF-2 Controls Expression and Intracellular Trafficking of the 2-Subunit of Na,K-ATPase in Hypoxic H9c2 Cardiomyocytes

The Na,K-ATPase (NKA) pump plays essential roles for optimal function of the heart. NKA activity decreases in necropsy materials from ischemic heart disease, heart failure and in experimental models. Cellular adaptation to hypoxia is regulated by hypoxia-induced transcription factors (HIF); we tested whether HIFs are involved in regulating the expression and intracellular dynamics of the alpha 2-isoform of NKA (alpha 2-NKA). HIF-1 alpha and HIF-2 alpha expression was suppressed in H9c2 cardiomyocytes by adenoviral infection, where cells were kept in 1% O2 for 24 h. The silencing efficiency of HIFs was tested on the mRNA and protein expression. We measured the mRNA expression of alpha 2-NKA in HIF-silenced and hypoxia-exposed cells. The membrane and intracellular expression of alpha 2-NKA was measured after labelling the cell surface with NHS-SS-biotin, immunoprecipitation and Western blotting. Hypoxia increased the mRNA expression of alpha 2-NKA 5-fold compared to normoxic cells in an HIF-2 alpha-sensitive manner. The plasma membrane expression of alpha 2-NKA increased in hypoxia by 2-fold and was fully prevented by HIF-2 alpha silencing. Intracellular expression of alpha 2-NKA was not affected. These results showed for the first time that in hypoxic cardiomyocytes alpha 2-NKA is transcriptionally and translationally regulated by HIF-2 alpha. The molecular mechanism behind this regulation needs further investigation.