Hypoxia and HIF-1 Regulate the Activity and Expression of Na,K-ATPase Subunits in H9c2 Cardiomyoblasts

The optimal function of the Na,K-ATPase (NKA) pump is essential for the heart. In ischemic heart disease, NKA activity decreases due to the decreased expression of the pump subunits. Here, we tested whether the hypoxia-inducible transcription factor (HIF-1 alpha), the key signaling molecule regulating the adaptation of cells to hypoxia, is involved in controlling the expression and cellular dynamics of alpha 1- and beta 1-NKA isoforms and of NKA activity in in-vitro hypoxic H9c2 cardiomyoblasts. HIF-1 alpha was silenced through adenoviral infection, and cells were kept in normoxia (19% O-2) or hypoxia (1% O-2) for 24 h. We investigated the mRNA and protein expression of alpha 1-, beta 1-NKA using RT-qPCR and Western blot in whole-cell lysates, cell membranes, and cytoplasmic fractions after labeling the cell surface with NHS-SS-biotin and immunoprecipitation. NKA activity and intracellular ATP levels were also measured. We found that in hypoxia, silencing HIF-1 alpha prevented the decreased mRNA expression of alpha 1-NKA but not of beta 1-NKA. Hypoxia decreased the plasma membrane expression of alpha 1-NKA and beta 1- NKA compared to normoxic cells. In hypoxic cells, HIF-1 alpha silencing prevented this effect by inhibiting the internalization of alpha 1-NKA. Total protein expression was not affected. The decreased activity of NKA in hypoxic cells was fully prevented by silencing HIF-1 alpha independent of cellular ATP levels. This study is the first to show that in hypoxic H9c2 cardiomyoblasts, HIF-1 alpha controls the internalization and membrane insertion of alpha 1-NKA subunit and of NKA activity. The mechanism behind this regulation needs further investigation.