Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA049

Poster Communications

HIF-1α controls the expression of Na/K-ATPase in hypoxic cardiomyocytes

E. Baloglu1, I. Ulus1, H. Mairbäurl2,3

1. Department of Pharmacology, School of Medicine, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey. 2. Medical Clinic VII, Sports Medicine, University of Heidelberg, Heidelberg, Germany. 3. Translational Lung Research Center (TLRC), part of the German Center of Lung Research (DZL), Heidelberg, Germany.

Na/K-ATPase is an integral membrane protein that functions as an ion pump by hydrolyzing ATP to pump three Na+ out the cell in exchange for two K+ per each pump cycle. Pump activity is important for regulating ion gradients, cell volume, resting membrane potential and electrical activity, and signal transduction in the heart. In ischemic heart and heart failure expression of the pump subunits decreases, contributing to decreased pump activity. Since in the ischemic heart cellular adaptation to hypoxia is regulated by hypoxia-inducible transcription factors (HIF), we tested whether the decreased Na/K-ATPase expression in hypoxic cardiomyocytes involves HIF signaling. Rat ventricular cardiomyocyte H-10 cells were used for the experiments. HIF-1α and HIF-2α were silenced in by adenoviral introduction of shRNAs at 200MOI (multiplicity of infection). Cells were exposed to normoxia (20% O2) and hypoxia (1.5% O2) for 24h. mRNA expressions of HIF-1α, HIF-2α, α1-and β1-Na/K-ATPase was measured by RT qPCR and normalized to 18S rRNA. The surface expression of α1-Na/K-ATPase subunit was measured after biotinylation and separation of biotin-labelled proteins with streptavidin coated beads. Proteins in the non-biotinylated fraction were considered intracellular. Protein expressions were measured by Western blot. Values are means ± S.D, compared by ANOVA or t-test. Hypoxia decreased the mRNA expression of α1-Na/K-ATPase (normoxia 1.08 ± 0.6 vs. hypoxia 0.43 ± 0.3, p=0.01) and β1-Na/K-ATPase (normoxia 0.89 ± 0.4 vs. hypoxia 0.42 ± 0.3, p=0.036). The decrease in Na/K-ATPase mRNA subunits was partially prevented by silencing HIF-1α but not HIF-2α (α1-Na/K-ATPase: hypoxia 0.43 ± 0.3 vs. hypoxia HIF-1α 0.68 ± 0.4, p=0.011; β1-Na/K-ATPase: hypoxia 0.42 ± 0.3 vs. hypoxia HIF-1α 0.79 ± 0.4, p=0.02). Hypoxia significantly decreased both the plasma membrane and intracellular expression of α1-Na/K-ATPase compared to normoxic cells (n=5, p<0.05); silencing HIF-1α fully prevented hypoxic inhibition of α1-Na/K-ATPase in both fractions (n=5, p<0.05). Results indicate that hypoxia decreased Na/K-ATPase in cardiomyocytes, which is due to inhibition of transcription and translation. Decreased mRNA-expression and amounts of α1-Na/K-ATPase protein inserted in to the plasma membrane and located intracellular in hypoxia is controlled by HIF-1α.

Where applicable, experiments conform with Society ethical requirements