Transcriptomic changes triggered by hypoxia: Evidence for HIF-1α -independent, [Na⁺]_i/[K⁺]_i-mediated, excitation-transcription coupling

This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na⁺_i/K⁺_i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na⁺]_i and 2-fold reduction of [K⁺]_i. Using global gene expression profiling we found that Na⁺, K⁺-ATPase inhibition by ouabain or K⁺-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na⁺, K⁺-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na⁺, high-K⁺ medium abolished amplification of the [Na⁺]_i/[K⁺]_i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na⁺ and K⁺ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na⁺, high-K⁺ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na⁺_i/K⁺_i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.