Hypoxia-inducible factor 1 alpha (HIF-1α) is emerging as a downstream target of ouabain and other cardiotonic steroids (CTS), the specific ligands of the membrane Na + /K + -ATPase (NKA) complex. The potential therapeutic implications of this signaling axis are broad and highly cell-type specific, spanning oncology, immune regulation, and cardiovascular disease. However, many aspects of the cellular mechanism of action remain unresolved. Cardiac NKAs are critical to cellular ion homeostasis to support metabolic stability and contractile function. In humans, their heterogeneous subunit composition includes three isoforms of the main catalytic α-subunit. Our recent gene-targeting studies in mice suggest that α1-containing NKA (NKAα1) acts as a receptor for endogenously produced CTS and transduces hypertrophic growth signals through kinase-dependent pathways that are distinct from its canonical ion-transporting function. Given the central role of HIF-1α in cardiac hypertrophy, we employed a genetic approach to assess the isoform-specific role of NKAα1 in HIF-1α activation by CTS (ouabain) and non-CTS (phenylephrine, PE) hypertrophic signals. Specifically, we used CRISPR-mediated knockout (KO) of NKAα1 in human cardiomyocytes (AC16). NKAα1 KO reduced α1 expression by 65% and Na + /K + -ATPase-specific hydrolytic activity by 80%. In wild-type (WT) cells, ouabain treatment (100 nM, 1 h) increased HIF-1α expression by 30% as revealed by western blotting (n=4, p< 0.05). In contrast, ouabain failed to activate HIF-1α in NKAα1 KO cells. Furthermore, in response to PE exposure (50 μM, 6 h), WT AC16 cells showed a robust HIF-1α accumulation of over 20% (p< 0.05), whereas NKAα1 KO cells did not. After 24 h of exposure to PE (50µM), the cell area of WT AC16 cells was increased by about 45% (3520 ± 99 μm 2 vs 2443 ± 58 μm 2 , n=3, p< 0.0001) as determined by ImageJ in at least 100 cells/group from 3 independent experiments following Alexa FluorTM 568 phalloidin staining. In contrast, NKAα1 KO AC16 cells failed to undergo hypertrophic growth (2454 ± 71 μm 2 vs 2563 ± 59 μm 2 , n=3, NS). Together, these results suggest that NKAα1 is critical for HIF-1α activation by CTS and other hypertrophic signals such as PE, as well as hypertrophic remodeling. Additional genetic and pharmacological approaches in AC16 and in mice shall reveal the role of other cardiac NKA isoforms in HIF-1α regulation and hypertrophic growth in response to CTS and other growth stimuli. Funding Source: NIH R15 HL145666 (PI: Pierre), AHA 22POST917776 (PI: Pessoa), APS William Townsend Porter Fellowship (PI: Strause), and AHA 25TPA1477521 (PI: Pierre). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Strause et al. (2026) studied this question.