Abstract Body: Cardiac hypertrophy is an adaptive stress response that can progress to pathological remodeling and heart failure. Hypoxia-inducible factor-1α (HIF-1α), a key regulator of cardiac remodeling in cardiomyocytes, has emerged as a downstream target of cardiotonic steroids (CTS). CTS exert their cellular effects through the Na⁺/K⁺-ATPase (NKA), whose α1 subunit functions as an ion pump and signaling receptor. At pharmacological concentrations that inhibit NKA-dependent ion transport and activate signaling, CTS stabilize HIF-1α in the intact rat heart. The mechanism was shown to include increased intracellular sodium (Na_i), but the involvement of other mechanisms of NKA α1 has not been ruled out. Because NKA α1 also activates signaling regulating cell growth, we hypothesized that NKA α1 regulates HIF-1α activation and hypertrophic signaling through mechanisms beyond ion transport. To assess this, we compared HIF-1α activation and hypertrophic response in AC16 human ventricular cardiomyocytes with intact (WT) or CRISPR-mediated knockout (KO) NKA α1.

In AC16 with a 65% reduction of NKA α1 expression (p<0.001, n=10), Na⁺/K⁺-ATPase enzymatic activity decreased by 80% (p<0.05, n=5), resulting in a two-fold increase in Na_i (126.3 ± 2.6 vs 243.7 ± 12.7 nmol/mg, p<0.01, n=4). CTS ouabain (100 nM, 1 h) increased Na_i in both WT and KO cells (170.6 ± 4.0 vs 411.3 ± 13.6 nmol/mg, p<0.01, n=3), leading to increased HIF-1α expression in WT (30%, p<0.05, n=4) but not in KO cells. Consistent with a role of NKA α1 in the control of HIF-1α and hypertrophy, phenylephrine (PE, 50 µM, 6 h) increased HIF-1α (40%, p<0.05, n=4) in WT but not in KO cells. Finally, PE (50 µM, 24 h) produced hypertrophic growth (2443 ± 58 μm² vs 3520 ± 99 μm², p<0.0001, n=3) in WT, but not in KO cells (2454 ± 71 μm² vs 2563 ± 59 μm², n=3).

In conclusion, these data suggest that NKA α1 integrity is a determinant of cardiac hypertrophic response through mechanisms that go beyond cellular ion homeostasis and may involve the regulation of HIF-1α. Understanding NKA α1–HIF-1α signaling axis may inform new paradigms in pathological cardiac remodeling.