Systemic levels of endogenous cardiotonic steroids (CTS) vary in response to physiological and pathophysiological stresses such as exercise, pregnancy, hypertension, and heart failure. Ample experimental evidence has shown that exposure to CTS modulates cardiac Na/K‐ATPase (NKA) α1 ion‐pumping and non‐ion‐pumping functions, which profoundly affects cardiac contractility and structure. However, the physiological role of CTS as a group of hormones with distinct but related modulatory effects on cardiovascular equipoise and dysfunction remains poorly understood. We used 2 genetically engineered mouse models to test the hypothesis that the endogenous CTS/NKA α1 interaction confers cardiac resilience during a sustained hemodynamic challenge.Wild type mice expressing the relatively CTS‐insensitive NKA α1 and the CTS‐sensitive NKA α2 (α1r/rα2s/s), mice expressing NKA α1 mutated to CTS‐sensitivity (α1s/sα2s/s), and mice with cardiomyocyte‐specific NKA α1 knockout (α1−/−α2s/s) were treated with a pressor dose of Angiotensin II (Ang II) of 1.5 mg.kg−1. d−1 for 14 days via osmotic minipumps to induce adverse cardiac remodeling.All mice were born with the expected Mendelian frequency and survived to adulthood. In the α1s/sα2s/s mouse, total cardiac expression of NKA α1 and α2 assessed by western blotting and ouabain‐sensitive ATPase activity were unchanged, and the expected increased affinity for the CTS ouabain was detected. NKA expression and activity in the α1−/−α2s/s heart was consistent with a nearly complete ablation of NKA α1, partially compensated by an upregulation of NKA α2. Ang II or vehicle treatment was administered to 3 month‐old males (n=6–8/group), and cardiac remodeling was assessed by gravimetry, histology, and qPCR. In contrast to wild‐type α1r/rα2s/s, the Ang II treatment did not induce any significant cardiac hypertrophy or fibrosis in α1s/sα2s/s hearts. Conversely, the Ang II treatment was lethal in α1−/−α2s/s mice, with a 70% mortality rate at 8 days. A shorter (7 days) Ang II treatment revealed massive cardiac fibrosis (50% increase compared to α1r/rα2s/s). Taken together, these results support a physiological role of endogenous CTS in cardiac resilience through an interaction with the signaling cardiac NKA α1 isoform.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.