Chronic stimulation of the β1-adrenergic pathway leads to cardiac hypertrophy and heart failure (HF). In mice overexpressing the β1-adrenergic receptor (β1), changes in Ca2+ handling precede the development of structural hypertrophy at an early stage of remodelling (8-12w). The Na+/K+ ATPase (NKA) is an important regulator of [Na+]i, and indirectly of Ca2+ homeostasis. Therefore we investigated putative changes in NKA activity, regulation and expression as well as mechanisms linked to the regulation of Na+ and Ca2+ in an early stage of HF. Single left ventricular myocytes were isolated from 8-12 weeks old male β1 mice and wild-type littermates (WT). For Na+-measurements, myocytes were loaded with SBFI-AM. Relaxation of Ca2+ transients (CaT, fluo-3 AM) was measured as the time constant of decay by exponential fitting (time constant tauStim for CaT during 0.5Hz stimulation; and tauCaff for caffeine-induced transients, 20 mM). For NKA pump current measurements (Ip) a Cs-Asp based pipette solution with 10 mM Na+ was used. To measure maximal Ip, Cs-Asp was replaced with Na-Asp to a final concentration of 100 mM [Na+]pip. Cells were superfused with normal Tyrode solution with additional, in mM: 2 BaCl2, 2 NiCl2 (pH=7.4 with NaOH), at 37°C. Ip was measured as the difference in outward current, at 0 mV, after rapid solution switch to K+-free solution. Currents were normalized to cell capacitance. Protein expression of NKA subunit α1 and α2, NHE 1, TRPC 1/3/6, PLM and PLM phosphorylation were determined using Western blotting in whole ventricle homogenates. Protein densities were normalized to GAPDH. In β1 myocytes, CaT showed a slower decline (tauStim 232±27ms in β1 vs. 180±17 ms in WT;mean±S.E.;n≥11/group;P<0.05). Ca2+ removal via forward NCX was significantly slowed. Cytosolic [Na+] was increased in resting cells (24.3±4.5 vs. 14.2±2.5 mM), and during stimulation (1Hz: 27.6±5.1 vs. 17.6±3.5 mM; 3 Hz:28.6±5.4 vs. 19.7±4.4 mM;n=14 and 8 respectively; all P<0.05). Protein levels of the NKA α1 subunit, NHE 1, TRPC 1,3 and 6 were unchanged, whereas the α2 subunit was reduced. Phospholemman (PLM), an inhibitory subunit of NKA, showed increased phosphorylation at Ser68 and the total amount was reduced in β1 mice. With [Na+]pip=10 mM, Ip density was significantly increased in β1 myocytes. Maximal Ip density was unchanged. In β1 mice, the slower Ca2+ removal via NCX is related to increased cytosolic [Na+]i. Proteins related to Na+ influx, NHE 1, TRPC 1/3 and 6, are not differentially regulated at this early stage of cardiac remodeling. At physiological [Na+]i, NKA current is increased, consistent with the higher phosphorylation degree of PLM, thus rather acting as a compensatory mechanism for the high Na+ levels during early remodeling in heart failure.