Na/K-ATpase α2-Subunit Preferentially Modulates Ca Transients and SR Ca Release in Cardiac Myocytes

Abstract

Na/K-ATPase (NKA) is essential in regulating [Na]i, and thus cardiac myocyte Ca and contractility via Na/Ca exchange. Different NKAα-subunit isoforms are present in heart and may differ functionally, depending on differential membrane localization. In smooth muscle and astrocytes, NKA-α2 is located at the junctions with endo(sarco)plasmic reticulum, where they could regulate local Na, and indirectly junctional cleft [Ca]. In contrast, NKA-α1 is ubiquitously distributed and may regulate bulk [Na]i. It is controversial whether this model holds for cardiac myocytes. We measured the effect of selective NKA-α2/NKA-α1 inhibition with low concentrations of ouabain on [Na]i, Ca transients and fractional SR Ca release in cardiac myocytes from wild-type (WT) mice and transgenic mice in which NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant (SWAP mice), respectively. Different [ouabain] (5 and 0.1 μM, respectively) were used to attain a similar level of total NKA inhibition in WT and SWAP mice. Ouabain increased Ca transients in WT (F/F0=1.84±0.13 vs. 1.54±0.12 under control conditions) but not in SWAP mice (1.49±0.15 vs. 1.46±0.12), despite a similar and modest increase in [Na]i (≤2 mM). Ca transient increase in WT mice was mediated specifically by NKA-α2 inhibition, because 5 μM ouabain had no effect in transgenic mice where both NKA-α1 and NKA-α2 are ouabain-resistant. Ouabain also significantly increased the fractional SR Ca release in WT mice (from 23±2% to 28±2%) but not SWAP mice (23±3% with and without ouabain). Dual-color immunofluorescence measurements coupled with spatial cross-correlation analysis revealed that NKA-α2 is co-localized to a significant extent with both Na/Ca exchanger (43% co-localization) and ryanodine receptors (43% co-localization) in cardiac myocytes. In conclusion, our data indicate that NKA-α2 has a more prominent role (vs. NKA-α1) in modulating Ca transients and SR Ca release in cardiac myocytes.