Abstract 11566: Distribution and New Function of Na, K-ATPase and Its Phosphoprotein Regulator Phospholemman in Cardiac Myocytes

Abstract

Introduction: Na, K-ATPase (NKA), regulated by phosphoprotein phospholemman (PLM), contributes to excitation-contraction via the Na,Ca-exchanger at cardiac T-tubules (TTs). It is known that the intercalated discs (IDs) contain concentrated specific Na channels and K channels, that rely on ion gradients set by NKA. Yet, the coordinated trafficking and localization of NKA and PLM in cardiomyocytes (CMs) remains incompletely understood, Hypothesis: NKA and PLM are concentrated in the ID and essential for cardiac conduction. Methods: We have made five antibodies (Abs) that specifically identify PLM (B8, PLM-N-term, and PLM-C-term) or NKA (NKA-α1 and Pan-α, recognizing both α1 and α2 subunits of NKA). Confocal immunofluorescence assays were used to determine the intracellular distribution of PLM and NKA. Optical mapping studies were performed in rabbit hearts. Results: These five Abs all displayed specific fluorescence signals in dog CMs that were more intense at the IDs than TTs. These signals were eliminated by competing peptides. B8 and NKA-α1 signals highly overlapped, with similar intensity ratios between ID and TT at ~3. Immunostaining of connexin 43, γ-catenin, or N-cadherin confirmed the co-localization of NKA and PLM to the ID. PLM and NKA co-enriched with ID protein markers in sarcolemma/ID membrane preparations. To corroborate the localization of PLM at the ID, we expressed dog PLM in rat CMs. By 30h post-infection, B8 detected newly synthesized dog PLM that paralleled the pattern of its steady-state distribution in tissue, with concentrated protein apparent at IDs (B8 fluorescence intensity ratio of ID/TT = 2.6 ± 0.5, 50CMs). Nocodazole prevented newly-made dog PLM trafficking to ID. Finally, optical mapping studies showed that the NKA inhibitor ouabain and digoxin significantly increased activation times in a dose-dependent manner (n=5 and 4 rabbits, respectively). Conclusions: The α1 subunit of NKA and PLM are each expressed at a ~3X higher concentration in ID compared to TT in CMs, contributing to cardiac conduction. Mechanisms of PLM and NKA protein trafficking to the two disparate subcellular sites, and associated effects of PLM regulation by adrenergic signaling, provide important new insights into the physiology of cardiac conduction.