The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes.
OBJECTIVE
The presence of both alpha1- and alpha2-isoforms of the Na+/K+-ATPase (NKA) in cardiomyocytes indicates different functions. We hypothesized that preferential localization of the alpha2-isoform to the t-tubules, locally controlling the Na+/Ca2+-exchanger (NCX), underlies a specific role in Ca2+ handling.


METHODS
We studied NKA isoform distribution in isolated cardiomyocytes from Wistar rats using immunocytochemistry. NKA pump and NCX currents (I(pump) and I(NCX)) were measured in control and detubulated cardiomyocytes. Intracellular Na+ concentration [Na+]i was assessed with the fluorescent dye SBFI.


RESULTS
The alpha2-isoform abundance was higher in the t-tubules than in the surface sarcolemma. We established that 0.3 microM ouabain specifically blocked the alpha2-isoform in isolated rat cardiomyocytes. This low concentration blocked 10.7+/-0.6% of I(pump) in control, but only 6.0+/-0.5% in detubulated cardiomyocytes. Moreover, measured and calculated alpha1-specific and alpha2-specific I(pump) in control (547+/-29 pA and 66 pA, respectively) and in detubulated cells (495+/-30 pA and 31 pA, respectively) showed that 53% of the alpha2-isoform, but only 9.5% of the alpha1-isoform, were localized to the t-tubules. Despite the small abundance of the alpha2-isoform (approximately 11% of total NKA), selective inhibition of this isoform induced a 40% increase in contractility in field stimulated cardiomyocytes, but no increase in global [Na+]i. However, inhibition of the alpha2-isoform increased I(NCX) indicating local subsarcolemmal accumulation of Na+ near NCX.


CONCLUSIONS
The alpha2-isoform of the NKA is functionally coupled to the NCX and can regulate Ca2+ handling without changing global [Na+]i.
Swift
Fredrik
F
Institute for Experimental Medical Research, Ullevaal University Hospital, University of Oslo, Oslo, Norway. fredrik.swift@medisin.uio.no
Tovsrud
Nils
N
Enger
Ulla H
UH
Sjaastad
Ivar
I
Sejersted
Ole M
OM
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism; D048429:Cell Size; D004558:Electric Stimulation; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000502:physiology*; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000032:analysis / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019831:Sodium-Calcium Exchanger; D010042:Ouabain; D012964:Sodium; C498532:Atp1a2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium