Downregulation of cardiac myocyte Na+-K+-ATPase by adenovirus-mediated expression of an α-subunit fragment

Cultured rat cardiac myocytes and A7r5 cells were transfected with an adenoviral vector used earlier for in vivo expression of functional alpha (2)-isoform of the catalytic subunit of rat Na+-K+-ATPase. Expressions of truncated forms of alpha (2), but little or no intact alpha (2), were detected, suggesting the rapid degradation of alpha (2) in these cultured cells. In neonatal myocytes normally containing the alpha (1)- and the alpha (3)-isoforms, expression of the alpha (2)-fragment led to 1) a significant decrease in the level of endogenous alpha (1)-protein and a modest decrease in alpha (3)-protein, 2) decreases in mRNAs of alpha (1) and alpha (3), 3) decrease in Na+-K+-ATPase function measured as ouabain-sensitive Rb+ uptake, 4) increase in intracellular Ca2+ concentration similar to that induced by ouabain, and 5) eventual loss of cell viability. These findings indicate that the alpha (2)-fragment downregulates endogenous Na+-K+-ATPase most likely by dominant negative interference either with folding and/or assembly of the predominant housekeeping alpha (1)-isoform or with signal transducing function of the enzyme. Demonstration of rise in intracellular Ca2+ resulting from alpha (1)-downregulation 1) does not support the previously suggested special roles of less abundant alpha (2)- and alpha (3)-isoforms in the regulation of cardiac Ca2+, 2) lends indirect support to proposals that observed decrease in total Na+-K+-ATPase of the failing heart may be a mechanism to compensate for impaired cardiac contractility, and 3) suggests the potential therapeutic utility of dominant negative inhibition of Na+-K+-ATPase.