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

Cultured rat cardiac myocytes and A7r5 cells were transfected with an adeno viral vector used earlier for in vivo expression of functional alpha (2)-is oform of the catalytic subunit of rat Na+-K+-ATPase. Expressions of truncat ed forms of alpha (2), but little or no intact alpha (2), were detected, su ggesting the rapid degradation of alpha (2) in these cultured cells. In neo natal myocytes normally containing the alpha (1)- and the alpha (3)-isoform s, 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 i n Na+-K+-ATPase function measured as ouabain-sensitive Rb+ uptake, 4) incre ase in intracellular Ca2+ concentration similar to that induced by ouabain, and 5) eventual loss of cell viability. These findings indicate that the a lpha (2)-fragment downregulates endogenous Na+-K+-ATPase most likely by dom inant negative interference either with folding and/or assembly of the pred ominant housekeeping alpha (1)-isoform or with signal transducing function of the enzyme. Demonstration of rise in intracellular Ca2+ resulting from a lpha (1)-downregulation 1) does not support the previously suggested specia l roles of less abundant alpha (2)- and alpha (3)-isoforms in the regulatio n of cardiac Ca2+, 2) lends indirect support to proposals that observed dec rease in total Na+-K+-ATPase of the failing heart may be a mechanism to com pensate for impaired cardiac contractility, and 3) suggests the potential t herapeutic utility of dominant negative inhibition of Na+-K+-ATPase.