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

Abstract

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 Ca(2+) 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 Ca(2+) 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 Ca(2+), 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.

The α1 Isoform of Na,K-ATPase Regulates Cardiac Contractility and Functionally Interacts and Co-localizes with the Na/Ca Exchanger in Heart

“…This is in agreement with previous studies attributing the increased contractility of isolated rat cardiac myocytes to reduced activity of the ␣1 isoform (16). In addition we demonstrate that the extent of the reduction in ␣1 isoform activity determines whether cardiac contractility will be enhanced or compromised.…”

“…The high expression of the ␣1 isoform in T-tubules of rat cardiac myocytes suggests that it has a major role in the regulation of cardiac contractility (14,15). An increased contraction of isolated rat cardiac myocytes has been attributed to a reduction in ␣1 isoform expression through dominant negative interference with a fragment of the ␣2 isoform (16). However, in these cardiac myocytes a reduction in the expression of the ␣3 isoform was also detected, which could contribute to the enhanced contractility.…”

Systems analysis of digoxin kinetics and inotropic response in the rat heart: effects of calcium and KB-R7943

“…ϩ -K ϩ -ATPase; Na ϩ /Ca 2ϩ exchanger; cardiac glycosides; mathematical modeling IT IS NOW WIDELY ACCEPTED that the cardiac glycosides exert their positive inotropic effect through an inhibition of Na ϩ -K ϩ -ATPase (Na ϩ pump): the Na ϩ pump controls the cytoplasmic [Na ϩ ], which, in turn, determines [Ca 2ϩ ] via the Na ] o ) are still a matter of debate (9,20,21,24,37). The suggestion that in mice the positive inotropic response to ouabain is solely mediated by the ␣ 2 -isoform (20) has thus been questioned by Schwartz and Petrashevskaya (37), who argued that the relatively high [Ca 2ϩ ] (2.5 mM) used in this study could have masked the contribution of ␣ 1 -receptors.…”

Digoxin Uptake, Receptor Heterogeneity, and Inotropic Response in the Isolated Rat Heart: A Comprehensive Kinetic Model

“…Despite fundamental new insights obtained in the last decades at the enzyme and cellular level by biochemical and electrophysiological studies, however, there is limited knowledge about the functional role of Na ϩ /K ϩ -ATPase isoforms in the intact heart. Although recent evidence suggests that the ␣ 2 -isoform may have a special function in the regulation of intracellular Ca 2ϩ (Blaustein and Lederer, 1999;James et al, 1999), implying that only this single isoform mediates the glycoside action, this view is still controversial (Gao et al,1995;Kometiani et al, 2001;Schwartz and Petrashevskaya, 2001). To understand the role of uptake kinetics and the contribution of Na ϩ /K ϩ -ATPase isoforms to cardiotonic effects of cardiac glycosides, we investigated the pharmacokinetics and pharmacodynamics of digoxin in the isolated perfused rat heart.…”