Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts

Abstract

Objective:The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in NdK-ATPase that might explain the observed higher ouabain sensitivity. Methods: For contractility studies, a total of 57 trabeculae were isolated from two nonfailing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total NdK-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three ci isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex. Results: Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic ECSo and the minimum toxic concentration were both reduced.[3H]Ouabain binding was significantly lower (p<<O.OOl) in failing than in non-failing hearts, at 293(SD 74) v 507(48) p m o l g ' wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K' , vanadate, and dihydro-ouabain. All three ci isoforms were expressed at the mRNA level in both normal and failing hearts. Conclusions: This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of NdK-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.

Changes in the expression of Na+/K+-ATPase isoenzymes in the left ventricle of diabetic rat hearts: effect of insulin treatment

“…While this manuscript was in preparation in agreement with our data, Gerbi et al [32] published that the activity of the low affinity isoenzyme is decreased in diabetes [32]. Since we found that the turnover rate of Na+/K+-ATPase (ATPase activity/ [3H]ouabain binding) does not change significantly during diabetes and insulin treatment, there is a possibility that the decrease in ATPase activity arises from the decrease in the amount of ATPase molecule [33], rather than inactivation or modification of the enzyme as suggested by others [32]. The results of our Western blot analysis of the samples supported the above assumption.…”

Transport and Pharmacological Properties of Nine Different Human Na,K-ATPase Isozymes

“…The differences in the ouabain binding kinetics of human Na,K-ATPase isozymes reported in this study may be reflected in studies on human cardiac plasma membranes where the rapid dissociation process described (t1 ⁄2 10 -13 min) (35,48) could correspond to ␣2-␤ complexes (t1 ⁄2 4 -5 min) while the slower process described (t1 ⁄2 39 -75 min) (35,48,49) could correspond to ␣1-␤ and/or ␣3-␤ isozymes (t1 ⁄2 30 -80 min). On the other hand, in human cardiac plasma membranes, only one association rate constant was described ranging from 0.12 ϫ 10 7 -0.7 ϫ 10 7 M Ϫ1 min Ϫ1 (35,49,50).…”

Intracellular Na + Concentration Is Elevated in Heart Failure But Na/K Pump Function Is Unchanged

“…This is somewhat surprising, considering that several biochemical studies revealed decreased expression and/or isoform shifts of the Na/K pump in failing or hypertrophied hearts. [13][14][15][16][17] However, most of these studies were performed in tissue homogenates and might reflect changes in nonmyocytes. Moreover, such measurements cannot differentiate between the internalized versus the sarcolemmal Na/K pumps 26 nor between functional and inactive pumps.…”

“…12 Higher [Na ϩ ] i could be explained by lower Na/K pump activity, consistent with reports of decreased Na/K pump expression and isoform shifts in some HF models. [13][14][15][16][17] However, functional studies in HF ventricular myocytes are sparse and contradictory. 8,15 Enhanced Na ϩ influx could also raise [Na ϩ ] i , and this explains the higher [Na ϩ ] i in rat versus rabbit ventricular myocytes.…”