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

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 Na/K-ATPase that might explain the observed higher ouabain sensitivity. Methods: For contractility studies, a total of 57 trabeculae were isolated from two non- failing (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 Na/K-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 α 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 EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p≪0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol·g−1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three α 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 Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition. Cardiovascular Research 1993;27:2229-223

Type 1 Phosphatase, a Negative Regulator of Cardiac Function

Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired β-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of β-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure