The Na⁺-K⁺-ATPase α₂-subunit isoform modulates contractility in the perinatal mouse diaphragm

Abstract

This study uses genetically altered mice to examine the contribution of the Na(+)-K(+)-ATPase alpha2 catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The alpha2 protein is reduced by 38% in alpha2-heterozygous and absent in alpha2-knockout mice, and alpha1-isoform is upregulated 1.9-fold in alpha2-knockout. Resting potentials are depolarized by 0.8-4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced alpha2 content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the alpha2-isoform at a step after membrane excitation, which cannot be restored simply by increasing alpha1 content. Na+/Ca2+ exchanger expression decreases in parallel with alpha2-isoform, suggesting that Ca2+ extrusion is affected by the altered alpha2 genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban, or plasma membrane Ca(2+)-ATPase. These results demonstrate that the Na(+)-K(+)-ATPase alpha1-isoform alone is able to maintain equilibrium K+ and Na+ gradients and to substitute for alpha2-isoform in most cellular functions related to excitability and force. They further indicate that the alpha2-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction.

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

“…On the basis of these data, it was proposed that the ␣2 isoform mediates positive cardiac inotropy, whereas the ␣1 isoform mediates the toxic effects of cardiac glycosides. A number of studies re-enforced the importance of the ␣2 isoform in the regulation of Ca 2ϩ transients during contraction (7)(8)(9)12). Also, we confirmed that the ␣2 isoform mediates the positive cardiac inotropy observed at low concentrations of ouabain, by comparing the effect of ouabain on cardiac contractility in wild type hearts expressing the ouabain-sensitive ␣2 isoform with that in hearts expressing a genetically modified, ouabain-resistant ␣2 isoform (13).…”

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

“…Accordingly, we crossed NKA␣2-heterozygous (Atp1a2 ϩ/Ϫ ) mice into the NCX1 TG background. The gene for the ␣2 isoform of NKA is the predominant gene expressed in skeletal muscle (50), and it is tightly coupled to NCX activity (51). We observed that NCX1 TG Atp1a2 ϩ/Ϫ mice had increased central nucleation and increased serum CK levels compared with NCX1 TG mice, as well as more obvious histopathology at 3 months of age (Fig.…”

The cardiac glycoside binding site on the Na,K-ATPase α2 isoform plays a role in the dynamic regulation of active transport in skeletal muscle

“…There was no significant group effect between genotypes for either ␣1 (P Ͼ 0.23) or ␣2 (P Ͼ 0.14) isoform. ␣-Isoform expression was measured by Western blot analysis using ␣1-and ␣2-specific antibodies, as described previously (20,37). The ␣3 Na,K-ATPase isoform is not detected in this preparation (20).…”

“…␣-Isoform expression was measured by Western blot analysis using ␣1-and ␣2-specific antibodies, as described previously (20,37). The ␣3 Na,K-ATPase isoform is not detected in this preparation (20). and produces large changes in Na,K-ATPase transport (18).…”

“…Our results suggest that the cardiac glycoside-binding site of the ␣2 Na,KATPase and an endogenous ligand(s) participate in the dynamic regulation of Na,K-ATPase transport activity in response to changing conditions of muscle use. Our previous studies with Na,K-ATPase ␣2 isoform knockout mice had shown that removal of ␣2 expression induces compensatory up-regulation of the ␣1 isoform in skeletal muscle (20). Fig.…”