Eplerenone repolarizes muscle membrane through Na,K-ATPase activation by Tyr10 dephosphorylation

Abstract

Eplerenone, an aldosterone antagonist, repolarizes muscle membrane in-vitro and increases strength in-vivo in channelopathies. In Duchenne dystrophy, it is administered for cardiomyopathy. We studied its mechanism of action on skeletal muscle to test its suitability for increasing strength in Duchenne dystrophy. Using membrane potential measurements, quantitative PCR, ELISA, and Western blots, we examined the effects of eplerenone on skeletal muscle Na,K-ATPase. The repolarizing effect of eplerenone in muscle fibres was counteracted by oubain, an ATPase blocker. In our experiment, ATPA1A mRNA and total ATPase protein were not elevated. Instead, Tyr10 of the α1 subunit was dephosphorylated which would agree with ATPase activation. Dephosporylation of the coupled Akt kinase corroborated our findings. We conclude that eplerenone repolarizes muscle membrane by Na,K-ATPase activation by dephosphorylation at Tyr10. Since ATPase protein is known to be compensatorily increased in Duchenne patients without activity change, eplerenone treatment may be beneficial.

Keywords: ATPase; Duchenne muscular dystrophy; eplerenone.

Figure 1.

(A-C) Smoothed histograms of relative frequency of resting membrane potentials of all myofibres of all rat diaphragm samples. For smoothing, three data histograms shifted by 1 mV each (class width of 3 mV) were added together (n = 182-290). (A) Depolarization in DMD model caused marked depolarization compared to control; (B) Eplerenone repolarised large portion of fibres; (C) Ouabain counteracted the eplerenone effect. (D) ELISA. Original blots for Extinction normalized to total protein and phosphorylated control respectively. Data are mean and standard deviation. Total Na,K-ATPase (82.2 ± 20.0%, n = 4) and phosphorylation of Ser16 (95.2 ± 24.9%; n = 4) and Ser943 (123.4 ± 24.9%; n = 4) remained unchanged, while phosphorylation of Tyr10 was significantly reduced (56.1 ± 29.5%; n = 4; p = 0.038) after eplerenone incubtation. (E) Western blot. Original blots of Akt (1:1.000) and phosphorylated Akt (1:500) with actin (1:10.000) as standard (left). Intensities normalized to actin and phosphorylated protein relative to total amount (right). Data are mean and standard deviation. Akt remained unchanged (100 ± 59%; n = 18), while phosphorylation at Ser473 was significantly reduced to 46 ± 57% (n = 14; p = 0.01) after 30 min eplerenone incubation.