Mice expressing ouabain-sensitive α1-Na,K-ATPase have increased susceptibility to pressure overload-induced cardiac hypertrophy

Abstract

The Na,K-ATPase is a ubiquitous transmembrane pump and a specific receptor for cardiac glycosides such as ouabain and digoxin, which are used in the management of congestive heart failure (CHF). A potential role for these so-called endogenous cardiotonic steroids (CS) has been explored, and it has become apparent that such compounds are elevated and may play an important role in a variety of physiological and pathophysiological conditions such as hypertension and CHF. Recent evidence suggests that the Na,K-ATPase may act as a signal transducer upon CS binding and induce nonproliferative cardiac growth, implicating a role for endogenous CS in the development of cardiac hypertrophy and progressive failure of the heart. In the present study, we tested whether hypertrophic responses to pressure overload would be altered in mutant mice that specifically express ouabain-sensitive or ouabain-resistant α1- and α2-Na,K-ATPase subunits, as follows: α1-resistant, α2-resistant (α1(R/R)α2(R/R)); α1-sensitive, α2-resistant (α1(S/S)α2(R/R)); and α1-resistant, α2-sensitive (α1(R/R)α2(S/S), wild-type). In α1(S/S)α2(R/R) mice, pressure overload by transverse aortic coarctation induced severe left ventricular (LV) hypertrophy with extensive perivascular and replacement fibrosis at only 4 wk. Responses in α1(R/R)α2(S/S) and α1(R/R)α2(R/R) mice were comparatively mild. Mutant α1(S/S)α2(R/R) mice also had LV dilatation and depressed LV systolic contractile function by 4 wk of pressure overload. In separate experiments, chronic Digibind treatment prevented the rapid progression of cardiac hypertrophy and fibrosis in α1(S/S)α2(R/R) mice. These data demonstrate that mice with a ouabain-sensitive α1-Na,K-ATPase subunit have a dramatic susceptibility to the development of cardiac hypertrophy, and failure from LV pressure overload and provide evidence for the involvement of endogenous CS in this process.

Fig. 1.

Comparison of cardiac weight in sham ouabain α1-resistant, α2-resistant (α1^R/Rα2^R/R) (n = 6), sham α1-sensitive, α2-resistant (α1^S/Sα2^R/R) (n = 6), sham α1-resistant, α2-sensitive (α1^R/Rα2^S/S) (n = 6), transverse aortic coarctation (TAC) α1^R/Rα2^R/R (n = 8), TAC α1^S/Sα2^R/R (n = 10), and TAC α1^R/Rα2^S/S mice (n = 5) at 4 wk postsurgery; values are means ± SE. A: whole heart weight (HW). B: heart weight normalized to tibia length (TL). C: transverse sections of hearts. Main effects and interaction terms from two-way ANOVA are shown in each panel. Gtype, genotype; Trt, treatment. Post hoc comparisons were as follows: *significant effect vs. respective sham (P < 0.05); †significant difference compared with TAC α1^R/Rα2^S/S and TAC α1^R/Rα2^R/R (P < 0.001).

Fig. 2.

Echocardiography measures of left ventricular (LV) structure and function of sham α1^R/Rα2^R/R (n = 5), sham α1^S/Sα2^R/R (n = 6), sham α1^R/Rα2^S/S (n = 3), TAC α1^R/Rα2^R/R (n = 5), TAC α1^S/Sα2^R/R (n = 6), and TAC α1^R/Rα2^S/S mice (n = 5) compared at 4 wk. values are means ± SE. Results from two-way ANOVA are shown in each panel. Post hoc comparisons are as follows: *significant difference vs. corresponding sham (P < 0.05); †significant difference vs. TAC α1^R/Rα2^S/S and TAC α1^R/Rα2^R/R (P < 0.05). LVEDD, LV end-diastolic diameter; LVESD, LV end-systolic diameter; AWth: LV anterior wall thickness; EF: ejection fraction.

Fig. 3.

Masons trichrome-stained sections at 4 wk after sham and TAC surgery. Heart sections showing degree of collagen accumulation; original magnification 100×. A: sham α1^R/Rα2^R/R. B: sham α1^S/Sα2^R/R. C: TAC α1^R/Rα2^R/R. D: TAC α1^S/Sα2^R/R. Immunohistochemistry on heart sections showing eosinophil accumulation in TAC α1^R/Rα2^R/R (E) and TAC α1^S/Sα2^R/R (F). G: quantification of total collagen content of heart sections: measurements from 4 sections per heart were averaged and then used to calculate mean values (± SE; n = 4 per group). Results from two-way ANOVA are shown. *Significant effect vs. corresponding sham (P < 0.001); †significant effect vs. TAC α1^R/Rα2^R/R (P < 0.05).

Fig. 4.

Representative photomicrographs of hematoxylin and eosin-stained LV sections from TAC α1^R/Rα2^R/R (A) and TAC α1^S/Sα2^R/R (B); original magnification 200×. C: summary data of myocyte diameter measurements (n = 2–3 hearts and 50–60 measurements/group); values are means ± SE. Results from two-way ANOVA are shown. Post hoc comparisons are as follows: *P < 0.001 vs. corresponding sham; †P < 0.001 vs. α1^R/Rα2^R/R.

Fig. 5.

Expression of Na,K-ATPase (NKA) α-isoforms in hearts of α1^R/Rα2^R/R (n = 3) and α1^S/Sα2^R/R (n = 3) mice, 4 wk after sham and TAC surgery. Proteins were resolved by 8% polyacrylamide gel electrophoresis. Immunoblots for α1-NKA (A) and α2-NKA (B) and densitometry analyses (C) are shown. Values are means ± SE.

Fig. 6.

Echocardiography measures of LV structure of Digibind-treated TAC α1^R/Rα2^R/R (n = 6) and TAC α1^S/Sα2^R/R mice (n = 7), compared at 4 wk. Values are means ± SE.