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. 2013 Apr 15;304(8):H1147-58.
doi: 10.1152/ajpheart.00594.2012. Epub 2013 Feb 22.

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction

Tara N Rindler  1 , Valerie M Lasko, Michelle L Nieman, Motoi Okada, John N Lorenz, Jerry B Lingrel
Affiliations

Knockout of the Na,K-ATPase α2-isoform in cardiac myocytes delays pressure overload-induced cardiac dysfunction

Tara N Rindler et al. Am J Physiol Heart Circ Physiol. .

Abstract

The α2-isoform of the Na,K-ATPase (α2) is the minor isoform of the Na,K-ATPase expressed in the cardiovascular system and is thought to play a critical role in the regulation of cardiovascular hemodynamics. However, the organ system/cell type expressing α2 that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of α2 to further define the tissue-specific role of α2 in the regulation of cardiovascular hemodynamics. To accomplish this, we developed a mouse model using the Cre/loxP system to generate a tissue-specific knockout of α2 in the heart using β-myosin heavy chain Cre. We have achieved a 90% knockout of α2 expression in the heart of the knockout mice. Interestingly, the heart-specific knockout mice exhibit normal basal cardiac function and systolic blood pressure, and in addition, these mice develop ACTH-induced hypertension in response to ACTH treatment similar to control mice. Surprisingly, the heart-specific knockout mice display delayed onset of cardiac dysfunction compared with control mice in response to pressure overload induced by transverse aortic constriction; however, the heart-specific knockout mice deteriorated to control levels by 9 wk post-transverse aortic constriction. These results suggest that heart expression of α2 does not play a role in the regulation of basal cardiovascular function or blood pressure; however, heart expression of α2 plays a role in the hypertrophic response to pressure overload. This study further emphasizes that the tissue localization of α2 determines its unique roles in the regulation of cardiovascular function.

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Figures

Fig. 1.
Fig. 1.
Schematic representation of the α2 locus (Atp1a2) of the Na,K-ATPase. Top: partial structure of the floxed α2 allele in which exon 1 was flanked by loxP sites. Bottom: deleted α2 allele following the tissue-specific expression of the β-myosin heavy chain (β-MHC) Cre.
Fig. 2.
Fig. 2.
Basal expression profile of the α1- and α2-isoforms of the Na,K-ATPase and Na/Ca-exchanger (NCX). Representative immunoblots and relative protein expression for α1, α2, NCX, and GAPDH or calsequestrin (CSQ) in the heart (A), aorta (B), brain (C), and kidney (D). Relative protein expression was determined by densitometry and normalized to the abundance of GAPDH or CSQ. The data represent an arbitrary ratio of protein expression in knockout mice compared with expression in control mice. The data were obtained from 3 independent experiments and are displayed as arbitrary units ± SE, n = 3. *P = 0.008.
Fig. 3.
Fig. 3.
Basal cardiovascular performance. Heart rate [in beats/min (bpm); A], mean atrial pressure (MAP; B), maximum rate of cardiac contraction (dP/dtmax; C), and rate of cardiac contraction at 40 mmHg (dP/dt40; D) were determined in anesthetized control (n = 7) and knockout (n = 7) mice using a pressure catheter advanced into the left ventricle. Data were collected at baseline (0) and following administration of increasing doses of dobutamine. Values represent means ± SE. Main effects and interactions from 2-way repeated-measures ANOVA are shown in each panel. Grp, group; Trt, treatment. Post hoc comparisons were as follows: *P = 0.015 compared with control.
Fig. 4.
Fig. 4.
ACTH-induced hypertension. Systolic blood pressure (SBP) was measured by the tail-cuff method in 3- to 6-mo-old mice following ACTH treatment (control, n = 6; and knockout, n = 4) or saline (control, n = 5; and knockout, n = 5). Baseline (BS) measurement was the average of 7 days of basal SBP measurements, followed by 3 days of ACTH or saline treatment. Values represent means ± SE. Main effects and interactions from 2-way repeated-measures ANOVA are shown. Post hoc comparisons were as follows: *P < 0.05 compared with saline treatment.
Fig. 5.
Fig. 5.
Expression profile of the α1- and α2-isoforms of the Na,K-ATPase and NCX following ACTH treatment. Representative immunoblots and relative protein expression in heart microsomes of α1 (A), α2 (B), and NCX (C) following 3 days of ACTH treatment are shown. Relative protein expression was determined by densitometry and normalized to the abundance of CSQ. The data represent a relative ratio of protein expression to that of saline control. The data were obtained from 3 independent experiments and are displayed as arbitrary units ± SE, n = 3. Main effects and interactions from 2-way ANOVA are shown in each panel. Post hoc comparisons were as follows: *P < 0.05 compared with control.
Fig. 6.
Fig. 6.
Morphology of hearts following 9 wk of transaortic constriction (TAC). Representative composite images of gross morphology of whole hearts (A) and longitudinal sections of the left ventricular free wall (B) from hearts of mice that underwent sham operation [control (i) and knockout (ii)] or TAC [control (iii) knockout (iv)] at 9 wk postsurgery. Longitudinal sections were stained with wheat germ agglutinin to visualize the cardiac myocyte membranes, and images were obtained at ×40 magnification. Comparison of whole heart weight normalized to body weight (HW/BW; C) were made following TAC: sham (0 wk; n = 4), 3 wk TAC (n = 3), 6 wk TAC (n = 2), and 9 wk TAC (control, n = 9; and knockout, n = 15). Cardiac myocyte cross-sectional area (D) was measured in 48–100 myocytes from each heart that underwent sham operation (control, n = 4; and knockout, n = 4) or TAC (control, n = 4; and knockout, n = 5). Values represent means ± SE. Main effects and interactions from 2-way ANOVA are shown in each panel.
Fig. 7.
Fig. 7.
mRNA expression profile of cardiac hypertrophy markers in isolated ventricles following TAC. Quantitative real-time PCR analysis of mRNA expression of atrial natriuretic peptide (ANP; A), brain natriuretic peptide (BNP; B), β-MHC (C), c-fos (D), and c-jun (E) normalized to GAPDH expression is shown. Data were collected from mice at 0 (sham), 3, 6, and 9 wk post-TAC surgery. The data represent a relative ratio of mRNA expression to that of the 0-wk control. Values represent means ± SE; n = 4. Main effects and interactions from 2-way ANOVA are shown in each panel.
Fig. 8.
Fig. 8.
Echocardiography in control and knockout mice following transaortic constriction. Echocardiographic measurements for end-systolic volume (ESV; A), end-diastolic volume (EDV; B), stroke volume (SV; C), ejection fraction (EF; D), left ventricular posterior wall thickness during diastole (LVPWd; E), and heart rate (HR; F) were made in control (n = 9) and knockout (n = 9) mice. Data were collected at baseline (0) and 3, 6, and 9 wk post-TAC surgery. Values represent means ± SE. Main effects and interactions from 2-way repeated-measures ANOVA are shown in each panel. Post hoc comparisons were as follows: *P < 0.05 compared with control.
Fig. 9.
Fig. 9.
Cardiovascular performance in control and knockout mice following TAC. Maximum rate of cardiac contraction (dP/dtmax; A), and minimum rate of cardiac contraction (dP/dtmin; B) were determined in anesthetized mice using a pressure catheter advanced into the left ventricle. Data were collected from mice at 0 (sham), 3, 6, and 9 wk post-TAC surgery (0 wk, n = 4; 3 wk TAC, n = 3; 6 wk TAC control, n = 2, and knockout, n = 1; 9 wk TAC control, n = 5, and knockout, n = 10). Values represent means ± SE. Main effects and interactions from 2-way ANOVA are shown in each panel.
Fig. 10.
Fig. 10.
Expression profile of the α1- and α2-isoforms of the Na,K-ATPase and NCX following TAC. Representative immunoblots and relative protein expression in heart microsomes of α1 (A), α2 (B), and NCX (C) following 9 wk of TAC. Relative protein expression was determined by densitometry and normalized to the abundance of CSQ. The data represent a relative ratio of protein expression to that of the 0-wk control. The data were obtained from 3 independent experiments and are displayed as arbitrary units ± SE; n = 3. Main effects and interactions from 2-way ANOVA are shown in each panel. Post hoc comparisons were as follows: *P < 0.05 compared with control.
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