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

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.

there are four known isoforms of the catalytic α-subunit of the Na,K-ATPase (α₁, α₂, α₃, and α₄), with each isoform displaying a unique tissue distribution and expression pattern (15, 16, 30). In the mouse, the α₂-isoform displays unique expression patterns in the brain, heart, skeletal muscle, and vascular smooth muscle, suggesting a tissue-specific role for the α₂-isoform (30). The α₂-isoform is the minor isoform of the Na,K-ATPase expressed in the vascular smooth muscle, with an α₁-to-α₂ protein expression ratio of ∼2.3 to 1 (29). In addition, the α₂-isoform is also the minor functional isoform of the Na,K-ATPase expressed in cardiac myocytes, with current generated by α₂ accounting for only ∼25% of total current generated by this enzyme (1, 11). Taken together, these reports demonstrate that the α₂-isoform composes only ∼5% of the total Na,K-ATPase protein expression in the cardiovascular system.

Although the α₂-isoform is expressed at low levels in the cardiovascular system, it is a critical regulator of cardiovascular function and blood pressure, as demonstrated in our previous studies using a global genetic knockout of one copy of α₂ (α₂^+/−). In one study we observed hypercontractility in α₂^+/− mouse heart, along with altered Ca²⁺ levels (15). In addition, Zhang et al. (37) reported an elevation of systolic blood pressure (SBP) in α₂^+/− mice accompanied by an increase in myogenic tone in the vasculature. Furthermore, using a global genetic mouse model, we have previously shown that the cardiac glycoside binding site of the α₂-isoform mediates ouabain and adrenocorticotropic hormone (ACTH)-induced hypertension (4–6, 8). To further evaluate the role of α₂ in the cardiovascular system, we recently reported on a smooth muscle-specific knockout of α₂ (SMα₂^−/−), in which the expression of α₂ is reduced by ∼90% in both the vascular smooth muscle and cardiac myocytes (25). These SMα₂^−/− mice failed to develop ACTH-induced hypertension, indicating that the cardiovascular expression of α₂ is required for the regulation of blood pressure in response to ACTH. In summary, it is generally accepted that α₂ is vital for the regulation and maintenance of cardiovascular hemodynamics, including the regulation of blood pressure. However, the organ system expressing the α₂ that is required for the regulation and maintenance of blood pressure has yet to be determined.

To further define the role of α₂ in the organ systems/cell types involved in the regulation of blood pressure, we evaluated the role of the cardiac expression of α₂. We hypothesized that α₂ expression in the heart is a critical regulator of blood pressure. To test this hypothesis, a tissue-specific knockout of α₂ in the heart was generated using the Cre/loxP system. The β-myosin heavy chain (β-MHC) Cre was used, which has been shown to drive efficient somatic recombination between loxP sites approaching 100% in the heart and skeletal muscle (20, 21).

MATERIALS AND METHODS

Animal model.

Homozygous α₂-floxed (α₂^Flox/Flox) mice have been previously described (25), in which the first exon of the α₂ locus (Atp1a2), containing the transcriptional start site, was flanked by loxP sites as depicted in Fig. 1. Homozygous α₂^Flox/Flox mice were mated to heterozygous β-MHC Cre mice [generous gift of Jeffery Molkentin (20, 21)] to generate a heart-specific knockout of α₂ (α₂^Flox/Flox Cre⁺). Genotyping was determined through allele-specific PCR from tail biopsies, as previously described (25). All experiments were approved by the University of Cincinnati Institutional Animal Care and Use Committee.

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.

Whole tissue preparations.

Aorta (pooled), brain, and kidney were homogenized in radioimmunoprecipitation assay buffer, consisting of 150 mM NaCl, 10 mM Tris, 5 mM EDTA (pH 7.4), 0.1% SDS, 1% Na-deoxycholate, and 1% Triton-X, containing 1:100 protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO), as previously described for Western blot analysis (25). Protein containing supernatant was removed and stored at −80°C. Protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL).

Microsomal preparations.

Cell membrane microsomal preparations were prepared from whole hearts as previously described for Western blot analysis (4, 25). Hearts were homogenized in homogenization buffer, consisting of (in mM) 250 sucrose, 30 imidazole, and 1 EDTA, containing 1:100 protease and phosphatase inhibitor cocktails (Sigma-Aldrich). To remove particulate matter, homogenates were then centrifuged at 5,000 rpm, and the supernatant was collected and ultracentrifuged at 200,000 g for 1 h. The pelleted microsomes were resuspended in resuspension buffer, consisting of (in mM) 1 imidazole, and 1 EDTA (pH 7.5), containing 1:100 protease and phosphatase inhibitor cocktails (Sigma-Aldrich), and protein concentration was determined with the BCA protein assay kit (Thermo Fisher Scientific).

Western blot analysis.

Western blot analysis were performed as previously described (4, 25). Protein samples were denatured at 37°C for 30 min in Laemmli loading buffer consisting of 125 mM Tris (pH 6.8), 6% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.1% bromophenal blue; separated by electrophoresis in 8% polyacrylamide gels; and transferred to polyvinylidene fluoride membranes. The membranes were blocked in blocking solution consisting of 5% nonfat dry milk in TBST, containing 150 mM Tris (pH 7.4), 100 mM NaCl, and 0.05% Tween-20 for 1 h at room temperature. The blots were incubated in primary antibodies overnight at 4°C in blocking solution as follows: α₁-isoform monoclonal antibody, 1:2,000 [α6F (31), University of Iowa Developmental Studies Hybrid Bank, Iowa City, IA]; and affinity purified α₂ polyclonal antibody, 1:1,000 [HERED, Pressley (23)]; Na/Ca exchanger (NCX) monoclonal antibody, 1:1,000 (MA3-926, Thermo Fisher Scientific). The primary antibodies were visualized with peroxidase-conjugated secondary antibodies (Calbiochem, San Diego, CA), following treatment with SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific), using HyBlot CL autoradiography film (Denville Scientific, Metuchen, NJ). Equal protein loading was confirmed by incubating the membranes in blocking solution containing primary antibodies for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) polyclonal antibody 1:6,000 (14C10, Cell Signaling Technology, Beverly, MA) for whole tissue preparations or calsequestrin polyclonal antibody 1:2,000 (PA1-913, Thermo Fisher Scientific) for microsomal preparations. Signal intensities were quantitated by densitometry using ImageQuant software 5.1 (Molecular Dynamics, Sunnyvale, CA).

Blood pressure analysis.

Tail-cuff measurements of SBP were obtained using the CODA-8 System (Kent Scientific, Torrington, CT) computerized apparatus, as previously described (2, 25). Mice were acclimated to the system for 5 consecutive days, immediately followed by 7 days of baseline measurements and 3 days of drug treatment measurements when applicable. Daily blood pressure measurements consisted of five acclimation readings, followed immediately by 15 SBP measurements, with little time elapse between readings, and measurements were accepted when SBP was determined by the computer in at least 7 of the 15 measurements and was between 80 and 200 mmHg.

Cardiovascular performance.

Cardiovascular measurements were performed in closed-chest mice as previously described (4, 25). Adult mice (3 to 6 mo old) were anesthetized with an intraperitoneal injection of ketamine (50 μg/g body wt) and thiobutabarbital (Inactin, 100 μg/g body wt, Research Biochemical International, Natick, MA). A 1.4-F Scisense pressure catheter (Scisense, London, Ontario, Canada) was advanced into the left ventricle through the right carotid artery to monitor cardiac performance. In addition, the right femoral artery and vein were cannulated to monitor blood pressure and to allow for the infusion of drugs, respectively. Pressure tracings were recorded and analyzed using PowerLab 4/s and Chart software (ADInstruments, Colorado Springs, CO) with values for heart rate (HR), blood pressure, and left ventricular pressure being measured before and 3 min following the intravenous infusion of dobutamine (2, 8, and 32 ng·g body wt⁻¹·min⁻¹). The maximum rate of cardiac contraction (dP/dt_max) and the rate of cardiac contraction at 40 mmHg (dP/dt₄₀) before and after infusion were calculated from the first derivative of the pressure waveforms.

Administration of ACTH.

Mice were administered 375 ng/g body wt Cortrosyn (Amphastar Pharmaceuticals, Rancho Cucamonga, CA), a synthetic peptide of ACTH or vehicle (saline), every 12 h by subcutaneous injection for 3 days, as previously described (25). SBP was obtained daily 3 h after administration of the a.m. dose, as described above. Following the final SBP recording, hearts were excised, dissected free from any connective tissue, and weighed. Hearts were then rapidly frozen in liquid N₂ and stored at −80°C until crude microsomal preparations were performed as described above.

Transverse aortic constriction.

Pressure overload of the left ventricle was induced in mice by transverse aortic constriction (TAC) as previously described (27, 34). Mice (3 to 4 mo old) were anesthetized with 1.5 to 2.5% isoflurane and intubated. The transverse aortic segment was dissected through a midsternal incision, and a 7-0 silk suture was tied against a 27-gauge needle around the transverse aorta between the innominate and left carotid arteries to produce a stenosis of uniform diameter. Sham-operated mice underwent a similar procedure except that the suture was left untied. Perioperative mortality from the procedure was <10% in TAC and sham-operated mice. Terminal measurements were made at 3, 6, and 9 wk. Additionally, noninvasive tail-cuff measurements of SBP were obtained at 0, 3, 6, and 9 wk postsurgery as described above.

Echocardiography.

Echocardiography was performed on TAC (0, 3, 6, and 9 wk) and sham-operated (9 wk) mice following TAC surgical procedure as previously described (34). Mice were anesthetized with <2% isoflurane to effect, and two dimensional-guided M-mode echocardiography was performed using a Philips HDI/5000 SONOS CT machine equipped with 15- to 7-MHz broadband transducer (Philips Ultrasound, Bothell, WA). M-mode measurements were used to determine the left ventricular end-systolic and end-diastolic length (L) and area (A) from two consecutive cardiac cycles. The left ventricular end-systolic volume (ESV) and end-diastolic volume (EDV) were calculated using the equation (0.85·A²)/L. The stroke volume (SV) was calculated using the formula SV = EDV − ESV, and the percent ejection fraction (EF) was calculated using the formula EF = SV/EDV.

Transstenotic pressure gradient.

Transstenotic pressure gradients were performed in all mice that underwent TAC or sham operation at 9 wk by simultaneous pressure readings from the right carotid and femoral arteries as previously described (34). Mice were anesthetized with isoflurane, and the right carotid artery was cannulated with a Scisense pressure catheter for the measurement of upstream arterial blood pressure; in addition, the right femoral artery was cannulated to monitor downstream arterial blood pressure. Following the measurement of the pressure gradient, the Scisense pressure catheter was advanced into the left ventricle through the right carotid artery to monitor cardiac performance. At the conclusion of the experiments, hearts were excised, dissected free from any connective tissue, and weighed. Hearts were then either fixed in a 10% buffered formalin solution and paraffin embedded for further analysis as described, or ventricles were snap frozen in liquid nitrogen for mRNA and protein expression analysis as described.

Morphometric and histological analysis.

Following fixation, whole heart images were taken using a dissecting microscope. Because of cardiac hypertrophy in TAC hearts, separate images were obtained of both the atria and ventricles, and the two images were then merged to generate a composite image of the whole heart. Longitudinal sections of 5-μm thickness were obtained to assess myocyte cross-sectional area. To visualize the myocyte membranes, membranes were stained with wheat germ agglutinin (WGA) as previously described (21). Sections were deparaffinized and rehydrated into phosphate-buffered saline (PBS), and the sections were then permeablized with 0.2% Triton-X in PBS (Sigma-Aldrich). Sections were blocked with 5% BSA in PBS (Thermo Fisher Scientific), and for us to visualize the myocyte membranes, the sections were incubated with 1:10 dilution of tetramethyl rhodamine isothiocyanate-labeled WGA (1 mg/ml, Sigma-Aldrich) for 20 min. The sections were then washed with PBS and countered stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen, Grand Island, NY) to visualize the nucleus. Coverslips were mounted on the sections using Fluoromount-G (Southern Biotech, Birmingham, AL). Images were acquired at ×40 magnification from 8 to 10 random fields within the left ventricular free wall from each heart. Myocyte diameter was determined from 6 to 10 myocytes per field (total of 48 to 100 myocytes per heart) using NIH ImageJ software.

Real-time PCR analysis.

Real-time PCR analysis was performed as previously described (24, 25). Briefly, total RNA was isolated from the ventricles using RNAzol RT (Molecular Research Center, Cincinnati, OH) according to the manufacturer's recommendations. SuperScript III first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) was used to generate first-strand cDNA from 5 μg of total RNA according to the manufacturer's recommendations for use with random hexamer primers. Real-time PCR reactions were performed in triplicate using Absolute Blue QPCR SYBR Green fluorescein mix (Thermo Scientific, Florence, KY) with atrial natriuretic peptide (ANP)-specific primers (forward 5′-GCTTCCAGGCCATATTGGAG-3′, and reverse 5′-GGGGGCATGACCTCATCTT-3′), brain natriuretic peptide (BNP)-specific primers (forward 5′-GAGGTCACTCCTATCCTCTGG-3′, and reverse 5′-GCCATTTCCTCCGACTTTTCT-3′), β-MHC-specific primers (forward 5′-TTCATCCGAATCCATTTTGGGG-3′, and reverse 5′-GCATAATCGTAGGGGTTGTTGG-3′), c-fos-specific primers (forward 5′-CGGGTTTCAACGCCGACTA-3′, and 5′-TTGGCACTAGAGACGGACAGA-3′), c-jun-specific primers (forward 5′-CCTTCTACGACGATGCCCTC-3′, and 5′-GGTTCAAGGTCATGCTCTGTTT-3′), and GAPDH-specific primers (forward 5′-TGACCACAGTCCATGCCATC-3′, and reverse 5′-GACGGACACATTGGGGGTAG-3′). The relative amount of ANP, BNP, β-MHC, c-fos, and c-jun mRNA was normalized to GAPDH.

Statistical analysis.

Data are presented as means ± SE, with statistical analysis performed using SigmaStat 3.5 software (Systat Software, San José, CA). Student t-test and one-way or two-way ANOVA were performed, with differences considered significant at P < 0.05.

RESULTS

Expression of the α₂-isoform of the Na,K-ATPase is reduced in the heart.

β-MHC Cre is highly expressed in the embryonic heart and has been shown to drive efficient somatic recombination between loxP sites approaching 100% (20, 21). Therefore, Western blot analyses were performed on the α₂^Flox/Flox Cre⁻ (control) and α₂^Flox/Flox Cre⁺ (knockout) mice to examine the tissue-specific expression of α₂. Representative immunoblots and relative protein expression levels for α₁, α₂, and the NCX are shown in Fig. 2. In heart microsomes, the abundance of α₂ was significantly reduced by 90% in the knockout mice (P = 0.008; Fig. 2A). In addition, the expression profiles of α₁ and NCX in heart microsomes were not significantly changed in knockout mice (Fig. 2A). Furthermore, there were no significant changes in the expression profiles of α₁ and α₂ in aorta (Fig. 2B) or brain (Fig. 2C) or in the expression profile of α₁ in kidney (Fig. 2D). These results demonstrate that we have generated a significant knockout of α₂ in the heart and that this knockout is specific for the heart within the cardiovascular system. Furthermore, the loss of the α₂ in the heart did not alter the expression of α₁ and NCX within the cardiovascular system.

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.

Mice with knockout of the α₂-isoform of the Na,K-ATPase in cardiac myocytes exhibit normal basal cardiovascular function.

Morphometric analysis of heart, kidney, and body weight were performed on mice at 4 and 8 mo of age (Table 1). There were no significant differences in the heart-to-body or kidney-to-body weight ratios in control and knockout mice at 4 or 8 mo of age. These results indicate that there are no gross abnormalities in the structure of the heart as a result of the heart-specific knockout of α₂. By tail-cuff, there was no significant difference in basal SBP between control and knockout mice (97 ± 1 and 99 ± 1, respectively, P = 0.164), indicating that the heart-specific expression of α₂ does not play a role in the regulation of basal SBP. Cardiovascular function in anesthetized close-chest mice is shown in Fig. 3. Under basal conditions there were no significant differences in HR, mean atrial pressure, dP/dt_max, and dP/dt₄₀ in knockout mice. Inotropic, chronotropic, and pressor responses to β-adrenergic stimulation with low doses of dobutamine were not different in knockout mice compared with control, but the inotropic response at the high dose of dobutamine (32 ng·g body wt⁻¹·min⁻¹) was slightly blunted (Fig. 3C). Taken together, these results indicate that the heart-specific expression of α₂ does not play a major role in the regulation of cardiac function at rest or in response to β-adrenergic stimulation.

Table 1.

Basal morphometric analysis of heart knockout mice

	4 mo		8 mo
	Control	Knockout	Control	Knockout
n	9	7	4	6
Body wt, g	26.3 ± 1.3	26.6 ± 1.0	34.4 ± 1.9	32.2 ± 1.5
Heart wt, mg	121.6 ± 7.9	124.9 ± 4.1	149.8 ± 12.7	141.5 ± 10.3
Kidney wt, mg	323.8 ± 29.2	355.3 ± 20.1	408.3 ± 55.9	381.7 ± 30.1
Heart wt:body wt	4.6 ± 0.1	4.7 ± 0.1	4.4 ± 0.4	4.4 ± 0.2
Kidney wt:body wt	13.1 ± 0.5	13.1 ± 0.4	12.2 ± 1.4	11.8 ± 0.6

Values are means ± SE.

Fig. 3.

Basal cardiovascular performance. Heart rate [in beats/min (bpm); A], mean atrial pressure (MAP; B), maximum rate of cardiac contraction (dP/dt_max; 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.

Mice with knockout of the α₂-isoform of the Na,K-ATPase in cardiac myocytes develop ACTH-induced hypertension.

Since the cardiac expression of α₂ resulted in no differences in basal cardiovascular hemodynamics as assessed by morphometric analysis, basal SBP, and cardiac functional analysis, we next examined whether cardiac α₂ participates in the pressor response to ACTH. Administration of ACTH has been shown to induce hypertension that is dependent on the cardiac glycoside binding site of α₂ and results from an increase in the concentration of circulating cardiac glycosides in the plasma (8, 19, 25). Therefore, we evaluated SBP by tail-cuff in control and heart-specific knockout mice in response to administration of ACTH. As shown in Fig. 4, there was no significant difference in the basal SBP in control and knockout mice, consistent with the basal blood pressure data. Administration of vehicle control (saline) resulted in no changes in SBP in control or knockout mice throughout the 3 days of treatment. However, control mice treated with ACTH developed hypertension by day 2 of ACTH treatment (101 ± 2 to 119 ± 3 mmHg; P < 0.05), and pressure remained elevated on day 3 of treatment. Similarly, knockout mice also developed hypertension by day 2 of ACTH treatment (99 ± 1 to 117 ± 6 mmHg; P < 0.05), and the pressure remained elevated on day 3 of treatment. These data indicate that the cardiac expression of α₂ does not participate in the development of ACTH-induced hypertension.

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.

There were no significant differences in the heart-to-body weight ratio in control and knockout mice treated with ACTH for 3 days (Table 2), indicating that ACTH did not cause any gross abnormalities in the structure of the heart in either control or knockout mice. Additionally, representative immunoblots and relative protein expression levels for α₁, α₂, and NCX are shown in Fig. 5. ACTH treatment did not result in any significant changes in expression profiles of α₁ (Fig. 5A), α₂ (Fig. 5B), or NCX (Fig. 5C) in control and knockout mice. However, consistent with the basal α₂ expression levels shown in Fig. 2A, there is a significant reduction in the expression level of α₂ in knockout heart microsomes. These results indicate that ACTH treatment did not change the expression profiles of α₁, α₂, or NCX in either control or knockout mice.

Table 2.

Morphometric analysis of heart knockout mice following 3 days of ACTH treatment

	Saline		ACTH
	Control	Knockout	Control	Knockout
Body wt, g	23.0 ± 1.6	22.7 ± 1.0	21.7 ± 0.7	21.4 ± 0.7
Heart wt, mg	106.2 ± 5.7	106.8 ± 4.6	106.4 ± 4.3	101.6 ± 2.0
Heart wt:body wt	4.6 ± 0.1	4.7 ± 0.1	4.9 ± 0.1	4.7 ± 0.2

Values are means ± SE; n = 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.

Mice with the knockout of the α₂-isoform of the Na,K-ATPase in cardiac myocytes display delayed onset of pressure overload-induced cardiac dysfunction.

Since cardiac expression of α₂ is not required for ACTH-induced hypertension, we then evaluated the role of cardiac expression of α₂ during chronic mechanical stress using transaortic constriction (TAC). Unlike ACTH-induced hypertension, which places stress on both the vasculature and the heart (32, 35), the mechanical stress induced by TAC is primarily directed toward the heart. Pressure gradients produced by TAC were comparable in both groups of mice at 3, 6, and 9 wk post-TAC (3 wk: control = 65.4 ± 4.1, knockout = 75.3 ± 5.8; 6 wk: control = 67.5 ± 11.4, knockout = 77.2 ± 2.4; and 9 wk: control = 63.6 ± 4.6, knockout = 63.1 ± 4.1). There were no appreciable gradients in sham-operated mice. There were no significant differences in body weight in control and knockout mice following TAC and sham-operation procedures (0 wk, 26.7 ± 0.8; 9 wk, 30.7 ± 0.9; group P = 0.217, treatment P < 0.001, interaction P = 0.832).

Representative composite whole heart images are shown in Fig. 6A, illustrating a substantial degree of hypertrophy in response to 9 wk of TAC in both groups of mice, but there were no gross differences between control and knockout mice 9 wk after TAC or sham operation. Gravimetric analysis of heart weight normalized to body weight at 0, 3, 6, and 9 wk post-TAC (Fig. 7C) indicated that the hypertrophic response to TAC was not different between control and knockout mice (significant treatment effect, but no group effect or interaction). To further examine the hypertrophic response, myocyte cross-sectional area was determined from control and knockout hearts at 9 wk post-TAC using WGA to stain myocyte membranes. Representative images of WGA-stained myocytes are shown in Fig. 6B, with the summary of myocytes cross-sectional areas in Fig. 6D. Interestingly, myocyte cross-sectional area was significantly higher in knockout mice compared with control mice, both under baseline conditions (sham operation) and following TAC; however, the increase in response to TAC was equivalent in both groups (significant group and treatment effect, but no interaction). Taken together, these results indicate that there are no major morphological differences in the hypertrophic response to TAC in knockout versus control hearts.

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.

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.

Cardiac hypertrophy is an adaptive response to increased hemodynamic demands on the heart, which is often accompanied by reactivation of fetal gene programs (3, 26, 27). Expression profiles of fetal gene programs including ANP, BNP, and β-MHC are shown in Fig. 7. There was increased ANP expression (Fig. 7A) at 3 wk post-TAC and increased BNP expression (Fig. 7B) at 6 wk post-TAC, consistent with previous reports (3, 26, 27). However, the increased expression of ANP and BNP in response to TAC was not different between control and knockout mice (significant treatment effect, but no group effect or interaction). Interestingly, there was decreased β-MHC expression (Fig. 7C) in both control and knockout mice following TAC. These results indicate that the heart-specific knockout of α₂ does not alter the reactivation of fetal gene programs in response to pressure overload.

The Na,K-ATPase has been shown to function as a signal transducer in response to low doses of ouabain, resulting in the activation of early response genes including c-fos and c-jun that are implicated in cardiac growth and hypetrophy (18, 22, 36). As shown in Fig. 7, there was decreased expression of both c-fos (Fig. 7D) and c-jun (Fig. 7E) in knockout animals at baseline (week 0; significant group effect). Furthermore, there was significant activation of c-fos expression (Fig. 7D) at 9 wk post-TAC in control mice; however, there was decreased activation in knockout mice (signifcant treatment and interaction). Taken together, these results indicate that the heart-specific knockout of α₂ decreases the activation of early response genes including c-fos and c-jun.

To assess whether the mechanical stress on the heart induced by TAC affected blood pressure regulation, SBP was evaluated at 0, 3, 6, and 9 wk post-TAC. As shown in Table 3, there was no significant difference in the basal (0) SBP in control and knockout mice, consistent with the basal blood pressure data and Fig. 4. Additionally, no significant differences in SBP were found in control and knockout mice at 3, 6, and 9 wk post-TAC. These results indicate that the pressure overload induced by TAC did not alter SBP and therefore supports that the mechanical stress induced by TAC is specific to the heart.

Table 3.

SBP following transaortic constriction

	SBP, mmHg
Post-TAC	Control	Knockout
0 wk	98.5 ± 0.8	97.0 ± 1.0
3 wk	96.4 ± 7.5	91.7 ± 2.6
6 wk	99.6 ± 5.6	91.4 ± 2.9
9 wk	93.2 ± 3.4	96.5 ± 3.5

Values are means ± SE; control (n = 5) and knockout (n = 10). Baseline (0), 3-, 6-, and 9-wk measurements are the average of 3 days of systolic blood pressure (SBP) measurements. Main effects and interactions from 2-way ANOVA are as follows: group, P = 0.200; treatment, P = 0.792; and group X treatment, P = 0.501.

To determine the effect of the heart-specific knockout of α₂ on cardiac function following TAC, echocardiographs were performed (Table 4 and Fig. 8). As shown in Table 4 and Fig. 8F, there were no significant differences in the HR between any groups; in addition, the HR was within the normal range for animals under isoflurane anesthesia. However, there was a significant decrease in the HR of both groups following 9 wk of TAC, consistent the development of cardiac dysfunction. Furthermore, as shown in Table 4, heart function was not different between sham-operated control and sham-operated knockout mice. In addition, before TAC, there were no significant differences in the ESV (Fig. 8A), EDV (Fig. 8B), SV (Fig. 8C), EF (Fig. 8D), or left ventricular posterior wall thickness during diastole (Fig. 8E) in control and knockout mice. Taken together, these results further support that the expression of α₂ in the heart does not play a major role in the regulation of basal cardiac function. Following TAC, knockout mice displayed a decreased severity of cardiac dysfunction at 3 and 6 wk postsurgery compared with control mice, as assessed through ESV (Fig. 8A), EDV (Fig. 8B), and EF (Fig. 8D). Furthermore, there were no significant differences in the SV (Fig. 8C) or left ventricular posterior wall thickness during diastole (Fig. 8E) between control and knockout mice following TAC. Interestingly, at 9 wk post-TAC, the severity of cardiac dysfunction in knockout mice reached control levels, as assessed through ESV (Fig. 8A), EDV (Fig. 8B), and EF (Fig. 8D). Interestingly, as shown in Fig. 9, there were no differences in cardiac contraction (dP/dt_max; Fig. 9A) or cardiac relaxation (dP/dt_min; Fig. 9B) in anesthetized mice following TAC. Taken together, these results indicate that the heart-specific knockout of α₂ delays the progression of cardiac dysfunction in response to pressure overload; however, the absence of α₂ is not sufficient to prevent the onset of cardiac dysfunction.

Table 4.

Functional echocardiography data for sham-operated mice at 9 wk

	Control	Knockout
End-systolic volume, ml	0.034 ± 0.002	0.029 ± 0.002
End-diastolic volume, ml	0.059 ± 0.005	0.058 ± 0.006
Stroke volume, ml	0.024 ± 0.003	0.029 ± 0.004
Ejection fraction, %	41.1 ± 2.7	49.7 ± 1.7
LVPWd, mm	1.299 ± 0.164	1.241 ± 0.038
Heart rate, beats/min	521.3 ± 11.3	528.8 ± 7.2

Values are means ± SE; n = 4. LVPWd, left ventricular posterior wall thickness diastole.

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.

Cardiovascular performance in control and knockout mice following TAC. Maximum rate of cardiac contraction (dP/dt_max; A), and minimum rate of cardiac contraction (dP/dt_min; 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.

Finally, to assess the possible role of altered Ca²⁺ handling in the delayed onset of cardiac dysfunction in knockout mice following TAC, the expression profile for α₁, α₂, and NCX were performed on mice following 9 wk of TAC. Representative immunoblots and relative protein expression levels for α₁, α₂, and NCX are shown in Fig. 10. The TAC surgical procedure did not result in any significant changes in expression profiles of α₁ (Fig. 10A), α₂ (Fig. 10B), or NCX (Fig. 10C) in control and knockout mice. However, consistent with the basal α₂ expression levels shown in Fig. 2A, there was significant reduction in the expression level of α₂ in knockout heart microsomes. These results indicate that TAC did not change the expression profiles of α₁, α₂, or NCX in either control or knockout mice. These results suggest that alterations in Ca²⁺ handling are not responsible for the delayed onset of cardiac dysfunction.

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.

DISCUSSION

Although the α₂-isoform of the Na,K-ATPase is a minor isoform of the Na,K-ATPase expressed in the cardiovascular system, the α₂ plays a critical role in the regulation of cardiovascular function (1, 11, 15, 25, 29, 37). However, the organ system expressing the α₂ that is required for this regulation has not been fully defined. The present study uses a heart-specific knockout of the α₂-isoform of the Na,K-ATPase to further examine the tissue-specific role of α₂ in the regulation of cardiovascular hemodynamics. The β-MHC Cre was used to generate a heart-specific knockout of α₂, and from quantitative Western blot analysis, there was a ∼90% reduction in α₂ expression in the heart. In addition, the β-MHC Cre has been shown to drive efficient recombination in skeletal muscle; however, this not expected to affect cardiovascular hemodynamics (21).

Consistent with our previous report on the vascular SMα₂^−/− (25), the heart-specific knockout mice also exhibit normal basal cardiac function and SBP. This is in striking contrast to the basal hypertension and cardiac hypercontractility observed in mice with the global genetic knockout of one copy of α₂ (α₂^+/−) (37) and further suggests that the α₂-isoform of the Na,K-ATPase in another tissue is responsible for the regulation of basal cardiovascular function. Interestingly, several reports have shown that increased levels of endogenous brain ouabain activate the renin-angiotensin system (13, 17, 38). Furthermore, Hou et al. (12) have shown that the activation of the renin-angiotensin system is increased in the brain of α₂^+/− mice, which may contribute to the basal hypertension. Additionally, increased salt (NaCl) in cerebrospinal fluid has been shown to result in an increase in the level of endogenous brain ouabain. The increased endogenous brain ouabain results in increased HR and hypertension that is dependent on the ouabain-binding site of α₂-isoform in the brain (33). Taken together, these results suggest that the chronic reduction in the activity/expression of α₂ in the brain of the α₂^+/− mice is likely responsible for the previously reported basal hypertension and hypercontractility.

Administration of ACTH, at doses similar to ours, results in a 1.5-fold increase in ACTH in circulating plasma (32). In addition, we have shown that these doses result in a 2.5-fold increase in circulating endogenous cardiac glycosides in mice (7, 8, 32). Therefore, ACTH administration acts as a stress on mice, resulting in a significant increase in blood pressure, which is associated with changes in cardiac output and total peripheral resistance (10, 28, 32, 35). Surprisingly, the heart-specific knockout mice developed ACTH-induced hypertension in response to 3 days of ACTH administration that was not quantitatively different from that seen in control mice. This is in contrast to our previous reports on the SMα₂^−/− mice, which failed to develop ACTH-induced hypertension in response to 5 days of ACTH administration. The SMα₂^−/− mice exhibit knockout of α₂ in both the heart and vascular smooth muscle (25). Taken together, these findings suggest that ACTH-induced hypertension is primarily dependent on vascular smooth muscle α₂ expression. Furthermore, these results suggest that α₂ expression in the heart and vascular smooth muscle are playing distinct roles in the regulation of cardiovascular hemodynamics.

Cardiac hypertrophy and ensuing heart failure can result from an assortment of mechanical, hemodynamic, and hormonal stresses on the heart (9, 14, 27). During TAC, a model of pressure overload-induced hypertrophy, the heart undergoes a remodeling process that is initially characterized by hypertrophy of ventricular walls and cardiac myocytes, which is accompanied by a reactivation of fetal gene programs. The pressure overload ultimately leads to ventricular dilation and diminished cardiac function if left untreated (3, 9, 26, 27). Interestingly, the heart-specific knockout mice display a delayed onset of cardiac dysfunction compared with control mice at 3 and 6 wk post-TAC. However, at 9 wk post-TAC, cardiac dysfunction in the heart-specific knockout mice has deteriorated to control levels. These results suggest that cardiac expression of α₂ participates in the rapid onset of cardiac dysfunction in control mice exposed to pressure overload but that the absence of α₂ was not sufficient to prevent the onset of cardiac dysfunction.

Interestingly, the Na,K-ATPase is known to function as a signal transducer in response to low doses of ouabain, resulting in the activation of early response genes including c-fos and c-jun that are implicated in cardiac growth and hypetrophy (18, 22, 36). Furthermore, there was a significant decrease in expression of early response genes c-fos and c-jun in knockout mice at baseline. Additionally, there was a significant decrease in c-fos activation in knockout mice in response to TAC, indicating that knockout mice exhibited a diminished signaling response to TAC. Taken together, these results suggest that the activation of signaling cascades through the α₂-isoform in the heart is required for the rapid onset of cardiac dysfunction in control mice exposed to pressure overload.

In summary, the present study refines the roles of α₂ expression in the brain, vascular smooth muscle, and heart in the regulation of cardiovascular function. Though vascular smooth muscle expression of α₂ appears to be required for ACTH-induced hypertension, cardiac expression does not. By contrast, cardiac expression of α₂ seems to participate in the hypertrophic response to pressure overload though the activation of early response genes. Based on these and previous findings, we surmise that the expression of α₂ in the brain is required for the basal regulation of cardiovascular function, whereas the vascular smooth muscle and heart expression of α₂ play distinct roles in the regulation of cardiovascular function in response to mechanical stress. These data support the hypothesis that the tissue localization of the α₂-isoform of the Na,K-ATPase determines its unique roles in the regulation of cardiovascular function.

GRANTS

This research was supported by National Heart, Lung, and Blood Institute Grant R01-HL-28573(to J. B. Lingrel).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

T.N.R., J.N.L., and J.B.L. conception and design of research; T.N.R., V.M.L., M.L.N., and M.O. performed experiments; T.N.R., V.M.L., M.L.N., and J.N.L. analyzed data; T.N.R. and J.N.L. interpreted results of experiments; T.N.R. prepared figures; T.N.R. drafted manuscript; T.N.R., V.M.L., M.L.N., J.N.L., and J.B.L. edited and revised manuscript; T.N.R., V.M.L., M.L.N., M.O., J.N.L., and J.B.L. approved final version of manuscript.