The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart.

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.

Active Na ϩ transport across the cardiac sarcolemma, driven by the Na,K-ATPase, is an important regulator of cardiac function (1,2). The intracellular Na ϩ concentration affects a number of physiological processes in cardiac myocytes, including intracellular Ca 2ϩ handling, contraction-relaxation processes, pH regulation, energy metabolism, and cell growth (1)(2). Alterations in the maintenance of normal intracellular Na ϩ ho-meostasis result in heart failure (1).
Na,K-ATPase is a heterodimer composed of ␣ and ␤ subunits (3). The ␣ subunit is the catalytic subunit, and it binds translocating cations and ATP. The ␣ subunit is also the pharmacological receptor for cardiac glycosides. These compounds inhibit Na,K-ATPase activity and are used in the treatment of congestive heart failure. There are four isoforms of the ␣ subunit, each with a distinct tissue distribution and developmental pattern of expression, suggestive of their differential and tissue-specific functional roles (4 -10). Depending on the species, different combinations of these ␣ isoforms are present in heart. The ␣1 and ␣2 isoforms are expressed in rodent heart, whereas three isoforms (␣1, ␣2, and ␣3) are expressed in human heart (5,10,11). As multiple isoforms are expressed in heart, it is possible that they play different biological roles.
Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the ␣1 or ␣2 isoform gene exhibit opposite phenotypes (6). The hearts from ␣2 ϩ/Ϫ animals, in which ␣2 levels were reduced by 50%, were hypercontractile, whereas those of the ␣1 ϩ/Ϫ animals, in which ␣1 levels were reduced by 40%, were hypocontractile. Calcium transients were increased in ␣2 ϩ/Ϫ cardiac myocytes but were unchanged in ␣1 ϩ/Ϫ myocytes, suggesting that reduced levels of the ␣2 isoform, but not ␣1 isoform, affect Ca 2ϩ handling. Furthermore, administration of ouabain to the hypocontractile ␣1 ϩ/Ϫ hearts led to a positive inotropic response. 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).
Several reports suggest that the ␣1 isoform modulates cardiac contractility differently than that proposed based on the hypo-contractile cardiac phenotype in the isolated ␣1 ϩ/Ϫ hearts (14 -18). The high expression of the ␣1 isoform in T-tubules of rat cardiac myocytes suggests that it has a major role in the regulation of cardiac contractility (14,15). An increased contraction of isolated rat cardiac myocytes has been attributed to a reduction in ␣1 isoform expression through dominant negative interference with a fragment of the ␣2 isoform (16). However, in these cardiac myocytes a reduction in the expression of the ␣3 isoform was also detected, which could contribute to the enhanced contractility. Also a biphasic ouabain dose response in isolated rodent heart preparations is well established, and the positive inotropic effect of high concentrations of ouabain has been accounted for by inhibition of the low affinity ␣1 isoform (17,18). However, as this second phase of inhibition always occurs following inhibition of the ␣2 isoform, it is uncertain whether this pre-dispositions the heart to further contraction following inhibition of the ␣1 isoform.
To address directly and unambiguously the role of the ␣1 isoform, we developed animals in which the ␣1 isoform is ouabain-sensitive and the ␣2 isoform is ouabain-resistant, and we analyzed cardiac contractility following administration of ouabain that would inhibit only the ␣1 isoform without altering the ␣2 isoform. The present study demonstrates that the ␣1 isoform regulates cardiac contractility and is functionally and physically coupled with the Na/Ca exchanger.

EXPERIMENTAL PROCEDURES
Generation of a Mice Expressing the Cardiac Glycoside-sensitive ␣1 and ␣2 Isoforms of Na,K-ATPase-The R111Q and D122N amino acid substitutions were introduced into the ␣1 isoform by 2-bp mutations in exon 4 by using PCR site-directed mutagenesis, as shown in Fig. 1. Furthermore, 2 silent base pair substitutions were made to introduce an EcoRI site. The LoxP-neomycin-LoxP cassette was cloned into intron 3 at a site 750 bp upstream of exon 4. The thymidine kinase gene was inserted downstream of the cloning sequence. The presence of the desired mutation in the targeting vector was verified by both sequence analysis and EcoRI restriction digestion. The Duffy ES cell line was transfected with the targeting vector by electroporation, and successful homologous recombination was confirmed by Southern blot analysis. The 5Ј probe was derived from intron 3, and the 3 Ј probe was derived from intron 4 of the ␣1 isoform gene. The presence of the EcoRI site and desired mutation in exon 4 was determined in successfully targeted ES cells by PCR using the P1 (5Ј-AGCTCAGGACATTCTGG) and the P2 (5Ј-CTCCTAACCACGCTCCTAG) primers, and the amplified exon 4 fragments were treated with EcoRI restriction enzyme. Successfully targeted ES cells were subjected to the second electroporation with the Cre-recombinase encoding vector. Following the transfection, ES clones were duplicated, and one set was grown in media supplemented with 250 g/ml G418. Genomic DNA was extracted from the neomycinsensitive ES cells, digested with EcoRI, and analyzed by Southern blot using the 3Ј and 5Ј probes described above. The presence of the EcoRI site, and therefore the presence of the desired mutation, in exon 4 was analyzed in targeted ES cells by PCR, as described above. Chimeric mice were generated via blastocyst injection of the positive ES cell clone and were bred to Black Swiss mice for two generations to establish a hybrid line. Homozygous knock-in mice were obtained by heterozygous mating. Genotyping was performed by allele-specific PCR analysis across the LoxP site in intron 3 of the genomic DNA. The presence of the mutation in homozygous ␣1 S/S ␣2 S/S mice was verified by sequence analysis of exon 4 using the P1 and the P2 primers described above.
Crude Microsomal Preparations-Crude microsomal preparations were used for both Western blot analysis and [ 3 H]ouabain binding assays and were prepared as described previously (19), omitting NaI treatment. Briefly, for each preparation, tissues were pooled from three animals and homogenized twice on ice for 30 s in 2 ml of homogenization buffer (250 mM sucrose, 1 mM imidazole, and 1 mM EDTA). Homogenates were centrifuged at 5,000 rpm for 15 min. The supernatant was collected and ultracentrifuged at 40,800 rpm for 1 h by using an SW 50.1 Beckman rotor. Following ultracentrifugation, the pellet was resuspended in 1 mM imidazole, 1 mM EDTA buffer and stored at Ϫ80°C. Protein concentrations were determined using a BCA protein assay kit (Pierce).
Immunoprecipitation-The membranes from heart were solubilized with the non-ionic detergent IGEPAL CA-630 as described previously (20). The immunoprecipitation was also performed as described previously (20). Briefly, appropriate monoclonal or polyclonal antibodies were incubated with magnetic beads coated with the appropriate secondary antibodies (Dynabeats; M-450 anti-mouse goat and M-280 antirabbit sheep) overnight at 4°C. The following specific primary antibodies were used: mouse anti-Na/Ca exchanger, NCX (Affinity Bioreagents), mouse anti-␣1 isoform (␣6F, University of Iowa Developmental Studies Hybrid Bank), and rabbit anti-␣2 isoform (Dr. McDouhgna). Following this incubation, antibody attached beads were washed (three times) with phosphate-buffered saline (PBS). Following washing, the antibody-coated beads were incubated with 1 mg of the detergentsoluble fraction of heart plasma membrane for 4 h at 4°C. As a negative control, 1 mg of the detergent-soluble fraction of heart plasma membrane was incubated for 4 h at 4°C with Dynabeats coated with the appropriate secondary IgG. Following this incubation, the supernatant was saved and designated as post-immunoprecipitated fraction. The bead complex was washed four times with PTA (145 mM NaCl, 10 mM sodium azide, 10 mM NaH 2 PO 4 , and 0.5% Tween 20, pH 7.0). Immunoprecipitated proteins were extracted with Laemmli Sample Buffer (50 mM Tris (pH 6.9), 5% SDS, 1% ␤-mercaptoethanol, 10% glycerol, and 0.05% (w/v) bromphenol blue) by incubation at 37°C for 30 min.
Western Blot Analysis-Western blot analysis was performed via standard methods (13). Briefly, protein samples were incubated for 30 min at 37°C in Laemmli Sample Buffer and separated by electrophoresis in 10% polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes and blocked in 1% nonfat dry milk in TBST (5 mM Tris (pH 7.4), 150 mM NaCl, 0.01% Tween 20) for 1 h at room temperature. The blots were incubated in TBST containing ␣1 isoform-specific monoclonal antibody (␣6F, University of Iowa Developmental Studies Hybrid Bank), ␣2 isoform-specific monoclonal HERED antibody (Dr. Alicia McDonough), ␣3 isoform-specific monoclonal MA3-915 antibody (Affinity Bioreagents), and KETTY polyclonal antibody that recognized the C-terminal KETTY sequence of all ␣ isoforms (Dr. Jack Kyte) at 4°C overnight. For Western blot analysis of the immunoprecipitated proteins, additional antibodies were used as follows: mouse anti-Na/Ca exchanger, NCX (Affinity Bioreagents), mouse anti-plasma membrane Ca-ATPase 1 (Affinity Bioreagents), and mouse anti-dihydropyridine receptor ␣2, DHPR 1 ␣2 (Affinity Bioreagents). After incubation with peroxidase-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch), immunoreactivity was visualized following treatment with the ECL TM Western blotting Detection Reagents (Amersham Biosciences) by using the Kodak BioMax MR x-ray film.

Analysis of Total [ 3 H]Ouabain
Binding-Total [ 3 H]ouabain binding was performed as described previously (13). Briefly, 100 -500 g of crude membrane preparations were incubated at 37°C for 6 h in a reaction mixture containing 20 M [ 3 H]ouabain, 10 mM sodium metavanadate, 5 mM MgCl 2 , 50 mM Tris-HCl (pH 7.4), 5 mM Tris-PO 4 , and either 0 or 30 M unlabeled ouabain. Following incubation, the crude membrane preparations were filtered through the 1.2-m glass microfiber Whatman GF/C filter paper and washed five times with 7 ml of water using a Brandel cell harvester. The filter-associated radioactivity was determined by liquid scintillation counting. Nonspecific [ 3 H]ouabain for each microsomal preparation was determined using an excess of unlabeled ouabain (1 mM), and this value was subtracted from total binding obtained using 30 M ouabain. Subtraction of nonspecific binding from total binding obtained in kidney samples from both genotypes and in heart and skeletal muscle samples from targeted animals resulted in a value of zero.
Na,K-ATPase Activity-The Na,K-ATPase activity was performed as described previously (21). Briefly, the assay medium for Na,K-ATPase activity contained 130 mM NaCl, 20 mM KCl, 50 mM choline-Cl, 3 mM MgCl 2 , 3 mM ATP, 0.5 mM EGTA, 50 mM imidazole, pH 7.2 (20°C), ϳ1 g/ml of membrane protein, and different concentrations of ouabain. Assays were performed at 37°C for 30 min, and the released P i was determined by the colorimetric method at 660 nm, using malachite green/ammonium molybdate solution. Total ATPase activity was calculated as the difference in activity observed in the presence of 0 and 0.1 mM ouabain. The ouabain dose-response curve was obtained at increasing concentrations of ouabain, ranging from 0 to 0.1 mM.
Analysis of Cardiovascular Performance in Intact Closed-chest Animals-The analysis in intact animals was performed as described previously (13). The mean arterial pressure was measured using a lowcompliance pressure transducer (COBE Cardiovascular; Arvada, CO). A high fidelity, 1.8-French Millar Mikro-Tip transducer (model SPR-612; Millar Instruments, TX) in the left ventricle was used as the monitor. Dobutamine and ouabain were infused through the cannulated right femoral vein. Mean arterial pressure and intraventricular pressure from the COBE transducer and the Miller transducer were analyzed using the MacLab4/s data acquisition system connected to a Macintosh 7100/80 computer. Average values for heart rate, mean arterial pressure, and systolic left ventricular pressure were measured from the pressure waveforms and were determined for each animal from at least 50 consecutive beats during the final 30 s of each 3-min dosage period. Maximum dP/dt and dP/dt at 40 mm Hg (dP/dt 40 ) for dobutamine and ouabain infusions were calculated from the first derivative of the pressure waveforms.
Work-performing (Anterograde) Mouse Heart Analysis-Cardiac contractile parameters were also obtained using a work-performing heart apparatus as described previously (22). All cardiac measurements were taken with mean aortic pressure fixed at 50 mm Hg and venous return fixed at 5 ml/min (left ventricular minute work ϭ 250 mm Hg ϫ ml/min). Coronary flow was measured with a Transonic flowmeter (model T206). Temperature was continuously monitored via a Physitemp probe (Thermalert model  and was kept at 37.7°C. Hearts were perfused with oxygenated Kreb's-Henseleit solution containing 2.0 mM Ca 2ϩ (standard) or 1.5 mM Ca 2ϩ (low), and base-line functional parameters were obtained at the different Ca 2ϩ concentrations.
The effects of ouabain on cardiac contractility and of KB-R7943 on ouabain-induced positive inotropy were assessed at 1.5 mM Ca 2ϩ . Increasing concentrations (1 ϫ 10 Ϫ9 to 8 ϫ 10 Ϫ4 M) of ouabain, to produce a cumulative concentration response curve, were infused and mixed into the perfusate via catheters inserted into the aortic tubing 8 cm above the heart. The hearts were exposed to each concentration until the maximal effect was reached (usually a period of 5 min). The final concentration of ouabain delivered to the heart was calculated based on the stock concentration, infusion rate, and cardiac output. For analysis of with KB-R4973, hearts were perfused continuously with 10 M KB-R7943 for 45 min, following which the ouabain dose-response curve was obtained.
Statistical Analysis-Two-way analysis of variance for time and treatment with repeated measurements was performed, and post-hoc test of Fisher's least significant difference was obtained.

RESULTS
Generation of Mice Expressing a Cardiac Glycoside-sensitive ␣1 Isoform of Na,K-ATPase-The ouabain sensitivity of the mouse ␣1 isoform was increased by introducing R111Q and D122N amino acid substitutions into the first extracellular domain of this isoform (Fig. 1). The Gln-111 and Asn-122 residues are naturally present in the high affinity human and sheep ␣1 isoforms and were shown previously to confer sensitivity to cardiac glycosides (23,24). The R111Q and D122N substitutions were introduced by 2-bp mutations in exon 4 of the ␣1 isoform gene. In addition, an EcoRI restriction enzyme site was introduced as a silent mutation for easy identification of the targeted allele.
The ouabain-sensitive ␣1 isoform mice were generated by the Cre/LoxP gene targeting strategy, as depicted in Fig. 1B. The strategy consists of two sequential ES cell transfections; the first transfection introduced the targeting sequence into one of the wild type alleles by homologous recombination. Homologous recombination resulted in heterozygous ES cell clones having a targeted allele containing the LoxP-flanked neomycin resistance cassette and the desired R111Q and D122N mutations (data not shown). The second transfection introduced Cre-recombinase, which excised the neomycin resistance-conferring cassette from the targeted allele by recombination of two LoxP sites. Recombination generated heterozygous ES cells that retained the desired mutation in exon 4 (data not shown).
Chimeric animals were generated by injecting the targeted ES cell clone into blastocysts that were then implanted into pseudopregnant SVJ/129 mice. The chimeric offspring were FIG. 1. Generation of mice expressing the cardiac glycoside-insensitive ␣1 Na,K-ATPase (␣1 S/S ␣2 S/S ). A, introduction of the R111Q and D122N substitutions in the mouse ␣1 isoform of Na,K-ATPase. Two base pair mutations were introduced into exon 4 by PCR amplification, resulting in the R111Q and D122N amino acid substitutions. Two silent base pair mutations were also introduced to form a unique EcoRI site. B, Cre-LoxP targeting strategy. Top, a restriction map of the region in one of the wild type alleles that would be involved in homologous recombination. Middle, targeting vector and a schematic of the targeted allele following successful homologous recombination. Bottom, targeted allele following successful excision of the neomycin cassette by Cre-recombinase. The 5Ј and 3Ј probes were used for screening of targeted ES cells. Primers P1 and P2 were used in PCR amplification of exon 4 for verification of EcoRI site presence. Primers P3 and P4 were used for genotyping of the offspring animals. C, sequence histogram of the partial exon 4 sequence from the ␣1 S/S ␣2 S/S animals. Introduced R111Q and D122N amino acid substitutions and an EcoRI site were retained in genomic DNA of the ␣1 S/S ␣2 S/S animals. crossed to Black Swiss females to yield heterozygous ␣1 R/S ␣2 S/S animals. Through mating of heterozygous mice, wild type (␣1 R/R ␣2 S/S ), heterozygous (␣1 R/S ␣2 S/S ), and homozygous (␣1 S/S ␣2 S/S ) offspring were generated. These genotypes were born in expected Mendelian ratios. The ␣1 S/S ␣2 S/S animals retained the desired R111Q and D122N substitutions as shown by the sequence histogram of their genomic DNA (Fig. 1C).
Total [ 3 H]ouabain binding in tissue preparations from the ␣1 S/S ␣2 R/R mice demonstrated an increase in the ouabain affinity of the ␣1 isoform and ouabain resistance of the ␣2 isoform (Fig. 2B). As expected, no [ 3 H]ouabain binding was detected in kidney from wild type mice as only the low affinity ␣1 isoform is expressed. In contrast, high levels of ouabain binding were detected in kidney preparations expressing the modified ␣1 isoform. The ouabain binding was detected in wild type skeletal muscle and heart as these tissues express the ouabain-sensitive ␣2 isoform of Na,K-ATPase. The decrease in ouabain binding in skeletal muscle preparations from the ␣1 S/S ␣2 R/R mice reflects the reduction in the ouabain sensitivity of the ␣2 isoform, which is the major isoform in this tissue. This is consistent with our previous study demonstrating that the modification of the ␣2 isoform to a ouabain-resistant form completely abolishes ouabain binding in skeletal muscle from ␣1 R/R ␣2 R/R mice (13). Although the ␣2 isoform is resistant to ouabain in double-knock-in animals, i.e. ␣1 S/S ␣2 R/R mice, more ouabain binding was detected in heart preparations from ␣1 S/S ␣2 R/R mice than from wild type. This reflects the increase in ouabain sensitivity of the ␣1 isoform and indicates that this isoform contributes more to the total levels of the Na,K-ATPase than the ␣2 isoform.
By comparing the total ouabain binding in skeletal muscle and heart between the wild type and targeted mice, we determined the relative abundance of the ␣1 and ␣2 isoforms in these tissues. This is possible as in these tissues the total ouabain binding is contributed by the ␣2 isoform in wild type mice and by the ␣1 isoform in targeted mice. There was four times more ouabain bound in wild type skeletal muscle than in skeletal muscle from ␣1 S/S ␣2 R/R mice. Therefore, the ␣1 isoform comprises 20% and the ␣2 isoform 80% of total Na,K-ATPase in skeletal muscle. In contrast, there was 20 times more ouabain bound in ␣1 S/S ␣2 R/R heart than in wild type ␣1 R/R ␣2 S/S . Hence, the ␣1 isoform comprises 95.2% and the ␣2 isoform comprises 4.8% of total Na,K-ATPase in heart.
Western blot analysis of the tissue from the wild type and ␣1 S/S ␣2 R/R mice demonstrated that the expression and tissue FIG. 2. A, schematic of generation of the ␣1 S/S ␣2 R/R animals through mating of ouabain-sensitive ␣1 isoform (␣1 S/S ␣2 S/S ) mice with ouabain-resistant ␣2 isoform (␣1 R/R ␣2 R/R ) mice. This is possible as the genes for the ␣1 (ATPA1) and ␣2 (ATPA2) isoforms are located on different chromosomes in mice. B, total [ 3 H]ouabain binding in crude membrane preparations of kidney, skeletal muscle, and heart from wild type (␣1 R/R ␣2 S/S ) and targeted (␣1 S/S ␣2 R/R ) animals. Data are represented as mean Ϯ S.E. of three independent experiments, each analyzing pooled tissues from three animals of each genotype. C, Western blot analysis of the total Na,K-ATPase by C-terminal KETTY antibody and the individual ␣1, ␣2, and ␣3 isoforms in membrane preparations from kidney, heart, brain, and skeletal muscle from wild type (␣1 R/R ␣2 S/S ) and targeted (␣1 S/S ␣2 R/R ) animals. Data are representative of three independent experiments, each analyzing pooled tissues from three animals of both genotypes. The amount of protein is represented in g at the top of each blot. D, Na,K-ATPase activity at increasing concentrations of ouabain in kidney membrane preparations from wild type and targeted animals. Modification of the ␣1 isoform resulted in leftward shift in the ouabain dose-response curve. The IC 50 for wild type ␣1 is 1 ϫ 10 Ϫ4 M ouabain, and the IC 50 for targeted ␣1 isoform is 1 ϫ 10 Ϫ6 M ouabain. Data are represented as mean Ϯ S.E. of three independent experiments, each analyzing pooled tissues from three animals of each genotype. distribution of the ␣1 and ␣3 isoforms are normal in targeted animals (Fig. 2C). Although the tissue distribution of the ␣2 isoform was normal, there was a slight, but not significant, up-regulation in its expression in targeted heart preparations. This may be due to variability in the genetic background of these animals, as expression of the ␣2 isoform is normal in hearts from ␣1 R/R ␣2 R/R (13) and ␣1 S/S ␣2 S/S (data not shown) mice. However, this slight alternation in the expression of the modified/ouabain-resistant ␣2 isoform could have not affected the analysis because we determined the difference in the contractile parameters before and after selective inhibition of only the ouabain-sensitive ␣1 isoform.
To determine whether modification of ouabain sensitivity altered enzymatic activity of the ␣1 isoform, we analyzed the total ouabain inhibitable Na,K-ATPase activity in kidney preparations from ␣1 S/S ␣2 R/R and wild type mice. There was no difference in total Na,K-ATPase activity between the two genotypes (data not shown). This was expected as amino acids Leu-111 and Asn-122 are naturally present in the human ␣1 isoform that is sensitive to ouabain. A 100-fold increase in sensitivity of the modified ␣1 isoform was observed (Fig. 2D). Whereas 1 ϫ 10 Ϫ4 M ouabain inhibited 50% of the total Na,K-ATPase in wild type, the same effect was reached at 1 ϫ 10 Ϫ6 M ouabain in targeted kidney preparations.
Basal Hemodynamic Parameters Are Normal in Intact Closed-chest ␣1 S/S ␣2 R/R Mice-Basal cardiovascular function in wild type and ␣1 S/S ␣2 R/R mice was evaluated in vivo by cardiac catheterization (Table I). There were no significant differences in basal heart rate, mean arterial pressure, systolic blood pressure, left ventricular pressure, maximum rate of force of contraction, minimum rate of force of contraction, and the rate of force of contraction at 40 mm Hg between the two genotypes. This demonstrates normal physiological hemodynamics and heart function in targeted mice. This also indicates that slight down expression of the ␣2 isoform has not altered normal hemodynamics.
␤-Adrenergic Cardiac Response Is Normal in ␣1 S/S ␣2 R/R Mice-To determine whether modification of the ␣1 and ␣2 isoforms of Na,K-ATPase alerted the normal Ca 2ϩ handling in heart, we analyzed cardiac contractility in intact wild type and ␣1 S/S ␣2 R/R mice following administration of dobutamine, a ␤-adrenergic agonist. There were no significant differences in dP/dt max , dP/dt min , and dP/dt 40 following the ␤-adrenergic stimuli between the two genotypes (Table II). This indicated that cardiac contraction and its response to the ␤-adrenergic stimuli are normal in targeted animals.
Inhibition of the ␣1 Isoform of Na,K-ATPase Results in Positive Cardiac Inotropy-The effect of ouabain on contractility in isolated work-performing ␣1 S/S ␣2 R/R hearts was analyzed to determine the consequence of selective inhibition of the ␣1 isoform on cardiac contractility. Administration of ouabain increased the maximum rate of heart contraction (ϩdP/dt) and relaxation (ϪdP/dt) in both wild type and targeted isolated hearts (Fig. 3, A  and C). The initial increase in cardiac function of wild type hearts was observed at 8 ϫ 10 Ϫ7 M ouabain and gradually increased with increasing concentrations of ouabain. This positive cardiac inotropy in rodent heart at low concentrations of ouabain is well established and was shown to be mediated by the ␣2 isoform of Na,K-ATPase (13). Ouabain increased cardiac contractility in isolated ␣1 S/S ␣2 R/R hearts and resulted in greater positive inotropy than in wild type hearts. The maximum increase in cardiac contractility of targeted hearts occurred at 8 ϫ 10 Ϫ6 M ouabain. At this concentration of ouabain, the total change from base line in ϩdP/dt (Fig. 3B) and ϪdP/dt (Fig. 3D) for each targeted heart was 5-fold greater than in wild type. This indicates that the activity of the ␣1 isoform is functionally linked with cardiac contractility.
The maximum increase in cardiac contraction was reached at a concentration of ouabain (8 ϫ 10 Ϫ6 M) that inhibited 40% of the modified ␣1 isoform activity in kidney preparations from ␣1 S/S ␣2 S/S mice (Fig. 2D). However, if we take into account that in membrane preparations the ouabain-binding site is more exposed than in intact tissue, we can assume that inhibition of less than 40% of the ␣1 isoform resulted in maximum positive cardiac inotropy in the perfused ␣1 S/S ␣2 R/R hearts. Perfusion of higher concentrations of ouabain, which inhibited more than 40% of total Na,K-ATPase activity in isolated kidney preparations from ␣1 S/S ␣2 S/S mice, led to cardiac failure in targeted hearts (data not shown).
Inhibition of the ␣1 Isoform by Ouabain Increases Cardiac Contractility in Intact Animals-Cardiac contractility as a function of ouabain was also analyzed in intact anesthetized animals. Measurements of the maximal (dP/dt max ) and minimal rate (dP/dt min ) of contraction and relaxation and rate of contraction at 40 mm Hg (dP/dt 40 ) under basal conditions and following infusion of ouabain were measured.
One minute following the infusion of 0.48 nmol/g of ouabain dP/dt max (Fig. 4A), dP/dt min (data not shown), and dP/dt 40 (Fig.  4B) increased in both the wild type and ␣1 S/S ␣2 R/R mice. The increase in cardiac contractility was greater in targeted than in wild type mice. The increase in dP/dt max (Fig. 4C), and dP/dt 40 (Fig. 4D) was 3-fold greater in targeted than in wild type mice. The administration of a higher concentration of ouabain (1.77 nmol/g) significantly increased cardiac contractility in wild type but caused cardiac failure in ␣1 S/S ␣2 R/R mice, 15 min following infusion (data not shown).
The Inhibitor of Reverse Na/Ca Exchanger Abolishes Positive Cardiac Inotropy Caused by Inhibition of the ␣1 Isoform of Na,K-ATPase-We next determined whether the ␣1 isoform regulates cardiac contractility through the reverse action of the Na/Ca exchanger. Pretreatment of hearts with 10 M KB-R7943, a specific inhibitor of the reverse mode of the Na/Ca exchanger, did not alter basal contractile parameters in isolated wild type and targeted hearts (Fig. 5). However, it completely inhibited the ouabain-induced increase in cardiac contraction of both wild type and targeted hearts. Inhibition of the Na/Ca exchanger completely abolished a ouabain-induced increase in ϩdP/dt and ϪdP/dt of both wild type (Fig. 5, A and B)  and ␣1 S/S ␣2 R/R (Fig. 5, C and D) isolated hearts. Thus, the reverse mode of the Na/Ca exchanger is necessary for contraction to occur following inhibition of the ␣1 isoform in ␣1 S/S ␣2 R/R hearts and the ␣2 isoform in wild type hearts. The ␣1 Isoform and Na/Ca Exchanger Form a Protein-Protein Complex in the Heart Plasma Membrane-We next deter-

FIG. 3. Contraction and relaxation parameters of the isolated ␣1 R/R ␣2 S/S and ␣1 S/S ␣2 R/R hearts at low doses of ouabain.
A, maximum rate of contraction, ϩdP/dt. B, maximum rate of relaxation, ϪdP/dt, in the wild type (n ϭ 6) and targeted hearts (n ϭ 9) was determined at increasing concentrations of ouabain. The average of the total change in maximum rate of contraction, ⌬ϩdP/dt (C), and maximum rate of relaxation, ⌬Ϫ dP/dt, for each ␣1 R/R ␣2 S/S and ␣1 S/S ␣2 R/R heart (D) was determined following perfusion of 8 ϫ 10 Ϫ6 M ouabain. *, p Ͻ 0.01 versus wild type. Values are represented as mean Ϯ S.E.
FIG. 4. Analysis of cardiac contractility in intact ␣1 R/R ␣2 S/S and ␣1 S/S ␣2 R/R animals following infusion of ouabain. The effect of ouabain on the maximum rate of contraction, dP/dt max (A), and on the maximum rate of contraction at 40 mm Hg, dP/dt 40 (C), was determined in ␣1 R/R ␣2 S/S (n ϭ 9) and ␣1 S/S ␣2 R/R (n ϭ 10) mice. ⌬dP/dt max (B) and ⌬dP/dt 40 (D) were determined following infusion of 0.48 nmol/g body weight ouabain. Increasing ouabain concentrations raised the dP/dt max (A) and dP/dt 40 (C) in both intact wild type and targeted mice. ⌬dP/dt max (B) and ⌬dP/dt 40 (D) were 3-fold greater in targeted mice than in wild type. *, p Ͻ 0.01 versus wild type. Values are represented as mean Ϯ S.E. mined whether the ␣1 isoform of the Na,K-ATPase forms a protein complex with the Na/Ca exchanger in heart plasma membranes. Previous immunoprecipitation analysis from brain demonstrated that only the ␣2 isoform forms a complex with the Na/Ca exchanger (20). The immunoprecipitates of the Na/Ca exchanger (NCX) and the ␣1 isoform were prepared from the detergent-soluble fraction of mouse heart plasma membranes. In addition, we prepared immunoprecipitates of the ␣2 isoform as a positive control. As a negative control, the immunoprecipitates of mouse and rabbit IgG were also prepared.
The immunoprecipitation with NCX-specific antibody removed all the Na/Ca exchangers from the detergent-soluble fraction of heart membranes. As shown in Fig. 6A, a predominant NCX band of 140 kDa was detected in the precipitated fraction, and no band was detected in the post-immunoprecipitated, detergent-soluble fraction. No NCX immunoprecipitated with mouse IgG. Both the ␣1 and ␣2 isoforms of Na,K-ATPase co-immunoprecipitated to a significant extent with NCX. Most of the ␣2 isoform was bound by NCX, and thereby a majority of it was recovered in the immunoprecipitate. Although a significant amount of the ␣1 isoform co-immunoprecipitated with NCX, some of it was recovered in the post-immunoprecipitated soluble fraction. The negligible amounts of the dihydropyridine receptor, DHPR ␣2, co-immunoprecipitated with NCX. In contrast, the plasma membrane Ca 2ϩ -ATPase 1 (PMCA 1) did not co-immunoprecipitate with NCX. The immunoprecipitation with mouse IgG did not remove any of these plasma membrane proteins from the detergent fraction, and thereby none was recovered in the immunoprecipitate.
As shown in Fig. 6B, immunoprecipitation with the ␣1 isoform-specific antibody removed all the ␣1 isoform from the detergent-soluble fraction, and all the ␣1 isoform was recovered in the immunoprecipitate. A control immunoprecipitation with mouse IgG did not remove any the ␣1 isoform. The NCX coimmunoprecipitated with the ␣1 isoform. However, not all NCX was bound by the ␣1 isoform, and some was recovered in the post-immunoprecipitated supernatant. The ␣2 isoform, DHPR ␣2, and PMCA 1 did not co-immunoprecipitate with the ␣1 isoform, and these proteins were recovered in the post-immunoprecipitated supernatant. The nonspecific immunoprecipitation with mouse IgG did not pull down any of the analyzed proteins and was recovered in the IgG immunoprecipitate.
The immunoprecipitation with the ␣2 isoform-specific antibody removed a significant amount of the ␣2 isoform from the solubilized heart membranes (Fig. 6C). However, some of the ␣2 isoform was recovered in the post-immunoprecipitated supernatant. The immunoprecipitation with rabbit IgG did not remove the ␣2 isoform. As expected, NCX co-immunoprecipitated with the ␣2 isoform. Small amounts of the NCX were bound by this isoform, and the majority of NCX was recovered in the post-immunoprecipitated supernatant. The ␣1 isoform did not co-immunoprecipitate with the ␣2 isoform. Similarly, both DHPR ␣2 and PMCA 1 were fully recovered in the postimmunoprecipitated supernatant. The immunoprecipitate of rabbit IgG did not contain any of the analyzed proteins.
In summary, both the ␣1 and ␣2 isoforms of Na,K-ATPase co-localized with the Na/Ca exchanger in cardiac sarcolemma. However, protein complexes formed between the ␣1 isoform and the Na/Ca exchanger are distinct from the Na/Ca exchanger complexes containing the ␣2 isoform. DISCUSSION Studies in mice lacking one copy of the ␣1 or ␣2 isoform gene suggested that these two ␣ isoforms of Na,K-ATPase play a differential role in the regulation of cardiac contractility (6). The hearts from ␣2 ϩ/Ϫ animals are hypercontractile, whereas those of the ␣1 ϩ/Ϫ animals are hypocontractile. Although this was expected for the ␣2 isoform, it was quite surprising for the ␣1 isoform, as several studies (14 -18) suggested that reduction in the activity of the ␣1 isoform results in positive cardiac inotropy. We have used an approach that makes it possible to examine the functional role of each isoform of Na,K-ATPase separately. We have confirmed that the ␣2 isoform of Na,K-ATPase is coupled to cardiac contraction and mediates ouabain-induced positive inotropy in mouse heart. This was accomplished by comparing the effect of ouabain on ouabain-sensitive (wild type) and ouabain-resistant ␣2 isoform hearts (13). In the present study, we directly examined the functional role of the ␣1 isoform of Na,K-ATPase in the regulation of cardiac contractility by using genetically engineered animals where the ␣1 isoform was modified to a ouabain-sensitive form, and the ␣2 isoform was modified to a ouabain-resistant one. This enabled us to selectively inhibit only the ␣1 isoform without altering the activity of the ␣2 isoform. Hence, any alteration in cardiac contractility in these knock-in mice, following administration of ouabain, can only be accounted for by the reduction in the activity of the ␣1 isoform. This approach bypassed the embryonic lethality associated with complete genetic elimination of the ␣1 isoform (6). In addition, as cardiac contractility was analyzed immediately after administration of ouabain, we avoided the occurrence of genetic compensatory mechanisms that may accompany a long term down-regulation of the ␣1 isoform and mask the actual cardiac phenotype. Taken together, this represents a novel approach for analyzing the functional role of a specific Na,K-ATPase isozyme.
In the present study we demonstrate that a reduction in ␣1 isoform activity enhances cardiac contractility. This is in agreement with previous studies attributing the increased contrac-tility of isolated rat cardiac myocytes to reduced activity of the ␣1 isoform (16). In addition we demonstrate that the extent of the reduction in ␣1 isoform activity determines whether cardiac contractility will be enhanced or compromised. Whereas inhibition of modest amounts of the ␣1 isoform activity at low concentrations of ouabain increased cardiac contractility, cardiac failure occurred following significant reduction in the ␣1 isoform activity at high concentrations of ouabain. This is expected if we consider the amounts of the ␣1 isoform in heart. In agreement with previous findings, our study demonstrates that the ␣1 isoform comprises a majority of the Na,K-ATPase in mouse heart (26,27). Thus, it is expected that inhibition of significant amounts of Na,K-ATPase activity would result in cardiac failure. This may explain why mice lacking one copy of the ␣1 isoform gene have depressed cardiac contractility.
The present data suggest that the ␣1 and ␣2 isoforms play a similar role in the regulation of cardiac contractility. It is quite clear that inhibition of either the ␣1 or ␣2 isoform enhances cardiac contraction. This is of importance as human hearts express essentially equal amounts of three ␣ isoforms (␣1, ␣2, and ␣3), which are all sensitive to ouabain (10,28). Thus, in patients with congestive heart failure, inhibition of the ␣1 isoform along with the ␣2 isoform by cardiac glycosides will contribute to increased heart contraction. In addition, the ␣1 isoform is not a major contributor to the total Na,K-ATPase in human heart, and it is unlikely that a significant reduction in its activity will result in cardiac failure as it does in mouse hearts. Thus, the present data should help in understanding the role of the ␣1 isoform in failing human hearts, where a significant down-regulation of the ␣1 isoform occurs (29 -32).
The present study also demonstrates that the ␣1 isoform FIG. 6. Both the ␣1 and ␣2 isoforms co-localize with the Na/Ca exchanger in heart. Western blot of immunoprecipitates generated with antibodies (Ab) raised against NCX (A), the ␣1 isoform of Na,K-ATPase (B), and the ␣2 isoform of Na,K-ATPase(C). Control immunoprecipitates (IP) were generated with mouse IgG (A and B) and with rabbit IgG. The immunoprecipitates were probed with antibodies against, NCX, the ␣1 isoform, the ␣2 isoform, PMCA1, and DHPR ␣2. Lanes labeled Input (detergent-soluble homogenate), IP S (post-immunoprecipitated supernatant), and IP P (immunoprecipitate) were loaded with equal volumes. Lane Heart is nondetergent-soluble homogenate. regulates cardiac contractility through the reverse mode of the Na/Ca exchanger as does the ␣2 isoform. This is in contrast to previous studies suggesting that only the ␣2 isoform of Na,K-ATPase regulates the flux mode of the Na/Ca exchanger (6,12). However, these studies did not directly analyze the role of the ␣1 isoform in regulating cardiac contractility and the Na/Ca exchanger transport mode. Nevertheless, our data show that the ␣1 isoform, as well as the ␣2 isoform, regulates cardiac contractility through the Na/Ca exchanger.
The current study also demonstrates that in cardiac sarcolemma both the ␣1 and ␣2 isoforms form distinct protein complexes with the Na/Ca exchanger. In contrast, a recent study demonstrated that in brain the Na/Ca exchanger formed a complex with the ␣2 isoform and not the ␣1 isoform of Na,K-ATPase (20). Thus, it is possible that the functional role of the ␣1 isoform in brain may be different from that in contractile tissue, such as heart.
In summary, the present study represents a direct analysis of the functional role of the ␣1 isoform of Na,K-ATPase. We used a novel approach that allowed us to bypass the embryonic lethality associated with the complete genetic elimination of the ␣1 isoform and to directly analyze the role of the ␣1 isoform in the regulation of cardiac function. We demonstrate that the ␣1 isoform of Na,K-ATPase regulates cardiac contractility through the reverse mode of the Na/Ca exchanger, as does the ␣2 isoform. This finding demonstrates that the reversal of the Na/Ca exchanger occurs subsequent to the inhibition of the Na,K-ATPase and results in increased cardiac contractility. This is in agreement with the previous analysis of the ouabaininduced inotropy in the Na/Ca exchanger-deficient embryonic heart tubes (33). Our studies also demonstrate that both the ␣1 and ␣2 isoforms form a protein complex with Na/Ca exchanger in the plasma membrane of heart. The present study also confirms that the ␣1 isoform mediates ouabain-induced positive cardiac inotropy. Recently, we confirmed that the ␣2 isoform of Na,K-ATPase mediates ouabain-induced positive inotropy in mouse heart by comparing ouabain-sensitive (wild type) and ouabain-resistant ␣2 isoform hearts (13). Thus, inhibition of either the ␣1 or ␣2 isoform enhances cardiac contractility. Hence, the present data argue against the ␣2 isoform being the only Na,K-ATPase isoform regulating cardiac contractility. Although our study demonstrates that both the ␣1 and ␣2 isoforms have the same role in heart, our data cannot address whether there are subtle differences in mechanisms underlining the function of these two ␣ isoforms in this tissue.