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(The FASEB Journal. 2005;19:53-61.)
© 2005 FASEB

Evidence that the H1-H2 domain of {alpha}1 subunit of (Na++K+)-ATPase participates in the regulation of cardiac contraction

Kai Y. Xu1, Eiki Takimoto*, George J. Juang*, Qi Zhang, Holly Rohde{dagger} and Allen C. Myers{dagger}

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, USA;
* Department of Medicine, Division of Cardiology,
{dagger} Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

1Correspondence: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene St., Room 308, Baltimore, MD 21201, USA. E-mail: kxu002{at}umaryland.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
(Na++K+)-ATPase (NKA) plays an important role in ion homeostasis and regulates cardiac contraction. To understand the molecular basis of its cardiac regulatory functions, we investigated whether the primary structure of the H1-H2 domain in {alpha}-1 ({alpha}1) subunit of the enzyme plays a role in myocardial contractile regulation. Here we show that site-specific binding to this {alpha}1 H1-H2 domain with a targeted antibody (SSA78) markedly augments intracellular Ca2+ transients and contraction of rat ventricular cardiomyocytes without inactivating NKA. In vivo SSA78 infusion in mice results in a positive inotropic effect with enhanced contractile function yet no change in relaxation, indicating a direct cardiac effect linked to the H1-H2 domain. Competitive immunofluorescent staining and flow cytometry reveal that SSA78 binding is antagonized by ouabain, supporting the interaction of SSA78 at one of the glycoside-effecter sites. These new findings suggest that the H1-H2 domain of {alpha}1 subunit of NKA is a critical determinant of enzyme biologic activity, which couples to enhanced myocyte calcium transient and inotropic action.—Xu, K. Y., Takimoto, E., Juang, G. J., Zhang, Q., Rohde, H., Myers, H. C. Evidence that the H1-H2 domain of {alpha}1 subunit of (Na++K+)-ATPase participates in the regulation of cardiac contraction.


Key Words: structure and function • antibody • positive inotropic effect • molecular regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SODIUM AND POTASSIUM ion-activated adenosinetriphosphatase [(Na++K+)-ATPase (NKA)] (1) catalyzes active transport of Na+/K+ across the plasma membrane in virtually all animal cells (2) and contributes to the regulation of excitation and contraction of heart muscle (3 4) . Although valuable information has been obtained by experiments designed to study the structure-function relationships of native NKA, the precise structural features of the enzyme that regulates myocardial contraction remain unclear. Inhibition of NKA by cardiac glycosides has long been used to treat heart failure as this leads to accumulation of intracellular sodium that is exchanged for calcium (5 6 7 8 9) to enhance cardiac contraction. Site-directed mutagenesis has shown that the first extracellular loop (H1-H2 domain) of the {alpha}1 subunit of NKA potently affects ouabain binding affinity (10) . However, whether binding to this domain itself is coupled to NKA inhibition and/or cardiac inotropy is unknown. To test this, we developed a site-specific antibody (SSA78) against the H1-H2 region of {alpha}1 subunit of rat NKA and tested the hypothesis that targeted binding can itself enhance cardiac contraction in isolated heart muscle cells and intact ventricles. The results support a primary link between H1-H2 binding and calcium-associated positive inotropy revealing in vivo efficacy of the antibody inotrope in mouse hearts, and suggesting an important functional role of the {alpha}1 subunit H1-H2 sequence to NKA-contractile modulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Reagents were from Sigma Chemical unless otherwise specified. Purified site-specific anti-peptide antibody (SSA78, 0.5 mg/mL) against the H1-H2 region of rat {alpha}1 NKA was generated by Genemed (South San Francisco, CA, USA). The Ca2+ probe Indo-1/acetoxymethyl-ester (Indo-1/AM) was purchased from Molecular Probes (Eugene, OR, USA). Protein A/G-agarose was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN., USA). Color developing reagent was from Promega Corporation (Madison, WI, USA). The anti-SERCA2 (MA3-919) and anti-NKA {alpha}3 (MA3-915) antibodies were from Affinity BioReagents (Neshanic Station, NJ, USA). The anti-NKA {alpha}2 antibody was provided by Dr. Alicia McDonough. Highly purified dog kidney NKA was a gift from Dr. Jack Kyte. Mice (CD-1) and rats (Sprague Dawley) were obtained from Charles River Laboratories (Wilmington, MA, USA). The Animal Care and Use Committees of the Johns Hopkins University and the University of Maryland School of Medicine approved animal protocols.

Antibody preparation
The RSATEEEPPNDD peptide (11 , 12) was synthesized and polyclonal antibody SSA78 against this sequence was generated in New Zealand white rabbits using KLH as a peptide carrier. SSA78 was further purified through an affinity column directed against the same synthetic peptide. Purified SSA78 recognized denatured (Western blot) and native NKA (immunostaining). Synthetic peptide was also used as specific peptide blocker (PB78) for the study.

Immunoprecipitation and Western blot
Rat cardiac myocytes were used for immunoprecipitation. All steps described below were performed at 4°C. Cells were washed in three changes of PBS, then suspended in 1 mL cold lysis buffer containing 150 mM NaCl, 10 mM EDTA, 1% NP-40 (Sigma-Aldrich Corp. St. Louis, MO, U SA), 1 mM Na3VO4, and 1 x protease inhibitor cocktail (Roche Applied Science) for 60 min. This mix was sonicated 8 times (10–15 s each) on ice by an ultrasonic converter and centrifuged at 14,000 rpm for 2 min. The supernatant was carefully collected and transferred to another tube. To lower the amount of nonspecific contaminants in the cell lysate and remove proteins with high affinity for protein G or protein A, preclearing of cell lysate was performed as follows: the freshly prepared whole cell lysate (1 mL) was incubated with 0.25 µg control rabbit IgG (corresponding to the host species of the primary antibody SSA78), together with 20 µL protein A/G-agarose beads for 30 min. The mixture was then centrifuged at 2500 rpm for 30 s and the supernatant was transferred to another Eppendorf tube. Immunoprecipitation was started by adding SSA78 (0.1 mg/mL) to the Eppendorf tube containing cold precleared lysate (1 mg/mL) and continued for 8–10 h, then protein A/G-agarose (40 µL) was added to the sample and left overnight on a rocker platform. The agarose beads were pelleted by centrifugation at 2500 rpm for 5 min and washed 3 times for 5 min each with PBS. The pellets were resuspended in 40 µL of 2x electrophoresis sample buffer. The samples were boiled for 5 min and up to 30 µL were loaded to a 7% SDS gel. Proteins were transferred from the SDS gel to a nitrocellulose membrane using an electroblotting apparatus. The nitrocellulose membrane was blocked with BSA for 1 h and incubated with SSA78 or other control antibodies overnight. The membrane was incubated with alkaline phosphatase-conjugated secondary antibody (1:2500) for 1 h and washed 3 times for 5 min each with TBS, 0.05% Tween-20, and once for 5 min with TBS. The color was developed using a reagent containing a mixture of NBT and BCIP (Promega) for visual analysis of the immunoprecipitated protein.

Immunofluorescent staining
Rat cardiac myocytes were prepared as described previously (13) . Primary incubation with SSA78 antibody alone (1:1000 dilution) or SSA78 antibody plus peptide blocker (1:1 ratio) was carried out overnight at 4°C in PBS. Secondary antibody was incubated with samples at room temperature for 1 h using anti-rabbit Alexa 488. Imaging was carried out with a Nikon Diaphot 300 inverted epifluorescence microscope. Cultured CV-1 cells were frozen and cut on a cryostat. Sections (8 µm) of each tissue were blocked with 10% goat serum with the 1% bovine serum albumin (BSA) and incubated with SSA78 (1:100) for 60 min in the presence or absence of 1mM ouabain or strophanthidin. Washed slides were evaluated after incubation with a fluorescein-isothiocyanate (FITC) -conjugated goat anti-rabbit Alexa 488/fluorescein antibody (1:75, Molecular Probes).

Flow cytometric analysis
Membrane surface expression of the RSATEEEPPNDD region of {alpha}1 NKA was analyzed using indirect immunofluorescence and flow cytometry. Cultured CV-1 cells were washed and incubated with SSA78 (0.18 µM) or with SSA78 + PB78 (90 µM) for one hour in the presence or absence of ouabain (20 µM). The control CV-1 cell sample was incubated with rabbit IgG (0.18 µM). Washed cells were then incubated with FITC-conjugated goat anti-rabbit IgG (Jackson Laboratories) for 1 h and analyzed using a Becton Dickinson FACS Calibur flow cytometer after excitation at 488 nm.

In vivo assessment
In vivo assessment of the cardiovascular effects of SSA78 were examined in anesthetized male wild-type mice (CD1, Charles River, 32–40 g) using miniature pressure-volume catheter technique as described previously in detail (14) . Animals were anesthetized with i.p. urethane (300–500 mg/kg), etomidate (5 mg/kg), and morphine (0.5 mg/kg), intubated, and ventilated with supplemental oxygen. The cardiac apex was exposed by thoracotomy and a pressure-volume catheter (SPR-839, Millar Inc., Houston, Texas) was inserted along the long axis. Atrial pacing (600 bpm) was achieved by intraesophageal catheter (NuMed, Nicholville, NY) and the volume signal was calibrated. Animals received 5 µL/min PBS for 10 min (control), then SSA78 (5 µL/min) for 30 min, followed by a 20–40 min washout with PBS. Control studies showed that PBS had no cardiovascular effect at this infusion rate. Cardiac function was assessed at steady–state and during transient preload reduction by inferior vena cava obstruction. Systolic function was assessed by load-independent indices including end-systolic elastance (Ees), preload-recruitable stroke work (PRSW), and maximal rate of pressure rise normalized to instantaneously developed pressure (dP/dtmax/IP) (15) , diastolic function by negative dP/dtmin and time constant of pressure relaxation, preload by end-diastolic volume, and afterload by effective arterial elastance.

Measurement of cell contraction
Cardiac myocytes were isolated from adult Sprague-Dawley rats using standard enzymatic methods (16 , 17) and suspended in buffer containing (in mM) 137 NaCl, 5.4 KCl, 15 dextrose, 1.3 MgSO4, 1.2 NaH2PO4, 1 CaCl2, and 20 HEPES, pH 7.4. Cells were placed on an inverted microscope (Zeiss model IM-35) and electrically stimulated at 0.5 Hz at 27°C. Cell length was monitored by optical edge tracking using a photodiode array (model 1024 SAQ, Reticon) with 3 ms temporal resolution. Contraction amplitude was indexed by the percent shortening of cell length.

Intracellular Ca2+ transients
Myocytes were loaded with 50 µg of Indo-1/AM for 10 min, washed and resuspended in HEPES-buffered solution in the presence of 1 mM Ca2+,then stored in the dark at room temperature for 60 min before use (18) . Cells were placed on the stage of a modified inverted microscope equipped for simultaneous recording of Indo-1 fluorescence and cell length (optical edge tracking). Cells were studied at room temperature at 0.5 Hz stimulation rate, excited at 350 nm and the ratio of 410:490 emission was determined to quantify intracellular calcium. Background autofluorescence was subtracted.

Isolation of sarcolemmal vesicles and purification of NKA
Cardiac sarcolemmal (SL) vesicles were isolated from rat hearts as reported previously (19) . The SL vesicles were tested with saponin (20) and found to be predominately right-side out in orientation. NKA was further purified as described (20) . Briefly, the rat SL vesicles (4.4 mg/mL) were titrated with 0.58 mg/mL of SDS in the presence of 2 mM ATP at 20°C for 30 min, loaded on the top of a sucrose (W/W) step gradient (15%, 28.8% and 37.3%) in a Ti 60 tube, and centrifuged at 40,000 rpm for 90 min. The fractions that contain NKA were collected and stored at –70°C.

Isolation of cardiac SR vesicles
Cardiac muscle sarcoplasmic reticulum (SR) vesicles were prepared from rat hearts according to the methods of Eletr and Inesi (21) and Chu et al. (22) . The final vesicles were resuspended in 10 mM Tris/HCl and 0.29 M sucrose buffer, pH 7.4, and stored at –70°C.

NKA activity
Enzymatic activity was determined as described previously (23) with modifications. Briefly, purified rat or dog NKA was incubated with or without SSA78 or ouabain ranging from 10, 50, 100, 250, 500, and 1000 nM in the presence of 100 mM Na+for 30 min at room temperature. The reaction was initiated by adding 3 mM MgATP and 20 mM K+in a final volume of 0.25 mL at 37°C for 30 min and terminated by adding 0.75 mL quench solution and 0.025 mL developer. Color was developed for 30 min at room temperature, then the concentration of phosphate was determined at 700 nm using a spectrophotometer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Binding specificity of SSA78
It is generally accepted that the H1-H2 domain 118RSATEEEPPNDD129 of rat NKA is expressed on extracellular side of the membrane. Amino acid sequence comparisons showed this region to be highly conserved in ouabain-resistant [rat (11 , 12) ] and -sensitive [human (24) , dog (25) , horse (26) , and sheep (27) ] NKA and identical among the {alpha}1 subunit of ouabain-sensitive enzymes (Fig. 1 ). To determine the binding specificity of SSA78, immunoprecipitation and Western blot were used to test the interaction between SSA78 and its antigenic site on the NKA. SSA78 specifically bound to and precipitated NKA (Fig. 2 C) from whole rat heart cell lysates (Fig. 2D ). To further assess the binding specificity, we contrasted SSA78 to control antibody SSA95 (Fig. 2E ), which targeted a different region of NKA (28) . SSA95 recognized the {alpha}-subunit band of purified rat NKA (Fig. 2E ), in which the molecular size of the band matched the SSA78 immunoprecipitated {alpha}-subunit (Fig. 2F ). Western blot experiments detected a very light {alpha}2 band (Fig. 2I ) that was coimmunoprecipitated with {alpha}1 by SSA78, but not {alpha}3 (Fig. 2J ). SERCA2 antibody did not recognize the SSA78 immunoprecipitated {alpha}-subunit of NKA (Fig. 2H ), while specifically recognizing Ca2+-ATPase from cardiac SR vesicles (Fig. 2K ). No detectable cross activity was found between SSA78 and Ca2+-ATPase (Fig. 2L ).



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  		Figure 1. H1-H2 domain of the {alpha} subunit of rat NKA. Primary amino acid sequence of H1-H2 region, the SSA78 binding site, is highly conserved in rat (11 , 12) , human (24) , dog (25) , horse (26) and sheep (27) NKA.



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  		Figure 2. Binding specificity of SSA78 to the {alpha}-subunit of NKA. Whole rat heart cell lysate was used for immunoprecipitation followed by electrophoresis and Western blot. For SDS gel: A) 30 µg of highly purified dog kidney (Na++K+)-ATPase, B) 30 µg of partially purified rat cardiac NKA, C) ~10 µg of immunoprecipitates, D) rat heart cell lysate. For Western blot: E) 30 µg of rat NKA stained with SSA95, F) 10 µg of immunoprecipitates stained with SSA78, G) secondary antibody control for immunoprecipitates, H) 10 µg of immunoprecipitates stained with SERCA2, I) 10 µg of immunoprecipitates stained with anti-{alpha}2 antibody, J) 10 µg of immunoprecipitates stained with anti-{alpha}3 antibody, K) cardiac SR vesicles stained with SERCA2, L) SR vesicles stained with SSA78. The 55,000 Da band in Western blots F–J are the denatured heavy domains of IgG that were liberated from the SSA78-proteins A and G complex during the preparation of immunoprecipitation. SSA78 specifically binds to {alpha}1 NKA and coimmunoprecipitated {alpha}2, but does not recognize {alpha}3 of NKA and SR Ca2+-ATPase. Each of the data represents one of 10 similar experiments.

To visualize the specific interaction between SSA78 and membrane location of H1-H2 domain of NKA, immunofluorescent staining was performed using isolated rat heart cells and CV-1 cells. Immunofluorescence microscopy revealed that SSA78 interacted with its antigenic site on the surface of rat myocytes (Fig. 3 A) and this interaction was abolished by coincubation with the specific peptide blocker PB78 (Fig. 3B ). Similar membrane localization of SSA78 staining was demonstrated in CV-1 cells (Fig. 3D, E ). Ouabain (Fig. 3G ) or strophanthidin (Fig. 3H ) eliminated CV-1 cell immunofluorescent staining, suggesting that SSA78 binds to an extracellular site on NKA that at least partially overlaps with the cardiac glycoside binding site. This was further confirmed by flow cytometry. Incubation with rabbit IgG and FITC-labeled secondary antibody did not alter fluorescence intensity (Fig. 4 A), whereas incubation with SSA78 increased fluorescence labeling (Fig. 4B ) and fluorescence intensity shift. Preincubation with PB78 or ouabain eliminated fluorescent labeling (Fig. 4C, D ) confirming specific antibody-antigen interaction and cotargeting of ouabain and SSA78 on the same NKA site.



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  		Figure 3. Immunofluorescent staining of SSA78 in rat cardiac myocytes and African green monkey CV-1 cells. Confocal images of rat myocytes: A) with SSA78, B) with SSA78 and PB78, and C) with secondary antibody. Images of CV-1 cells: D) a group of cells at a magnification of 400X, E) a single CV-1 cell at 3000X, F) secondary antibody control for panel D, E staining G) with 1 mM ouabain, H) with 1 mM strophanthidin, I) secondary antibody control for panel G, H staining. Results reveal that ouabain and strophanthidin compete with the SSA78 binding, consistent with SSA78 binding to NKA on extracellular surface of cell membrane. Each of the data represents one of 6 similar immunofluorescent stainings.



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  		Figure 4. Flow cytometric analysis of SSA78 binding on intact CV-1 cells. Histograms were generated by flow cytometry after labeling with or without SSA78. A) Untreated CV-1 cells incubated with rabbit IgG (0.18 µM) as control. B) Cells incubated with SSA78 (0.18 µM); C) Same as panel B except in the presence of PB78 (90.0 µM). D) Same as panel B but in the presence of ouabain (20 µM). The results show that ouabain antagonizes the binding of SSA78 to NKA and confirm that SSA78 binds at the ouabain interacting site on NKA. Each of the data represents one of 3 similar measurements.

SSA78 enhances myocyte shortening and calcium transient
We next investigated whether binding of SSA78 to the H1-H2 domain of NKA affects cardiac contractility. SSA78 (85 nM) increased isolated rat myocyte shortening by 2.1±0.3 times (P<0.01), initiating a response within 10 min after exposure, a reversible peak effect observed after 30 min (Fig. 5 E). In contrast, no significant changes were observed in the presence of ouabain at the same concentration (85 nM, Fig. 5B ). Control studies without SSA78 (Fig. 5A ) or with rabbit IgG (85 nM, Fig. 5D ) did not alter myocyte contraction. SSA78 increased contraction in a dose-dependent manner (Fig. 5G ), with a half effective concentration (EC50) of 41.0 nM. In separate experiments using calcium indicator Indo-1/AM, calcium transient amplitude was increased 1.3-fold (Fig. 6 C, n=12, P<0.00002) by SSA78 (0.25 µM), and cell contraction increased 1.6 times (Fig. 6D, n =12, P<0.000004). Coincubation with PB78 largely abolished increases in both Ca2+transient and contraction (Fig. 6) .



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  		Figure 5. Representative time courses of rat heart cell contraction with or without SSA78. Column 1: cell contraction baseline. Columns 2, 3, and 4: cell contraction at 10, 20, and 30 min after administration of different reagents. Column 5: cell contraction after 40 min wash at the rate of 1.5 mL/min. A) Control cell, B) with 85 nM ouabain, C) with 20 µM ouabain, D) with 85 nM rabbit IgG, E) with 85 nM SSA78, F) NKA activity was monitored in cell homogenates under the same experimental conditions as in panels A, B, E. Values of enzyme activities are means of 3 measurements for each cell under the specific conditions indicated. Each of the original recordings of cell contraction represents one of the 8 similar results for each cell. R show that SSA78 enhanced rat heart cell contraction without inhibiting NKA activity. G) Dose-dependent contractile response of SSA78 in rat ventricular myocytes. SSA78 concentrations were 1, 3, 5, 16, 25, 34, 51, 84, and 101 nM and the corresponding cell responses were 100, 100, 105±6.0, 120±8.0, 163±11, 214±19, 320±68, 420±28, and 424±23%. The half-maximal contractile response (EC50) of SSA78 is ~41.0 nM. Each data point in panel G represents the mean values of 4 cells.



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  		Figure 6. Effects of SSA78 on Ca2+ transient amplitudes and contraction in Indo-1/AM-loaded rat heart cells. A) Simultaneously recorded contractions (upper) and Ca2+ transient (lower) in the absence (left) or presence (right) of 0.25 µM SSA78 from the same rat myocyte. B) Representative recordings from control cell under the same experimental conditions as panel A except in the presence of 0.3 mM peptide blocker. C) Average changes of Ca2+ transient. D) Average changes of cell contraction. Data are presented as % of control based on 6 to 12 independent measurements of different cells for each group. The overall SSA78 effects are significant at P< 0.00002 for Ca2+ transient and P < 0.000004 for cell contraction.

SSA78 induces a positive inotropic effect on mouse heart in vivo
We further tested the effect of SSA78 on in vivo cardiac function in mice. SSA78 (0.5 µM, i.v.) induced a significant increase in cardiac contractility in intact heart (Table 1 ). Global systolic parameters such as ejection fraction (EF) and cardiac output (CO), and other contractile parameters such as dP/dtmx/IP, Ees and PRSW all increased with SSA78 (Table 1) . SSA78 did not alter cardiac preload volume (EDV) or afterload end-systolic pressure and Ea, suggesting that enhanced myocardial performance is a direct cardiac effect of the antibody. Cardiac diastolic function was not significantly affected by SSA78 although relaxation was slightly prolonged after its discontinuation (Table 1) . Representative pressure-volume loops are shown in Fig. 7 . Maximal response to SSA78 was achieved after ~15 min, and gradually washed out over 20–40 min after cessation, which is consistent with reversible antibody binding (Fig. 7C ).


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  		Table 1. Cardiovascular effects of SSA78 on mouse heart (n = 5)a



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  		Figure 7. Representative pressure-volume loops during preload reduction by IVC occlusion showing the effect of SSA78 on intact heart. SSA78 (0.5 µM) was administered intravenously at the rate of 5 µL/min. A) Control background, B) presence of SSA78 (peak response), C) after 20–40 min wash with PBS. Results show that SSA78 induced a positive inotropic effect as demonstrated by the leftward shift of the PV loop with increased end-systolic elastance (Ees, slope of upper left relation). These changes gradually disappeared after discontinuing use of antibody. Data represent 1 of 5 similar independent determinations.

Effect of SSA78 on NKA activity
NKA enzymatic activity in cardiac myocytes was monitored in cell homogenates under the same experimental conditions as in the control cell (Fig. 5A ), with 85 nM ouabain (Fig. 5B ) or 85 nM SSA78 (Fig. 5E ). No loss of enzyme activity was detected in control cell and in the presence of SSA78 (Fig. 5F ), except the activity of NKA decreased 13% after 40 min wash in the presence of ouabain (Fig. 5F ). The effect of SSA78 on purified rat and dog NKA activities was further tested in the presence of 100 mM Na+, 20 mM K+, 3 mM MgATP, and varying concentrations of SSA78 or ouabain. Figure 8 shows that no inactivation of ouabain-resistant rat NKA (147 nM) was observed over a concentration range of 10–1000 nM for either SSA78 or ouabain (Fig. 8A ). To rule out the possibility that inability of SSA78 to inhibit NKA activity might be related to the ouabain-resistant nature of rat NKA (Fig. 8A ), we tested SSA78 on ouabain-sensitive NKA (18 nM) purified from dog kidneys under the same conditions. Figure 8B shows that ouabain, not SSA78, significantly inhibited the ATP hydrolysis of the enzyme. Dog NKA activity decreased to 31±3% of baseline with 1 µM ouabain, whereas SSA78 had no significant effect on NKA activity over the same concentration range. This supports independence of the SSA78 effect from NKA inhibition under our conditions. To further verify this, we tested whether the interaction between SSA78 and enzyme affected ion binding sites on NKA. SSA78 produced no changes in the optimal binding of Na+ and K+ ions to the enzyme (Fig. 8C ).



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  		Figure 8. Effect of SSA78 and ouabain on NKA function. A) Ouabain-resistant rat NKA (147 nM). B) Ouabain-sensitive dog NKA (18 nM). Black circles: with SSA78. Open circles: with ouabain. Note that SSA78 inactivated neither, regardless of cardiac glycoside sensitivity. C) NKA hydrolysis of ATP as a function of Na+ and K+ concentrations. Enzyme activity of purified rat NKA was tested in the presence of 3 mM Mg-ATP under the different Na+/K+ concentrations indicated. Black circles: with 0.5 µM SSA78. Open circles: as control without SSA78. Results show that the binding of SSA78 to NKA did not affect Na+/K+ ion binding or the catalytic function of NKA. Data represent mean values of 5 experiments for each data point.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The experimental results reported here show that binding of SSA78 to the H1-H2 region of {alpha}1 NKA markedly increases rat heart cell contraction in vitro (Figs. 5 , 6) and generates a positive inotropic effect in mouse heart in vivo (Fig. 7) . These data provide the first evidence to demonstrate that the H1-H2 domain of {alpha}1 NKA participates in the regulation of cardiac contraction.

Binding specificity of SSA78 is crucial to our investigation. Immunochemical analyses reveal that SSA78 1) immunoprecipitates NKA from whole rat heart cell lysates (Fig. 2C ), 2) recognizes the {alpha}-subunit of NKA (Fig. 2F ), 3) competes with ouabain for binding on the enzyme (Figs. 3 , 4) , and 4) does not cross-react with SR Ca2+-ATPase (Fig. 2L ). These results establish the specificity of SSA78 binding to its antigenic site on the {alpha}1 subunit of NKA. By specifically binding to and directly interacting with the H1-H2 domain of NKA, SSA78 markedly enhances the myocyte Ca2+ transient and contraction (Figs. 5 , 6) . These data provide an important mechanistic link between the molecular structure of the H1-H2 domain of NKA and contractile function of heart cells. Peptide blocker PB78 significantly eliminated the biological activity of SSA78, suggesting that the binding of SSA78 to its specific antigenic site was necessary and sufficient for SSA78 to increase myocyte contraction and alter intracellular Ca2+ handling (Fig. 6) . Since nonspecific rabbit IgG did not affect cell contraction (Fig. 5C ), it is unlikely that SSA78-induced changes of Ca2+ transient and contraction are due to nonspecific binding to other proteins.

There are two {alpha} isoforms ({alpha}1 and {alpha}2) of NKA in rodent heart (28 , 29) and three {alpha} isoforms ({alpha}1, {alpha}2, and {alpha}3) in human heart (30 , 31) . There is a 50% identity of the H1-H2 domain of the enzyme between {alpha}1 and {alpha}2, and 30% between {alpha}2 and {alpha}3. To test whether SSA78 interacts with {alpha}1 and {alpha}3 isoforms of NKA, we examined the binding activity of SSA78 using {alpha}2 and {alpha}3 isoform specific antibodies. Western blot experiments detected a very light {alpha}2 band (Fig. 2I ) that was immunoprecipitated with {alpha}1 by SSA78, but not {alpha}3 (Fig. 2J ). The results of Area-density Calculation (LabWorks analysis software, Ultraviolet Products Bioimaging Inc., Upland, CA, USA) show that ~5% {alpha}2 was coprecipitated with {alpha}1 (data not shown), suggesting that SSA78 may react with {alpha}2 and that the {alpha}2 isoform of cardiac NKA may also participate, along with {alpha}1, in the regulation of cardiac contraction. SSA78 immunoprecipitates 95% {alpha}1 and 5% {alpha}2, suggesting that {alpha}1 NKA is mainly responsible for the SSA78 induced positive inotropy.

Our results show that rat heart cell contraction increased ~2-, 3-, and 4-fold after 10, 20, and 30 min incubation with 85 nM SSA78 (Fig. 5E ). These results provide a functional link between the molecular sequence of the H1-H2 domain of NKA and contractile property of rat heart cells. In contrast, 20 µM ouabain enhanced cell contraction by 2-fold (Fig. 5C ) suggesting that SSA78 is more sensitive than ouabain to induce cell contraction. Using calcium indicator Indo-1/AM, cell contraction was found to have increased 1.6 times (Fig. 6D ) in the presence of SSA78 (Fig. 6D ). The differences in increasing cell contraction between Fig. 5E and Fig. 6D, is presumably due to the characteristics of Indo-1 in binding intracellular Ca2+. Importantly SSA78 increased cardiac systolic performance and induced a reversible positive inotropic effect in vivo in the intact heart (Fig. 7) . Global systolic parameters, such as EF and CO, and specific contractile parameters including dP/dtmax, dP/dtmx/IP, PMX/EDV, Ees, and PRSW, all increased after administration of SSA78 (Table 1) , demonstrating a dramatic change in mouse heart presumably caused by interaction between the antibody and the H1-H2 domain of NKA. As SSA78 did not alter cardiac preload as indexed by EDV or afterload by ESP and Ea (Table 1) , the enhanced cardiac systolic performance is a direct effect of SSA78 on the heart. These in vivo and in vitro results are mutually consistent and provide the first evidence for involvement of the H1-H2 domain of NKA with myocardium contractile regulation. Moreover, the inotropic effect of the SSA78 may lead to a new therapeutic strategy for heart failure.

The H1-H2 domain of NKA {alpha}1 subunit is thought to be important to the glycoside binding affinity (10) . Price and Lingrel have demonstrated that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the {alpha}1 H1-H2 region is responsible for the ouabain-resistant character of rat NKA (10) . However, controversy still exists (32) regarding the role of the H1-H2 loop in the cardiac glycoside binding site; for example, a monoclonal antibody VG4 exceptionally enhances rather than impedes ouabain binding to NKA (33) . Our observations that ouabain and strophanthidin (Figs. 3 , 4) completely inhibited SSA78 binding to NKA are consistent with and further support the assumption that the H1-H2 domain of NKA is one of the cardiac glycoside target regions expressed on extracellular surface of the membrane.

Kinetic analysis shows that SSA78 induces a dose-dependent positive inotropic effect with an EC50 of 41.0 nM (Fig. 5G ). Under our experimental conditions, SSA78 did not inhibit enzyme activity at a concentration associated with the maximum increase in cellular contraction (Figs. 5 , 8) . Moreover, neither enzyme activity nor Na+/K+ ion binding was affected by the binding of SSA78 to NKA (Fig. 8) . These results reveal that SSA78-induced increase in cardiac inotropy is independent of the inactivation of NKA and suggest that interaction with the H1-H2 domain of the enzyme may not directly contribute to the inhibition of NKA function. We are not ruling out the possibility of inhibitory effect of SSA78 that may occur at higher concentrations. Other laboratories have also observed related phenomenon of low concentrations of cardiac glycosides stimulating rather than inhibiting NKA activity (34) . Recent findings of Wasserstrom’s laboratory also show that ouabain induces positive inotropic effects in isolated cat ventricular myocytes in sodium-free conditions independent of NKA inhibition, Na/Ca-exchanger, and changes in Na concentration (35) . It is not known whether the inhibition of NKA and the inotropic action provoked by SSA78 or ouabain are two parallel events. Nevertheless, the observations from other laboratories and ours suggest that inhibition of NKA may not be the sole factor to induce a positive inotropic effect.

Calcium ions play an essential role in excitation-contraction coupling of the heart (36) . Our findings show that administration of SSA78 enhances the intracellular Ca2+ transient amplitude by 30% (Fig. 6A, C ). The peptide blocker PB78 inhibited SSA78-induced alteration of [Ca2+]i (Fig. 6B ), indicating that Ca2+ is essentially involved in the molecular mechanism of SSA78 enhanced cardiac contraction. Cardiac glycosides increase [Ca2+]i and contraction by inactivating NKA and affecting Na+/Ca2+ exchange (3 , 5 , 7 , 11) . Since SSA78 did not inactivate NKA (Fig. 5E ) while increasing the force of contraction of the rat heart cells, it is not clear whether Na+/Ca2+ exchanger is involved in the alteration of [Ca2+]i through the binding of SSA78 to NKA. Mechanistic pathways of SSA78-induced cardiac contraction without inhibiting NKA activity may be explained by recent studies on NKA as a signal transducer (37) . Xie and Askari have demonstrated that the binding of nontoxic concentrations of ouabain to cardiac NKA activates multiple signaling pathways that regulate [Ca2+]i (38 , 39) . Whether binding of SSA78 to the H1-H2 site of the enzyme activates the Ras/MAPK cascade (38 , 39) or induces a channel activity (40) of NKA to cause increase in [Ca2+]i remains to be determined.

In summary, we examined the role of H1-H2 domain of {alpha}1 NKA in relation to cardiac contractile function at the enzyme, cellular and animal levels. Our experimental results provide direct evidence demonstrating that the H1-H2 domain of NKA participates in the regulation of cardiac contraction. The ability of low concentrations of SSA78 to modulate heart contraction without inactivating NKA activity may be therapeutically useful in the treatment of congestive heart failure. More detailed investigations on whether Na+/Ca2+ exchanger, ion channels, and signal transduction pathways are potentially linked to the {alpha}1 H1-H2 domain regulated cardiac contraction, should increase our understanding of the molecular basis of biological processes mediated by NKA.


   ACKNOWLEDGMENTS
 
We thank Drs. E. G. Lakatta, D. Kass, and G. F. Tomaselli for their important advice and help in rat myocytes studies and mouse heart measurement, J. Kyte for providing purified dog NKA, A. A. McDonough for anti-NKA {alpha}2 antibody, J. Sham for confocal imaging, Mr. B. Ziman for isolating rat myocytes, and Mr. D. Lewis for area density calculation. Drs. P. L. Pedersen, B. S. Bochner, H. Spurgeon, S. Wang, R. P. Xiao, M. P. Blaustein, K. J. Sweadner, and W. Zhu for helpful discussions and suggestions. This research was supported by grants HL52175 (to K.Y.X. and HL48198 (to A.C.M.) from the National Heart, Lung and Blood Institute.

Received for publication May 14, 2004. Accepted for publication September 7, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Skou, J. C. (1998) The identification of the sodium pump. Biosci. Rep. 18,155-169[CrossRef][Medline]
  2. Kyte, J. (1981) Molecular considerations relevant to the mechanism of active transport. Nature (London) 292,201-204[CrossRef][Medline]
  3. Allen, D. G., Eisner, D. A., Wray, S. C. (1985) Birthday present for digitalis. Nature (London) 316,674-675[CrossRef][Medline]
  4. Eisner, D. A., Smith, T. W. (1992) The Na-K pump and its effectors in cardiac muscle. Fozzard, H. A. Haber, E. Jennings, R. B. eds. The Heart and Cardiovascular System ,863-901 Raven Press. Ltd. New York.
  5. Forbush, B., III, Hoffman, J. F. (1979) Direct photoaffinity labeling of the primary region of the ouabain binding site of Na,K-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin. Biochemistry 18,2308-2315[CrossRef][Medline]
  6. Smith, T. W. (1988) Digitalis, Mechanisms of action and clinical use. N. Engl. J. Med. 318,358-365[Medline]
  7. Forbush, B., III (1983) Cardiotonic steroid binding to Na,K-ATPase. Curr. Top. Memb. Trans. 19,167-201
  8. Kaplan, J. H. (2002) Biochemistry of Na,K-ATPase. Annu. Rev. Biochem. 71,511-535[CrossRef][Medline]
  9. Hauptman, P. J., Kelly, R. A. (1999) Digitalis. Circulation 99,1265-1270[Abstract/Free Full Text]
  10. Price, E. M., Lingrel, J. B. (1988) Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27,8400-8408[CrossRef][Medline]
  11. Schneider, J. W., Mercer, R. W., Caplan, M., Emanuel, J. R., Sweadner, K. J., Benz, E. J., Levenson, R. (1985) Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA. Proc. Natl. Acad. Sci. USA 82,6357-6361[Abstract/Free Full Text]
  12. Shull, G. E., Greeb, J., Lingrel, J. B. (1986) Molecular cloning of three distinct forms of the Na,K-ATPase alpha-subunit from rat brain. Biochemistry 25,8125-8132[CrossRef][Medline]
  13. Sham, J. S., Song, l. S., Chen, Y., Deng, L. H., Stern, M. D., Lakatta, E. G., Cheng, H. (1998) Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes. Proc. Natl. Acad. Sci. USA 95,15096-15101[Abstract/Free Full Text]
  14. Georgakopoulos, D., Kass, D. A. (2001) Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J. Physiol. (London) 534,535-545[Abstract/Free Full Text]
  15. Kelly, R. P., Tunin, R., Kass, D. A. (1992) Effect of reduced aortic compliance on cardiac efficiency and contractile function of in situ canine left ventricle. Circ. Res. 71,490-502[Abstract/Free Full Text]
  16. Wang, S. Q., Song, L. S., Lakatta, E. G., Cheng, H. (2001) Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells. Nature (London) 410,592-596[CrossRef][Medline]
  17. Xu, K. Y., Wang, S. Q., Cheng, H. (2001) Site-Specific antibody of Na,K-ATPase augments cardiac myocyte contraction without inactivating enzyme activity. Biochem. Biophys. Res. Commun. 289,167-172[CrossRef][Medline]
  18. Jo, S.-H., Leblais, V., Wang, P. H., Crow, M. T., Xiao, R.-P. (2002) Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent G(s) signaling during beta2-adrenergic stimulation. Circ. Res. 91,46-53[Abstract/Free Full Text]
  19. Jones, L. R. (1988) Rapid preparation of canine cardiac sarcolemmal vesicles by sucrose flotation. Methods Enzymol. 157,85-91[Medline]
  20. Kyte, J. (1971) Purification of the sodium- and potassium-dependent adenosine triphosphatase from canine renal medulla. J. Biol. Chem. 246,4157-4165[Abstract/Free Full Text]
  21. Eleter, S., Inesi, G. (1972) Phospholipid orientation in sarcoplasmic membranes: spin-label ESR and proton MNR studies. Biochim. Biophys. Acta 282,174-179[Medline]
  22. Chu, A., Dixon, M. C., Saito, A., Seiler, S., Fleischer, S. (1988) Isolation of sarcoplasmic reticulum fractions referable to longitudinal tubules and junctional terminal cisternae from rabbit skeletal muscle. Methods Enzymol. 157,36-36[Medline]
  23. Kyte, J., Xu, K. Y., Bayer, R. (1987) Demonstration that lysine-501 of the alpha polypeptide of native sodium and potassium ion activated adenosinetriphosphatase is located on its cytoplasmic surface. Biochemistry 26,8350-8360[CrossRef][Medline]
  24. Shull, M. M., Lingrel, J. B. (1987) Multiple genes encode the human Na,K-ATPase catalytic subunit. Proc. Natl. Acad. Sci. USA 84,4039-4043[Abstract/Free Full Text]
  25. Xie, Z., Li, H., Liu, G., Wang, Y., Askari, A., Mercer, R. W. (1994) Cloning of the dog Na,K-ATPase alpha 1 subunit. Bamberg, S. Schoner, W. eds. The Na Pump ,49 Springer-Verlag New York.
  26. Kano, I., Nagai, F., Satoh, K., Ushiyama, K., Nakao, T., Kano, K. (1989) Structure of the alpha 1 subunit of horse Na,K-ATPase gene. FEBS Lett. 250,91-98[CrossRef][Medline]
  27. Shull, G. E., Schwartz, A., Lingrel, J. B. (1985) Amino-acid sequence of the catalytic subunit of the Na,K-ATPase deduced from a complementary DNA. Nature (London) 316,691-695[CrossRef][Medline]
  28. Lucchesi, P. A., Sweadner, K. J. (1991) Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity. J. Biol. Chem. 266,9327-9331[Abstract/Free Full Text]
  29. McDonough, A. A., Zhang, Y., Shin, V., Frank, J. S. (1996) Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes. Am. J. Physiol. 270,C1221-C1227
  30. McDonough, A. A., Welotta, J. B., Schwinger, R. H., Philipson, K. D., Farley, R. A. (2002) The cardiac sodium pump: structure and function. Basic Res. Cardiol. 97,19-24
  31. Wang, J., Velotta, J. B., McDonough, A. A., Farley, R. A. (2001) All human Na,K-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides. Am. J. Physiol. Cell Physiol. 281,C1336-C1343[Abstract/Free Full Text]
  32. Repke, K. R. H., Megges, R., Weiland, J., Schon, R. (1995) Location and properties of the digitalis receptor site in Na,K-ATPase. FEBS Lett. 359,107-109[CrossRef][Medline]
  33. Arystarkhova, E., Gasparian, M., Modyanov, N. N., Sweadner, K. J. (1992) Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding. J. Biol. Chem. 267,13694-13701[Abstract/Free Full Text]
  34. Adams, R. J., Schwartz, A., Grupp, G., Grupp, I., Lee, S. W., Wallick, E. T., Powel, T., Twist, V. W., Gathiram, P. (1982) High-affinity ouabain binding site and low-dose positive inotropic effect in rat myocardium. Nature (London) 296,167-169[CrossRef][Medline]
  35. Nishio, M., Ruch, S. W., Wasserstrom, J. A. (2002) Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions. Am. J. Physiol. 283,2045-2053
  36. Bers, D. M. (2002) Cardiac excitation-contraction coupling. Nature (London) 415,197-205[CrossRef]
  37. Xie, Z. (2003) Molecular mechanisms of Na,K-ATPase-mediated signal transduction. Ann. N.Y. Acad. Sci. 986,497-503[Abstract/Free Full Text]
  38. Liu, J., Tian, J., Haas, M., Shapiro, J. I., Askair, A., Xie, Z. (2000) Ouabain interaction with cardiac Na,K-ATPase initiates signal cascades independent of changes in intracellular Na and Ca concentrations. J. Biol. Chem. 275,27838-27844[Abstract/Free Full Text]
  39. Tian, J., Gong, X., Xie, Z. (2001) Signal transducing function of Na,K-ATPase is essential for ouabain’s effect on intracellular Ca2+ in rat cardiac myocytes. Am. J. Physiol. 281,H1899-H1907
  40. Gadsby, D. C., Rakowski, R. F., De Weer, P. (1993) Extracellular access to the Na,K pump: pathway similar to ion channel. Science 260,100-103[Abstract/Free Full Text]




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