AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 292: H1301-H1312, 2007. First published October 20, 2006; doi:10.1152/ajpheart.00449.2006
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Cardiac hypertrophy in anion exchanger 1-null mutant mice with severe hemolytic anemia

Bernardo V. Alvarez,1 Dawn M. Kieller,2 Anita L. Quon,1 Murray Robertson,3 and Joseph R. Casey4

1Department of Physiology and 4Department of Physiology and Department of Biochemistry, CIHR Membrane Protein Research Group, 2Department of Biological Sciences, and 3Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada

Submitted 3 May 2006 ; accepted in final form 17 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl–/HCO3– exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1–/–) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1–/– mice than in wild-type (AE1+/+) littermates at both 1–3 days postnatal (3.01 ± 0.38 vs. 1.45 ± 0.04) and at 7 days postnatal (9.45 ± 0.53 vs. 4.13 ± 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1+/–) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1–/– mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1–/– hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1+/+ hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1–/– mutant mice was indistinguishable from AE1+/– and AE1+/+ mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1–/– mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.

AE1; intracellular pH; Cl–/HCO3– exchange

ANEMIA IMPLIES A REDUCTION in the O2-carrying capacity of the blood and is usually characterized by low hematocrit level. Excessive loss of erythrocytes, deficiency in the hemoglobin content of erythrocytes, and a decreased rate of erythropoiesis cause anemia. Hemolytic anemia is caused by rupture of excessive numbers of circulating erythrocytes. Hemolysis occurs because erythrocytes are defective (16, 20) or because otherwise normal cells are induced to rupture by external factors (13).

Although anemia is a common clinical problem, the chronic manifestation of the disorder normally leads to hyperkinetic circulation, chamber dilation, and finally the development of left ventricular hypertrophy (27). Hence, severe anemia may lead to the deterioration of the heart ventricle function seen in heart failure, a major cause of mortality (15).

The anion exchanger 1 (AE1) Cl–/HCO3– exchanger (band 3) is the predominant (106 copies per erythrocyte) integral membrane protein of the erythrocyte that mediates two distinct biological functions (2, 18, 23, 39). The 55-kDa COOH-terminal domain, which spans the plasma membrane 12–14 times, catalyzes the Cl–/HCO3– exchange activity of AE1. The 43-kDa NH2-terminal domain of AE1 extends into the cytoplasm and mediates binding to the membrane skeleton components ankyrin, protein 4.1, and protein 4.2. Cl–/HCO3– exchange increases the capacity of the blood to transport CO2 from respiring tissues to the lungs, by performing Cl–/HCO3– exchange (39, 42), and contributes to the maintenance of blood acid-base homeostasis (19).

AE1 has a central role in maintenance of mechanical properties of erythrocytes by forming the connection between the spectrin-actin complex and the plasma membrane through association with ankyrin (26, 41). Therefore, AE1 is believed to be critical to the biosynthesis and mechanical properties of the erythrocyte membrane through its association with the membrane skeleton complex, a multiprotein network underlying and attached to the plasma membrane. The erythrocyte cytoskeleton is crucial to the elasticity and mechanical integrity of the erythrocyte (5). Genetic defects in spectrin, ankyrin, AE1, and protein 4.1, result in loss of erythrocyte elasticity and deformability (30). Severe and life-threatening hemolytic anemias are a consequence of erythrocyte fragility and cellular fragmentation, which produce oddly shaped erythrocytes (30). In recent years, hereditary disorders (28, 37, 40) and incomplete deficiencies of human red cell band 3 (17) have been reported. Two independent lines of AE1–/– mice have been established (33, 38), both of which displayed severe hemolytic anemia and altered erythrocyte morphology (spherocytosis) resulting from loss of plasma membrane-cytoskeletal coupling.

The role of AE1 in cardiac function has not been well studied. Members of the solute carrier family 4 (SLC4) (AE1, AE2, and AE3) function in an acidifying pathway against alkaline loads to maintain steady-state intracellular pH (pHi) (25, 35, 43). More recently, a member of the SLC26 family, the slc26a6 Cl–/HCO3– and Cl–/OH– exchanger, was identified as the predominant anion exchanger of the mouse myocardium (4). (Note that by convention SLC and slc refer to human and nonhuman orthologs, respectively.) AE1 is expressed in the heart as a truncated transcript called nAE1 (3, 35); however, on the basis of its level of expression in the murine heart, AE1 does not contribute to cardiac Cl–/HCO3– exchange in a major way (4). Nonetheless Cl–/HCO3– exchange in the heart is proposed to be involved in prohypertrophic pathways, by working counter to the cardiac alkalinizing Na+/H+ exchanger 1 (NHE1) (3, 8). Establishing which Cl–/HCO3– exchanger is involved in hypertrophy is thus important. In the present study, we have characterized the cardiac performance of AE1-null mice (33, 38) with severe spherocytosis and hemolytic anemia and observed concomitant cardiac hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
AE1 knockout mice. Adult mice heterozygous for the disrupted AE1 gene, obtained from Jackson Laboratories (Bar Harbor, ME), have been previously described (33). Although mice homozygous for the disrupted gene display severe hemolytic anemia, AE1–/– mice were born with the expected Mendelian ratio.

Genotyping of mice. Genomic DNA was extracted from 3.0-mm tail samples with a Qiagen kit and used to genotype progeny by PCR. For the detection of the wild-type allele (+), the oligonucleotides AE1wt.for and AE1wt.rev were used as forward and reverse primers, respectively, and amplified a 400-bp fragment (Table 1). The forward primer corresponds to a portion of the deleted AE1 gene; thus the mutant allele was not amplified. The AE1ko.for primer and AE1ko.rev primer amplified a 520-bp fragment of the mutant allele (–) (Table 1).


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  		Table 1. Properties of oligonucleotides used in real-time PCR

 
RNA isolation. Total RNA was isolated from 1- to 3-day-old neonate mouse heart ventricles with TRIzol reagent (GIBCO-BRL, Life Technologies), according to the manufacturer's instructions. Samples of isolated RNA were incubated with DNase (0.5 U/10 µg of RNA; GIBCO-BRL) at 37°C for 5 min, to digest contaminating DNA. DNase-treated RNA was reextracted with TRIzol reagent. RNA integrity was confirmed by denaturing agarose gel electrophoresis, and RNA was quantified spectrophotometrically at 260 nm.

cDNA synthesis. For RT-PCR, single-strand cDNA synthesis was carried out with SuperScript first-strand synthesis for RT-PCR (Invitrogen, Life Technologies), according to the manufacturer's instructions. In each PCR reaction, 2 µl of cDNA synthesis were added to 50 µl of "master mix" containing 5 µl of 10x PCR buffer, 1 µl of 10 mM dNTP mix, 1.5 µl of 50 mM MgCl2, 1 µl of 1 M KCl, 0.4 µl (5 U/µl) of Taq DNA polymerase (New England Biolabs), and 37.1 µl of H2O. To each reaction was added 1 µl of 10 µM of each primer.

PCR primers. cDNA sequences were obtained from the public GenBank sequence database of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), and primers were designed with the oligo software of the DNA Star program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). Mouse NBC3 and NBC4 (Na+-HCO3– cotransporters) have not yet been reported. To prepare primers, the NBC4 (slc4a5) and NBC3 (slc4a7) mouse genes were identified by homology search of the mouse genome (Blast search, www.ncbi.nlm.nih.gov/genome/seq/), using the human NBC4 and NBC3 sequences. In conventional RT-PCR, all primers generated only one amplification band visualized by agarose gel electrophoresis on 1% agarose gels stained with ethidium bromide, demonstrating specificity. Sequences for all PCR primers are shown in Table 1.

SYBR green real-time PCR. Real-time PCR was performed in an ABI Prism 7900H sequence detection system (Applied Biosystems). Each real-time RT-PCR reaction contained 50 mM KCl, 3 mM MgCl2, 0.08% (vol/vol) glycerol, 0.001% (vol/vol) Tween 20, 0.02% (vol/vol) DMSO, 1/40,000 dilution SYBR green (Molecular Probes), 0.03 U/µl Jumpstart Taq (Sigma), 3.2 µM of each primer, 5 µl of template diluted (0, 1/4, 1/16, and 1/64), and 1 mM Tris, pH 8.3. Template was prepared by reverse transcription of 2 µg of total RNA in a reaction volume of 20 µl. Replicate samples were pipetted into Axygen 384 well reaction plates with the use of a Biomek Fx pipetting robot (Beckman Coulter). Results were presented as cycle threshold (CT), that is, the PCR cycle number at which exponential PCR-generated fluorescence is first detected. The CT values obtained for each gene were individually normalized to CT values obtained for parallel samples probed for GAPDH expression.

Histological analysis. Histological analysis of adult mouse hearts was performed as previously described (29). Serial longitudinal 4-µm-thick heart sections were formalin-fixed, paraffin-embedded, and stained with hematoxylin-eosin for cell morphometry, with trichrome Masson staining (TMS), or with Picrosirius red staining (Direct red 80; Aldrich) for collagen identification and quantification. Images were captured with a Nikon digital camera (DXM 200) mounted on top of a Nikon Eclipse E600 microscope. Polarized lenses were utilized to capture images when heart sections were stained with Picrosirius red.

M-mode echocardiography. Transgenic and control mice were anesthetized with methoxyflurane and maintained at 37°C on a heated pad to prevent hypothermia. For cardiac imaging, a 12-MHz phase array sector transducer with frequency fusion technology (SONOS 5500; Hewlett-Packard, Andover, MA) was used. M-mode images were obtained in the parasternal short and long views at the papillary muscle level (29). For M-mode quantification: From the M-mode tracing using an off-line analysis system the left ventricular end-diastolic and end-systolic dimensions were measured, and a shortening fraction was calculated as the difference between the end-diastolic and end-systolic dimensions divided by the end-diastolic dimension. The leading edge-to-leading edge technique was used.

Isolation of mouse cardiomyocytes. Adult mice were anesthetized with euthanyl (pentobarbital sodium; 150 mg/kg ip). Animal protocols were approved by the University of Alberta Animal Policy and Welfare Committee and performed in accordance with Canadian Council on Animal Care guidelines. The hearts were rapidly removed, and ventricular myocytes obtained by enzymatic dissociation, using standard protocols (4).

Immunostaining of mouse cardiac myocytes and analysis by confocal microscopy. Freshly single dissociated myocytes were plated onto 22 x 22-mm laminin (25–50 µg/ml)-coated glass coverslips and incubated at 37°C for 30 min to allow attachment. Cells were rinsed with PBS and fixed in 4% (wt/vol) paraformaldehyde for 15 min at room temperature, followed by methanol fixation-permeabilization [ice-cold 100% (vol/vol) methanol, 5 min at –20°C]. Myocytes were then washed with PBS and permeabilized with 0.1% Triton X-100 (vol/vol) in PBS for 15 min at room temperature. After washing (2 x 5 min with PBS) and blocking (5% BSA in PBS, 20 min) were completed, the cells were incubated with primary antibodies (1 h at room temperature, in a humidified chamber), washed (3 x 5 min in PBS containing 0.2% gelatin), and incubated with secondary antibody. Primary rabbit polyclonal anti-AE1 antibody (1658), rabbit polyclonal anti-human SLC26A6 antibody (4), and rabbit polyclonal anti-mouse AE3 (AP3) antibody (22) were used at 1:100 dilution. Secondary chicken anti-rabbit conjugated to Alexa fluor 488 was used at 1:100 dilution. Coverslips were washed three times in PBS containing 0.2% gelatin and two times in PBS and mounted and viewed with a confocal microscope. Immunostained cells were mounted in Prolong anti-fade solution (Molecular Probes, Eugene, OR) and imaged with a Zeiss LSM 510 laser-scanning confocal microscope imaging system mounted on an Axiovert 100M controller. Images were collected with an oil immersion x63 objective (numerical aperture 1.4, plan Apochromat) at a resolution of 0.5- to 0.7-µm field depth. Filtering was used to integrate the signal collected over four frames to decrease noise (scan time of 7 s/frame).

Double immunostaining of mouse cardiac myocytes and analysis by confocal microscopy. Freshly isolated adult mouse cardiomyocytes were fixed to laminin-coated coverslips and permeabilized as described above. Myocytes were incubated with a combination of primary rabbit polyclonal anti-SLC26A6 antibody and goat anti-calreticulin antibody (gift from Dr. M. Michalak, University of Alberta), rabbit anti-SLC26A6 and goat polyclonal anti-vinculin antibody (N-19; Santa Cruz), or a combination of rabbit anti-mouse AE3 (AP3) antibody and goat anti-calreticulin antibody or rabbit anti-AE3 and goat anti-vinculin antibody. Combined primary antibodies were used at 1:100 dilutions. Secondary chicken anti-rabbit conjugated to Alexa fluor 488 and chicken anti-goat conjugated to Alexa fluor 594 were used at 1:100 dilutions. Collected images were quantified by MetaMorph software. MetaMorph compared the images of slc26a6 and either calreticulin or vinculin or AE3 and either calreticulin or vinculin, pixel by pixel (pixel size 0.18 x 0.18 µm), and determined the percentage of overlapping fluorescent signals. In these experiments, a combination of Alexa fluor 488 and Alexa fluor 594 fluor probes, which have a low degree of spectral overlap and minimal bleed through, was used in colocalization experiments. Detector gain was adjusted to acquire images on a 0–256 gray scale without saturation (50 ± 5% intensity). Background signal (offset) was adjusted to a low but readable intensity level.

Immunostaining of mouse ventricular muscle and analysis by confocal microscopy. Serial longitudinal 4-µm-thick heart sections mounted on glass microscope slides were deparaffinized by sequential washes with Fisher Scientific Citri-Solv solvent (twice for 5 min), 100% ethanol (2x, 5 min), 70% ethanol (2 min), 50% ethanol (2 min), distilled H2O (2 min), and PBS (2 min). Sections were prepared essentially as in the previous section. Images were collected with an oil-immersion x40 objective (numerical aperture 1.3, Neofluar oil) at a resolution of 0.7-µm field depth. Filtering was used to integrate the signal collected over four frames to decrease noise (scan time of 6 s/frame).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of cardiac hypertrophy in AE1–/– mice. Mouse genotyping at birth revealed the expected Mendelian frequency (26%) of homozygous mutant (AE1–/–) offspring from heterozygous (AE1+/–) mating pairs (Fig. 1A). Two bands were detected by PCR for AE1+/– (Fig. 1A), whereas only one band was detected for wild type (AE1+/+) or AE1–/–.


Figure 1
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  		Fig. 1. Genotype and appearance of AE1–/– mutant mice. A: PCR analysis of offspring of an AE1+/– breeding revealing all 3 Mendelian genotypes: AE1 knockouts (AE1–/–), heterozygotes (AE1+/–), and wild type (AE1+/+). In each pair of lanes, right lane is diagnostic for wild-type (+) alleles, whereas left lane indicates knockout (–) alleles. Specific primers used for genotyping are described in Table 1. B: appearance of 2-day-old AE1–/– mouse compared with an AE1+/+ littermate.

 
At birth, AE1–/– mice were readily distinguished by their extreme paleness, particularly in the prominent white abdominal band (Fig. 1B). As reported (33), no fetal loss occurs, but there is high mortality in the neonatal period with >90% of AE1–/– mice dying within the first week. After the first postnatal week, survival improves dramatically and some of the remaining mice reach adulthood. Adult AE1–/– mice are severely anemic and smaller than their normal littermates (not shown). Weights of neonate (Table 2) and adult (not shown) heterozygous (AE1+/–) mice were normal and indistinguishable from homozygous (AE1+/+) littermates.


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  		Table 2. Body and heart weight data in neonate mice

 
The AE1 Cl–/HCO3– exchanger constitutes 50% of the integral protein of the erythrocyte (2, 18). Loss of AE1 in the erythrocytes of AE1–/– mice led to spherocytosis and severe hemolysis, resulting in hemolytic anemia (33). This is reflected in the hematocrit of AE1–/– homozygotes, which drops to 18%, compared with 48% in AE1+/+ mice. We speculated that the hearts of the AE1–/– mice would compensate by hypertrophic growth to meet cardiovascular demands.

To investigate this possibility, heart ventricles from AE1–/– mutant and AE1+/– and AE1+/+ littermates were dissected and separated from blood vessels and atria, and morphological parameters were then obtained (Table 2). Hearts excised from AE1–/– mice were misshapen and occupied most of the chest cavity (not shown). AE1–/– body weight was reduced to 50% (1–3 days old) and 55% (1 wk old) when compared with AE1+/+ littermates. Thus, given similar heart weights, the heart weight-to-body weight ratio was increased by ~50% in the AE1–/– mice (Table 2). AE1+/– erythrocytes are more spherocytic and more susceptible to lysis by osmotic change than those of AE1+/+ mice, but AE1+/– hematocrit are reported as normal (33). We found no heart weight-to-body weight differences between AE1+/– mice and AE1+/+ littermates, indicating that cardiac hypertrophy did not occur in AE1+/– mice.

Cardiac hypertrophy was further characterized by histological analysis of hearts from 15-wk-old mice. Longitudinal cryostat section images of whole hearts stained with hematoxylin-eosin revealed nuclei in blue and muscle tissue in red. Left ventricles of AE1–/– mice were enlarged compared with AE1+/+ littermates (Fig. 2). At higher magnification, the left ventricular free wall at the basal level did not reveal remarkable pathology in the AE1–/– mice (Fig. 2C), compared with AE1+/+ (Fig. 2D).


Figure 2
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  		Fig. 2. Histological examination of mouse hearts. AE1–/– (A and C) and AE1+/+ (B and D) mouse hearts at 15 wk of age were hematoxylin-eosin stained. Shown are longitudinal cryostat sections of the whole heart of AE1–/– mouse (A) and AE1+/+ littermate (B) (x2.5). Higher magnification of the left ventricular free wall at x5 (C and D) allows comparisons with greater detail. Dashed box indicates the region magnified. Bars = 400 µm (A, B) and 200 µm (C, D).

 
On the basis of heart weight-to-body weight ratio and enlarged heart size, AE1–/– mutant mice presented with marked cardiac hypertrophy from postnatal day 1. Myocardial fibrosis, as characterized by an increase in the collagen fibers surrounding muscle fiber, usually accompanies cardiac hypertrophy. TMS was used to examine collagen deposition in heart sections. Collagen content of mouse ventricles was determined from the blue stain density normalized to the red stain density of the same portion of the image. Picrosirius red staining eliminates cytoplasmic staining, revealing thin septa and collagen fibers. Picrosirius red detected interstitial and perivascular myocardial fibrosis, which combined with polarizing lenses allowed detection of birefringent collagen fibers. Birefringence of thick (appearing yellow under polarization microscopy) and thin (appearing green under polarization microscopy) colocalized collagen fibers (36) was detected in the endocardial surface of the right ventricle and in hearts of both AE1+/+ (Fig. 3B) and AE1–/– mice (Fig. 3D), with more extensive staining present in the AE1 knockout animals. No fibrosis was detected in the right ventricle of normal AE1+/+ and AE1–/– mutant mice by TMS (Fig. 3, A and C, respectively). Conversely, disperse interstitial fibrosis, manifested by the finding of a diffuse increase in the thickness of collagen fibers (likely type III and type IV collagen fibers) surrounding the bundle of muscle fibers, was detected in the left ventricle of AE1–/– mice (Fig. 3H) but not in the left ventricle of AE1+/+ littermates (Fig. 3F). In addition, a higher level of fibrosis was present in the left ventricle of AE1–/– mice by TMS as seen by blue collagen staining (Fig. 3G), compared with AE1+/+ littermates (Fig. 3E). Fibrosis in human appendix tissue was used as a positive control for the TMS staining (Fig. 3I). Light blue staining of collagen fibers, purple/black staining of nuclei, and red staining of muscle and erythrocytes are evident. The lightly blue-stained collagen fibers indicate the ability to detect fibrosis with blue stain. Human lung tissue, showing alveolar structures, was used as control for the Picrosirius red technique. Fibrosis here presented as yellow-orange birefringent staining of collagen fibers under polarized microscopy (Fig. 3J).


Figure 3
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  		Fig. 3. Fibrosis in the hypertrophic heart of 15-wk-old AE1–/– mutant mice. Longitudinal cryostat heart sections were stained with trichrome Masson (A, C, E, and G) and Picrosirius red (B, D, F, and H) techniques and observed with light microscopy or polarization microscopy, respectively. A, B, E, and F: AE1+/+ mice. C, D, G, and H: AE1–/– mice. A, B, C, and D: right ventricle. E, F, G, and H: left ventricle. Human appendix (I) and lung samples (J) were stained with trichrome Masson and Picrosirius red, respectively. Bars = 300 µm.

 
Cardiac dysfunction in the Cl–/HCO3– exchanger 1 (AE1) mutant mice. AE1–/– mutant mice presented with significant cardiac hypertrophy, accompanied by abnormal architecture of the left ventricle with intense fibrosis. These findings are often associated with contractile dysfunction, so the cardiac function of the AE1–/– mice was studied by echocardiography. To evaluate cardiac function, we performed M-mode echocardiography on 4-mo-old AE1–/– and wild-type littermates. Echocardiography revealed ventricle dilation in the AE1–/– mutant mice, in both the systolic and diastolic phases (Fig. 4). There was an increase in the left ventricular end-diastolic dimension and an increase in the left ventricular end-systolic dimension in AE1–/– vs. control littermates (Table 3). Shortening fraction was dramatically (twofold) decreased in the AE1–/– animals compared with AE1+/+ littermates (Table 3). During echocardiography of the AE1–/– mutant heart, aortic Doppler echo found that heart rates in the mutant mice were faster (535 beats/min) than in AE1+/+ mice (461 beats/min). Consistent with heart weight/body weight measurements, echocardiography also revealed a significant increase in left ventricular size in AE1–/– mutant compared with control (Fig. 4). We conclude that, at 4 mo, the AE1–/– mutant mice had significant abnormalities in cardiac function.


Figure 4
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  		Fig. 4. Impaired cardiac function in 4-mo-old AE1–/– mutant mice. M-mode echocardiography showing dilated left ventricle end diastole (dashed arrow) and systole (solid arrow) and expanded left ventricular volume in the AE1–/– heart compared with the heart from AE1+/+ littermate.

 

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  		Table 3. Echocardiographic analysis of AE1+/+ and AE1–/– mice

 
Expression and location of the Cl–/HCO3– exchangers in mouse heart. To examine the normal role of AE1 in hearts, we performed confocal microscopy studies. The localization of AE1, AE3, and slc26a6 was explored in longitudinal heart sections of AE1+/+ and AE1–/– mice by confocal laser scanning microscopy (Fig. 5A). Antibodies against AE1 revealed labeling surrounding the isolated wild-type cardiomyocytes, consistent with sarcolemmal staining (Fig. 5A, top) but with distinct regions of elevated intensity. Although the hearts were perfused to remove blood, these regions may represent capillary-associated erythrocytes that stained because of the high abundance of erythrocyte AE1 protein. At the same intensity of exposure, only faint nonspecific fluorescence was evident in sections of AE1–/– hearts (Fig. 5A, bottom), indicating specificity of the signal. Consistent with the data from ventricular sections, in isolated myocytes, AE1 clearly displayed a sarcolemmal localization (Fig. 5B). Anti-AE3 antibody recognized both full-length AE3 and cardiac AE3 variants. AE3 and slc26a6 localizations were similar to each other, with widespread myocyte labeling at the sarcolemma and with apparent intracellular localization. No difference in expression level or localization of AE3 and slc26a6 was evident between the AE1+/+ and AE1–/– mice, suggesting that neither protein dramatically alters to compensate for loss of AE1.


Figure 5
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  		Fig. 5. Localization of Cl–/HCO3– exchangers in mouse ventricular sections and isolated cardiomyocytes. A: heart sections (top: AE1+/+; bottom: AE1–/–) were labeled with rabbit polyclonal anti-AE1 antibody, rabbit polyclonal anti-AE3 antibody, or rabbit polyclonal anti-slc26a6 antibody. Immunofluorescence signals were visualized by an Alexa fluor 488-conjugated anti-rabbit IgG antibody (green, 1:100 dilution). Images were collected with a Zeiss LSM 510 laser-scanning confocal microscope with x40/1.3 oil immersion objective (Neofluar oil differential interference contrast). Bars = 20 µm. B: AE1+/+-isolated cardiomyocytes were immunostained by rabbit polyclonal anti-AE1 antibody. Labeling was visualized by an Alexa fluor 488-conjugated anti-rabbit IgG (green, 1:100 dilution). DICM, differential interference contrast microscopy. Bars = 20 µm.

 
The suggestion that AE3 and slc26a6 differ from AE1 in their cellular localization was explored by confocal immunofluorescence of isolated wild-type cardiomyocytes. Analysis of confocal projection images built from 24 sequential sections at an optical resolution of 0.2 µm in the z direction (z-stack) revealed intensely stained AE1 at the sarcolemma, especially clear in the myocyte cross-sections displayed in the xz and yz projections (Fig. 6, top). Three-dimensional reconstruction of AE3 showed invaginations that ran toward the center of the cardiomyocyte. Such a distribution is consistent with the presence of AE3 in the sarcolemma and along the T tubules, as previously suggested for AE1 (34) (Fig. 6, middle). The z-stack revealed predominant expression of slc26a6 in the sarcolemma, with additional localization of the protein in longitudinal structures (Fig. 6, bottom).


Figure 6
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  		Fig. 6. Confocal immunofluorescence analysis of the distribution of AE1 and AE3 in mouse myocytes. Samples were prepared as described in Fig. 5. As indicated, cells were probed with antibodies against AE1, AE3, and slc26a6. Images are 3-dimensional displays of 24 confocal planes, taken at a distance of 0.2 µm. Conventional xy-plane together with xz-plane (top) and yz-plane (right) are for the points indicated by the thin lines (red, blue, and green). Images were collected with a confocal microscope as above with a x63/1.4 oil immersion objective (plan Apochromat). Bars = 10 µm.

 
The identity of the intracellular structures containing slc26a6 and AE3 was further explored in double-labeling experiments of freshly isolated mouse cardiomyocytes (Fig. 7). AE3 and slc26a6 proteins were found with transverse rib-like patterns and scattered longitudinal bands staining, respectively, suggesting specific location of this proteins in the transverse tubular system (T tubules) and/or sarcoplasmic reticulum (SR) of cardiomyocytes. The T tubules of mammalian cardiac cells consist of tubular invaginations of the sarcolemma at the level of the Z band, extending deeply into the cell. The SR of cardiomyocytes, the main calcium storage compartment of the cardiac cell, which plays a role in excitation-contraction coupling, also typically presents a striated patter, converging at the level of Z bands. AE3 showed intense colocalization with vinculin, a cell-matrix focal adhesion molecule present in the T system (24), at the level of costamers (Fig. 7A, top, and 7B, top left), whereas slc26a6co-localized with vinculin with a more intense, punctuate cross-striated labeling pattern (Fig. 7A, bottom, and 7B, bottom left). The calcium storage protein, calreticulin, is a marker of SR (29). Coimmunofluorescence revealed that that AE3 and slc26a6 colocalize with calreticulin to a similar degree (Fig. 7B, right). Colocalization of fluorescence signals was performed with MetaMorph software, which compared the images of slc26a6 and either calreticulin or vinculin or AE3 and either calreticulin or vinculin, pixel by pixel, and determined the degree of overlap. Slc26a6 showed stronger colocalization with vinculin (82 ± 1%) than AE3 colocalization with vinculin (67 ± 2%). slc26a6 and AE3 showed a similar degree of colocalization with calreticulin (59 ± 4% and 66 ± 3%, respectively; Fig. 7C). However, the ability to measure colocalization was limited by the resolution of the optical system.


Figure 7
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  		Fig. 7. Double-labeling images of adult mouse cardiomyocytes. A: freshly isolated adult mouse cardiomyocytes were double stained with rabbit anti-AE3 antibody (top) or with rabbit anti-slc26a6 antibody (bottom), as indicated in the panels and in green staining. Cells were double stained with anti-vinculin antibody, as indicated in red labeling. Dapi staining visualized nuclei in blue. Colocalization of AE3 (top) or slc26a6 (bottom) and vinculin is indicated as merge and with yellow staining. Images were collected with a Zeiss LSM 510 laser-scanning confocal microscope. Bars = 20 µm. B: merged images of higher magnification of isolated mouse cardiomyocytes showing colocalization of AE3 and calreticulin and AE3 and vinculin or colocalization of slc26a6 and calreticulin or slc26a6 and vinculin, as indicated. Colocalization is shown in yellow (merge). Labeling of costamers (arrows) and intracellular punctate cross-striated (arrowheads) is indicated. Bars = 20 µm (top) and 40 µm (bottom). C: images were analyzed with MetaMorph software to quantify the degree of calreticulin (open bars) or vinculin (red bars) colocalization with either AE3 or slc26a6 (n = 9–12 cells).

 
Together, AE1, AE3, and slc26a6 are broadly expressed across the population of ventricular myocytes; however, within myocytes, the proteins differ in their expression pattern. AE1 is predominately sarcolemmal. AE3 localizes to both T tubules and to some degree to the SR. slc26a6 associates strongly with the T tubule but also has at least weak localization to SR.

pHi-regulatory mechanisms in the hypertrophic hearts of AE1–/– mice. Quantitative real-time RT-PCR was used to characterize the abundance of mRNA encoding pHi-regulatory transports in the normal (AE1+/+ and AE1+/–) and the hypertrophic (AE1–/–) mouse myocardium. Total mRNA, extracted from mouse ventricle, was reverse transcribed and used as template in RT-PCR. Expression of Na+-independent anion exchangers/transporters of the slc4 family (AE1, AE2, AE3c, and AE3fl) and slc26 family (slc26a3 and slc26a6) was found, as previously established (4). Expression of the Na+-dependent Cl–/HCO3– exchanger (NCBE; slc4a10), which was not detected in Northern blots of the heart (12), was also revealed. Expression of alkaline-loading Na+-HCO3– cotransporters (NBC1, NBC3, NBC4, and kNBC3) and expression of NHE1 were measured in the myocardium. We also measured expression of mRNA encoding monocarboxylate transporter 1, a protein that catalyzes the cotransport of lactate-H+ during ischemic conditions of the heart (14).

Quantification of mRNA by quantitative real-time RT-PCR was performed with the use of SYBR green reagent, which binds double-stranded DNA generated by PCR. Thus nonspecific products could generate a signal. To verify the specificity of the real-time RT-PCR data, we performed RT-PCR using the designed pair primers (Table 1) and ran the products on 1% agarose-ethidium bromide gel. A single band was found for each product, for the AE1+/+, AE1+/–, and AE1–/– mutant neonate mouse ventricle samples (Fig. 8A). The expression of transcripts, as measured by CT in real-time PCR, was corrected for individual variation with GAPDH standard curves. A difference of one in the CT between two samples corresponds to a twofold difference in abundance of the starting template. A lower CT value represents a larger amount of template cDNA (Fig. 8B).


Figure 8
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  		Fig. 8. Analysis of expression of pH-regulatory transporters by PCR. mRNA was isolated from AE1–/–, AE1+/–, and AE1+/+ mouse hearts. Samples of mouse ventricle (AE1–/–, AE1+/–, or AE1+/+, as indicated) were reverse transcribed, and the resulting cDNA was used as template for PCR (see primers, Table 1). A: RT-PCR analysis of the expression levels of mRNA encoding the intracellular pH-regulatory transporters (indicated at top) in neonate mouse ventricle. PCR products were analyzed on a 1% agarose-ethidium bromide gel. B: mRNA expression (gene names shown at bottom and defined in Table 1) was quantified from ventricles of neonatal mutant AE1–/– (filled bars), AE1+/– (gray bars), or AE1+/+ (open bars) mice, using real-time quantitative RT-PCR. Data were corrected for individual variation with GAPDH standard curves, and results are expressed as cycle threshold. Cycle threshold values of 32 or higher were interpreted as indicating no significant expression of the transcript (dashed line). {gamma}Differences in AE1 mRNA expression for AE1–/– mutant mice compared with AE1+/– and *differences of AE1 gene expression for AE1–/– mutant mice compared with AE1+/+, P < 0.05, one way-ANOVA (n = 5–7 separate neonate mouse hearts supplying ventricular samples).

 
Expression levels of pH-regulatory transporter mRNA was quantified by quantitative real-time RT-PCR. As expected, AE1 message was not significant in the AE1–/– mouse hearts, as indicated by a CT value >32. AE1 mRNA levels of AE1+/– mice were intermediate between the AE1+/+ and AE1–/– hearts. Expression of AE2, AE3c, and AE3fl in the heart was indistinguishable between AE1+/+, AE1+/–, and AE1–/– hearts. Similarly, expression of slc26a6, the predominant Cl–/HCO3– and Cl–/OH– exchanger of the mouse heart (4), was also comparable in the three groups. slc26a3 and NCBE were also identified in the neonate mouse heart, with no difference of mRNA levels between the normal and the hypertrophic myocardium of mutant AE1–/– mice. NHE1 and NBCs showed minor differences in mRNA expression levels in the three groups of hearts. Similarly, monocarboxylate transporter 1, which exhibited a robust band of 172 bp, was equally expressed in AE1+/+, AE1+/–, and AE1–/– mutant mice hearts. We could find no evidence that loss of AE1 and associated cardiac hypertrophy in AE1–/– mice was compensated by increased expression of the other cardiac Cl–/HCO3– exchangers or pHi-regulatory mechanisms.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Hypertrophic growth of the heart is a crucial step in the development of heart failure. AE1–/– mice were anemic (33) and developed substantial cardiac hypertrophy, with ventricular collagen deposition and cardiac dysfunction reminiscent of heart failure. This finding indicates that AE1 in the heart does not have an indispensable role in the progression of cardiac hypertrophy. Moreover, the observation that expression of key pH-regulatory transporters did not alter in their expression level after loss of AE1 suggests that the role of AE1 in the heart is dispensable. Other evidence points toward AE3 and slc26a6 as the key Cl–/HCO3– exchangers, and here for the first time we examined intracardiomyocyte localization of these transporters, finding significant differences in localization of AE1, AE3, and slc26a6.

Severe anemia, a clinical condition characterized by low O2-carrying capacity of the blood, may be followed by development of left ventricular hypertrophy. Anemia is also common in patients with congestive heart failure, and its progression contributes to the worsening of heart failure by deterioration of contractile performance (15). Although the AE1–/– mice are very sick and usually die within a few days of birth, some survived homozygous AE1 deficiency to reach adulthood. Therefore, AE1 mutant mice represent a valuable model to investigate human hereditary spherocytosis and other blood-related disorders in which AE1 is implicated. Among various human hereditary abnormalities of erythrocytes that make cells very fragile and are accompanied by heart disease, sickle cell anemia is the best-known example (11). Echocardiographic abnormalities in sickle cell disease attributed to chronic hemolytic anemia (1) include left ventricular hypertrophy, left ventricular dilation, and increased stroke volume, which resemble the cardiac performance of adult AE1–/– mice (Fig. 4). AE1-related human spherocytosis is manifested in the heterozygous condition with ~25% reduction in hematocrit, hemolysis, spherocytosis, and little or no anemia (17). AE1+/– mice displayed erythrocyte spherocytosis (33), but we found no associated abnormalities of cardiac phenotype on the basis of morphological features (Table 2). This is consistent with cardiac hypertrophy in AE1–/– mice, which results from anemia. We cannot, however, rule out the possibility that loss of AE1 in the heart is itself a prohypertrophic event.

Little information on the localization of Cl–/HCO3– exchangers in the heart is available. Here, we examined the localization of AE1, AE3, and slc26a6. AE1 in adult cardiomyocytes showed a restricted localization, with AE1 primarily found at the sarcolemma, as assessed by confocal microscopy (Figs. 5B and 6). slc26a6, the Cl–/OH– and predominant Cl–/HCO3– exchanger of the heart (4), displayed a different location pattern, with longitudinal punctuate bands arranged along the cardiomyocyte, indicating localization beyond the sarcolemma. Furthermore, AE3 also showed a more widespread longitudinal distribution, with some transverse costamers on the isolated cardiomyocyte (Fig. 6).

This is the first localization of AE3 variant and slc26a6 in normal adult cardiac muscle cells. Although we do not have evidence for the functional significance of the isoform-specific localization of AE1, AE3, and slc26a6 in adult cardiomyocytes, these may be involved in anion exchange under different conditions in different cell compartments. AE3 associated predominantly with costamers (Fig. 7A, top), defined by the concentration of vinculin among other proteins in a series of sarcolemmal rib-like bands overlaying the Z band (31). Costamers anchor the myofibrils to the sarcolemma and are implicated in the transduction of contractile force from the myocyte to the extracellular matrix. AE3 may therefore assist in regulation of pH at these specific locations, which may be important given the sensitivity of contractile function to changes in pH (10). slc26a6 is even more strongly associated with T-tubular structures than AE3 (Fig. 7). T tubules are important in excitation-contraction coupling, suggesting a pH-regulatory role for both AE3 and slc26a6; however, particularly slc26a6 may be involved in the T tubules of cardiomyocytes. On the basis of colocalization with calreticulin, both AE3 and slc26a6 associated with the SR (Fig. 7), which implies a role for these proteins in buffering the pH of the SR, maintaining the constant pH required for the normal activity of calcium channels and pumps. Because SR is a specialized form of endoplasmic reticulum, it is also possible that AE3 and slc26a6 are present in SR as they pass through the membrane protein biosynthetic pathway.

The expression of a NCBE, which contributes to the pHi regulation of the central nervous system (12), was also first identified here by RT-PCR, in the neonate mouse heart. The previous failure (12) to detect NCBE on Northern blots of heart tissue suggests that, although NCBE message is present in heart, its expression level is low.

If AE1 is a key pH regulator in the heart, we would expect that its loss would be compensated by altered expression of other pH-regulatory genes, in particular other Cl–/HCO3– exchanger isoforms. The hypertrophic myocardium of the AE1–/– mutant mouse, however, did not compensate for the loss of alkaline extruder activity of AE1, by altering expression of any other gene in the heart ventricle, as assessed by quantitative real-time RT-PCR (Fig. 8B). There is thus sufficient Cl–/HCO3– exchange capacity in the heart that loss of AE1 does not require compensation by altered expression of other anion exchange proteins. Although AE1 was been proposed as the predominant Cl–/HCO3– exchanger of the heart (35), more recent studies showed that slc26a6 is the most abundant Cl–/HCO3– exchanger (4). Similarly, we found that inhibition of AE3 with an isoform-selective inhibitory antibody blocked 50% of recovery from alkaline load in cardiac muscle, consistent with a major role for AE3 in cardiomyocyte acid loading (7). The present work, showing that the heart does not significantly alter gene expression to compensate for loss of AE1, is consistent with the determination of slc26a6 as the predominant Cl–/HCO3– exchanger of heart.

The present work also provides insight into the role of Cl–/HCO3– exchangers in progression of cardiac hypertrophy. Considerable evidence has emerged indicating that hyperactivation of cardiac NHE1 has a central role in development of cardiac hypertrophy (6, 9, 21, 44). This has led to the realization that hyperactive NHE1 must work against an acid load (3, 8). Other work indicates that the acid-loading mechanism is Cl–/HCO3– exchange (32); in particular, there is evidence suggesting that AE3fl is responsible (3, 8). The finding that AE1–/– mice develop substantial hypertrophy indicates that AE1 does not have an indispensable role in progression of cardiac hypertrophy and is consistent with the model that AE3fl is the prohypertrophic Cl–/HCO3– exchanger.

AE1-null mice represent a valuable model of clinical conditions characterized by cardiac hypertrophy secondary to hemolytic anemia and other inherited diseases. AE1-null mice develop cardiac hypertrophy, likely secondary to severe anemia and without elevated expression of other pH-regulatory transporters. Progression of cardiac hypertrophy does not depend on AE1 activity, providing support for the idea that other Cl–/HCO3– exchangers hold this role. The restricted cellular localization of AE1, at the sarcolemma, was distinct from other major cardiac Cl–/HCO3– exchangers (slc26a6 and AE3). Yet the unique function of cardiac AE1 remains elusive.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
J. R. Casey is a Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). AHFMR and Canadian Cystic Fibrosis Foundation of Canada supported B. V. Alvarez. This work was supported by the Heart and Stroke Foundation of Alberta.


   ACKNOWLEDGMENTS
 
Dr. Luanne L. Peters (The Jackson Laboratory, Bar Harbor, ME) kindly provided AE1-null mice (National Heart, Lung, and Blood Institute Grant HL-64885). The authors thank Dr. H. Idikio of the Laboratory Medicine and Pathology, University of Alberta Hospital, for assistance with histological study of heart specimens.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Casey, Dept. of Physiology, Dept. of Physiology and Dept. of Biochemistry, Membrane Protein Research Group, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7 (e-mail: joe.casey{at}ualberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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