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Circulation Research. 1995;77:556-564

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(Circulation Research. 1995;77:556-564.)
© 1995 American Heart Association, Inc.

Articles

Cl--HCO3- Exchange in Developing Neonatal Rat Cardiac Cells

Biochemical Identification and Immunolocalization of Band 3–Like Proteins

Irina Korichneva, Michel Pucéat, Robert Cassoly, Guy Vassort

From the Laboratoire Physiopathologie Cardiovasculaire (I.K., M.P., G.V.), INSERM U-390, Hôpital Arnaud de Villeneuve, Montpellier, France, and Institut Jacques Monod (R.C.), CNRS UMR 9922, Universite Paris-7.

Correspondence to Dr Irina Korichneva, Laboratoire Physiopathologie Cardiovasculaire, INSERM U-390, Hôpital Arnaud de Villeneuve, 371, Avenue du Doyen Gaston Giraud, 34295 Montpellier Cedex 5, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract The Cl--HCO3- exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl--HCO3- exchanger activity concomitantly with the expression and intracellular localization of the band 3–like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pHi in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl--HCO3- exchanger at early stages of development. Neither basal pHi nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti–whole erythroid band 3 antibody, we found both the 80- and the 120-kD band 3–like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti–cardiac AE3 antibody. Thus, we propose that the proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3–like proteins at an early stage of development and their appearance within myofilaments after cell maturation. Colocalization of band 3–like proteins with specific markers of the Golgi apparatus, wheat germ agglutinin and CTR433 antibody, suggests the presence of these proteins in the Golgi area. The later decoration of repetitive striations between each sarcomere indicates that the band 3–like proteins are assigned in development within a compartment similar to costameres, areas of attachment of myofibrils to sarcolemma. These areas are likely to play a major role in signal transduction of neurohormonal stimuli.


Key Words: pH regulation • costamere • Golgi complex


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Intracellular pH (pHi) is a major regulator of diverse cellular processes.1 pHi regulation is particularly important in cardiac cells, because changes in pHi affect most steps of excitation-contraction coupling, including ionic conductances, Ca2+ homeostasis, Ca2+ sensitivity of myofilaments, and, in turn, cell contractility.2 pHi has also been known for many years to play a role in cell growth and division. More specifically, growth factors modulate pHi in various cell types3 4 ; they induce an alkalinization mediated by Na+-H+ antiport activation,3 or an acidification when the activity of the Na+-independent Cl--HCO3- exchanger overcomes the stimulation of the alkalinizing ionic transporters.4 5 6 Intracellular acidification triggers immediate-early gene expression in embryo cells,7 an event also known to be the earliest one in the hypertrophic process that occurs in the terminally differentiated neonatal cardiomyocytes.8

To restore their pHi from an acidic load, cardiomyocytes switch on both a Na+-H+ antiport9 10 and a Na+- and HCO3--dependent acid extruder.11 12 13 The activity of a Na+-independent Cl--HCO3- transporter that exchanges intracellular HCO3- for extracellular Cl- allows myocytes to regulate their pHi after an intracellular alkalinization. The properties of such an exchanger have been well characterized in adult cardiac Purkinje fibers,14 and the ion exchange activity has been also measured in adult rat isolated ventricular myocytes.15 So far, the Na+-independent Cl--HCO3- exchanger has been poorly investigated in growing cells, including neonatal cardiomyocytes. Only one study has suggested its presence in neonatal rat cardiomyocytes,16 but little is known about its properties.

The erythroid Cl--HCO3- exchanger is the best characterized anionic transporter. The ion exchange is mediated by a 100-kD membrane protein named band 3.17 18 The band 3 protein is encoded by the AE1 gene.19 Other band 3–related proteins encoded by different genes19 (ie, AE2 and AE3) were found in various cell types,20 21 including cardiomyocytes.22 In adult cardiac tissue, two membrane proteins of 80 and 120 kD immunologically related to erythroid band 3 are likely to mediate the anion transport.

The expression of many proteins, including contractile proteins, ionic channels, or GTP-binding proteins,23 24 25 26 has been shown to change during cardiac cell development. The properties of the Na+-H+ antiport have also been reported to depend on the stage of development in muscle cells.27 Moreover, in fetal neurons,28 although a band 3–related protein is already expressed, it does not function as an anion transporter. Accordingly, we used the model of neonatal rat cultured cardiomyocytes to determine whether a Cl--HCO3- exchanger is functional in developing heart and, if so, what proteins mediate the anion exchange. Microspectrofluorometric monitoring of pHi demonstrates that cultured neonatal cardiac cells possess a functionally active Cl--HCO3- exchanger; the anion exchange activity does not significantly change with different stages of cell culture. By use of an antibody raised against whole erythroid band 3 and antibodies specific to AE1 or expected AE3 translation products, Western blot analysis shows that both the 80- and the 120-kD band 3–like proteins are expressed in neonatal cardiac cells. Distribution of the proteins in the myocytes was also assessed by cell fractionation and confocal microscopy of immunostained cells. Our data show that arrangement of band 3–like proteins in growing cardiomyocytes is related to sarcomeric unit assembling.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Preparation of Whole-Heart Homogenates and Subcellular Fractions From Total Organs
Hearts from 10 to 12 neonatal and 2 adult rats were quickly removed from anesthetized animals. Adult hearts were perfused for 5 minutes in a Ca2+-containing solution to wash out blood. Then the organs were minced, extensively washed, and homogenized by using a glass-glass homogenizer in a Tris-HCl buffer (pH 7.5) containing 50 mmol/L Tris, 2 mmol/L EDTA, 10 mmol/L EGTA, 5 mmol/L dithiothreitol, 250 mmol/L sucrose, 1% Triton X-100, 1 mmol/L PMSF, 10 µg/mL leupeptin, and 100 µmol/L E-64. The homogenates were spun down for 15 minutes at 15 000g to discard the myofilaments. The supernatants were supplemented with 4x Laemmli electrophoresis buffer and boiled for 5 minutes.

Hearts were fractionated into cytosolic, membrane, and myofibril fractions after homogenization in a buffer containing 50 mmol/L glycerophosphate, 1 mmol/L EGTA, and 250 mmol/L sucrose, along with 1 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin, adjusted to pH 7.4. The homogenate was centrifuged for 30 minutes at 12 000g, and then the supernatant was purified by spinning for 90 minutes at 120 000g. The supernatant after ultracentrifugation was saved as cytosolic fraction. The pellet was extracted on ice for 30 minutes with glycerophosphate buffer containing 1% Triton X-100 and supplemented with the protease inhibitors. The myofilaments were spun down at 12 000g for 30 minutes. The supernatant was saved as membrane fraction. The myofibrils were washed three times with the Triton X-100 containing buffer. In some experiments designed to investigate the presence of band 3–like proteins in the cytoskeleton, the pellet was incubated for 10 minutes in a high ionic strength buffer (Triton X-100 containing buffer supplemented with 0.6 mol/L KCl).

Cell Culture
Cardiomyocytes were isolated from 3- to 4-day-old Wistar rats as described previously.29 Briefly, hearts were removed from anesthetized animals, minced, and dissociated with 0.1% (wt/vol) trypsin (Difco) in a Ca2+- and Mg2+-free PBS added to 0.4% EDTA and adjusted to pH 7.4. Myocytes and fibroblasts were separated by differential attachment to plastic plates. The cells were maintained in Eagle's MEM supplemented with antibiotics, 10% newborn calf serum (all from GIBCO), 5.5 mmol/L glucose, and 25 mmol/L NaHCO3. For pHi measurements and immunostaining, myocytes were plated at a density of 0.4x105 cells per square centimeter on glass coverslips. For biochemical experiments, cells were plated at a density of 1.75x105 cells per square centimeter in 25-mL culture flasks. To prevent fibroblast proliferation, 1 µmol/L arabinofuranosylcytosine (Sigma Chemical Co) was added 48 hours after plating and subsequently every 48 hours. Under these experimental conditions, most cells were spontaneously beating. Cells were kept for 17 days for pHi measurement and immunostaining and for 6 days for biochemical experiments. Cells were serum-starved 24 hours before any experiment.

Cell Fractionation
Cytosol, membrane, and myofibril fractions were separated from cell monolayers from 12 culture flasks as previously described.30 The cells were washed twice with cold PBS and scraped from the flasks in the buffer containing 20 mmol/L HEPES, 250 mmol/L sucrose, and 1 mmol/L EDTA and adjusted to pH 7.4. Cells were spun down and resuspended in glycerophosphate containing 1 mmol/L PMSF, 10 µg/mL aprotinin, and 10 µg/mL leupeptin. After homogenization with a glass-glass homogenizer, the homogenate was centrifuged for 30 minutes at 15 000g. In some other experiments, the cells were incubated with gentle shaking for 10 minutes on ice in glycerophosphate buffer containing 0.05% (wt/vol) digitonin and spun down for 2 minutes at 10 000g. The supernatant in both cases was centrifuged for 90 minutes at 120 000g to prevent contamination of the cytosolic fraction with light membranes. After mechanical homogenization or digitonin treatment, the pellet was resuspended in 1% Triton X-100 containing glycerophosphate buffer and the same protease inhibitors. After 30 minutes of incubation on ice, the homogenate was spun for 30 minutes at 12 000g. The supernatant was saved as membrane fraction, and the pellet was further washed three or four times with the Triton X-100 containing buffer and saved as the detergent-insoluble myofibril fraction. In some experiments, this fraction was further extracted with a high ionic strength buffer as described above.

Nuclei were isolated from the myocytes according to the method of Allo et al.31 Namely, after washing the cells with PBS they were rinsed once with the buffer containing 10 mmol/L Tris, 10 mmol/L NaCl, and 2.5 mmol/L MgCl2, pH 7.9 (buffer I), scraped from the flasks in this buffer containing protease inhibitors, and supplemented with 0.3 mol/L sucrose and 0.3% Triton X-100. The cells were homogenized, and the homogenate was laid on an equal volume of buffer I containing 0.6 mol/L sucrose and centrifuged for 10 minutes at 1500g. The pellet was resuspended in buffer I, washed, and saved as nuclear fraction.

Laemmli buffer (4x) was added to every fraction. The samples were then boiled for 5 minutes before electrophoresis. To check contamination of the cytosolic fraction by membrane proteins or the nuclear fraction by myofilaments, the proteins from both fractions were submitted to Western blotting. The blots were probed with either an anti–Na+,K+-ATPase {alpha}-subunit (McK1) antibody32 or an anti–troponin T antibody, respectively. Contaminating immunoreactive band was detected in none of the blots.

Electrophoresis and Western Blot Analysis
Electrophoresis and Western blotting analysis were carried out as previously described.22

Immunostaining and Confocal Microscopy
For immunostaining, the cells attached to glass coverslips were quickly rinsed with PBS and fixed for 10 minutes at room temperature with 2% (vol/vol) paraformaldehyde in PBS. The cells were then quenched in 0.1 mol/L glycine in PBS for 15 minutes at room temperature, washed with PBS, and permeabilized with 0.1% Triton X-100 (wt/vol) in PBS containing 1% bovine serum albumin for 10 minutes at room temperature. After washing and blocking of nonspecific binding sites by 1% bovine serum albumin, the cells were probed overnight at 4°C with the anti–whole band 3 antibody (1/50). After washing, the cells were incubated for 30 minutes at 37°C with Texas red–conjugated affinity-purified goat anti-rabbit IgG antibody (1/100). A monoclonal anti–myosin light chain antibody (1/200, Sigma) or a monoclonal anti–Golgi apparatus (CTR433) antibody33 were then used (1 hour at room temperature) as the second primary antibody to stain myosin or the Golgi complex, respectively. Fluorescein-conjugated sheep anti-mouse immunoglobulin served as a corresponding secondary antibody. Some cells were double-stained with the anti–whole band 3 antibody and with WGA (Molecular Probes) used at a concentration of 50 µg/mL for 40 minutes at room temperature. WGA was added either before or after the primary and secondary antibodies; the cells were extensively washed between each incubation. To stain nuclei, the cells were incubated for 20 minutes at room temperature with 10 µg/mL propidium iodide. After the cells were washed, the coverslips were mounted on glass slides in Citifluor (UKC Chem Lab).

The immunostained cells were observed in confocal laser scanning as described previously.22

pHi Measurements
pHi was measured in single isolated cells attached to coverslips and loaded with the pH-sensitive fluorescent indicator SNARF 1-AM as previously described,15 with minor modifications. Namely, only one ND 16 filter (Nikon) was used to attenuate the excitation light provided by a xenon lamp. In some cases, to increase the fluorescence signal limited by the low cell volume, pHi was monitored in a cluster of two or three myocytes. For such experiments, the cells were bathed in a control solution containing (mmol/L) NaCl 120, KHCO3 5.7, NaH2PO4 1.2, MgSO4 1.7, NaHCO3 1, CaCO3 1, and HEPES 20 adjusted to pH 7.4 at 32°C. In the Cl--free solution used to reverse the Cl--HCO3- exchanger, sodium gluconate replaced NaCl. pHi was calibrated in situ by the nigericin method,15 assuming that [K+]i was 140 mmol/L in all cells. All the experiments were performed in the presence of 1 µmol/L EIPA (Research Biochemicals Inc) to prevent the Na+-H+ antiport activity. Buffering capacity of the cells was estimated by using either the ammonium pulse protocol in the presence of EIPA or the alkaline load method in the presence of both DIDS and EIPA according to Roos and Boron.34 Both protocols gave similar results.

Statistical Analysis
Results are expressed as mean±SEM and compared by Student's t test.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Functional Characterization of the Cl--HCO3- Exchanger in Cultured Neonatal Cardiac Myocytes
The activity of the anion exchange was measured by monitoring pHi in SNARF 1–loaded cells after the removal of Cl- ions from external medium. Under this experimental condition, the reversal of the Cl--HCO3- exchanger induced a rapid alkalinization due to the influx of HCO3- ions into the cells. The alkalinization was not dependent on external Na+, since it was still observed when Na+ ions were substituted by N-methyl-D-glucamine (data not shown). Readdition of the Cl- ions into the external buffer triggered an acidification leading the cells to their initial pHi (Fig 1ADown, insert).



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  		Figure 1. Functional characterization of the Cl--HCO3- exchanger in neonatal cardiomyocytes. A, Cl- dependence of the anion exchange activity calculated from the slopes of pHi recovery after readdition of Cl- ions at different concentrations into the external Cl--free solution. Inset, Original pHi recording on SNARF 1-AM–loaded neonatal cardiomyocytes, superfused with Cl--free buffer as indicated. pHi was calibrated in situ in K+ buffer containing 10 µg/mL nigericin (see "Materials and Methods"). B, DIDS inhibition of the anion exchange activity estimated after incubation of the cells for 5 minutes at 32°C with increasing concentrations of DIDS. The drug was removed between each DIDS concentration tested. The Cl--HCO3- exchange activities were calculated from the rate of alkalinization after superfusion with Cl--free buffer. Results are expressed as mean±SEM of at least three cells for each Cl- or DIDS concentration.

pHi recovery depended on the concentration of external Cl- ions. The Cl- dependence of anion exchange activity calculated from the slopes of pHi recovery after readdition of Cl- ions at different concentrations into the external solution is shown in Fig 1AUp. Half-maximal anion exchange activity was reached in the presence of 14 mmol/L external Cl-. Full recovery of pHi required 120 mmol/L Cl-. To check further whether the change in pHi induced by Cl- removal could be fully attributed to Cl--HCO3- exchange activity, the cells were bathed for 5 minutes at 32°C with increasing concentrations of DIDS. The excess of drug was removed between each concentration tested, and the experiments were repeated as described above. The inhibition of the anion exchange was dependent on DIDS concentration in the range 0.5 to 30 µmol/L. The half-maximal inhibition was observed at 2.5 µmol/L DIDS. Full inhibition required 30 µmol/L DIDS (Fig 1BUp). Both the Cl- and the DIDS dose dependencies were similar to those observed in adult isolated Purkinje fibers or cardiac cells.22 35 The anion exchange activity was no longer observed when the cells were treated by 1 mmol/L probenecid or 0.1 mmol/L ethacrynic acid (data not shown), two specific inhibitors of the Na+-independent Cl--HCO3- exchanger.36 Thus, neonatal cardiac cells possess a functional Na+-independent Cl--HCO3- exchanger.

Basal pHi and the activities of the anion exchange during the development of neonatal cardiomyocytes in culture are summarized in the TableDown. Basal pHi, buffering capacity, and the activity of the exchanger appeared not to significantly change within 17 days of culture.


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  		Table 1. Basal pHi and Cl--HCO3- Exchange Activity During Cell Culture of Neonatal Cardiomyocytes

Identification of Band 3–Like Proteins in Neonatal Rat Hearts and Cultured Neonatal Cardiac Myocytes
To identify the proteins accounting for anion exchange activity in neonatal rat heart, we looked at the presence of band 3–like proteins known to mediate Cl--HCO3- exchange in other tissues. Since the members of the band 3 gene family share high homology in their amino acid sequences,19 Western blotting analyses with anti–whole human erythrocyte band 3, anti-mouse AE1, and anti–rat heart AE3 antibodies were used. The anti–whole erythrocyte band 3 antibody recognized two proteins in total homogenate prepared from adult (Fig 2ADown, lane 1) and neonatal (Fig 2ADown, lane 2) rat hearts. These proteins migrated with apparent molecular masses of 80 and 120 kD, respectively. They were abundant in the membrane fraction prepared from neonatal hearts (Fig 2BDown, lane 1). A weak 80-kD protein immunoreactivity was also detected in the cytosolic fraction (Fig 2BDown, lane 2). No proteins reacted with the anti–human whole band 3 antibody in the myofibril fractions prepared from neonatal hearts (Fig 2BDown, lane 3).



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  		Figure 2. Identification and subcellular distribution of band 3–like proteins in rat hearts. A, Western blot of proteins (200 µg per lane) of adult (lane 1) and neonatal (lane 2) rat heart homogenates using the anti–whole band 3 antibody. B, Western blot of proteins of membrane (lane 1), cytosolic (lane 2), and myofibril (lane 3) fractions prepared from neonatal rat hearts. Protein (100 µg) was loaded on the gel. The blot was probed with the anti–whole band 3 antibody. The proteins were revealed by using the extravidin-biotin system and ECL detection. Molecular weights were estimated from prestained standards. The different parts of the figure are representative of three to five experiments.

The same 80- and 120-kD proteins were revealed with the anti–whole band 3 antibody in immunoblotting of membrane fraction from isolated and cultured neonatal cells (Fig 3ADown, lane 1). In addition, the proteins were also found in myofibril (Fig 3ADown, lane 3) and nuclear (Fig 3ADown, lane 4) fractions. In every blot probed with the anti–human whole band 3 antibody, immunoreactivity of the 80-kD protein was higher than the one of the 120-kD protein. Even after extraction with a high ionic strength buffer (see "Materials and Methods"), the pellet containing the cytoskeletal proteins still showed the 80-kD band in Western blot with the anti–whole band 3 antibody (not shown). Immunoreactivity of the 80-kD protein appeared to be the same in these three fractions, whereas immunoreactivity of the 120-kD protein was weaker in the myofibril and nuclear fractions compared with membrane fraction. Some traces of immunoreactivity could be detected in cytosolic fraction prepared from neonatal cells (Fig 3ADown, lane 2).



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  		Figure 3. Distribution of band 3–like proteins in subfractions prepared from 6-day cultured neonatal rat cardiac cells. A, Western blot of proteins of membrane (lane 1), cytosolic (lane 2), myofibril (lane 3), and nuclear (lane 4) fractions prepared from neonatal rat cardiomyocytes. The blot was probed with the anti–whole band 3 antibody. B, Western blot of proteins of membrane (lane 1), myofibril (lane 2), and nuclear (lane 3) fractions prepared from cultured neonatal rat cardiomyocytes. The blot was probed with an anti-mouse AE1 antiserum. The proteins (A and B) were revealed by using the extravidin-biotin system and ECL detection. C, Western blot of proteins of membrane (lane 1), myofibril (lane 2), and nuclear (lane 3) fractions prepared from cultured for neonatal rat cardiomyocytes. The blot was probed with the antibody raised against an amino-terminal domain of rat cardiac AE3. The proteins were revealed with peroxidase-conjugated anti-rabbit IgG antibody and visualized by ECL detection. Protein (100 µg) was loaded in every lane (A through C). Molecular weights were estimated from prestained standards. The different parts of the figure are representative of three to five experiments.

When the blots were probed with an antiserum raised against a specific external domain of mouse AE1 (amino acids 571 to 58337 ), which is not shared by AE3, Western blotting of membrane, myofibril, and nuclear fractions from cultured neonatal cardiomyocytes showed only a 80-kD band (Fig 3BUp; lanes 1, 2, and 3, respectively). In contrast, an antibody raised against the peptide corresponding to the predicted amino terminal sequence of rat cardiac AE3 did not recognize the 80-kD but only the 120-kD band (Fig 3CUp). It was detected in the membrane fraction prepared from neonatal cells (lane 1) but not in the myofibril (lane 2) or in the nuclear (line 3) fractions.

Subcellular Localization and Visualization of Band 3–Like Proteins in Neonatal Cardiac Cells at Different Stages of Development
To specify the subcellular location of band 3–like proteins in neonatal cardiomyocytes in relation to cell growth, we used double immunostaining with the anti–whole erythrocyte band 3 and the anti–myosin light chain antibodies and confocal microscopy approaches. At day 3 of cell culture, the two antibodies revealed perinuclear distribution of both myosin and band 3–like proteins (Fig 4ADown). This pattern remained the same for band 3–like proteins at day 6 of neonatal cardiac cell culture, whereas the anti–myosin light chain antibody decorated repetitive small square structures spreading from the nucleus toward the cell periphery (Fig 4BDown). In fact, in these cells, myosin was already assembled into sarcomeric units. At this stage of development, the length of the sarcomeres was 0.82±0.05 µm (n=8). The sarcomeric structures developed with cell growth. At day 9 of cell culture, the assembly of myosin into sarcomeres was fully achieved, as shown by clear linear striations stained by the anti-myosin antibody. Fig 4CDown presents images of one cell immunostained with both the anti–myosin light chain antibody (panel C-a) and the anti–whole band 3 antibody (panel C-b) and the double immunostaining of the same cell (panel C-c). At the same stage of development, in addition to perinuclear staining the anti–whole band 3 antibody decorated thin repetitive riblike bands that complemented the anti–myosin light chain antibody staining. The secondary antibodies alone elicited weak diffuse staining uniformly distributed within the cells (not shown). Fig 4DDown presents a high magnification image of one myofibril of a cell cultured for 9 days and immunostained with both the anti–whole band 3 and the anti–myosin light chain antibodies. The striations decorated by the anti–whole band 3 antibody clearly showed a sarcomeric distribution. The distance between two striations equals 2.23±0.07 µm (n=20), which is similar to the adult sarcomere length.



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  		Figure 4. Immunolocalization of band 3–like proteins at different stages of development of neonatal cardiac cells in culture. The cells were double-immunostained with both the anti–whole band 3 and an anti–myosin light chain antibodies; the labeling was visualized by Texas red–conjugated and fluorescein-conjugated secondary antibodies, respectively. A, Confocal image of the cell cultured for 3 days. B, A field of three cells after 6 days in culture. C, A cell cultured for 9 days and immunostained with the anti–myosin light chain antibody (a) and anti–whole band 3 antibody (b) and double immunostaining of the same cell (c). The confocal projection images present one section at an optical resolution of 0.5 µm in the Z direction. The focal plan (A through C) passes close to the center of the nucleus. D, High-magnification confocal view of one myofibril of the cell cultured for 9 days and double-immunostained with the anti–whole band 3 and anti–myosin light chain antibodies. Bars=10 µm.

The observation of riblike bands colocalized with the dark lines that were not stained by the anti–myosin light chain antibody suggests the presence of the band 3–like proteins in particular structures similar to costameres in adult cells. To further define the perinuclear staining, double staining with both the anti–whole band 3 antibody and WGA, known to specifically bind lectins of the Golgi membrane,38 was used. Panels A and B of Fig 5Down show two optical sections (one of them, Fig 5BDown, crossing the nucleus) of the cells double-stained with these markers. The Golgi complex is limited to the perinuclear region. It should be noted that at early stages of development, some cells displayed polar staining with WGA (not shown). The green staining of the band 3–like proteins associated with the red staining of WGA overlapped, giving a yellow color. Fig 5CDown presents the image of a cell doublet immunostained with both the anti–whole band 3 antibody and CTR433, an anti–Golgi apparatus monoclonal antibody recognizing an antigen of the medium compartment of the Golgi complex.33 The optical section, crossing the nuclei, displays a perinuclear distribution of both immunological markers. Overlapping of the two colors results in a yellow color. To distinguish the observed staining from the nuclear labeling itself, the cells were probed with propidium iodide, a nuclear marker. The cell presented (Fig 5DDown) was cultured for 9 days. The anti–myosin light chain antibody stained the sarcomeres; propidium iodide strongly labeled the nucleus. This staining underlines the sharp border of the nucleus in contrast to the perinuclear anti–whole band 3 antibody staining. Thus, the band 3–like proteins were detected in a perinuclear compartment likely to be the Golgi apparatus of cultured neonatal cardiomyocytes.



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  		Figure 5. Comparison of perinuclear staining of band 3–like proteins with the Golgi apparatus and nuclei labeling. A, The cell was double-stained with both the anti–whole band 3 antibody and Texas red–conjugated WGA. Band 3–like proteins were visualized by fluorescein-conjugated secondary antibody. B, The same conditions were present as in panel A, but the focal plan crosses the nucleus. C, Confocal image shows a cell doublet immunostained with both the anti–whole band 3 antibody and an anti–Golgi apparatus antibody (CTR433, see "Materials and Methods"). The labeling was visualized by Texas-red–conjugated and fluorescein-conjugated secondary antibodies, respectively. Overlapping of the red and green fluorescence gives a yellow color. D, The cell was labeled with both the anti–myosin light chain antibody and propidium iodide; myosin was visualized by fluorescein-conjugated secondary antibody. Bars=10 µm.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we demonstrate that cultured neonatal rat cardiac cells possess a functionally active Na+-independent Cl--HCO3- exchanger regulating pHi from alkalosis at the early stages of the development. Western-blot analysis using the anti–whole band 3 antibody shows the presence of both 80- and 120-kD band 3–like proteins in neonatal cardiac tissue. We have recently identified the same two proteins in adult cardiac cells22 that were found only in the crude membrane fraction in Western blotting experiments, and their location was attributed to the level of T tubules. Moreover, we showed that either one or both proteins perform an anion exchange in adult cardiac cells. In neonatal cultured cells, the anion exchange activity exhibits the same Cl- dependence and DIDS sensitivity as found in adult cells. These results indicate maturation of the exchanger at least on day 3 of cell culture and suggest that the same proteins mediate the Cl--HCO3- exchange.

Subcellular fractionation of neonatal whole hearts demonstrates that the band 3–like proteins are mainly present in the membrane fraction. However, in neonatal cardiac cells kept in culture for 6 days, membrane, myofibril, and nuclear fractions contained band 3–like proteins. This difference most probably results from the particular experimental conditions used in cell culture. Indeed, to develop and maintain the features of cardiac phenotype, the cells attached to a plastic substratum need to spread and to be in contact with the neighbors. To keep this contact, the cells greatly developed their cytoskeleton.39 The presence of band 3–like proteins in the myofibril fraction prepared from cultured neonatal cardiomyocytes raises the possibility that besides exchanging anions, the band 3–like proteins could also exert an anchorage function for the contractile apparatus and the cytoskeleton to the membrane, as observed in erythrocytes.40 This could explain that even after three extractions with 1% Triton X-100 and 0.6 mol/L KCl treatment, the band 3–like proteins were still bound to the cytoskeleton. Furthermore, in our experiments the nuclei were most likely copurified with the prenuclear intracellular membranes such as endoplasmic reticulum membrane or/and the Golgi stack. The distribution of band 3–like proteins found in the subcellular fractionation experiments is in good agreement with the results obtained in confocal microscopy of immunostained cells. Such a localization of band 3–like proteins appears to be specific for cultured neonatal cardiomyocytes.

In the myofibril and nuclear fractions, only the 80-kD protein was present to the same extent as in the sarcolemma. It should be noted that at day 6 of culture (when cells were fractionated), no anti–band 3 antibody staining was detected in the myofibrils. This could be related to a weaker immunoreactivity of the native protein labeled in immunofluorescence compared with the denatured protein investigated in Western blotting. Since the anti-AE1 antibody used in the experiments (Fig 3BUp) was raised against a specific amino acid sequence of AE1 protein that is not shared by AE3,19 41 we consider that the 80-kD protein is most probably the product of one of the AE1 transcripts expressed in heart.42 Similarly, the 120-kD protein is proposed to be the translation product of heart AE3 gene because it is recognized by the antibody raised against a peptide corresponding to the predicted specific cardiac amino-terminal domain of rat AE3 protein, which is not shared by brain AE3.43 Interestingly, the 120-kD protein was much less detected in the myofibril and nuclear fractions than in the membrane fraction. Immunoblotting with the anti–rat AE3 antibody indicated the presence of the 120-kD protein in the membrane fraction, but the protein was barely detected in the nuclear fraction (Fig 3CUp). No band could be detected with this antibody in the myofibril fraction. This finding indicates that either the 120-kD protein is less strongly associated with the myofibril and nuclear fractions, or it is in a lower amount in these fractions. These data would support the hypothesis that the 80- and 120-kD proteins encoded by two different genes exert different and specific functions.

In every immunoblotting experiment with neonatal tissue, we observed a weak immunoreactivity of the 80-kD protein in the cytosolic fractions prepared from both hearts and cells. It is likely that both digitonin treatment and mechanical homogenization released the protein from a compartment particularly developed in neonatal cardiomyocytes or cultured cells, eg, the Golgi stack.

The differences found in immunoblotting experiments with neonatal and adult cardiac cells agree with the dissimilarity of localization of band 3–like proteins as observed in confocal microscopy of immunostained cells. At the same time, we found in neonatal cells, like in adult cells, that the anti–whole band 3 antibody decorated striations between each sarcomere labeled by the anti–myosin light chain antibody. This observation raises the question of the presence of T tubules in neonatal cardiac cells at early stages of development, since they have been considered to be among the last structural elements of the mammalian myocyte to develop in vivo. In fact, morphometric studies have indicated that in rat heart, T tubules become apparent at {approx}10 days postpartum.44 In the same line, the photomicrographs obtained by electron microscopy45 reveal T tubules in cultured neonatal cardiomyocytes by their continuity with the sarcolemma, relatively large diameter, and regular entry at the level of the Z line.

It was suggested that one role of the T tubules may be to divide the myofibrillar matrix of ventricular heart muscle into functional units.46 Structures similar to those we observed with the anti–whole band 3 antibody staining (ie, transversal riblike bands) were named costameres.47 These areas were defined as regions of attachment of myofibrils to the sarcolemma and T tubules. Costameres can be compared with focal contacts, the regions where microfilament bundles associate with the plasma membrane in nonmuscle cells. The focal contact proteins vinculin and talin were shown to colocalize with protein kinase C-{alpha} in normal rat embryo fibroblasts.48 Many other molecules involved in signal transduction, including phospholipase C,49 phosphatidylinositol kinases, diacylglycerol kinase, pp60c-src,50 and phosphatidylinositol 4,5-diphosphate,51 were also found in focal contacts. In neonatal cardiac myocytes, immunostaining with anti–protein kinase C antibodies closely resembles that of the costamere proteins.52 Among ion transporters, the focal accumulation of the Na+-H+ antiporter was described in fibroblasts and Chinese hamster ovary cells.53 In adult rat cardiomyocytes, the Na+-Ca2+ exchanger was also recently found to be distributed along the sarcolemma and the T tubules.54 55 The physiological significance of such a distribution would be to extend the exchange surface between the cell and the external medium. The costameres could play a role of functional subunits of myocardial cells where external signals are transduced and transferred to targets such as cytoskeletal and contractile proteins. A fine regulation of pH is thus required in this area. Localization of band 3–like proteins depends on the cell development. Thus, the above results complement our knowledge of the development of the structural organization in cardiac cells.

In addition to staining of structures that resemble costameres, shared by both neonatal and adult cells, significant immunoreactivity of band 3–like proteins was found in the perinuclear area in neonatal myocytes. Colocalization of the labeling with the anti–whole band 3 antibody and the staining of WGA or CTR433, an antibody that turned out to be a valid marker of the Golgi apparatus in muscle cells,33 suggests that the band 3–like proteins are in part associated with the Golgi stack. They could be either in the process of transport after polypeptide synthesis or could participate in the functioning of this compartment by transporting anions and regulating organelle internal pH. Data obtained in transiently transfected human embryonic kidney cells indicate that AE1 and AE2 proteins acquire the capacity to catalyze anion exchange while still in an early compartment of the secretory pathway.56

In conclusion, neonatal myocytes possess a functionally active Cl--HCO3- exchanger from the early stages of development. The ion transporter allows for a rapid anion transport required by the high metabolism of the growing cells. Such an anion exchange function is likely to be performed by the same proteins found in adult cardiac cells. We further propose that the association of band 3–like proteins with the structures that resemble costameres suggests an important role of the exchanger in the specific functional compartment responsible for signal transduction and regulation of myofibril matrix.


*    Selected Abbreviations and Acronyms
 
AE = anionic exchanger
ECL = electrochemiluminescence
EIPA = 5-(N-ethyl-N-isopropyl)amiloride
PMSF = phenylmethylsulfonyl fluoride
SNARF 1-AM = seminaphthorhodafluor acetoxy methyl ester
WGA = wheat germ agglutinin


*    Acknowledgments
 
This study was supported in part by the Fondation pour la Recherche Médicale and Fondation de France. We are grateful to Dr E. Millanvoye-van Brussel for helpful assistance in cell culture, to Dr S. Alper for the kind gift of the anti-AE1 antiserum, and to Drs I. Lebbar and C. Deprette for preparing the anti–erythroid band 3 antibodies and for skillful help in preparing the anti–rat AE3 antibodies. We also thank Dr M. Bornens for providing anti–Golgi apparatus antibodies and critical reading of the manuscript and G. Geraud for expert assistance in confocal microscopy.

Received November 18, 1994; accepted May 15, 1995.


*    References
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