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(Circulation Research. 1997;81:128-135.)
© 1997 American Heart Association, Inc.

Articles

Regional Localization of ERG, the Channel Protein Responsible for the Rapid Component of the Delayed Rectifier, K⁺ Current in the Ferret Heart

Mulugu V. Brahmajothi, Michael J. Morales, Keith A. Reimer, , Harold C. Strauss

From the Departments of Medicine (H.C.S.), Pathology (K.A.R.), and Pharmacology (M.V.B., M.J.M., H.C.S.), Duke University School of Medicine, Durham, NC.

Correspondence to Michael J. Morales, Department of Pharmacology, Box 3845, Bell Building, Room 345, Duke University Medical Center, Durham, NC 27710. E-mail mmorales{at}acpub.duke.edu

	Abstract

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Abstract Repolarization of the cardiac action potential varies widely throughout the heart. This could be due to the differential distribution of ion channels responsible for repolarization, especially the K⁺ channels. We have therefore studied the cardiac localization of ERG, a channel protein known to play an important role in generation of the rapid component of the delayed rectifier K⁺ current (I_Kr), an important determinant of the repolarization waveform. Cryosections of the ferret atrium and ventricle were prepared to determine the localization of ERG by fluorescence in situ hybridization (FISH) and immunofluorescence. We found that in the ferret, ERG transcript and protein expression was most abundant in the epicardial cell layers throughout most of the ventricle, except at the base. In the atrium, we found that ERG is most abundant in the medial right atrium, especially in the trabeculae and the crista terminalis of the right atrial appendage. It also is present in areas within the sinoatrial node. In all regions studied, FISH and immunofluorescence showed concordant localization patterns. These data suggest that repolarization mediated by I_Kr is not uniform throughout the ferret heart and provide a molecular explanation for heterogeneity in action potential repolarization throughout the mammalian heart.

Key Words: in situ hybridization • immunolocalization • K⁺ channel • ventricle • atrium

	Introduction

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Repolarization of the cardiac action potential is dominated by the outward flow of K⁺ ions. It is well established that action potential repolarization varies widely among regions of the heart, largely because of differences in K⁺ currents. Many distinct K⁺ currents have been identified in cardiac myocytes isolated from various anatomic regions, including transient outward and delayed rectifier K⁺ currents, yet the exact number of different K⁺ currents in the heart is unknown.^{1 2 3} The unambiguous identification of the channel proteins responsible for each current would greatly facilitate our understanding of the basis of the diversity of cardiac K⁺ current expression.

The general approach to molecular identification of these K⁺ channels has been to compare the phenotype of currents found in isolated native myocytes with those of heterologously expressed K⁺ channels thought to be present in the heart.¹ Although this approach has been successful in two cases in which human mutations have led to the correlation of channels with known currents,^{4 5 6 7} it has not led to an unequivocal identification of the remainder of the K⁺ channel proteins. This is primarily a reflection of the similarity in many of the functional properties of cloned channels,⁸ the unresolved effects of K⁺ channel ancillary subunits,^{9 10 11 12 13} and the possibility of heteromeric assembly of channel subunits.^{14 15} An additional difficulty is that electrophysiological characterizations are necessarily performed on small numbers of isolated myocytes, whereas molecular biological approaches commonly rely on material (ie, RNA) isolated from >10⁶ cells.

To bring molecular biological techniques to the level of the single cell, we used fluorescent in situ hybridization to detect mRNA of individual K⁺ channels in isolated myocytes from seven different anatomic regions of the heart.¹⁶ In contrast to previous studies,^{17 18 19} our results suggested that the transcript for almost all of the known K⁺ channel subunits is expressed in myocytes from the different regions but that very few were expressed in >60% of all myocytes. Our observation suggested that K⁺ channels in the heart, like those in the brain,^{20 21 22} may show very distinct patterns of localization. We therefore have performed FISH and immunolocalization experiments on the mRNA transcript and protein product of ERG to test the hypothesis that this K⁺ channel has a nonuniform distribution in the heart.²³ We chose ERG because of the high probability of its association with a known K⁺ current, I_Kr.^{4 5} Individuals with mutations in HERG have a form of familial long-QT syndrome.²⁴ It is also the primary molecular target of several selective and nonselective antiarrhythmic drugs, such as dofetilide, quinidine, and flecainide.^{25 26 27 28}

	Materials and Methods

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Sectioning and In Situ Hybridization of Ferret Hearts
Ferret hearts procured from 8- to 11-week-old ferrets were dissected and perfused with modified Ringer's solution, and where necessary, myocytes were isolated, as previously described.²⁹ They were then perfused with 4% paraformaldehyde in phosphate-buffered saline for 45 minutes at room temperature to fix the hearts. The anatomic regions of interest were dissected from the remainder of the heart and further incubated in paraformaldehyde for 1 hour, followed by overnight incubation at 4°C in 40% sucrose in PBS. The tissue was then incubated in OCT medium (10.24% polyvinyl alcohol, 4.26% polyethylene glycol) for 4 (atrium) to 8 (ventricle) hours, embedded in OCT medium, and frozen in a solid CO₂/isobutanol bath. The block was sectioned using a Leica Jung Frigocut 2800E cryostat in the locations and at the thicknesses described in Figs 1 and 2. The sections were laid out on gelatin-coated slides, fixed for 30 minutes in paraformaldehyde, and dehydrated by sequential 5-minute incubations in 50%, 75%, and 95% ethanol. After being allowed to air dry, in situ hybridization was performed as previously described.¹⁶ After the hybridization washes, the sections were rehydrated by sequential 5-minute incubations in 95%, 75%, and 50% ethanol, followed by deionized water and then PBS. Immunological detection was performed as previously described.¹⁶ In all experiments, secondary antibodies conjugated with FITC were used to detect binding, except in Fig 2I6 and Fig 3N through 3Z, where secondary antibodies conjugated with rhodamine were used. All experiments were performed independently on a minimum of three different ferret hearts. The images were scanned on a Zeiss LSM 410 inverted confocal microscope equipped with a class 3B laser. Digitized images were obtained using LSM 3.9v (Zeiss), processed for publication using Adobe Photoshop (Adobe) or IPlab Spectrum (Signal Analytics Corp), and printed using a Tektronix color phaser IISDX V2013-102 printer.

View larger version (60K): [in this window] [in a new window]   Figure 1. Localization of ERG in the ferret ventricle. All the sections were 10 µm thick, and the magnification factor of the micrographs is x5, unless otherwise specified. In panel A, the schematic representation of the ferret ventricle depicting the location of the five cross sections is shown. Sections are as follows: 1, through apex; 2, 3 mm above the apex; 3, 1.5 cm above the apex; 4, 3 cm above the apex; and 5, 3.3 cm above the apex. Panels B through E show bright-field (B) and immunofluorescence (C through E) detection of transfected HEK293 cells (magnification x40). Cells expressing ERG cDNA and sham-transfected cells probed with anti-ERG antibody and ERG-transfected cells probed with preimmune antisera are shown in panels C through E. In contrast to panel C, panels D and E show little detectable fluorescence. Panels F through J are bright-field micrographs of cross sections 1 through 5 (apex to base), respectively. Panels K and L show FISH using the sense ERG probe on the sections adjacent to those shown in panels F and G to serve as negative controls. Panel M is the FISH of the section adjacent to that shown in panel H using a cardiac form of the troponin I (troponin IC) antisense probe to serve as a positive control. Panels P through T show FISH using ERG antisense probes on sections shown in panels F through J. Panels U through Y are x40 views of the epicardial areas represented in the white boxes in panels P through T. Panel U corresponds to panel P, V to Q, W to R, X to S, and Y to T. Panels N (x40) and O (x63) are enlargements of the midmyocardial wall encompassed by the red box in panels S and T. ERG transcript expression is more abundant in the epicardial region than in the remainder of the ventricular wall, where it is present in fewer cells. This is in contrast to the uniform expression of the troponin IC transcript in panel M. Panels Z through NN represent immunofluorescent studies of ERG localization. Panels EE through II represent immunofluorescence of sections adjacent to sections depicted in panels P through T. Panels Z through DD are x40 enlargements of the epicardial regions represented in the white boxes in panels EE through II. Panel Z corresponds to EE, AA to FF, BB to GG, CC to HH, and DD to II. Panels JJ and KK show little detectable fluorescence in sections incubated with preimmune serum, which are adjacent to those shown in panels EE and FF, and serve as negative controls. Panels LL (x40), MM (x40), and NN (x63) are magnified images from the midmyocardial regions represented in the red boxes of panels GG through II. Note the correlation between immunofluorescence and FISH, showing concordance between protein and transcript expression.

View larger version (77K): [in this window] [in a new window]   Figure 2. Localization of ERG in the ferret right atrium. The sections all were 6 µm thick, except for panels H through I6, which were 5 µm thick. The magnification factor of the micrographs is x10, unless otherwise specified. Panel A shows a top view of the right atrium (RA) with the dotted lines representing the mediolateral incision to endocardial surface of the RA. IVC and SVC indicate inferior and superior vena cava, respectively. Panel B shows the endocardium of the medial RA appendage, with the path of one section (pink bar) perpendicular to the crista terminalis (CT) crossing from the top to the bottom through a small portion of the lateral RA appendage, atrial trabeculae, CT, and intercaval area, including the sinoatrial (SA) nodal region (H through I) and the other section (yellow bar) parallel to the CT (D through G). Panel C shows the endocardium of the lateral RA appendage, with the path of the section (yellow bar) (J through N); AVR indicates atrioventricular ring. Bright-field images are shown in panels D, H, I4, and J. FISH using ERG antisense probes on sections are shown in panels E and K, with the corresponding x40 images representing the areas within the white squares (E1 through E3 and K1 through K3). Panels F, I, and M are immunofluorescence studies of ERG localization, with anti-ERG antibody, on sections adjacent to those shown in panels E and K. Panels F1 through F3, F1 through F3, and M1 through M3 are x40 images representing the areas within the squares of panels F, I, and M, respectively. Panel I4 is a light micrographic image (at x100) of the SA node (SAN), along with the adjacent crista (CT) and intercaval region (top). Panel I5 is the immunofluorescence image of the section shown in I4 using anti-ERG antibody. Panel I6 is FISH performed using an oligonucleotide probe to the slow-twitch form of troponin I (TnIs) using secondary antibody conjugated with rhodamine. Panels G and N are immunofluorescence of sections incubated with preimmune serum that are adjacent to those shown in Panels F and M and serve as negative controls. Note the relative abundance of ERG transcript and protein in CT, trabecular region of the medial RA, and presumed SAN region within the intercaval region and near absence of transcript and protein in the lateral RA and remainder of the intercaval region. The FISH signals obtained using a TnIS-specific probe confirms the identification of the SA nodal region. The correlation between the immunofluorescence and FISH shows that protein and mRNA expression are concordant.

View larger version (28K): [in this window] [in a new window]   Figure 3. Optical Z sections of ferret right ventricular myocytes. Panels A through L show 0.75-µm-thick optical Z sections through two myocytes probed with anti-ERG antibody. Panel M is a composite of panels A through L. The presence of signal in only the outer sections (A through D and I through L) suggests that ERG is most abundant at the cell surface. Panels N through Y show optical sections using FISH of ERG mRNA with secondary antibody conjugated with rhodamine. Panel Z is a composite of panels N through Y. Unlike the ERG protein, the mRNA is most abundant in the center of the myocyte.

Preparation of ERG Anti-Peptide Antibodies
Peptides in HERG were selected on the basis of their predilection for ß-turn motifs as predicted by PREDITOP,³⁰ which was kindly provided by J.L. Pellequer (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France). The peptide sequences are GLLNSTSDSDLVRYR (amino acids 321 to 335 in HERG²³ ) and ARPGKSNGDVRALTY (amino acids 820 to 834). These residues are conserved in ferret ERG, but the valine (underlined) in the first peptide is a methionine in the ferret (M.J. Morales, unpublished data, 1996). The peptides were synthesized commercially (Genosys) using the "multiple antigenic peptide" approach.³¹ Peptide (1.0 mg) was emulsified with either Freund's complete (primary immunization) or incomplete (boosts) adjuvant, and polyclonal antibodies were generated in rabbits and purified on a protein-G column as previously described.³² Antibodies generated independently in two animals showed identical specificity (data not shown). Sham-immune serum was generated by inoculating a third rabbit with adjuvant emulsified with PBS on a schedule identical to that for rabbits inoculated with ERG peptide.

Characterization of Anti-ERG Antibodies
Anti-ERG antibodies were characterized both by peptide ELISA and by immunofluorescence assay of transfected cells. Peptide ELISA was performed by the indirect sandwich method on affinity-purified IgG, essentially as previously described,³² using 1 µg/mL peptide in carbonate/bicarbonate buffer to coat an Immunolon plate (Nunc). Antibodies were tested against each peptide independently, and in both cases, the secondary antibody was horseradish peroxidase–conjugated goat anti-rabbit IgG (Jackson Immunological Research). Horseradish peroxidase activity was detected by absorbance at 450 nm after incubation with 3,3',5,5'-tetramethyl benzidine, followed by addition of 2 mol/L H₂SO₄. This assay showed the enriched IgG fraction was 64 000-fold more specific than IgG from control rabbits (data not shown).

To test antibody specificity in an immunofluorescence assay, a plasmid containing the H-ERG cDNA⁵ (generously provided by M. Keating, University of Utah Health Sciences Center, Salt Lake City) was digested with Kpn I and BamHI and cloned into those sites in pCEP4 (Invitrogen) to give phERG/CEP4. pCEP4 contains a cytomegalovirus promoter to ensure high-level expression. phERG/CEP4 or pCEP4 (as a sham-transfected control) was transfected into the EBNA variant of human embryonic kidney 293 cells (Fig 1B)³³ using lipofectamine (Life Technologies) according to the manufacturer. Cells were fixed, and immunofluorescence was performed by standard techniques³² using 1.0 µg/mL of anti-ERG IgG affinity-purified primary antibody and goat anti-rabbit FITC-conjugated IgG as the secondary antibody (Fig 1C through 1E). Cells transfected with ERG cDNA show an immunofluorescence pattern consistent with the detection of the ERG protein by the antibodies (Fig 1C). This reaction is specific, because cells transfected with plasmid alone (Fig 1D) or transfected with ERG plasmid but probed with sham-immune IgG (Fig 1E) showed no detectable response.

	Results

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Ventricle
To test the hypothesis that ERG is localized within the ventricle, five 10-µm sections were prepared to give a cross-sectional view of the ventricle, and ERG transcript was localized by FISH. A schematic drawing of the ventricle with the lines indicating the levels of the five cross sections is shown in Fig 1A. Two of the cross sections (Nos. 1 and 2) were obtained from the apex, one was midway between the apex and base (No. 3), and two (Nos. 4 and 5) were near the base of the heart (Fig 1A). In situ hybridizations performed on these five sections are illustrated in rows 3 through 5 of Fig 1 and show that ERG transcript is apparently present throughout the ventricle (Fig 1N through 1Y). However, the density of ERG transcript in sections 1 through 4 (Fig 1P through 1S) is most abundant in the outer cell layers, with less transcript expression in the remainder of the ventricle. Sense probes used as a negative hybridization control in sections (Nos. 1 and 2) closest to the apex (Fig 1K and 1L), as well as the other sections (data not shown), had no detectable fluorescence, showing that the hybridization was specific. Control experiments with an antisense probe for the cardiac form of troponin I (Fig 1M)¹⁶ in sections adjacent to those in Fig 1H showed dense and evenly distributed fluorescence throughout the ventricle, reflecting a previously observed pattern in rats.³⁴ This experiment shows that the entire section was competent for hybridization and that ERG localization patterns shown are not artifactual. Sense probes of these transcripts gave no detectable signal (data not shown). Similar findings were observed in three hearts.

Although protein expression can often be inferred from in situ hybridization data, protein localization may not follow the same pattern as mRNA expression. Therefore, we used antibodies to peptides derived from the ERG cDNA sequence to test the localization of the ERG protein in the ferret ventricle. Immunofluorescence performed on these sections is illustrated in rows 6 through 8 of Fig 1. As shown in adjacent sections (Fig 1Z through 1II and 1LL through 1NN), ERG protein appears to correspond to that of the transcript throughout the ventricular sections. These data indicate that the protein is preferentially distributed in the epicardial cell layers throughout most of the ventricle. In the remainder of the ventricular wall, ERG protein expression is less dense. It is interesting that the transmural distribution of both transcript and protein is not uniform in sections 3 through 5 (Fig 1R through 1T and 1GG through 1II), suggesting that other interesting localization patterns may be present. Similar results were obtained in three hearts.

Atrium
ERG expression also was nonuniform in the ferret right atrium (Fig 2). The right atrial appendage (Fig 2A) was dissected to expose both the medial and lateral walls of the right atrium. Cuts used to prepare sections from the medial and lateral right atrium are shown (Fig 2B and 2C). Both in situ hybridization and immunolocalization show considerable ERG expression in the medial right atrium (Fig 2D through 2I). ERG is especially abundant in the atrial trabeculae and crista terminalis of the medial right atrial appendage (Fig 2E through 2F and 2I). This expression is markedly reduced toward the caval margin of the crista terminalis and in the intercaval area except for a small area in the SA nodal region (Fig 2I1 through 2I6). Two criteria were used to identify the SA nodal region: cell morphology (Fig 2I4)³⁵ and the presence of transcript for the slow-twitch form of troponin I³⁴ (Fig 2I6). Presence of ERG in the SA node corresponds to data demonstrating I_Kr in isolated SA nodal myocytes.³⁶ In contrast, ERG expression is much lower in the lateral right atrial appendage (Fig 2K through 2N). Similar results were obtained in additional sections (one in the medial and one in the lateral wall of the atrial appendage) parallel to those shown in Fig 2D and 2J, each 5 mm toward the edge of the atrial appendage (data not shown). Similar observations were obtained from experiments in four hearts.

Localization of ERG Within Myocytes
Although the correspondence in expression of ERG protein and mRNA suggests that active ERG/I_Kr channels are expressed in these cells, it may still be possible that the ERG protein produced in these cells may not form active channels but be sequestered in intercellular compartments. To examine this possibility, we used the confocal microscope to perform optical sections on right ventricular myocytes on which immunofluorescence or FISH had been performed (Fig 3). Each optical section was 0.75 µm thick and was in the Z plane of the figure, perpendicular to the page. In Fig 3A through 3L, immunofluorescent detection of ERG proteins is shown. These sections indicate that ERG is most abundant in the outer portions of the cell, suggesting that it was present in the plasma membrane. In contrast, the ERG transcript was, as expected, present in the interior portions of the cell (Fig 3N through 3Y). In Fig 3M and 3Z, composites of the optical sections taken from the immunofluorescence and FISH experiments, respectively, are shown.

	Discussion

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Our data demonstrate that the spatial distribution of ERG is complex and suggest that its biochemical anatomy may be an important determinant of its functional properties in the ferret heart. The heterogeneous pattern of expression is most striking throughout much of the ventricle, where ERG is uniformly expressed in the epicardial cell layers, but is much less abundant in the remainder of the ventricular wall. Although this is the first detailed demonstration of a varied distribution of ion channel proteins within a mammalian heart, several lines of evidence indicate complex distribution patterns for other K⁺ channels. Regional differences in the magnitude of the transient outward K⁺ current in ventricular myocytes have been shown in dogs,³⁷ humans,^{38 39} and other species,⁴⁰ with the current being more abundant in epicardium than endocardium. Transcripts for Kv4.2, a K⁺ channel clone thought to participate in the transient outward K⁺ current,¹ also seem to follow a concentration gradient from endocardium to epicardium in dogs and rats.^{17 18} I_Ks also is more abundant in the endocardium and epicardium than in the midmyocardium of dogs,⁴¹ although there is no molecular correlate to this observation.

It has been suggested that I_Kr is an important current contributing to the end of phase 2 and much of phase 3 of the cardiac action potential.^{27 42 43} If ERG is the molecular basis of I_Kr, our data suggest that this current and its contribution to repolarization must be greatest in the ventricular epicardium. Other studies have documented ERG mRNA^{24 44} and I_Kr^{41 45 46} in the hearts of humans and other species. However, only in dogs, where I_Kr is evenly distributed in the ventricular wall,⁴¹ has the question of organization of I_Kr been addressed systematically. This observation may suggest that our data may not be generalizable to other species.

Given its importance to repolarization, it is somewhat surprising that I_Kr may not be found in all ventricular cells. However, even a small current localized to a single cell may influence repolarization in neighboring cells not expressing this current. This is because the membrane resistance during the plateau phase of the action potential is relatively large, making the DC length constant correspondingly large. As a result, this high impedance enables this small current to influence repolarization in nearby cells.³

Our data displaying the biochemical anatomy of ERG in the ferret heart have profound mechanistic implications and may provide a potential explanation for several observations concerning repolarization in human hearts. First, the importance of I_Kr to repolarization in human hearts has been inferred from sensitivity to dofetilide as well as genetic studies.⁵ Yet this current could not be detected in some isolated human myocytes (M.W. Veldkamp, unpublished data, 1996).^{38 45} It has been suggested that this may be a reflection of the cell-isolation technique.⁴⁶ However, sampling differences in the face of spatial heterogeneity may account for some of the reported differences between studies from Veldkamp et al,⁴⁵ Konarzewska et al,³⁸ and Li et al.⁴⁶ Second, our demonstration of ERG in the SA node may explain why one form of sinus bradycardia occurs in patients with familial long-QT syndrome linked to chromosome 7 and in patients receiving dofetilide.⁴⁷ Thus, although these results have not yet been extended to humans, localization of this important channel in the ferret may be predictive of its distribution in the human heart. Should such patterns be present in human hearts, they may be valuable in understanding the molecular basis of repolarization and in predicting the efficacy of selective K⁺ channel blockers in treating those cardiac arrhythmias for which the vulnerable region has been identified and localized within the heart. As more selective K⁺ channel blockers are developed, knowledge of the spatial distribution of corresponding K⁺ channels may enable more sophisticated strategies for antiarrhythmic therapy to be developed.

	Selected Abbreviations and Acronyms

 

ELISA = enzyme-linked immunosorbent assay ERG = ether-à-go-go–related gene FISH = fluorescence in situ hybridization(s) HERG = human ERG IKr, IKs = rapid and slow components of delayed rectifier K+ current SA = sinoatrial

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL-19216 and National Institutes of Health equipment grant BIR-9318118. The authors wish to thank Dr James O. McNamara, Sr, for use of equipment; Dr Paul Dolber for assistance with the preparation of the light microscopic photographs; Anne Crews for excellent technical assistance; and Randall L. Rasmusson, Shimin Wang, Shuguang Liu, and Don L. Campbell for valuable discussions.

Received January 7, 1997; accepted May 6, 1997.

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