Articles

In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes

Mulugu V. Brahmajothi, Michael J. Morales, Shuguang Liu, Randall L. Rasmusson, Donald L. Campbell, Harold C. Strauss
https://doi.org/10.1161/01.RES.78.6.1083
Circulation Research. 1996;78:1083-1089
Originally published June 1, 1996

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Abstract

Abstract The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by Kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.

Atrial tachyarrhythmias are an important cause of morbidity in patients with heart disease. The mechanisms underlying these arrhythmias and their sites of origin or reentrant paths vary substantially.1 2 3 4 5 Although pharmacotherapy with Na+ and K+ channel blockers can be effective against many different atrial arrhythmias, shortcomings resulting from toxicity and limited efficacy underscore the current limitations in antiarrhythmic therapy.6 Since many current antiarrhythmic agents are primarily targeted at refractoriness and/or repolarization, these shortcomings ultimately reflect our limited understanding of the molecular basis of the K+ currents generating repolarization.

Until recently, the K+ currents that dominate atrial repolarization were thought to be the transient outward current (Ito)7 and two delayed rectifier currents: a rapidly activating inwardly rectifying current (IKr) and a slower activating current (IKs).8 9 10 11 12 13 However, other K+ currents could potentially contribute to repolarization, eg, the ultrarapidly activating current (IKur).14 15 Despite extensive physiological characterization, the molecular basis of each of these currents is still debated. Assignment of currents to specific channel cDNAs is complicated by their similar functional properties and the wide molecular diversity of the voltage-gated K+ channel family.16 An additional confounding factor is the difference between functional properties of native human cardiac myocyte K+ currents versus those from other mammalian species (rabbit, dogs, rats, and guinea pigs). Recent analyses of the functional properties of the different K+ currents in ferret ventricular myocytes demonstrate that the phenotypes of ferret K+ currents closely resemble those of human ventricular myocytes,7 17 18 19 suggesting that the ferret is a particularly appropriate animal model in which to study mammalian cardiac K+ currents.

Originally discovered in Drosophila, the evolutionarily conserved Shaker family of K+ channels consists of six distinct subclasses: Shaker (Kv1), Shab (Kv2), Shaw (Kv3), Shal (Kv4), Kv5, and Kv6. Four of these (Kv1 through Kv4) have several mammalian homologues, whereas Kv5 and Kv6 each have only one known member.16 Although K+ channels in the Kv1 through Kv4 families have received the most experimental attention, additional attention will undoubtedly be focused on two channels implicated in familial forms of long-QT syndrome.20 21 One of these channels, ERG, is a member of the ether-à-go-go family,22 which are structurally similar to Shaker-type channels. Heterologously expressed ERG produces currents similar to that of the cardiac IKr.23 24 The other, KvLQT1, is a physiologically uncharacterized channel that represents a new class of the Shaker family.21

Two K+ channel families that lack the six membrane-spanning domains of the Shaker-type channels probably play a role in the generation of cardiac K+ currents. The IRK family of inwardly rectifying K+ channels has a structure with only two membrane-spanning domains on either side of a pore structure conserved with other members of the Shaker superfamily.25 The minK channels have a structure completely unrelated to other channels, with only one membrane-spanning segment.26 Heterologous expression of this protein results in a slowly activating delayed rectifier current.27 28

To identify K+ channels expressed in heart, Dixon and McKinnon29 used an RNase protection assay to detect the expression of 15 different K+ channels in rat atrial and ventricular muscle. Of these, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2 were expressed at relatively high levels. The abundance of the remaining mRNAs ranged from low to undetectable. Any attempt to draw conclusions about transcript expression in heart cells using this method is predicated on the assumption that the tissue samples used consisted solely of myocardial cells and that expression was uniform between cells. However, it is widely recognized that only 70% of the cells isolated from heart consist of cardiac myocytes.30 In addition, some genes of interest were not included in this analysis.

To further our understanding of the molecular basis of cardiac K+ currents, we set out to determine the following: (1) the identity of voltage-sensitive K+ channel mRNAs expressed in cardiac myocytes, (2) the differences in voltage-gated K+ channel expression between major anatomic regions of the heart (ie, left and right atria, left and right ventricles, sinoatrial [SA] node, and ventricular and atrial septa), and (3) the uniformity of gene expression between myocardial cells in major anatomic regions of the heart. The present study used in situ hybridization on large numbers of enzymatically isolated single myocytes obtained from the major anatomic regions of the ferret heart, ensuring that our sample population was essentially free of contamination by nonmyocyte cell types.

Materials and Methods

Cell Preparation

Single cardiac myocytes were isolated from the hearts of male ferrets (10 to 20 weeks old) using a Langendorff perfusion technique as previously described.31 After 25 to 40 minutes of perfusion with proteolytic enzymes, the heart was removed from the Langendorff apparatus, and the left ventricle, right ventricle, ventricular septum, left atrium, right atrium, atrial septum, and SA node were dissected free. Each dissected region was then placed in a separate 15-mL conical tissue culture tube containing fresh proteolytic enzyme solution to which an additional 10 mg/mL of BSA (essentially fatty acid free, Sigma Chemical Co) was added. The tubes were then gently shaken (≈0.5 to 1 cycle per second) on a rocker table at 37°C. Aliquots of myocytes were collected at 10- to 15-minute intervals, gently centrifuged, and the pellets were directly resuspended in normal 1.8 mmol/L Ca2+–containing control saline. After isolation, the myocytes were fixed for 30 minutes in 3% paraformaldehyde, subsequently washed twice with PBS, and then used directly for in situ hybridization.

Preparation of Probe and Labeling

K+ channel subunit sequences from both the GenBank library and our previous cloning studies in ferret heart were used to design oligonucleotide probes of 45 to 50 nucleotides (see Table 1⇓). Where possible, ferret or human sequences were used. When computer analysis (Gene Runner v3.0, Hastings Software) predicted extensive secondary structure, inosines were used to disrupt the potential structure. The uniqueness of the probes was confirmed by comparison with the entire GenBank database using BLAST32 and FASTA33 and by direct comparison with known channel sequences. A probe was judged to be suitable if (1) no other channel appeared in the top 50 matches of the BLAST or the FASTA search and (2) the individual searches of the antisense probe of related channel sequences showed no more similarity than the corresponding sense probe. The sequence of the probes, location in the GenBank entry, and the family from which they are derived are presented in Table 1⇓. It was not possible to design unique probes to members of the IRK family; the IRK probe34 has 67% sequence identity with rat B-IRK235 and may cross-react with other unknown members of that family. Oligonucleotides were synthesized and gel-purified by Genosys Inc. The oligonucleotide probes were 3′ end-labeled with digoxigenin-ddUTP using terminal transferase as described by the manufacturer’s protocol (Boehringer-Mannheim).

View this table:
Table 1.

Probes Used for In Situ Hybridization

In Situ Hybridization

In situ hybridization was performed by a modification of established techniques.36 The cells were suspended in 0.1 mol/L triethanolamine-OH, pH 7.2, 0.15 mol/L NaCl (tetraethylammonium buffer), incubated for 3 minutes at 37°C, treated with acetic anhydride (0.25% in tetraethylammonium buffer) for 10 minutes at 37°C (to inactivate residual protease), washed in PBS for 5 minutes at 37°C, and incubated in 2× SSC (1× SSC contains 150 mmol/L NaCl and 15 mmol/L Na3 citrate, pH 7.0) for 10 minutes at room temperature. The cells were prehybridized with 50% deionized formamide, 4× SSC, 1× Denhardt’s solution, 0.5 mg/mL salmon sperm DNA, 0.5 mg/mL yeast tRNA, and 10% dextran sulfate for 30 minutes at room temperature. The digoxigenin-labeled probe (100 ng/mL) was added and incubated overnight at 42°C. The samples were washed with 4× SSC for 10 minutes at room temperature, 1× SSC for 10 minutes at room temperature, 0.1× SSC at 42°C for 30 minutes, and 0.1× SSC for 10 minutes at room temperature. Cells were then washed in 100 mmol/L Tris-HCl plus 150 mmol/L NaCl (pH 7.5) and incubated for 30 minutes in the same buffer containing 2% normal sheep serum and 0.3% Triton X-100. The cells were then incubated with fluorescein isothiocyanate–conjugated anti-digoxigenin antibody (1:500 dilution in blocking buffer, Boehringer-Mannheim) for 1 hour at room temperature. After incubation, the reactions were stopped using 100 mmol/L Tris-HCl (pH 9.5), 100 mmol/L NaCl, and 50 mmol/L MgCl2. The cells were centrifuged and laid on gelatin-coated slides.

Fluorescence was detected with a Nikon UFX IIA microscope using an excitation range of 450 to 498 nm and an emission range of 550 to 560 nm. The relative percentage of positive isolated myocytes was evaluated visually, and the totals were calculated using counts from a hemocytometer. Between 1000 (for SA node) and 40 000 (ventricular) cells were individually evaluated in each experiment. All numbers in Table 2⇓ represent the results of at least three different experiments. Because of differential yields of cells from various anatomic regions, cells from different ferrets were mixed in some experiments; however, each value in Table 2⇓ is derived from cells isolated from at least three different ferret hearts. Where appropriate, confidence limits between experiments were calculated using an unpaired Student’s t test. Differences were taken to be significant at P≤.01. Images were photographed on 400 ASA color film. Photographs were scanned using a Hewlett-Packard ScanJet IIC and reproduced for publication using Adobe Photoshop.

View this table:
Table 2.

Abundance and Distribution of K+ Channel Subunit mRNA in Cardiac Myocytes

Additional controls were run with some of the probes. Preincubation of the fixed cells with 100 μg/mL RNase A (37°C for 30 minutes) abolished the hybridization signal with Kv1.2, Kv1.3, Kv1.4, Kv1.5, and the cardiac form of troponin I (TnIC) antisense probes. To test the fluorescent signal for quenching, cells were hybridized with the TnIC, Kv1.4, Kv1.5, ERG, and IRK antisense probes, stained, and incubated with reticulocyte lysate as described previously.37

Results

The prevalence and distribution of voltage-gated K+ channel subunit mRNA transcripts in cardiac myocytes from different regions of the ferret heart is shown in Table 2⇑, and examples of probed cells are shown in the Figure⇓. Several control experiments were performed to ensure the validity of our results. All experiments included the corresponding sense probe. In no experiment did any sense probe give a visually detectable signal (panels A through D), suggesting that the observed signals from the antisense probe (panels E through R) were due to specific hybridization. Additionally, several antisense probes were hybridized after treatment of the myocytes with RNase A. These also gave no detectable signal, showing that the hybridization signals were RNA dependent (data not shown).

Figure 1.
Figure 1.

In situ hybridization of isolated ferret myocytes. A through D, sense probes; E through R, antisense probes. Panels A and E show Kv1.1 with cells from SA node; B and F, Kv1.3 (right atrial cells); C and G, Kv1.4 (right atrial cells); and D and H, Kv1.5 (right atrial cells). Cells in panels I through N were derived from right ventricle. Panel I shows Kv3.1; J, Kv3.2; K, Kv3.3; L, Kv3.4; M, IRK; and N, ERG. Panels O and P show the TnIC probe on cells from left ventricle (O) and SA node (P). Panels Q and R show the TnIS probe on cells from right ventricle (Q) and SA node (R). Arrows show cells counted as negative for hybridization.

To validate our approach further, probes to TnIC and the slow-twitch form of troponin I (TnIS) were used as positive controls. Gorza et al38 showed that TnIC was present in anatomic regions of the mature rat heart rich in working myocytes (atrium and ventricle) and in the SA and atrioventricular nodes as well. In contrast, TnIS was present only in cells of the conduction system.38 Our results (Table 2⇑; Figure⇑, panels O through R) confirmed these observations in the ferret, where TnIC was present in ≈90% of myocytes (except in the septa) and TnIS was present in the SA node (54.1%) but virtually absent from other anatomic regions. Contrary to expectations, we were not able to detect TnIC in ≈10% of myocytes from the atrium or ventricle. Although it is possible that some myocytes do not contain this message, a more likely explanation is that some cells were compromised during preparation, resulting in a few false negatives.

The most intensively studied cardiac K+ channels are those of the Kv1 family. Our data (Table 2⇑) show interesting patterns of transcript expression among the Shaker family of K+ channels. The most widely distributed K+ channel transcripts in the ferret heart are Kv1.5 (69% to 86%) and Kv1.4 (46% to 94%). Both Kv1.5 and Kv1.4 (Figure⇑, panels C and D and panels G through H) are fairly evenly distributed among myocytes from different regions of the heart, except for the relatively low abundance of Kv1.4 in the atrial and ventricular septa. Kv1.2 is also widely distributed in the atrium and ventricle. Cells expressing Kv1.1 (Figure⇑, panels A and E), Kv1.3 (panels B and F), and Kv1.6 were all rare in atrium and ventricle, with the most abundant being Kv1.3 in the right atrium (16.5%). The pattern of Kv1.3 channel mRNA expression differed markedly from the free walls of the atrium and ventricle to septum and SA node (P≤.01). Both Kv1.2 and Kv1.4 are expressed at lower levels in the septa, whereas Kv1.3 expression is higher in the septa than in the free walls of the atria and ventricles (P≤.01). More dramatically, Kv1.1 and Kv1.6, which are very rare elsewhere in the heart, are quite common in the SA node.

Transcripts from the three other classes of Shaker-type channels were also expressed in ferret myocytes. Myocytes containing Kv2 channel mRNA were uniformly distributed throughout the heart, with myocytes expressing Kv2.1 being twofold to threefold more abundant than those expressing Kv2.2. Surprisingly, the four known members of the Kv3 (Figure⇑, panels I through L) family are also expressed in myocytes throughout the heart. Kv3.4 shows the most striking regional variation; ventricular myocytes expressing Kv3.4 are roughly threefold more common in the ventricle than in the atrium (P≤.01). All three known members of the Kv4 family are expressed in the ferret heart, with Kv4.2 being the most abundant (P≤.01). The transcript of Kv5.1 was most common in the right atrium (46.4%) and rarest in the atrial septum (21.5%). Kv6.1 was less abundant. It was present in 28.4% of SA nodal cells and in <16% of cells in the other anatomic regions.

Recent studies have shown that besides the Shaker-type K+ channels, several other proteins may participate in the generation of certain cardiac K+ currents. These include members of the IRK family of inward rectifying channels (two membrane-spanning segments)34 and the minK protein (with only a single membrane-spanning segment).27 28 Essentially all ventricular myocytes express some member of the IRK family (Figure⇑, panel M), which is also common in the atrium; interestingly, it is nearly absent (6.6±0.6%) from the SA node, consistent with the virtual absence of inward rectifying current (IK1) in this region.39 The minK protein, which has been proposed to be responsible for IKs27 28 or a possible cofactor of ERG,40 is most common in the SA node, followed by atrium, and then ventricle.

Two channels deserving of special attention are ERG and KvLQT1; mutations in each of these channels are responsible for two of the forms of familial long-QT syndrome.20 21 Our results show that ERG (Figure⇑, panel N) is uniformly expressed in about half the myocytes throughout the heart, whereas KvLQT1 is somewhat more common, present in roughly 70% of right atrial and ventricular myocytes and in 50% to 60% in the other regions.

Discussion

The results of the present study suggest that mRNAs encoding most known voltage-gated K+ channels can be found in a substantial portion of ferret cardiac myocytes. We have also shown that the pattern of expression can vary considerably between anatomic regions. Our approach to detection of K+ channel gene expression was to use in situ hybridization on enzymatically isolated myocytes obtained from different regions of ferret heart. This approach affords several advantages. The probes are inexpensive and easy to design. Transcripts can be examined in many individual identifiable myocytes isolated from different regions of the heart. Use of the ferret permitted us to conduct these studies on an animal model whose cardiac K+ currents are very similar to those of the human. However, only one of the probe sequences was available from ferret. This necessitated extensive use of heterologous probes, which carry an increased risk of nonspecific hybridization. To minimize this risk, most of our probes were designed against human channels (Table 1⇑), which seem to have a high degree of sequence similarity with ferret channels17 (D.R. Lamson, M.C. Comer, D.L. Campbell, M.J. Morales, and H.C. Strauss, unpublished data, 1995). It was necessary to use rat sequences for five of the probes. Yet even with these heterologous probes, detecting mRNA with the antisense probe was possible, whereas the sense probes never gave a detectable signal, supporting the validity of our approach.

In a previous study, Dixon and McKinnon determined K+ channel mRNA levels expressed in different regions of the rat heart.29 However, we detected myocytes containing mRNA for channels not previously thought to exist in the heart, notably members of the Kv3 family. Unfortunately, results obtained by these two methods are not directly comparable. Dixon and McKinnon29 studied RNA isolated from whole hearts by RNase protection assay. This approach is very specific and potentially quantitative. Yet the RNA is necessarily isolated from a large population of cells in whole tissue; it presumably contains all the myocyte mRNA and RNA from other cell types. Our approach of fluorescent in situ hybridization to isolated myocytes has the important advantage of probing for RNA that exists only in single myocytes isolated from specific anatomic regions of the heart. Since we do not know how many myocytes are present in each region and cannot quantify this signal, one cannot draw conclusions about the overall abundance of an individual RNA species. Despite these differences, our results seem to be in general agreement with those of Dixon and McKinnon; the most obvious difference is the presence of Kv3 channel message, which was of “very low” abundance in rat heart when assayed by RNase protection assay. One explanation of this possible discrepancy is a species difference in mRNA expression between ferret and rat. It is also possible that no discrepancy exists. A transcript, expressed at a moderate level in 10% to 20% of individual myocytes, might be of very low abundance when assayed by RNase protection.

The present data suggest that cellular variability in transcript expression may be an important variable in determining the strength of the mRNA signal detected in studies performed on homogenates of whole heart. A subsequent study was performed by Barry et al,41 who used antibodies to the five channels whose RNA was most abundant. They found that all these proteins were expressed in the rat heart except Kv1.4. Their study highlights an important caution when interpreting in situ hybridization data: mRNA expression may not show a direct correlation with protein expression, although lack of message generally predicts lack of protein expression.

Physiological and Therapeutic Implications

Although our hybridization data do not address whether the variability in transcript expression is cell-to- cell or is due to regional sublocalization, it clearly suggests that differences in K+ channel transcript expression could be a significant determinant of differential K+ current expression in isolated cardiac myocytes. Rational design of antiarrhythmic drugs will require knowledge of the structure of the channel responsible for generating a target current. Our data suggest that a wider range of potential molecular targets for antiarrhythmic drug action may exist in the heart than previously suspected. Further, the discovery of heteromultimerism among K+ channels,42 43 the discovery of a cardiac voltage-gated K+ channel β subunit,44 45 46 and the realization that non–Shaker-type channels may play an important role in generation of cardiac currents23 24 27 28 34 all serve to complicate the process of identification. The traditional criteria for establishing this connection include correlation of physiological and pharmacological properties between cloned channels and native currents.47 We propose that understanding the full complexity of the molecular basis of K+ current expression in the heart will require coordination of electrophysiological measurements with in situ detection methods for channel subunits in the cell or region of the heart where the current of interest resides.

In conclusion, the present study has used an in situ hybridization technique on myocytes isolated from different regions of the heart. This approach has enabled us to localize mRNA expression of voltage-sensitive K+ channels to individual cells, although our data do not predict to what extent the mRNA detected is translated into protein. However, the present study suggests that differences in transcript expression could account for the experimentally observed differential K+ current expression between myocytes.48 For example, there are significant differences in K+ currents between atrial cells,49 in the inward rectifier K+ current between the ventricle,50 51 atrium, and sinus node, and in the Ito between the subepicardium and subendocardium of the human ventricle.52 Finally, the present data suggest that molecular information concerning regional and cell-to-cell variability of potential therapeutic targets may ultimately facilitate development of safer and more efficacious antiarrhythmic therapy.

Acknowledgments

This work was supported by NIH grants HL-19216, HL-52874, and HL-54314 and NSF grant CDR8622201. We gratefully acknowledge the invaluable advice provided by Dr J.O. McNamara and J.E. Kraus and the excellent technical assistance of Anne L. Crews.

Footnotes

  • This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

  • Received October 2, 1995.
  • Accepted March 21, 1996.

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  • In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes
    Mulugu V. Brahmajothi, Michael J. Morales, Shuguang Liu, Randall L. Rasmusson, Donald L. Campbell and Harold C. Strauss
    Circulation Research. 1996;78:1083-1089, originally published June 1, 1996
    https://doi.org/10.1161/01.RES.78.6.1083
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