Europace Advance Access originally published online on September 27, 2006
Europace 2006 8(11):1011-1015; doi:10.1093/europace/eul099
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GENETICS
A method for the simultaneous analysis of mRNA levels of multiple cardiac ion channels with a multi-probe RNase protection assay
1 The First Department of Internal Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo 1138603, Japan; 2 The Cardiovascular Institute, Tokyo, Japan
Manuscript submitted 6 February 2004. Accepted after revision 16 May 2006.
* Corresponding author. Tel: +81 3 3822 2131 ext. 6743; fax: +81 3 5685 0987. E-mail address: iwasaki{at}nms.ac.jp
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Abstract |
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Aims Various pathological conditions can alter cardiac electrophysiological properties not only by physiological responses but also by modifying the gene expression of ion channels (electrical remodelling). To investigate the underlying mechanisms of the latter, electrophysiological alterations would require a simultaneous and comprehensive analysis of the mRNA level of the ion channel genes.
Methods and results We designed 19 cardiac ion channel cDNA templates to analyse the corresponding mRNAs and classified them into three template sets. Those sets were a voltage-dependent K+ channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying K+ channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, 
1C, ß2, 2
2 of cardiac L-type Ca2+ channel and 1G). These cDNA templates were used to synthesize antisense digoxigenin-labelled RNA probes. An amount of the total RNA of 25 µg was adequate to analyse simultaneously the mRNA levels of the ion channel genes with the use of multi-probe RPA, and these three multi-probe template sets enabled us to evaluate the profile of the spatial and temporal transcripts of the cardiac ion channels.
Conclusion The newly developed ion channel multi-probe RPA templates provide an aid in the comprehensive analysis of the electrical remodelling of the heart.
Key Words: RNase protection assay, Cardiac ion channel, Gene expression, Electrical remodelling, Simultaneous detection
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Introduction |
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Many pathological conditions including atrial fibrillation,1
cardiac hypertrophy, and heart failure2 have been reported to alter the cardiac electrophysiological properties that may lead to arrhythmogenesis, i.e. ‘electrical remodelling’.3 This remodelling is mainly believed to result from the alteration of ionic currents through modification of gene expression. As cardiac ion channels are diverse, in order to discuss the substrates for arrhythmogenesis, it is necessary to obtain comprehensive information about the gene expression of many of the ion channels.
The RNase protection assay (RPA) is known as a highly sensitive method for the quantification of specific mRNAs.4 Recently, RPA has been used for the simultaneous detection of multiple target genes using multi-probes.5 In fact, the simultaneous quantification of many chemokines has been reported to be useful. In cardiac electrophysiology, RPA has been used to analyse a single target gene.6,7 One of the reasons is that the mRNA of cardiac ion channels has a high homologous sequence to each other, which makes the simultaneous analysis difficult. However, a method for the simultaneous analysis of multiple genes with limited samples would provide great help also in cardiac electrophysiology. We developed three multi-probe RPA template sets that allowed for the simultaneous and comprehensive analysis of the mRNA levels of cardiac voltage-dependent potassium channels, inwardly rectifying potassium channels, and sodium and calcium channels.
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Methods |
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Animal and total RNA preparation
Sprague-Dawley rats aged 10 weeks were used in the present study. The hearts were removed, and the atria and ventricles were excised and quickly frozen in liquid nitrogen and stored at –80°C. To avoid contamination of the tissues, only right and left appendages were used as the atrial samples. The total RNA was extracted using the acid guanidinium isothiocyanate method.8
The total RNA concentration was measured with a spectrophotometer at a wave length of 260 nm.
Multi-probe templates
We classified 19 cardiac ion channel genes into three groups according to the characteristic of each gene. They were a voltage-dependent K+ channel series (rat erg, KvLQT1, Kv4.3, Kv4.2, Kv2.1, Kv1.5, Kv1.4, Kv1.2), an inwardly rectifying K+ channel series (rat Kir6.2, SUR2A/B, Kir3.4, Kir3.1, Kir2.2, Kir2.1), and an inward cationic ion channel series (rat SCN5A, 1C, ß2, 22 of cardiac L-type Ca2+ channel and 1G), referred to as the Kv series, Kir series, and Cin series, respectively. All the cardiac ion channel genes were known to be expressed in the rat heart. Cardiac troponin T was used as internal control.
Alignment confirmation
For an optimal setting of the multi-probe templates, we carefully selected the sub-cloning site for specific RNA probes. First, for genes already known to have splicing variants, i.e. Kv4.3 and Kir3.1,9,10 variant sites were excluded to avoid making a ladder band in the RPA. Secondly, the maximum highly homologous nucleotide sequences in the channel genes were detected and excluded. To evaluate the homologous alignment within the ion channel genes in each group, cluster analysis of those genes was preformed using an alignment programme (clustal X).11 Thirdly, only protected fragments that had at least a 10% difference in length were selected to differentiate well between each protected fragment. Thereafter, the nucleotide sequence of the primers was determined for the sub-cloning site of the RNA probes (Table 1 and 2).
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To prepare the cDNA templates of the 19 cardiac ion channel genes, a reverse transcription–polymerase chain reaction (RT–PCR, 30 cycles at 94°C for 30 s, at 60–62°C for 30 s, and at 72°C for 90 s, with an Access RT–PCR system, Promega, WI, USA), using the total RNA isolated from the rat atria as a template, was performed. The fresh PCR products were cloned using a pCR II vector (Invitrogen, Carlsbad, CA, USA). All the nucleotide sequences and the orientation of the insert were confirmed by a sequencer (ABI PRISM 3100, Applied Biosystems, CA, USA). The plasmid-containing cDNAs were linearized by a restriction enzyme (XbaI or SpeI) according to the orientation of the insert.
RNA probe preparation
Antisense RNA probes were prepared using a MAXI script (Ambion, Austin, TX, USA) with a digoxigenin-labelling mix (Roche, Mannheim, Germany). To equalize the protected fragment intensities, optimal amounts of the unlabelled RNA probes were used to dilute the corresponding digoxigenin-labelled RNA probes, because the protected fragment intensities tended to be various according to the length of the digoxigenin-labelled RNA probes and their expression levels. RNA probes from each multi-probe template were diluted in a probe-elution buffer containing (0.5 M NH4 acetate, 1 mM EDTA, and 0.2% SDS) 500 pg/0.5 µL for ready use.
RNase protection assay
RNA amounts of 5–15 µg, each with 500 pg RNA probes, were used for the hybridization with a RPA III kit (Ambion). The mixture was denatured at 95°C for 5 min and subsequently hybridized at 48–52°C for 12 h. After the hybridization, the non-protected fragment RNAs were digested with RNase A/T1 (1:100 dilutions) at 37°C for 30 min. After the protected RNAs were purified and denatured, the samples were run on a denaturing polyacrylamide/8M urea gel and were transferred to a positively charged nylon membrane and fixed by UV crosslinking. The membranes were incubated with a 1:10 000 anti-digoxigenin antibody (Roche) conjugated with an alkaline phosphatase. Subsequently, the protected fragments were detected using a CDPstar (Roche). Chemiluminescent signals were quantified by a cool saver AE6955 with ATTO Lane Analyzer software (ATTO, Tokyo, Japan).
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Results |
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Development of the multi-probe RPA template sets for cardiac ion channels
We developed three multi-probe template sets for the quantification of the mRNA levels of rat cardiac ion channels. These multi-probe templates allowed the simultaneous quantification of Kv series, Kir series, and Cin series mRNA levels. The amount of the total RNA required for the optimal protected fragment intensity was 15, 5, and 5 µg for Kv series, Kir series, and Cin series, respectively. Because
80 µg of total RNA was obtained from a rat atrium, it allowed triple quantification of cardiac ion channel gene screening from one sample. Figure 1 represents the simultaneous analysis of the mRNA expression of the Kv, Kir, and Cin series in the rat atrium. Band level and density of each protected fragment of three multi-probe templates were compared with that of each single-probe method, validating that each multi-probe protected fragment was identical to that of a single probe.
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Detection of a heterogeneous distribution of the cardiac ion channel mRNA in the heart by the multi-probe RPA
The Kv4.2 mRNA has been known to have a dominant expression in the epicardium of rat left ventricles, which underlies the different action potential duration between the epicardium and the endocardium.12
Actually, using our method, the Kv series multi-probe RPA identified a higher expression of the Kv4.2 mRNA in the left ventricular epicardium compared with that in the endocardium, whereas the other voltage-dependent K+ channels did not show any differences (Figure 2A). Similarly, Figure 2B shows the heterogeneous expression of the Cin series between the rat atrium and ventricle. A quite obvious dominant expression of 1G and 22, the components of calcium channels, was observed in the rat atrium over the ventricle with a similar expression of the mRNA levels of other channels.
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Detection of the effect of glucocorticoids on the voltage-dependent K+ channels in the rat ventricle using the multi-probe RPA
Many neuroendocrine factors are considered to play an important role in electrical remodelling. We investigated the effect of glucocorticoids, one of the neuroendocrine factors that exert various transcriptional regulation effects in various organs, on the ion channel mRNA expression, using the newly developed multi-probe RPA.
Figure 2C is a representative recording from the RPA showing the effect of dexamethasone on voltage-dependent K+ channels in the rat ventricle. Six hours after the injection of dexamethasone at a dose of 0.5 mg/kg, the total RNA extracted from the ventricular apex was subject to analysis. The figure clearly identifies that dexamethasone induced upregulation of Kv1.5 mRNA and downregulation of Kv4.2 mRNA, whereas the other voltage-dependent K+ channels were not altered. The glucocorticoid-induced upregulation of Kv1.5 mRNA was consistent with a previous report,13 supporting the validity of this new method. Moreover, our method exhibited advantages in the simultaneous analysis of many cardiac ion channels, clarifying a new finding that the procedure downregulated Kv4.2 mRNA. As shown in Figure 2, our method was useful in analysing the mRNA expression of ion channels simultaneously with different region- and time-dependent samples.
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Discussion |
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Main advantage of RPA using multiple probes corresponding to ion channels
This newly developed multi-probe RPA could quantify the mRNA levels of voltage-dependent K+ channels, inwardly rectifying K+ channels, Ca2+, and Na+ channels simultaneously and thus would be suitable for the comprehensive evaluation of the mRNA levels of cardiac ion channels from limited samples with less cost and total procedural time compared with the conventional single-probe RPA.
Most of the cardiac ion channels exist as multi-gene families that share structural features common to each other with highly homologous sequences. Actually, Kv4.3 had a highly homologous sequence identity to that of Kv4.2, making it difficult to develop a multi-probe template. To solve this problem avoiding inappropriate hybridization between the RNA probe and non-targets, we used an alignment programme and consequently were able to develop these newly developed multi-probe templates.
Other advantages of the multi-probe RPA
The amount of total RNA obtained from limited samples is usually quite low. However, as shown in Figure 2, the present multi-probe RPA needed only 25 µg of total RNA to screen most of the cardiac ion channels, whereas the single-probe RPA would require more than 150 µg of the total RNA. Therefore, even limited samples from a restricted area such as the sinus node region or pulmonary veins would be adequate for the simultaneous analysis of the expressed ion channels. Moreover, there is another advantage to this method: evaluation of mRNA levels of many cardiac ion channels relative to each other, the relative expression of many ion channels. This analysis of relative expression of many cardiac ion channel genes would assure a more accurate estimation of the gene expressions compared with the method using only the internal control.
Comparison with the alternative quantitative methods for mRNA
There are several alternative methods for quantifying the mRNA levels: northern blotting, reverse RT–PCR, and DNA tips/microarray. For the mRNA levels of the cardiac ion channels, their relative low expression is one of the problems. Although the northern blot analysis can be used for quantifying multiple target mRNAs by stripping and reprobing the membranes, the sensitivity would decrease with each stripping step. Moreover, the northern blot analysis would require large amounts of total RNA for hybridization and long procedural times compared with the RPA. The real-time RT–PCR method is a quite sensitive method. However, a preliminary examination is required to establish the optimal amplification efficacy for each cardiac ion channel and thus would not be suitable for the simultaneous evaluation of multiple ion channel genes that are quite diverse in their level of expression. Now, microarray analysis allows for the simultaneous analysis of the gene expression levels of more than thousands of genes and could be applied to cardiac electrophysiology.14 In general, however, the sensitivity of the microarray technology should be relatively low. Alterations in the cardiac ion channel gene expression have been reported to be restricted to relatively small ranges. Therefore, at present, the microarray analysis requires other final confirmation steps. Moreover, it should be pointed out that all these methods, including ours, have an inherent limitation as a true quantitative method, the dependence on the selection of internal controls.
Previously reported multi-probe templates of cytokine
The two multi-probe RPA sets for the detection of murine chemokines have recently been reported.5 These templates each contain six chemokine sequences (CXCL12/SDF-1, XCL1/lymphotactin, CCL20/exodus-1, CCL25/TECK, CX3CL1/fractalkine, CXCL1/KC, and CCL20/MDC, CXCL9/MIG, CCL9/10/MIP-1 g, CXCL13/BLC, CCL12/MCP-5, CCL19/ELC, respectively) and allow for the simultaneous analysis of chemokine expression. Various murine models have demonstrated their validity for analysis. Similarly, our method will provide an aid in the simultaneous analysis of the expression of the cardiac ion channels. However, in our analysis, the mRNA levels of the cardiac ion channels, including 1H, ß3, 21, and minK, could not be included because of their extremely low expression in rats, which would require a more sophisticated method for analysis.
In conclusion, our newly developed multi-probe system with RPA method was applied to the simultaneous analysis of mRNA levels of the cardiac ion channels in various pathophysiological conditions in rats. This simultaneous analysis of the mRNA levels of the diverse cardiac ion channels will provide great help in evaluating the arrhythmogenic substrates from a molecular biological viewpoint even with limited samples.
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Acknowledgements |
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We thank Mr John Martin for his linguistic assistance.
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References |
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