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(Circulation Research. 2003;92:234.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Microarray Analysis Reveals Complex Remodeling of Cardiac Ion Channel Expression With Altered Thyroid Status

Relation to Cellular and Integrated Electrophysiology

Sabrina Le Bouter^*, Sophie Demolombe^*, Arnaud Chambellan, Chloé Bellocq, Franck Aimond, Gilles Toumaniantz, Gilles Lande, Sepideh Siavoshian, Isabelle Baró, Amber L. Pond, Jeanne M. Nerbonne, Jean J. Léger, Denis Escande, Flavien Charpentier

From INSERM U533 (S.L.B., S.D., A.C., C.B., G.T., G.L., S.S., I.B., J.J.L., D.E., F.C.), Physiopathologie et Pharmacologie Cellulaires et Moléculaires, Faculté de Médecine, Nantes, France; and the Department of Molecular Biology and Pharmacology (F.A., A.L.P., J.M.N.), Washington University School of Medicine, Saint-Louis, Mo.

Correspondence to Flavien Charpentier, PhD, INSERM U533, Faculté de Médecine, 1 rue G. Veil, 44035 Nantes cedex, France. E-mail flavien.charpentier{at}nantes.inserm.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits ("IonChips") were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in I_to,f and I_K,slow densities, and increased I_Ks density in hypothyroid myocytes. In addition to effects on K⁺ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the 1C Ca²⁺ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na⁺, Cl^-, and inwardly rectifying K⁺ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel - and ß-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

Key Words: ion channel • cDNA microarray • repolarization • ionic remodeling

	Introduction

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Electrical remodeling refers to changes in cardiac electrophysiological function caused by heart disease. Various pathologies are associated with electrophysiological remodeling including cardiac hypertrophy and failure,¹ chronic arrhythmia (eg, atrial fibrillation),² ischemic injury,³ or altered thyroid status,⁴ and several lines of evidence suggest that the underlying molecular mechanisms are complex and may well be model-dependent. In addition, most studies in the remodeled myocardium have focused on examination of individual ionic currents and/or expression levels of the channel subunits generating these currents.

The application of genomic techniques, however, holds the promise of allowing the expression levels of thousands of genes to be examined simultaneously.⁵ We have developed cDNA microarrays ("IonChips") containing probes for a large subset of genes encoding ion channel subunit proteins. With the objective to validate this novel tool, we have investigated cardiac ion channel remodeling associated with altered thyroid hormone status in the mouse heart. Previous studies have clearly demonstrated that changes in thyroid status dramatically impact cardiac electrical functioning. Hypothyroidism produces sinus bradycardia and prolongs cardiac repolarization, whereas hyperthyroidism accelerates sinus rate and shortens repolarization.^4,6 In addition, changes in the expression and/or the functioning of cardiac ion channels have been documented in animals with altered thyroid hormone levels. In the rat, for example, hypothyroidism reduces the transient outward current (I_to) density,^7,8 an effect attributed to decreased expression of KCND2, the gene encoding the voltage-dependent K⁺ channel protein, Kv4.2.⁹ The expression levels of other genes encoding voltage-gated K⁺ channel proteins, notably KCNA5 (Kv1.5) and KCNB1 (Kv2.1), have also been shown to be decreased, whereas KCNA2 (Kv1.2) and KCNA4 (Kv1.4) expression is increased in hypothyroid (rat) ventricles.^9–11 Recent studies have also documented thyroid hormone-dependent effects on the expression of HCN2 and HCN4, two genes encoding pacemaker channel proteins.¹²

Nevertheless, the observations available in the literature are conflicting due to different experimental animals and methodologies to manipulate thyroid hormone levels and to assess the physiological and molecular consequences of these manipulations. In mice in which thyroid hormone receptors have been deleted, for example, expression of KCNB1 and KCND2 is reportedly unaffected.¹² In addition, because previous studies have focused on individual genes,^9–11 it is unclear whether additional genes are also affected and important in the observed pathophysiology.

The use of cDNA microarrays here reveals that the QT prolongation seen in hypothyroid animals is caused by a more complex remodeling of cardiac ion channels than was previously thought. The results demonstrate that the mRNA levels of several voltage-gated K⁺ channel subunits are decreased, whereas others are increased. Similar observations were made in hyperthyroidism. Importantly, the observed changes in the expression of mRNAs encoding ion channel subunits are reflected in alterations in protein levels and in the functional expression of the channels encoded by these subunits. Taken together, our results demonstrate that cDNA microarrays offer new opportunities to investigate ion channel remodeling in detail in highly integrative pathophysiological models.

	Materials and Methods

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An expanded Materials and Methods section containing details of cDNA microarray and semiquantitative RT-PCR studies, Western blots, ECGs, and electrophysiological recordings is available in the online data supplement, which can be found at http://www.circresaha. org. All animal experiments were performed in accordance with institutional guidelines for animal use in research.

Ten-week-old male C57BL/6 mice (IFFA CREDO, L’Arbresle, France) were assigned to 2 control groups (24 animals in each), 1 hypothyroid group (n=24), and 1 hyperthyroid group (n=24). Hypothyroidism was obtained by treating the animals with 5-propyl-2-thiouracil (PTU 0.15% in low-iodine food; Harlan) for 5 weeks. One group of control animals had the same food without PTU and supplemented with iodine. Mice were weighed before and every week during the treatment. Mice assigned to the hyperthyroid group were treated with daily intraperitoneal injections of T3 hormone in saline solution with NaOH 1 mmol/L (1 µg/g; Sigma) during 8 days. Control animals received intraperitoneal injections of the vehicle. Mice were weighed before treatment and 12 hours after the last injection.

Our home-made cDNA microarrays contained 116 probes representing most mouse voltage-gated Na⁺, Ca²⁺, Cl^-, and K⁺ channel subunits, inward rectifier and 2-pore domains K⁺ channels, epithelial Na⁺ channels, and connexins cloned so far, and also genes encoding proteins involved in Ca²⁺ homeostasis and thyroid hormone receptors (see online Table 1 of the online data supplement). Microarrays also contained 93 probes for several human genes encoding Na⁺ and K⁺ channel subunits (see online Table 2). The clones were obtained by PCR amplification of genomic DNA. The 100 to 700 bp amplified products were subcloned into the pCRII-TOPO plasmid vector (Invitrogen) and sequenced.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Functional Effects of Altered Thyroid Status
T3 and T4 plasma levels were evaluated using radio immunoassay in 4 pools of 3 mice in each group. In control mice, T3 and T4 levels were 10.8±0.3 and 3.3±0.2 pg/mL, respectively. In mice receiving PTU for 5 weeks, T4 was undetectable and T3 expression was reduced to 0.6±0.1 pg/mL (P<0.001). In mice injected daily with T3, the level of plasma T4 (1.3±0.4 pg/mL) was reduced compared with control mice (9.3±0.3 pg/mL; P<0.01), resulting from negative feed-back control. As shown in the Table, hypothyroid mice had decreased body and heart weights. Inversely, hyperthyroid mice had increased body and heart weights. In addition, heart to body weight ratios were significantly decreased in hypothyroid mice and increased in hyperthyroid mice.

View this table: [in this window] [in a new window]   Table 1. Effects of Thyroid Status on Body and Heart Weights

Representative ECG recordings from hypo- and hyperthyroid mice are shown in Figure 1. As is evident, hypothyroidism significantly (P<0.001) decreased heart rate and conduction velocity at both the atrial (P-wave duration=32±1 ms compared with 22±1 ms in control animals) and atrioventricular (PR interval duration=42±1 ms compared with 36±1 ms in control animals) levels. In hypothyroid animals, the corrected QT interval (QTc) was 86±2 ms, longer than seen in control animals (50±1 ms; P<0.001). Both rapid and slow components of the T wave were prolonged. Hyperthyroidism had opposite effects, inducing on average a 25% increase in heart rate and 19% and 17% decreases in atrial and atrioventricular conduction times, respectively (P<0.001 for each effect). In addition, QTc was shortened by 12% (P<0.05). In contrast to hypothyroidism, hyperthyroidism did not affect the rapid component of the T wave when corrected for heart rate. Figure 1 also shows QT/RR plots in control mice IV injected with tedisamil, a drug that blocks the transient outward (I_to) and the delayed rectifier (I_K,slow) currents in rat ventricular myocytes,¹³ as well as heterologously expressed Kv1.5-encoded K⁺ currents.¹⁴ Although the effects of hypothyroidism on repolarization were similar to those observed with tedisamil, the bradycardic effects were more pronounced.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of thyroid status on ECG. A, Typical ECG traces (lead I) recorded in control (top), hypothyroid (middle), and hyperthyroid (bottom) mice. Horizontal bar=200 ms. B, QT-RR (top) and QTr-RR (bottom) relationships for control mice (open squares), hyperthyroid mice (filled squares), hypothyroid mice (filled circles), and mice injected with 5 mg/Kg (T5; inverted triangles) and 15 mg/Kg (T15; triangles) of tedisamil. For clarity, control mice for the hypothyroidy and hyperthyroidy studies were pooled because there were not significantly different. Data expressed as mean±SEM.

Effects of Thyroid Status on Cardiac Levels of Ion Channel Transcripts
In order to appreciate the variability of the gene expression ratios, 4 control experiments were performed. Each experiment consisted of extracting RNA from a pool of 3 mouse hearts and splitting it into 2 samples that were then reverse-transcribed and labeled, one with Cy3 and the other with Cy5. The 2 labeled cDNA samples from the same hearts were then hybridized on one microarray. Figure 2A illustrates such an experiment. All spots (592) but 11 had Cy5/Cy3 normalized ratio values ranging between 0.66 to 1.5. As expected, variation was larger for genes expressed at lower levels. Figure 2B shows the average percentage of change in expression for the 98 mouse genes from this series of 4 microarrays. Results were obtained and analyzed with the method used for the hypo-/hyperthyroidism study (see online data supplement). The large Y-axis scale is comparable to those used in Figures 3 and 4. Variations, ranging from -9.5±6.0% and +12.3±5.7%, were not statistically significant.

View larger version (24K): [in this window] [in a new window]   Figure 2. cDNA microarray control experiments obtained by hybridizing Cy3- and Cy5-labeled cDNA reverse-transcribed from the same cardiac preparation (see text for further details). A, Scatter plot showing a typical example of cCy5/Cy3 relation (expressed in Log2; y-axis) vs Cy3 fluorescence intensity (x-axis). Each data point represents one spot. cCy5 indicates value for Cy5 fluorescence after normalization. Dashed lines represent the Log2 of 0.66 and 1.5. B, Bar graph showing the percentage of variation in expression (y-axis) for 98 mouse ion channels encoding genes. Labeled cDNAs were obtained from the same cardiac preparations. Data are mean±SEM of 3 to 4 values obtained from 4 different microarrays (see expanded Materials and Methods section in online data supplement). y-axis scale is enlarged for direct comparison with Figure 3. There was no significant variation, as assessed with Cyber T and SAM.

View larger version (34K): [in this window] [in a new window]   Figure 3. Effects of hypothyroidism on cardiac ion channel transcripts. Bar graphs show the percentage of variation in cardiac expression (y-axis) evaluated by cDNA microarrays of genes selected for their cardiac expression (x-axis) in hypothyroid mice vs control mice. Na+ ch indicates Na+ channel subunits; Ca2+ channels, Ca2+ channel subunits; Ca2+ reg, proteins involved in Ca2+ regulation; Cl- channels, Cl- channel subunits; Cnx, connexins; K+ channels, K+ channel subunits; and T3R, receptors to thyroid hormones. KCND3L (AF107781) and KCND3M (AF107782) are 2 alternatively spliced isoforms. Data are mean±SEM of 4 to 8 values obtained from 8 different chips (see expanded Materials and Methods section in online data supplement). *P<0.05, as evaluated with Cyber-T; P<0.05, as evaluated with SAM.

View larger version (34K): [in this window] [in a new window]   Figure 4. Effects of hyperthyroidism on cardiac ion channel transcripts. Bar graph shows the percentage of variation in cardiac expression (y-axis) evaluated by cDNA microarrays of genes selected for their cardiac expression (x-axis) in hyperthyroid mice vs control mice. Data are mean±SEM of 4 to 7 values obtained from 7 different chips. See Figure 3 legend for abbreviations and statistics.

cDNA microarrays were then used to evaluate the effects of thyroid status on ion channel cardiac expression. For clarity, only genes that are consistently expressed in the normal mouse heart according to the literature and to RT-PCR experiments performed in our laboratory (data not shown) are illustrated on graphs (Figures 3 and 4). Results concerning genes that are either not expressed in heart or expressed at very low levels can be found at http://www.U533.org.¹⁵ Expression of these latter genes was not modified by altered thyroid status, with the very exception of ATP2A1, the gene encoding the skeletal muscle sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1), which was found to be upregulated by 166% in hyperthyroid hearts.

The subset of genes normally expressed in the mouse heart behave differently. cDNA microarrays revealed marked alterations in the expression of genes encoding ion channel subunits and proteins involved in cardiac Ca²⁺ homeostasis in both hypothyroid and hyperthyroid hearts (Figures 3 and 4). In hypothyroid hearts, the mRNA levels of SLC8A1, the gene encoding the sarcolemmal Na⁺-Ca²⁺ exchanger (NCX1), and of PLN (phospholamban) were increased, whereas those of ATP2A2, the gene encoding the cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) were significantly decreased. Hypothyroidism also increased by about 60% the expression of CaCNA1C, the gene encoding the cardiac L-type _1C Ca²⁺ channel protein. In contrast, hyperthyroidism was associated with a significant decrease in phospholamban expression and a small nonsignificant decrease in SLC8A1 expression (Figure 4).

The cardiac expression of several genes encoding K⁺ channel proteins was also markedly affected by thyroid status. Hypothyroidism (Figure 3) led to a decreased expression of KCNA5 (Kv1.5), KCNB1 (Kv2.1), and KCND2 (Kv4.2) genes. These voltage-gated K⁺ channel -subunits contribute to the main repolarizing K⁺ currents, I_to,f (Kv4.2)and I_K,slow (Kv1.5 and Kv2.1) in adult mouse ventricle.^16–19 The expression of the two-pore domain K⁺ channel TREK-1 (KCNK2 gene) was also reduced. More surprising was the finding that mRNA levels for KCNQ1 (KvLQT1) and its regulator KCNE1 (minK), which generate the slow component of cardiac delayed rectification, I_Ks, were increased by hypothyroidism. Microarray analysis also showed that hypothyroidism did not alter the expression of Na⁺, Cl^-, and inward-rectifier K⁺ channels, and of connexins transcripts.

The changes in ion channel mRNA levels found in hyperthyroidism were opposite to those associated with hypothyroidism (Figure 4). The expression of both KCNA5 (Kv1.5) and KCNB1 (Kv2.1) was increased, whereas the expression of KCNQ1 (KvLQT1) and KCNE1 (minK) was decreased. However, unlike hypothyroidism, the expression levels of KCND2 (Kv4.2) and of CaCNA1C (Ca²⁺ channel _1C-subunit) in hyperthyroid ventricles were indistinguishable from those measured in control hearts. The expression of GJA1, which encodes connexin 43, which was not modified by hypothyroidism, was decreased in hyperthyroid mice. The expression of genes encoding Na⁺, Cl^- and inward-rectifier K⁺ channel subunits was not affected by hyperthyroidism.

Validation of Microarray Data by Real-Time RT-PCR
To validate further the microarray data, we used relative quantitative RT-PCR (SYBRGreen) to measure mRNA expression of 15 genes, selected as either unchanged or up- or downregulated in hypothyroidism or hyperthyroidism, and GAPDH, used as a reference house-keeping gene (Figure 5). Relative quantitative RT-PCR largely confirmed, with only one exception, KCND3, the results obtained with microarrays. In one case, however (SLC8A1), decreased expression in hyperthyroidism reached significance in relative quantitative RT-PCR experiments although not in the microarray studies. As for KCND3, the variation observed with real-time RT-PCR in hypothyroid hearts was modest. Genes that did not vary in microarray experiments (eg, KCNA4, KCNJ12, ClCN4, GJA5) were not identified as variants in quantitative PCR. Finally, real-time RT-PCR also revealed that the expression of HCN2, a cardiac pacemaker channel, was downregulated, whereas the expression of another member of this family, HCN4, was upregulated in hypothyroid animals. Inversely, hyperthyroidism increased HCN2 expression and decreased HCN4 expression. Those 2 genes were not represented on our microarrays.

View larger version (34K): [in this window] [in a new window]   Figure 5. Relative quantitative RT-PCR evaluation of the effects of hypothyroidism (top) and hyperthyroidism (bottom) on cardiac expression of 16 selected genes. Bar graph shows the percentage of variation in hypothyroid and hyperthyroid mice vs control mice. Data are mean±SEM of 5 to 16 measurements. **P<0.01 and ***P<0.001 vs GAPDH.

Effects of Thyroid Status on Cardiac Levels of Ion Channel Proteins
To investigate whether the changes in mRNA levels observed in microarray and SYBRGreen techniques led to alterations in protein levels, western blot analysis was performed for selected voltage-gated K⁺ channel subunits (Figure 6). As expected from gene expression studies, hypothyroidism decreased KCNA5, KCNB1 and KCND2 protein levels, and increased KCNQ1 protein. No change in KCNAB1 (Kvß₁) was observed, a result consistent with microarray studies. On the other hand, there was a significant decrease in KCND3 protein levels despite small variations of the mRNA levels which was detected only with real-time PCR. All changes in protein levels induced by hyperthyroidism were consistent with mRNA changes: KCNA5 and KCNB1 proteins were increased while KCNQ1 was decreased; KCND2, KCND3 and KCNAB1 protein levels were not significantly affected by hyperthyroidism.

View larger version (41K): [in this window] [in a new window]   Figure 6. Western blot analysis of the cardiac expression of K+ channel subunits. A, Representative Western blots using specific polyclonal or monoclonal antibodies against KCND2, KCND3, KCNA5, KCNB1, KCNQ1, and KCNAB1 (Kvß1.2 splice variant) in control (Ct), hypothyroid (Hypo), and hyperthyroid mice (Hyper). B, Bar graph shows the percentage of variation in cardiac protein levels evaluated by Western blot (y-axis) of KCND2, KCND3, KCNA5, KCNB1, KCNQ1, and KCNAB1 in hypothyroid (white bars) and hyperthyroid (gray bars) mice vs controls. All experiments were performed in duplicate. Data are mean±SEM of 4 hypothyroid and 3 hyperthyroid hearts. *P<0.05 vs control mice.

Functional Consequences of Voltage-Gated K⁺ Channel Remodeling Induced by Hypothyroidism
To determine the cellular consequences of the observed alterations in K⁺ channel subunit expression, whole-cell K⁺ currents were recorded in ventricular myocytes from control or hypothyroid mouse hearts. As shown in Figure 7A, the amplitude of I_to was reduced significantly in hypothyroid myocytes, consistent with the downregulation of KCND2 and KCND3 expression. Similarly, consistent with the reduced expression of KCNA5 and KCNB1, the density of the current remaining at the end of 300-ms pulses, which reflects I_K,slow and I_ss,^18,19 was also significantly decreased. In contrast, the inwardly rectifying K⁺ current, I_K1, was not modified (Figure 7B), a finding consistent with the lack of measurable effects on the expression of the underlying genes, KCNJ2 (Kir2.1) and KCNJ12 (Kir2.2; see Figures 3 and 5).

View larger version (23K): [in this window] [in a new window]   Figure 7. Functional consequences of hypothyroidism-induced remodeling of K+ channels expression. A, Current-voltage relationships of the peak transient outward current (Ito) and the sustained current (Isus, a mix of IK,slow and Iss) measured at the end of 300-ms voltage steps in control (n=12; circles) and hypothyroid mice (n=8; triangles). *P<0.05, **P<0.01, and ***P<0.01 vs corresponding value in control mice. B, Current-voltage relationship of the inwardly rectifier IK1 current in control (n=12; circles) and hypothyroid mice (n=8; triangles). Voltage steps were 300-ms long. C, Typical tail currents recorded at -40 mV after a 3-second step to +40 mV in absence (filled symbols) and in presence of HMR1556 10-6 mol/L (open symbols) in a control (left) and a hypothyroid ventricular myocyte (right). Horizontal bar=200 ms; vertical bar=30 and 40 pA for, respectively, left and right traces.

Perhaps the most striking result obtained with microarrays was the observed upregulation of KCNE1 and KCNQ1 expression by hypothyroidism. Because previous studies suggest that KCNQ1 and KCNE1 encode the channel subunits generating the slow component of cardiac delayed rectification, I_Ks, voltage-clamp experiments were also performed on isolated myocytes to determine if I_Ks density was affected in hypothyroid myocytes. In these experiments, I_Ks was measured as the HMR 1556-sensitive tail current (Figure 7C). ²⁰ It is well known that wild-type adult mouse ventricular myocytes express high levels of KCNQ1,²¹ whereas KCNE1 expression is low, having declined markedly during the first week after birth.^21,22 As a result, I_Ks is not a prominent repolarizing current in adult mouse ventricle.²² As expected, HMR1556-sensitive tail currents were recorded only in a small subset (3/16) of control myocytes. In contrast, I_Ks current was recorded in 7/7 hypothyroid myocytes tested; I_Ks tail current density at -40 mV after a 3-second step to +40 mV=0.22±0.5 pA/pF.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
cDNA Microarray Analysis of Cardiac Remodeling
In the present work, we aimed to validate recently developed cDNA microarrays containing probes for a large collection of genes encoding ion channel proteins (IonChips) by determining the substrates for the electrical remodeling associated with altered thyroid hormone status. The genomic scale of our approach shows that a subset only of the ion channel families is regulated by thyroid hormones.

Because the effects of thyroid hormones on cardiac contractile activity and the expression of proteins involved in intracellular Ca²⁺ homeostasis are well-documented, these genes were used as positive controls. Consistent with previous reports in hypothyroid hearts,^23–26 IonChips analysis revealed that expression of ATP2A2 (SERCA2) was decreased, whereas the mRNA levels of phospholamban and the sarcolemmal Na⁺-Ca²⁺ exchanger were increased. In contrast, hyperthyroidism was associated with decreased phospholamban and Na⁺-Ca²⁺ exchanger mRNA expression, whereas ATP2A2 expression was unaffected. Importantly, the changes in the expression observed with the microarrays were of the same order of magnitude as those previously reported by others using more conventional experimental approaches.^23–26

IonChips analysis also revealed that altered thyroid hormone status is accompanied by a complex remodeling of ion channel subunits, which concerns a specific subset of the channel subunits. The expression levels of genes encoding Na⁺, Cl^-, and inward rectifier K⁺ channel proteins, for example, were unaffected in either hypothyroid or hyperthyroid ventricles. To our knowledge, the only information available before the present study on these channels was that KCNJ2 (Kir2.1) was not regulated by thyroid hormones.¹² Although most of the two-pore domain K⁺ channels were unaffected, KCNK2 (TREK-1) expression was reduced in hypothyroidism, but not modified by hyperthyroidism. In addition, CaCNA1C expression (_1C Ca²⁺ channel protein) was increased in hypothyroid animals, a finding in line with previous results obtained in rat with binding studies.^27,28 Finally, although genes encoding pacemaker channels were not present on our microarrays, real-time PCR studies show that HCN2 and HCN4 were, respectively, down- and upregulated in hypothyroidism and inversely regulated in hyperthyroidism. Our results for HCN2, but not for HCN4, confirm those obtained recently by Gloss and coworkers.¹² Changes in HCN2 expression, rather than HCN4, might explain in part the effects of thyroid hormones on heart rate because HCN4 does not seem to be regulated at the atrial level.¹² This hypothesis implies that HCN2 is expressed in the mouse sinus node, which has not been demonstrated yet.

Complex Remodeling of Cardiac Voltage-Gated K⁺ Channels
Consistent with previous findings,^4–8 prolongation of ventricular repolarization was evident in the hypothyroid mice. Microarray analysis revealed marked reductions in KCNA5, KCNB1, and KCND2 expression, the genes encoding Kv1.5, Kv2.1, and Kv4.2 K⁺ channel -subunit proteins, respectively. In the rat, expression levels for KCNA5, KCNB1, and KCND2 are affected similarly in hypothyroidism.⁹ In the rat, however, KCNA2 and KCNA4 expressions are upregulated in hypothyroidism, changes that were not observed in hypothyroid mice. However, KCNA2 is almost undetectable in the mouse heart and KCNA4 expression is limited to the septal area where it generates the slow transient K⁺ current, I_to,s.¹⁶ Importantly, in the mouse, it is possible to relate changes in Kv subunit expression with electrophysiological consequences because the functional roles of these subunits in the generation of K⁺ currents have been well defined.^16–19 It has been demonstrated, for example, that Kv4 -subunits generate the fast transient K⁺ current, I_to,f, in mouse ventricles,^16,17 whereas there are 2 components of the delayed rectifier current, I_K,slow, one generated by Kv1.5 (KCNA5 gene)¹⁹ and the other by Kv2.x (KCNBx) -subunits.¹⁸ Previous studies have shown that attenuation of either I_to,f^16,17 or the Kv2-generated I_K,slow¹⁸ prolongs action potential and QT duration. Electrophysiological experiments here reveal that I_to,f density is attenuated in parallel with the reductions in KCND2 and KCND3 expression, a result that confirms the very recent demonstration that functional mouse ventricular I_to,f channels reflect the heteromeric assembly of KCND2 and KCND3.²⁹ Electrophysiological experiments also reveal that the reduction of I_K,slow density parallels that of KCNA5 and KCNB1 expression. These observations suggest that hypothyroid mice have prolonged QT intervals because of decreased KCND2 and KCND3 mRNA levels, accounting for the rapid initial component of the QT interval, and KCNA5 and KCNB1 mRNA levels, accounting for the late component of the QT interval. One could expect downexpression of KCNA5, KCNB1, and KCND2 to be partially counterbalanced by overexpression of KCNQ1 and KCNE1. Similar results for KCNE1 have been previously reported.¹² However, although electrophysiological experiments revealed a small increase in I_Ks tail current density, it is unlikely to influence consistently the repolarization process, because the participation of I_Ks to repolarize adult mouse ventricle is small if any.³⁰ This may not be the case in other species such as human in which I_Ks plays a more prominent role in ventricular repolarization. On the other hand, because KCNE1 is preferentially expressed in the conduction system,³¹ changes in KCNE1 and KCNQ1 expression by altered thyroid status might explain partially the observed changes in atrioventricular conduction. Whatsoever, prolongation of cardiac repolarization induced by hypothyroidism results from a complex interplay of ionic currents rather than a pure decrease in repolarizing currents.

Shortening of the QT duration during hyperthyroidism could result from increased KCNA5 and KCNB1 expression. That hyperthyroidism shortened the late phase but not the early phase of the QT interval is in line with the absence of regulation of KCND2 and KCND3 as previously shown in the rat heart.⁹ Our results also suggest that downregulation of KCNE1 expression during postnatal development might be partly caused by increased levels in plasmatic thyroid hormones, as previously proposed for KCND2 and KCND3 upregulation in the rat ventricle.³²

Study Limitations
It is usually admitted that the sensitivity of the microarray technology is relatively low and researchers only pay attention to at least 2-fold changes in gene expression. However, it is also usual to perform no more than two experiments, which is statistically irrelevant. Our study, which combines different techniques of molecular biology and physiology, shows that microarrays, as long as a sizable number of experiments are performed, can detect relatively low changes in mRNA levels.

It is also often argued that changes in mRNA expression are not systematically correlated with protein levels and that it is hazardous to conclude on the functional consequences of changes in mRNA levels. However, different studies investigating the effects of glucocorticoids or infarction on ion channel transcription have shown that the mRNA levels of KCNA5, KCNB1, and KCNB2 are closely correlated with their protein levels.^33,34 Western blot and patch-clamp studies performed on hypothyroid myocytes confirm this assumption and demonstrate that changes in KCNA5, KCNB1, KCND2, KCND3, and KCNQ1 mRNA levels also lead to similar changes in protein levels.

For specificity reasons, our probes were cloned in the 3'UTR regions; expression of transcripts resulting from alternative splicing in upstream regions cannot be evaluated. Such transcripts have been described in mouse for genes encoding ion channels such as KCNQ1.³⁵ To improve this aspect, we are currently preparing second generation IonChips utilizing long oligonucleotides (50 mer) specific for alternative transcripts, which will be spotted concomitantly with our PCR products.

A comparison of the results obtained with microarrays with those obtained with real-time RT-PCR show that real-time PCR was more potent to detect reduced expression of 2/15 genes (KCND3 and SLC8A1). It has been already shown that quantitative PCR is more powerful to detect downregulated genes rather than upregulated genes.³⁶ On the other hand, among all the genes tested with real-time PCR, none were shown as varying with microarrays but not with PCR (false positive).

Finally, the IonChips used in the present study did not contain every channel gene cloned so far but only a large, though partial, collection. At the time of this study, the 3'UTR sequence for various genes, was not publicly available. Since then, novel sequences have been published and our IonChips are continuously enriched with new probes as part of a permanent improvement process.

	Acknowledgments

 
This work was supported by grants from the Ministère de la Recherche (ACI "Biologie du Développement et Physiologie Intégrative" 2001) and the Association Française contre les Myopathies. The authors wish to thank Béatrice Le Ray, Marie-Jo Louérat, Sylvie Leroux, Martine Le Cunff, Emmanuelle Jugieau (INSERM U533), and Jean-Marie Heslan (INSERM U437) for expert technical assistance.

	Footnotes

 
*Both authors contributed equally to this work.

Received March 25, 2002; revision received November 1, 2002; accepted November 27, 2002.

	References

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