Effects of thyroid status on expression of voltage-gated potassium channels in rat left ventricle

Abstract

Objective: Thyroid hormone modifies cardiac action potentials and outward potassium currents directly and indirectly e.g. through β-adrenergic signaling pathway. We thus examined the expression of six voltage-gated potassium channel α-subunits in the rat left ventricle under hypo- and hyperthyroid status, and tested roles of β-adrenergic signaling pathway in their expressions under both status. Methods: Hypothyroidism and hyperthyroidism were induced by administration of methimazole (MMI) for 4 weeks and by injection of l-thyroxine (T₄) to the MMI-treated rats for the last 7 days, respectively. To distinguish the effects of T₄ and the β-adrenergic system, propranolol (Pro) was administered to the MMI-treated rats together with T₄, and isoproterenol (Iso) was injected to MMI-treated rats for the last 7 days. The mRNA levels of Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 in the left ventricles were determined by ribonuclease protection assay. Results: MMI treatment induced hypothyroidism and resulted in a significant decrease in the mRNA levels of Kv1.5, Kv2.1 and Kv4.2 (19%, 77% and 61% of control value, respectively; n=6, p<0.05). T₄ administration induced hyperthyroidism and cardiac hypertrophy, and it increased the Kv1.5 and Kv2.1 mRNA levels over the control value (212% and 140%, respectively; n=6, p<0.05). Kv4.2 mRNA level was restored to the control level by T₄. In contrast, the Kv1.2 and Kv1.4 mRNA levels increased in hypothyroid rats (161% and 186% of control value, respectively; n=6, p<0.01) and decreased in hyperthyroid rats (14% and 33% of control value, respectively; n=6, p<0.01). The Kv4.3 mRNA level was not altered by thyroid status. Pro did not inhibit the T₄-induced hypertrophy. Iso induced cardiac hypertrophy. Pro or Iso by itself did not alter Kv mRNA levels except for Kv1.2, the message of which was decreased by Iso. Conclusion: Thyroid hormone differentially regulates the expression of Kv1.4, Kv1.5, Kv2.1 and Kv4.2 mRNA levels in the rat left ventricle. This effect is not mediated through β-adrenergic signaling pathway. On the other hand, the reduction in Kv1.2 mRNA level was associated with cardiac hypertrophy induced by T₄ or Iso.

Adrenergic (ant)agonist, Gene expression, Hormones, Hypertrophy, K-channel, Rat

Time for primary review 31 days

1 Introduction

The heart is one of the major target organs for thyroid hormone [1]. In vivo administration of different doses of thyroid hormone to the thyroidectomized rats alters the electrophysiological properties of the cardiac myocytes measured in vitro. Di Meo et al reported that the action potential duration of ventricular myocytes was prolonged in the hypothyroid rats and was shortened in the hyperthyroid animals [2]. Shimoni et al showed that the amplitude of transient outward potassium current (Ito), the prominent potassium current in the rat ventricular myocytes, was reduced in the myocytes isolated from the hypothyroid rats [3]. These studies suggest that the thyroid hormone plays an important role in the regulation of voltage-gated potassium channels in the rat ventricle.

Our recent study has demonstrated that the expression of Kv1.5, one of the major voltage-gated potassium channel α-subunits expressed in the rat left ventricle, is altered by thyroid status [4]. To date, the expression of six α-subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3, has been demonstrated in the rat heart [5–11]. Among these subunits, Kv4.2 and Kv4.3 are abundantly expressed in the rat ventricular myocytes. When heterologously expressed in Xenopus oocytes, each channel produces the rapidly activating and inactivating current whose biophysical and pharmacological properties are similar to those of native Ito [9–11]. It is therefore possible that the reduction of Ito in the myocytes obtained from the hypothyroid rats is due to a decrease in expression of Kv4.2 and/or Kv4.3. We thus examined effects of thyroid status on expression of six voltage-gated potassium channel α-subunits in the rat left ventricle. We also examined the effects of a β-adrenergic blocker and a β-adrenergic agonist on the expression of the subunits, since it has been shown that thyroid hormone exerts its effects on cardiac myocytes directly and indirectly by increasing the number of β-adrenergic receptor [1, 12].

2 Materials and methods

2.1 Animal treatment

Four-week-old male Wistar rats were purchased from Nippon Bio-Supply Center (Tokyo, Japan) and were assigned to 5 groups: control, hypothyroid, hyperthyroid, hyperthyroid plus propranolol, and hypothyroid plus isoproterenol groups. Each group contained 6 rats. Rats were rendered hypothyroid by addition of 0.025% methimazole (2-mercapto-1-methyl-imidazole, MMI; Sigma Chemical Co., St. Louis, MO, U.S.A.) to the drinking water for 4 weeks [13]. Hyperthyroidism was induced in MMI-treated hypothyroid rats by daily intraperitoneal injection of 20 μg/100 g body weight/ day l-thyroxine (T₄; Sigma) for the last 7 days [14]. Propranolol (Sigma) was added to the drinking water at 750 mg/l and administered to the MMI-treated rats together with T₄ for the last 7 days [15]. Isoproterenol (Sigma) was injected intraperitoneally to the MMI-treated hypothyroid rats at a dose of 500 μg/100 g body weight/day for the last 7 days [16]. An additional injection of isoproterenol was performed 2 hours before the sacrifice. To manipulate rats in the same manner, non-treated (control) and hypothyroid rats were given daily intraperitoneal injections of vehicle (0.03% bovine serum albumin, 0.9% NaCl) for the last 7 days. All rats were allowed free access to standard rat chow (MF; Oriental Yeast Co., Ltd., Tokyo, Japan). Twelve hours after the last injection of T₄ or vehicle (2 h after the last isoproterenol injection), the rats were exsanguinated from the abdominal aorta and the left ventricles were dissected out under ether anesthesia. The free walls of the left ventricles were then excised, immediately frozen in liquid nitrogen and stored at −80°C until extraction of total RNA. Sera were separated and frozen at −30°C until the analysis of T₄ and 3, 3′,5-triiodothyronine (T₃). The hormones were measured by radioimmunoassays [T₄: Magnetic T₄ radioimmunoassay kit (Ciba-Corning, Tokyo, Japan), T₃: Gamma Coat T₃ radioimmunoassay kit (Baxter Healthcare Corp., Cambridge, MA, U.S.A.)]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Northern blot analysis

Total RNA was extracted from the frozen left ventricles after pulverization in liquid nitrogen using the protocol described by Chomczynski and Sacchi [17]. After fractionation of 15 μg of total RNA on 0.8% agarose gel, the RNA was transferred onto GeneScreen Plus membrane (Du Pont-New England Nuclear, Boston, MA, U.S.A.). The detailed procedures for Northern blotting was described in our previous report [18]. The membrane was hybridized with rat α- or β-myosin heavy chain (MHC) oligonucleotide probes (Calbiochem, San Diego, CA, U.S.A.) and with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA. The oligonucleotide probes were 5′-end labeled with [g-³²P]ATP (6000 Ci/mmol; Du Pont-New England Nuclear) using T₄ polynucleotide kinase. The hybridization and wash were performed according to the manufacturer's protocol (Calbiochem). The GAPDH cDNA was labeled with [α-³²P]dCTP (3000 Ci/mmol; New England Nuclear) using random primed labeling kit (Boehringer Mannheim, Mannheim, Germany). The cloning of GAPDH cDNA was described in our previous report [19]. After hybridization and wash, the membrane was exposed to XAR-5 film (Eastman Kodak, Rochester, NY, U.S.A.) at −80°C with an intensifying screen.

2.3 Cloning of Kv cDNAs

The cDNA fragments of Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 α-subunits were amplified from mRNA prepared from a normal rat left ventricle by reverse transcription (RT) and PCR as described in our previous report [20]. The nucleotide sequences of the primers and the amplified regions are described below. Nucleotide numbers for each primer correspond to those from the translation start site. Kv1.2: Sense 5′-AAGCTTTAACTGATGTCTGATTGAAACCTA-3′, Antisense 5′-GATGCTGGCTCCATGGGTGAC-3′, nucleotides 1487–1743 [5]. Kv1.4: Sense 5′-AAGCTTTCTACTTCTTCTTCCCTGGGGGAC-3′, Antisense 5′-TGCATCACTTATTTGATATGC-3′, nucleotides 1801–2132 [6]. Kv1.5: Sense 5′-CCGAGTATTTAAGCCCACCTG-3′, Antisense 5′-CTAAGCTTTTTAAAGTCAAATTTG-3′, nucleotides 1888–2144 [7]. Kv2.1: Sense 5′-AAGCTTGCTCTGGTTTCTTCGTGGAGAGTC-3′, Antisense 5′-CACGCTGTAGAGCAGCTGACC-3′, nucleotides 1931–2295 [8]. Kv4.2: Sense 5′-TACCGCACGGGGAAGCTTCACTAT-3′, Antisense 5′-TGGAACTGTTTCCACCACATTCGC-3′, nucleotides 295–624 [9]. Kv4.3: Sense 5′-AAGCTTGGCACCCCAGAAGAGGAGCACATG-3′, Antisense 5′-GTTGGAGTTGGGCAGGTGCGTGGT-3′, nucleotides 1372–1626 [11].

A Hind III site (AAGCTT) was introduced into the 5′ end of the sense primers of the Kv1.2, Kv1.4, Kv2.1 and Kv4.3 (italicized). In Kv4.2, a Hind III site is present in the coding region (italicized). The amplified cDNAs were cloned into pGEM-T vector using TA cloning system (Promega Corporation, Madison, WI, U.S.A.). The nucleotide sequences of the cloned cDNAs were verified by DNA sequencing.

2.4 Ribonuclease Protection Assay (RPA)

The plasmids containing Kv channel cDNAs were linearized by digestion with an appropriate restriction enzyme (Hind III for Kv1.2, Kv1.4, Kv2.1, Kv4.2 and Kv4.3; NcoI present in pGEM-T vector for Kv1.5). Antisense cRNA probes were synthesized using MAXIscript kit (Ambion Inc., Austin, TX, U.S.A.) and [α-³²P]UTP (Du Pont-New England Nuclear). The sizes of cRNA probes were slightly larger than those of the corresponding region of each mRNA because of the presence of vector sequence in the probe. The cyclophilin cRNA probe was also synthesized from the cDNA purchased from Ambion (pTRI-cyclophilin-rat antisense control template) to detect cyclophilin mRNA as an internal control. The sizes of cyclophilin cRNA probe and its protected region were 165 and 103 bases, respectively. RPA was performed using HybSpeed RPA kit (Ambion) according to the manufacturer's protocol. Hybridization of the two probes (2×10⁴ cpm channel cRNA and 2×10⁴ cpm cyclophilin cRNA) with 10 μg total RNA was carried out at 68°C for 10 min, followed by digestion with RNase A and RNase T1 at 37°C for 30 min. The reaction was terminated by the addition of SDS and proteinase K. After the phenol-chloroform extraction and ethanol precipitation, the protected RNA fragments were subjected to a 5% polyacrylamide/8 M urea gel electrophoresis. The gel was dried on a filter paper and subjected to Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film Co. Ltd., Tokyo, Japan) to perform quantitative analysis. The mRNA levels were normalized by the levels of cyclophilin mRNA and expressed in arbitrary units. The gel was then exposed to XAR-5 film (Eastman Kodak) at −80°C with an intensifying screen.

To evaluate the specificity of the Kv4.2 and Kv4.3 probes, Kv4.3 mRNA containing entire coding region was synthesized in vitro (MAXIscript kit) and subjected to RPA. With Kv4.3 probe, a single band of 312 bases was observed, while Kv4.2 probe was completely digested by RNase after hybridization with the Kv4.3 mRNA (data not shown). This result indicates that the Kv4.2 and Kv4.3 probes specifically detect corresponding mRNA.

3 Results

3.1 Effects of thyroid status, propranolol and isoproterenol on body and ventricular weights, heart rates and serum T₄ and T₃ concentrations

As shown in Table 1, induction of hypothyroidism in rats treated with MMI for 4 weeks was ascertained by a marked decrease in serum T₄ and T₃ levels. Administration of T₄ to the MMI-treated rats markedly increased the serum concentrations of T₄ and T₃. β-adrenergic blocker propranolol (Pro) and β-adrenergic agonist isoproterenol (Iso) had no effect on the altered thyroid hormone levels.

Table 1

Effects of thyroid status, propranolol and isoproterenol on body and ventricular weights, heart rates and serum T₄ and T₃ concentrations

	Body Weight (g)				Ventricle	Ventricle (mg) /	Heart rate	T4 (μg/dl)	T3 (ng/dl)
	Initial	3-week	4-week(sacrifice)		(mg)	Body weight(g)	(beats/min)
Control	142±3	283±5	314±9		1059±45	3.37±0.08	371±7	5.3±0.2	69±2
MMI	142+2	225±4*	221±5*		592±24*	2.68±0.08*	259±3*	<1.0*	43±2*
MMI+T4	138±2	217+4*	231±5*		845±10*†	366±0.06*†	403±7*†	17.2±1.0*†	159±14*†
MMI+T4+Pro	145±4	217±3*	227±3*		877±15*†	3.86±0.07*†	367±5#†	18.9±1.9*†	202±58*†
MMI+Iso	145+3	217+5*	222±3*		823±17*†	3.70+0.07*†	407±4*†	<1.0*	31±6*

Rats were assigned to five groups: control (Cont), hypothyroid (MMI), hyperthyroid (MMI+T₄), hyperthyroid plus propranolol (MMI+T₄+Pro), and hypothyroid plus isoproterenol (MMI+Iso) groups. Rats were rendered hypothyroid by MMI treatment for 4 weeks. Hyperthyroidism was induced in the MMI-treated rats by the administration of T₄ (20 μg/100 g body weight/day) for the last 7 days. Pro was added to the drinking water at 750 mg/liter and administered to the MMI-treated rats together with T₄ for the last 7 days. Iso (500 μg/l00 g body weight/day) was injected to the MMI-treated rats for the last 7 days. The initial body weights (Initial) and body weights before (3-week) and after (4-week) the administration of T₄, Pro and/or Iso were shown. The left ventricles and blood samples were obtained from 6 rats in each group. The data are expressed as mean±S.E. Statistical significance was determined by Student's t-test. *:p<0.01 vs Cont. †:p<0.01 vs MMI. #:p<0.01 vs MMI+T₄.

The initial body weights before MMI administration were similar in all groups. Treatment with MMI for 3 weeks significantly diminished the increase in body weights. Administration of T₄, Pro or Iso did not affect the body weights.

The ventricular weights in the MMI-treated rats were significantly lower than those of the control rats. Administration of T₄ or Iso to the MMI-treated rats resulted in a significant increase in ventricular weights when compared to those of the MMI rats. The ratio of ventricular weight relative to body weight in the T₄- or Iso-treated rats was significantly higher than that of the control rats. These results indicate that hyperthyroidism or β-adrenergic stimuli induces cardiac hypertrophy as previously reported [15, 16, 21, 22]. Administration of Pro to the T₄-treated rats did not inhibit the induction of hypertrophy, being consistent with the previous reports [23–25].

To evaluate effectiveness of Pro and Iso, we measured the heart rate before sacrifice. The heart rate was significantly decreased in the hypothyroid rats and increased in the hyperthyroid rats. Pro administration to the hyperthyroid rats significantly decreased the heart rate to the control level, indicating that the dose was sufficient to inhibit the T₄-induced increase in heart rate. Iso administration to the hypothyroid rats significantly increased the rate to the hyperthyroid level.

3.2 Effects of thyroid status on the expression of cardiac α- and β-myosin heavy chain (MHC)

To confirm the altered thyroid hormone status in the heart, the expression of α- and β-MHC isoforms was examined in the rat left ventricle. As shown in Fig. 1, α-MHC mRNA was detected as a single band of 7 kb. MMI treatment resulted in a disappearance of α-MHC mRNA. T₄ administration restored the mRNA levels. β-MHC mRNA was also detected as a single band of 7 kb. MMI treatment increased the mRNA level, whereas T₄ markedly reduced the mRNA level. These results are consistent with the previous study by Izumo et al [26]who demonstrated that MHC isoform is shifted from β to α by thyroid hormone.

Fig. 1

Effects of thyroid status on the expression of cardiac α- and β-MHC in rat ventricle. Aliquots of 15 μg total RNA extracted from the left ventricles of control, hypothyroid and hyperthyroid rats were subjected to Northern blot analysis using α- and β-MHC oligonucleotides and GAPDH cDNA as probes. The representative autoradiograms showing the result of two rats in each experimental group are presented. In the panel of MHC, the position of 28S ribosomal RNA is indicated by arrowhead. Note that the levels of GAPDH mRNA were not affected by MMI or T4 treatment.

3.3 The expression of Kv4.3 variant in the rat left ventricle

As shown in Fig. 2A, in the course of preparation of the Kv4.3 probe, we found a novel mRNA for Kv4.3 (Kv4.3B) which contains an in-frame insertion of 57 nucleotides after the codon for 487th amino acid in the previously described mRNA (Kv4.3A). Because the insertion contains no termination codon, the Kv4.3B product has additional 19 amino acids in the C-terminal intracellular region of the subunit. Interestingly, Kv4.3B is predominantly expressed in the rat left ventricle (Fig. 2B), whereas both Kv4.3B and Kv4.3A are expressed in the brain. We did not find any other insertion or deletion in the entire coding sequence of Kv4.3 mRNA by RT-PCR using mRNA prepared from the rat left ventricle.

Fig. 2

The Kv4.3 variant is predominantly expressed in the rat left ventricle. (A) The nucleotide and amino acid sequences of the variant, long form Kv4.3 (named Kv4.3B) and the previously described Kv4.3 (Kv4.3A) are shown. The additional 19-amino acid sequence in the Kv4.3B is underlined. The nucleotide numbers correspond to those from the translation start site. (B) Aliquots of 10 μg total RNA extracted from the rat left ventricle and the whole brain were subjected to RPA using ³²P-labeled Kv4.3B cRNA as a probe. In the right side of the figure, the sizes of the cRNA probe and the expected sizes of the Kv4.3B and Kv4.3A mRNA after RPA are depicted. The cRNA probe was 369 bases in length. It contains 312 bases corresponding to Kv4.3B mRNA with the 57-base insert (closed box) and an additional sequence derived from the vector (shaded box). Open and closed arrowheads indicate the positions of undigested and digested Kv4.3B cRNA probes, respectively. The sizes of the bands are also indicated.

3.4 Effects of thyroid status, propranolol and isoproterenol on the expression of cardiac voltage-gated potassium channel subunits

The cyclophilin mRNA levels were not altered by any treatments (Fig. 3). There was no significant difference when analyzed by one-way ANOVA. Therefore, the mRNA levels of Kv family members were normalized by those of cyclophilin.

Fig. 3

Effects of thyroid status, Pro and Iso on the expression of Kv channels in rat left ventricle. Rats were assigned to 5 groups: control (Cont), hypothyroid (MMI), hyperthyroid (MMI+T₄), hyperthyroid plus Pro (MMI+T₄+Pro), and hypothyroid plus Iso (MMI+Iso) groups. Animal treatments are detailed in Table 1. Aliquots of 10 μg total RNA extracted from the left ventricles were subjected to RPA using ³²P-labeled Kv and cyclophilin cRNAs as probes. A representative autoradiogram showing the result of two rats in each experimental group is shown in the upper panel. Open arrowheads indicate the positions of undigested Kv and cyclophilin (Cyclo) cRNA probes. The positions of protected cRNAs are indicated by the closed arrowheads. Autoradiogram for cyclophilin mRNA with shorter exposure (Cyclo^#) and its level relative to the control value are also presented in the middle panel. The abundance of Kv mRNA is normalized by cyclophilin mRNA level and is expressed as arbitrary units in the lower panel. Values are the mean±S.E. (n=6). *p<0.05 (vs control), **p<0.01 (vs control).

As shown in Fig. 3A, Kv1.2 mRNA level increased to 161% of control value (p<0.01) in the MMI-treated hypothyroid rats and decreased to 14% of control value (p<0.01) in hyperthyroid rats. Pro did not affect the alteration by T₄. However, treatment of the hypothyroid rats with Iso resulted in a decrease in the mRNA level (16% of control value, p<0.01).

Kv1.4 mRNA level also increased to 186% of control value (p<0.01) in the hypothyroid rats and decreased to 33% of control value (p<0.01) in hyperthyroid rats (Fig. 3B). Pro and Iso did not affect these changes.

In contrast, Kv1.5 mRNA levels were changed to the opposite directions. It decreased to 19% of control value (p<0.01) in the hypothyroid rats and increased to 212% of control value (p<0.01) in hyperthyroid rats (Fig. 3C). Pro and Iso had no effect on these changes. This indicates that Kv1.4 and Kv1.5 mRNA levels were altered by the thyroid status independent of the β-adrenergic stimuli.

The alterations in Kv2.1 mRNA level are shown in Fig. 3D. Kv2.1 mRNA decreased to 77% of control value (p<0.05) in the hypothyroid rats and increased to 140% of control value (p<0.05) in hyperthyroid rats. Pro and Iso did not affect these changes.

Kv4.2 mRNA also decreased significantly to 61% of control value (p<0.01) in the hypothyroid rats (Fig. 3E). Administration of T₄ to the MMI-treated rats increased its mRNA level to the control level. Neither Pro nor Iso affected these alterations. On the other hand, the mRNA levels of Kv4.3 were not significantly altered by the thyroid status and by the treatment with Pro or Iso (Fig. 3F).

4 Discussion

The present study demonstrated that hypothyroidism induced a significant decrease in the mRNA levels of Kv1.5, Kv2.1 and Kv4.2 in the rat left ventricle and that administration of T₄ to the hypothyroid rats increased the mRNA levels. These alterations were not affected by the concomitant treatment with β-adrenergic blocker or β-adrenergic agonist, indicating that thyroid hormone may exert its effects in a β-adrenergic receptor-independent manner. In addition, it is noted that the changes in these three mRNA levels in the T₄-induced cardiac hypertrophy are different from those observed in Iso-induced hypertrophy. This finding indicates that cardiac hypertrophy itself had no effects on the altered expression of these channels by thyroid status.

Recent studies have shown that the mRNA levels of these channels are correlated closely with their protein levels; glucocorticoid activates transcription of the Kv1.5 gene thereby increases the amount of Kv1.5 channel [27]; a decrease in the mRNAs for Kv2.1 and Kv4.2 is accompanied by a reduction of respective protein levels in the rat left ventricular myocytes obtained after the experimental myocardial infarction [28]. It is therefore likely that the changes in the mRNA levels by thyroid status consequently alters the protein levels of Kv1.5, Kv2.1 and Kv4.2 in the rat left ventricle.

The relation of cloned potassium channels to native ones has been extensively studied. When expressed heterologously in Xenopus oocytes, Kv4.2 and Kv4.3 produce the potassium currents with Ito-like gating kinetics [9, 11, 29, 30]. In addition, introduction of antisense oligonucleotides directed against either Kv4.2 or Kv4.3 to the rat ventricular myocytes is shown to reduce Ito current [31]; neonatal Ito development in the rat ventricular myocyte is reported to correlate positively with the Kv4.2 and Kv4.3 mRNA levels which are dependent on the serum thyroid hormone levels [32]. Therefore, it is likely that the decrease in Kv4.2 expression in the hypothyroid rat ventricle may cause the reduction of Ito which was reported by Shimoni et al [3]. On the other hand, the Kv4.3 mRNA level is not decreased in the hypothyroid adult rats, suggesting the difference in responsiveness of the Kv4.3 gene to thyroid hormone during growth.

Kv1.5, when expressed in Xenopus oocytes, produces a fast activating and slowly inactivating current [7]. It is also shown that coexpression of Kv1.5 and Kvβ3 subunit or other Kv1 subunit in the oocytes alters the functional properties of Kv1.5 [33–35]. Although the precise role of Kv1.5 in the native potassium current in the rat ventricle has not been established, the alteration in Kv1.5 mRNA levels by thyroid status may affect the outward potassium current in the repolarization phase of the cardiac myocytes.

Kv2.1 channel expressed in the oocytes produces a slowly activating current like Ik of adult rat ventricular myocytes [8, 36]. A decrease in the Kv2.1 expression is shown to correlate with reduction of Ik [28]. Thus, the alterations in Kv2.1 expression by thyroid status may influence the Ik in the rat left ventricular myocytes.

In contrast to the three mRNAs mentioned above, Kv1.4 mRNA increased in hypothyroid rats, while it decreased in hyperthyroid rats. This result is compatible with the finding that Kv1.4 decreases during the neonatal rat development with an increase in the serum thyroid hormone levels [32]. Neither β-adrenergic blocker nor agonist altered Kv1.4 mRNA level, indicating that thyroid hormone may affect the expression of Kv1.4 by a β-adrenergic receptor-independent mechanism. Although it has been reported that renovascular hypertension-induced cardiac hypertrophy is associated with an increase in Kv1.4 mRNA level [37], the present study shows a marked decrease in the mRNA in T₄-induced cardiac hypertrophy. This may indicate that Kv1.4 expression is regulated independent of intracellular signals leading to hypertrophy. It has been recently reported that Kv1.4 mRNA and protein are not detected in rat cardiac myocytes [38], but detected in vascular smooth muscle cells, endothelial cells and glia cells [39]. Thus, the observed changes in Kv1.4 mRNA in the rat left ventricle may not be associated with the functional change in cardiac myocytes.

Kv1.2 mRNA also increased under hypothyroid state, while it decreased under hyperthyroid state. Administration of β-adrenergic blocker to hyperthyroid rats did not affect the decreased mRNA level. However, administration of β-adrenergic agonist to the hypothyroid rats markedly decreased the mRNA level. Considering that both hyperthyroidism and administration of β-adrenergic agonist to the hypothyroid rats induce cardiac hypertrophy, the decrease in the Kv1.2 mRNA level may be related to cardiac hypertrophy. Recently, it is reported that the expression of Kv1.2 increases in parallel with an increase in Ito during postnatal development in rat and suggested that Kv1.2 in combination with another α and/or β subunit expresses the Ito [40]. However, our results show an increase in Kv1.2 mRNA level in hypothyroid rats in which Ito is reduced, suggesting that Kv1.2 is involved in the expression of outward potassium current other than Ito.

In conclusion, the present study demonstrates that the expression of four distinct voltage-gated potassium channel α-subunits, Kv4.2, Kv2.1, Kv1.5 and Kv1.4, in the rat left ventricle are differentially controlled by the thyroid status in a β-adrenergic receptor-independent manner. On the other hand, a decrease in Kv1.2 mRNA level was associated with cardiac hypertrophy induced by T₄ or β-adrenergic agonist. Although the precise role of each channel subunit in the native potassium channel has not been fully understood, the alterations in the expression of the channels, especially Kv4.2, Kv2.1 and Kv1.5, could account for the changes in the outward potassium current of ventricular myocytes caused by the thyroid status. Further studies with electrophysiological analysis must be performed to substantiate this conclusion.

5 Addendum

During the preparation of this manuscript, Takimoto et al (Circ Res 1997;81:533-539) have reported the same Kv4.3 variant as shown in this paper.

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture and Ministry of Health and Welfare, Japan.