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Division of Endocrinology, Department of Medicine, and Department of Pediatrics, North Shore University Hospital/New York University School of Medicine, Manhasset, New York 11030
Address all correspondence and requests for reprints to: Kaie Ojamaa, Ph.D., Division of Endocrinology, North Shore University Hospital, 300 Community Drive, Manhasset, New York 11030. E-mail: kojamaa{at}nshs.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies have described very rapid effects of T3 on the heart and systemic vasculature (15). These observations suggest nongenomic actions of thyroid hormone (16) or the presence of T3-inducible proteins with a very short half-life. Since the half-life of Kv1.5 mRNA has been shown to be approximately 30 min (12), a plausible hypothesis of a rapid response to thyroid hormone would encompass an effect on either Kv1.5 mRNA stability and/or gene transcription. In the present study, we examined the effect of experimental thyroid disease on Kv1.5 gene expression in the mammalian myocardium, both in the intact atria and ventricle as well as in cultured neonatal atrial and ventricular myocytes. The purpose of this work was to investigate possible molecular mechanisms that account for the effects of thyroid hormone on cardiac chronotropy (2).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a second experiment, hypothyroid male SD rats were treated with T3 (10 µg/0.5 ml PBS), injected sc every 6 h for 24 h. Two rats were killed at each hour from 1 h to 5 h, four rats were killed at 6 h, and three rats were killed at 12 and 24 h after T3 treatment was initiated. The LV and atria were rapidly removed and frozen for RNA analysis. Four rats of the same age served as euthyroid controls and four rats served as hypothyroid controls.
In a third series of experiments to measure Kv1.5 mRNA half-life, 12 euthyroid control and 12 hypothyroid male SD rats were injected with actinomycin D (1 mg/kg body wt, ip). Three animals were killed hourly between 0–4 h after drug administration, and their hearts were removed for RNA analysis. In another protocol, a subgroup of hypothyroid and euthyroid rats were injected ip with T3 (10 µg/0.5 ml PBS) either at 1 h or 2 h after actinomycin D treatment. Three to four animals were killed at each 2-h and 4-h time point after T3 administration for subsequent cardiac RNA extraction.
All chemicals used were of the highest purity available from Sigma Chemical Co. (St. Louis, MO).
RNA isolation
Total RNA was extracted from left ventricular and atrial tissues
by the acid-phenol method as previously described (4). The RNA was
quantified by OD at 260 nm, and its integrity was verified by ethidium
bromide intercalation of the 28S and 18S ribosomal RNA resolved by gel
electrophoresis. Northern blot analysis of the myosin heavy chain (MHC)
mRNAs used 5 µg of total RNA isolated from LVs as previously
described (17). 
-Tubulin, -MHC, and ß-MHC mRNAs were detected
by hybridization to unique 40-base oligonucleotide probes
(Oncogene Science, Inc., Uniondale, NY). Probe
hybridization and wash conditions were as previously reported (17).
Cultured neonatal rat cardiac myocytes
Ventricular and atrial myocytes were prepared as previously
described (4). Briefly, hearts were removed from 2-day-old neonatal
rats and trimmed free of the major vessels; under a magnifying scope
the atria and ventricles (left and right chambers not separated) were
separated by cutting along the atrioventricular groove. Tissues
were minced and incubated in collagenase-containing medium, and the
released cells were collected by centrifugation. After four rounds of
enzymatic digestion, the cells were preplated in noncoated T75 culture
flasks for 20 min. Nonadherent cells were then removed, resuspended in
PC-1 medium (Hycor Biomedical, Portland, ME), and plated onto
collagen-coated plates at a density of approximately 1.5 x
10-6/962 mm2 and were allowed to adhere for
18 h. The myocytes were subsequently maintained in DMEM/F12 medium
(Gibco BRL, Gaithersburg, MD) supplemented with insulin
(120 IU/liter), transferrin (5 mg/liter), selenium (5 µg/liter),
dexamethasone (10-7 M), with or without
T3 (10-8 M) for 48 h when
cells were harvested for RNA analysis. Reagents were tissue culture
grade from Sigma Chemical Co. The media were changed
daily.
Quantitative RT-PCR analysis
A competitive quantitative RT-PCR (QRT-PCR) method was developed
to determine the copy number of Kv1.5 mRNA per µg of total RNA from
cardiac tissue and in cultured cardiomyocytes (18). This competitive
PCR method used an homologous internal standard that amplified with
similar kinetics to the endogenous rat Kv1.5 cDNA since both DNA
sequences were identical except the standard was shorter by 215 bp. The
internal standard (189 bp) was developed by deletion of 215 bp of the
Kv1.5 cDNA sequence from nucleotide position 2171 to 2574 (GenBank
M27158).
The following primers were used for the amplification reaction: (upstream primer, 5'-GGCTATGGAGACATGAGACCCATC-3' and downstream primer, 5'-TCTCTTTACAAATCTGTTTCACGG-3'). In the RT reaction, 5 or 10 µg of total RNA were combined with 25 pmol of downstream PCR primer, and first-strand cDNA was synthesized using 200 U MMLV reverse transcriptase (RTase) (Stratagene, La Jolla, CA) in a reaction mixture containing 40 U of RNAsin, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, and 1 mM deoxynucleoside triphosphates (dNTPs). After incubation for 1 h at 37 C, the RTase was inactivated by heating the reaction mixture to 94 C for 6 min. Three microliters of the RT reaction were used for each amplification reaction in a final volume of 50 µl containing 2.5 U AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp., Norwalk, CT), 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 200 µM each dNTP and 500 nM each PCR primer. A series of five PCR reactions containing a fixed aliquot of RT reaction with decreasing mass amounts of standard cDNA (1.0 x 10-19, 3.33 x 10-20, 1.11 x 10-20, 3.70 x 10-21, or 1.23 x 10-21 mol) was amplified as follows: 45 sec denaturation at 94 C, 45 sec annealing at 55 C, 1 min extension at 72 C, for 30 cycles. PCR products (20 µl) were resolved by electrophoresis on 2% agarose gels and quantified by ethidium bromide intercalation. The intensity of UV fluorescence was quantified using a Molecular Imager system (Bio-Rad Laboratories, Inc., Hercules, CA).
Data analysis
Absolute amounts of Kv1.5 mRNA in moles per µg total RNA were
estimated by this equation: [mol wt factor x moles standard cDNA
at the equivalence point x dilution factor for the amount of RNA
used in the PCR]. The mol wt factor corrects for the difference in mol
wt between the endogenous and standard PCR products. The equivalence
point was reached when the competitor and sample DNA fragments yielded
equally intense ethidium bromide-stained bands. The log of the ratios
of sample to competitor PCR products was graphed as a function of the
log of the initial amount of standard added to each PCR reaction. The
molar amount of Kv1.5 mRNA was determined by linear regression analysis
of these data. All results were expressed as means ±
SEM. The unpaired t test was used for
statistical comparison of two groups.
The half-life of Kv1.5 mRNA was determined by calculating from the equation: t1/2 = ln2/k, where t1/2 is the half-life, and k is the first-order degradation rate constant (19). The number of moles Kv1.5 mRNA in each RNA sample was converted into a natural logarithm for use in this equation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of - and ß-MHC genes in rat ventricles was measured by
Northern blot analysis as previously published (21). The ß-MHC
isoform mRNA was expressed only in the hypothyroid state, whereas
-MHC was the exclusive MHC isoform expressed in euthyroid controls
and in the hyperthyroid animals that were treated daily with thyroid
hormone (+T4). Figure 1
shows
a representative Northern blot of ß-MHC and -tubulin mRNA in
animals from the three treatment groups. These data confirm the
validity of the experimental model for hypo- and hyperthyroidism, as we
have previously reported (4, 20, 21).
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, the moles of homologous Kv1.5
DNA standard that were added to each competitive PCR reaction fell
within the exponential phase of the reaction after 30 cycles.
Similarly, when total cardiac RNA was varied from 75–600 ng in the PCR
reaction, the amount of amplified product was within the exponential
phase of the reaction after 30 cycles as shown in Figure 2b. From these
data, we selected a PCR protocol of 30 cycles using an average of 300
ng total RNA and 10-19 to 1.23 x 10-21
mol of standard.
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shows the competitive QRT-PCR
analysis from euthyroid (panel A) and hypothyroid (panel B) ventricular
RNA samples, illustrating the ethidium bromide-stained amplified DNA
from a series of five competitive reactions. The slower migrating
404-bp fragment in each reaction is the amplified product of endogenous
Kv1.5 cDNA, while the 189-bp fragment is the internal standard added at
3-fold decreasing amounts to each of five reactions (shown left
to right). These data are plotted in Fig. 3b with each datum point
derived from the densitometric measurement of the standard and
endogenous Kv1.5 products from each RT-PCR reaction. Linear regression
analysis was used to calculate the absolute molar amount of
steady-state Kv1.5 mRNA in hypothyroid and euthyroid ventricles by
determining the molar equivalence point between standard and endogenous
PCR products (Fig. 3b). The correlation coefficient of the linear
regression curves for all data used in these studies was greater than
0.985.
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Cardiac Kv1.5 mRNA expression in thyroid disease states
Kv1.5 mRNA content in the euthyroid LV was 4.25 ± 0.60
x 10-20 mol/µg RNA. These levels were significantly
(P < 0.01) decreased by 70% to 1.27 ± 0.80
x 10-20 mol/µg total RNA in
6-n-propyl-2-thiouracil-treated hypothyroid animals (Fig. 4). Treatment of euthyroid animals with
T4 to render them hyperthyroid did not alter LV expression
of Kv1.5 mRNA (4.36 ± 0.53 x 10-20 mol/µg
RNA) compared with euthyroid (Fig. 4). Measurements of Kv1.5 mRNA in
atria from euthyroid animals were higher than in the LV at 6.10 ±
0.37 x 10-20 mol/µg RNA. In contrast to
ventricular myocardium, Kv1.5 mRNA content in the atria showed no
dependence on thyroid status. Atrial Kv1.5 transcript levels in
hypothyroid animals were 6.67 ± 0.88 x 10-20
mol/µg RNA, which did not differ significantly from euthyroid
controls (Fig. 4). Furthermore, administration of T4 to
euthyroid animals to render them hyperthyroid did not alter expression
of Kv1.5 in the atria (5.55 ± 0.50 x 10-20
mol/µg RNA).
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). Simultaneous analysis of
atrial tissue of these rats showed that hypothyroidism did not alter
steady-state levels of Kv1.5 mRNA (Fig. 4
), and that T3
treatment of the hypothyroid atrium had no effect over this time course
(data not shown).
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- and
ß-MHC genes. Northern blot analysis was used to measure steady-state
levels of - and ß-MHC mRNAs in ventricles of hypothyroid rats
treated with T3 for 6, 12, and 24 h. In the
hypothyroid ventricle (n = 12), only the ß-MHC mRNA isoform was
expressed (Fig. 1), which remained unaltered at 6 h and 12 h
of T3 treatment, but by 24 h the steady-state levels
had decreased by 51 ± 1%, similar to that previously reported by
our laboratory (22). This decrease of ß-MHC mRNA in response to
T3 treatment indicates an apparent half-life of
approximately 24 h. In contrast, the appearance of -MHC mRNA
was 25 ± 3% of euthyroid values at 12 h of T3
treatment and 170 ± 3% of euthyroid values at 24 h,
indicating a more rapid on-rate of appearance of this transcript with a
half-life of less than 24 h.
Effect of T3 on Kv1.5 mRNA half-life
To determine whether the rapid increase of ventricular Kv1.5 mRNA
in response to thyroid hormone was the result of an increase in
transcription or a change in mRNA half-life, we measured Kv1.5 mRNA
disappearance over a 4-h period in both euthyroid and hypothyroid rats
treated with actinomycin D, an inhibitor of transcription. Figure 6 shows ventricular Kv1.5 mRNA values
derived by QRT-PCR (average values of 3 rats per time point) after
actinomycin D treatment. The calculated mRNA half-life in the euthyroid
ventricles was 1.9 h, which was not significantly different from
the 2.0-h half-life in the hypothyroid ventricles. Furthermore, in
another series of experiments in which euthyroid and hypothyroid rats
were treated with T3 after transcription was inhibited by
actinomycin D, the half-life of Kv1.5 mRNA was not altered, suggesting
that the rapid increase in Kv1.5 mRNA in response to T3 in
the hypothyroid heart is unlikely to be mediated by a significant
increase in mRNA stability.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-MHC, sarcoplasmic reticulum
Ca2+-activated ATPase (SERCA2), ß-adrenergic
receptors, T3 nuclear receptors, and Na/K-ATPase, a recent
report suggests that Kv1.5 was induced by chronic thyroid hormone and
dexamethasone treatment of the hypothyroid-adrenalectomized rat (13).
Earlier reports have also identified changes in the expression of other
K+-channel genes, including Kv1.4, 4.2, and 4.3, in
response to T3 treatment (24).
In the present study we observed that the significantly lower content
of Kv1.5 mRNA in the hypothyroid ventricle was increased 4-fold to
euthyroid levels within 1 h of T3 treatment. This
response was more rapid than the induction of the cardiac-specific
-MHC gene, which required between 12 h and 24 h of thyroid
hormone treatment to reach euthyroid levels. Several molecular
mechanisms by which thyroid hormone regulates Kv1.5 expression can be
postulated. If transcription of the Kv1.5 gene is induced, then this
represents the most rapid nuclear effect of T3 reported to
date (23). This result is somewhat surprising in view of the lack of a
canonical thyroid response element within the Kv1.5 promoter (11).
Alternatively, T3 may be acting to preserve the stability
of the Kv1.5 transcript, similar to that shown for TSH-ß mRNA (14)
and for the -MHC gene in the heart (25). Using actinomycin D to
inhibit transcription, we determined that the half-life of ventricular
Kv1.5 mRNA was not statistically different between euthyroid and
hypothyroid rats. When either euthyroid or hypothyroid rats were
treated with T3, the half-life was not prolonged as would
be expected if mRNA stability was the primary mechanism by which
T3 rapidly increased Kv1.5 mRNA. However, due to the short
half-life and the small cellular pool of Kv1.5 mRNA, experiments to
inhibit transcription with actinomycin D while simultaneously treating
hypothyroid animals with T3 present a complex kinetic
model. It is also important to recognize that the apparent on-rate or
induction of transcription may differ from the off-rate of this
reaction so that measuring the rate of decrease in Kv1.5 mRNA after
actinomycin D treatment is likely to be different from its more rapid
increase in the hypothyroid animal treated with T3. Since
the present study measures cellular mRNA content, which depends not
only on the rate of transcription, RNA processing, and nuclear export,
but also on mRNA degradation, the effects seen by T3 may
well involve multiple sites of regulation.
We have reported that expression of T3 receptors in
the hypothyroid myocardium differs from that in the euthyroid heart,
providing an explanation for the apparent increased sensitivity of the
hypothyroid myocardium to T3 (22). In the hypothyroid
heart, the response of several T3-responsive genes,
including SERCA2 and -MHC, to T3 treatment results in an
increase in expression that is higher than euthyroid control levels,
suggesting enhanced sensitivity (20, 22, 26). Therefore, we postulate
that T3 treatment of the hypothyroid and the euthyroid
heart are not comparable, and this may explain in part the lack of
effect of T3 on Kv1.5 mRNA levels in the euthyroid
ventricle, whereas the hypothyroid ventricle responds rapidly.
Alternatively, the serum levels of T3 in euthyroid animals
may be sufficient to maximally stimulate Kv1.5 gene expression.
Previous studies have reported the level of expression of various
voltage-gated K+ channels in the rat heart (27). Those
studies and the present data show that expression of Kv1.5 mRNA is
approximately 30% higher in atrial compared with ventricular
myocardium. These data are in contrast to studies of Kv1.5 mRNA using
an RNA protection assay in which expression was shown to be more
abundant in ventricles than in atria, and only ventricular expression
was induced by glucocorticoids (28). Since the intact ventricle and
atrium consist of multiple cell types, we compared the expression of
Kv1.5 mRNA in purified cultures of atrial and ventricular myocytes. Not
only was Kv1.5 mRNA present in the purified myocytes, its expression
was modified by T3 similar to that observed in
vivo. As has been reported for the cardiac-specific -MHC gene
(29), expression of Kv1.5 in atrial tissue was unresponsive to thyroid
hormone. These data are also consistent with the observation that
atrial arrhythmias are a rare occurrence in the hypothyroid patient
(2).
Hypothyroidism is characterized by bradycardia and a long QT interval (2). Although this prolongation of the action potential may be the result of changes in a number of ion currents, including repolarizing K+ currents (30), the role of Kv1.5 in the repolarizing phase of the action potential within the mammalian ventricle is unclear. The ultrarapid delayed rectifier K+ current (Ikur) has been reported to be carried by the Kv1.5 channel (31). The Kv1.5 protein has been immunolocalized to human atrial and ventricular myocytes (31, 32) and, when studied in patients with atrial fibrillation, was shown to be altered, contrary to what would have been predicted based upon its proposed function (33). Western analysis of Kv1.5 protein in rat ventricles in the present study showed that the protein content decreased 20–60% in the hypothyroid hearts compared with euthyroid (data not shown). Whether regulation of Kv1.5 expression by thyroid hormone is similar in rat and human remains to be determined. However, if the marked decrease in the ventricular Kv1.5 gene product results in a decrease of the delayed rectifier K+ current, then this may in part contribute to the prolongation of the QT interval observed in hypothyroidism (2). Similarly, if the rapid response of Kv1.5 to thyroid hormone observed in the present experimental study occurs in humans, then this may have implications in the treatment of patients with varying degrees of hypothyroidism, as well as the potential utility of T3 therapy in cardiac surgery patients in which serum T3 levels are significantly reduced (15).
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Footnotes |
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Received December 9, 1998.
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-cardiac myosin heavy chain gene promoter. J Biol Chem 268:4331–4336This article has been cited by other articles:
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) and hypothyroid (•) ventricles.
The molar equivalence point (zero on y axis) between standard and
endogenous PCR fragments is indicated by the dashed
line, and the Kv1.5 mRNA is quantified from the curve.
