Time course of the in vivo effects of thyroid hormone on cardiac gene expression.

OOlS-7227/92/1304-2001$03.00/0 Endocrinology Copyright Q 1992 by The Endocrine

Vol. 130, No. 4 Printed in U.S.A.

Society

Time Course of the in Viuo Effects on Cardiac Gene Expression* CHERYL

BALKMAN,

KAIE

OJAMAA,

AND IRWIN

of Thyroid

Hormone

KLEIN

The Department of Medicine, North Shore University Hospital, Ma&asset, New York 11030;and The Department of Medicine, Cornell University Medical College,New York, New York 10021

ABSTRACT. The rate of response to thyroid hormone on cardiac growth, heart rate, and the relative changes in messenger RNA (mRNA) coding for a- and @-myosin heavy chain (MHC), slow sarcoplasmic reticulum calcium-adenosine triphosphatase, and thyroid hormone receptors in ventricular tissue of hypothyroid rate was investigated. Hypothyroid rate had significantly smaller hearta, with slower heart rates and expressed no (Y-MHC mRNA as analyzed by an Sl nucleate protection assay when compared to euthyroid animals that expressed 79% a-MHC. Twelve hours after treating hypothyroid rata with 20 ag of L-T,, detectable levels of a-MHC mRNA were present and the shift to ar-MHC mRNA was complete by 72 h of treatment. Northern blot analysis showed that hypothyroidism resulted in a 60%

decrease in the level of sarcoplasmic reticulum calcium-adenosine triphosphatase mRNA which increased after 12 h of T, adminietration and wae 2.bfold (P < 0.06) greater than euthyroid levels after 72 h. In contra&, thyroid hormone receptor mRNA levels measured in poly(A)+ RNA were elevated in hypothyroid rate and decreased to euthyroid levels within 24 h after thyroid hormone treatment. These changes in cardiac gene expression occurred simultaneously with changes in both cardiac size and heart rate. The current studies characterixe the coordinated changes and the time course for gene expression that occur in the hypothyroid heart after acute T1 administration. (E&timlo# 130: 2001-2006,1992)

T

HYROID hormones have profound effects on the cardiovascular system as evidenced by the changes which accompany both hypothyroidism and hyperthyroidism (1). Heart rate, cardiac output, myocardial contraction and relaxation, and systemic vascular resistance are all altered by thyroid status (2). Experimental hypothyroidism in rats leads to changes in the composition of specific contractile proteins found in cardiac ventricular tissue. This involves but is not limited to a shift in the myosin isoenxymes from the (Y-to the /3-myosin heavy chain (MHC) gene product (3,4). These changes, in part, explain the observation that thyroid hormone can influence the intrinsic systolic contractile properties of the myocardium (5). Thyroid hormone status can also affect the velocity of diastolic relaxation (6). One of the mechanisms to explain this change is the ability of T3 to alter the activity and cellular content of the cardiac-specific slow sarcoplasmic reticulum (SR) calcium-adenosine triphosphataae (ATPase) by altering the transcription of the gene coding for this protein (7, 8). Hypothyroid rat ventricles have significantly less messenger RNA (mRNA) coding for this enzyme while in the hyperthy-

roid ccndition SR calcium-ATPase mRNA levels are 4080% higher than in euthyroid rats (7,8). Many of the known cellular effects of thyroid hormone are mediated by specific nuclear receptor proteins which regulate the transcription of thyroid-responsive genes (9, 10). Thyroid hormone receptors are encoded by two distinct but closely related genes, c-erb-A-a and -/3 which are cellular homologs of the viral erythroblastosis A oncogene, v-erb-A (11). By differential splicing of these two genes four protein isoforms are synthesized. Biologically active thyroid hormone receptors, c-erb-A a-l and p-1, as well as the non-T3 binding variant c-erb-A (w-2, are expressed in cardiac tissue (12, 13). The levels of cerb-A a-l and (u-2 mRNA and the concentration of thyroid hormone receptors decreases with T3 treatment (14,X). The magnitude of the response or sensitivity of a tissue to thyroid hormone is likely to be influenced by the concentration and composition of thyroid receptors and other c-erb-A related proteins (16). To better understand the cellular mechanisms of the thyroid hormone effects on the heart we have examined the rate of the response to the hormone on cardiac growth, heart rate, and the relative changes in mRNA coding for (Y- and P-MHC, and the slow SR calciumATPase. In addition, we have correlated these changes with measurements of mRNA coding for nuclear thyroid hormone receptors in hypothyroid rats in response to T.+

Received September 3,199l. Address all correspondence and requests for reprints to: Kaie Ojamaa. Ph.D.. Laboratorv of Molecular EndocrinoloPv. North Shore University Hospital, Mkhasset, New York 11030. I* Supported in part by NIH Grant HL-41304. 2001

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 April 2015. at 19:21 For personal use only. No other uses without permission. . All rights reserved.

2002

THYROID Materials

Animal

HORMONE

and Methods

preparation

Male Sprague-Dawley rats weighing 140-160 g (Charles River Breeding Laboratories, Kingston, NC) were surgically thyroidectomized and nonoperatedanimalswere usedas agedmatched euthyroid controls. Animals were given standard rat chow and water ad libitum and maintained in a controlled 12h light, 12-h dark cycle. Thyroidectomized rats received water containing calcium chloride (4.5 mM). Animals were weighed every 7-10 days for 5 weeksafter surgery. Animals were divided into three groups;euthyroid, hypothyroid, and hypothyroid-T, treated. The treated animals were given daily SCinjections of T, (20 pg) and were killed either 12, 24, or 72 h after the first injection. Basalheart rates were obtained in consciousanimals using a Harvard photoelectric tail pulse detector and Harvard model59 pulseamplifier (Harvard Labs, Cambridge,MA) (17). Heart rates of animalstreated with Tq were monitored at 12, 24, 48, and 72 h postinjection. At the time of being killed, animalswere anesthetizedwith ketamine:xylazine (100 mg/kg body wt), blood was collected, and hearts were excised and rinsed in ice-cold PBS (pH 7.4). The atria and right ventricle wereremovedand the left ventricle, including the intraventricular septum, was immediately frozen in liquid nitrogen and stored at -80 C for subsequentRNA analysis. Total heart weights include the weight of left and right ventricles. Total serum T, and TB levels were measuredusing commercially availableRIA kits (Quantimune II T-4, Bio-Rad, Hercules,CA; Incstar Corp., Stillwater, MN). Total RNA and poly(A)+ RNA isolation

Total cellular RNA wasextracted from the left ventricles by the acidic phenol method as described by Chomczynski and Sacchi (18) with the following modifications. Left ventricles were pulverized in liquid nitrogen and then homogenizedin guanidinium isothiocyanate and the RNA extracted with phenohchloroform (1:l). The RNA was then precipitated from the aqueousphasewith an equal volume of isopropanol,kept at -20 C for 1 h, and collected by centrifugation. The pellet was washedwith 75% ethanol, air dried, and resuspendedin sterile water. Poly(A)+ RNA was isolated from pooledsamples of total cellular RNA by using oligo(dT) affinity chromatography (Pharmacia, Piscataway, NJ) (19). Both total RNA and poly(A)+ RNA were quantified spectrophotometrically. We have previously documentedthe recovery rate for left ventricle (LV) RNA extraction (20). Northern

blot analysis

Total cellular RNA or poly(A)+ RNA (5 rg) from eachsample was denatured in the presenceof 2.2 M formaldehyde, 33% formamide, 19.5 mM 3-[N-morpholinolpropane sulfonic acid, pH 7.0,5 mM sodiumacetate, 1 mM EDTA, at 65 C for 5 min and separatedby electrophoresison formaldehyde-l% agarose gels.The ribosomal RNA bands were visualized by ethidium bromide staining and the RNA transferred to Duralon UV membrane(Stratagene, San Diego, CA) by capillary blotting and then cross-linked to the membraneby exposure to UV light. Membranes were prehybridized at 42 C for 6-8 h in a

AND THE HEART

Endu - 199” Vol 130. No 4

solution containing 5~ SSPE [0.75 M NaCl, 50 mM Na-phosphate (pH 7.4), 5 mM EDTA], 5~ Denhardt’s solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 5% BSA), 50% formamide, 1% sodium dodecyl sulfate (SDS), and 100 Kg/ml denatured salmon sperm DNA. Hybridization with lo6 dpm/ml radiolabeled complementary DNA (cDNA) probes wascarried out in the samesolution at 42 C for 18 h. Membraneswere washedat high stringency in 0.2~ SSPE, 1% SDS at 65 C for 30 min, and then exposedto x-ray film with intensifying screensat -80 C. SR calcium-ATPase

and c-erb-A

cDNA probes

Slow SR calcium ATPase mRNA was detectedon Northern blots containing total cellular RNA by hybridization to a radiolabeled2.3-kb cDNA fragment (providedby W. Dilhnann (21)J. The thyroid hormone receptor isoforms,c-erb-A a-1 and a-2, were detected on Northern blots containing poly(A)+ RNA by a single 500 basepair cDNA probe [provided by R. M. Evans (22)]. The cDNA fragmentswere radiolabeledwith a-32P-dCTP and ‘M’P (New England Nuclear, Boston, MA) using random priming methodology to a specific radioactivity of 10Rdpm/ pmol and addedto the hybridization solution at lo6 dpm/ml. Hybridization and wash conditions were as describedabove, and the hybridizing mRNA bands were detected by autoradiography. To standardizefor gel loadingvariability on Northern blots containing total RNA, a genomic 18s ribosomal DNA fragment (20) wasrandomprimed, hybridized to Northern blots of total RNA, and washed stringently with 0.1X SSPE, 1% SDS for 30 min at 65 C. The mRNA speciesdetected on the autoradiogramswere quantified by laser densitometry (Ultroscan XL, Pharmacia LKB Biotechnology Inc., Piscataway, NJ) over a linear range and normalized to 18 S ribosomalRNA. In order to standardizec-erb-A mRNA values, the poly(A)+ RNA Northern blots were hybridized to a 5’-labeled deoxyoligonucleotide probe complementary to cY-tubulin(OncogeneScience Inc. Uniondale, NY) (23). C-erb-A a-l and (r-2 mRNA species were quantified by laser densitometry and normalized to (Ytubulin mRNA. Sl nud4wse protection

analysis

Alpha- and fl-MHC mRNA were quantified in the same sampleof total RNA by using a singleoligonucleotideprobe in an Sl nuclear mapping assay.The method wasas describedby Waspe et al. (24) with several modifications. A 61-baseoligonucleotide probe was synthesized on a Biosearch model 8600 DNA Synthesizer (San Rafael, CA) and purified by polyacrylamide gel electrophoresis.The probe was complementary to a 38-base coding sequencecommon to both CY-and B-MHC mRNAs (nucleotides 5763-5800) (25) and to an additional 8 basesunique to /3-MHC mRNA. The oligonucleotideprobe, 3’end labeled with cY-“‘P-Cordycepin5’ triphosphate (New England Nuclear) was hybridized in molar excessto 10 rg total cellular RNA in 60 ~1containing 50% formamide, 40 mM Pipes (pH 6.5), 400 mM NaCl, 1 mM EDTA, 10 pg yeast RNA for 15 h at 40 C. Sl nucleasedigestionwas conductedat 37 C for 2 h in 400 ~1final volume containing 280 mM NaCl, 30 mM sodium acetate (pH 4.6), 1 mM zinc acetate, 5% (vol/vol) glycerol, and 800 U enzyme (BethesdaResearchLaboratories,Gaithersburg, MD). The protected fragmentswere resolvedby electrophoresis on a 10% polyacrylamide/7 M urea gel and identified by auto-


THYROID

HORMONE

AND THE HEART

2003

radiography. The MHC mRNA specieswerequantified by laser densitometry scanningof the autoradiograms.

icantly lower than rates of euthyroid rats were increased with Tq treatment and by 72 h the heart rates were

Statistical analysis

significantly

Resultsare expressedas mean3~SEM. Statistical differences between mean values were evaluated by Student’s t test and Kruskal-WaIlis test. Significance was assumedat P lessthan 0.05. The data shown in Figs. l-5 were derived from one of three separateexperiments.

Results Animals

The thyroid status of the rats was determined by total serum T, and Ts levels which were undetectable in the thyroidectomized rats (Table 1). Twelve hours after Tq administration, serum Tq levels were 37.1 & 2.3 pg/dl, which subsequently fell to 23.1 f 0.8 pg/dl at 24 h. The value for the 72-h group of animals is the serum Tq concentration measured 24 h after the last injection. Serum T3 in thyroidectomized rats reached euthyroid levels by 12 h after T., treatment and continued to rise until the next injection at 24 h. Body weights were monitored during the 6-week experimental period. Thyroidectomized rats did not gain weight which was in contrast to normal controls (Table 1). Although T4 administration did not increase body weight, the heart weight/body weight ratio was increased significantly from 2.33 & 0.28 to 3.26 f 0.35 (X 10e3) after 72 h of treatment. Data from additional studies including a larger group (n = 10) of T, treated hypothyroid rats also showed significant (P < 0.01) increases in heart wt/body wt ratios at 72 h. At the time of killing, the weights of the whole heart and LV were measured (Table 1). Both total heart and LV weights of the hypothyroid animals were 63% lower than the age-matched euthyroid animals. Administration of Tq to the hypothyroid rats resulted in a 23% and 27% increase in LV and total heart weight, respectively, by the third day of treatment. Heart rates of the hypothyroid rats which were signif-

higher than pretreatment

(Table 1).

Expression of a- and /3-MHC mRNA To quantitate the relative levels of expression of a-

and P-MHC genes, analysis by Sl nuclease protection showed that hypothyroid rat ventricles expressed only flMHC mRNA (Fig. 1). In order to quantitate the relative changes in CY-and P-MHC mRNA, the autoradiograms were scanned by laser densitometry and expressed as percent total (Y-plus P-MHC mRNA as shown in Fig. 2. Treatment of these animals with Tq resulted in the appearance of a-MHC mRNA which was 10 k 1% of the total MHC mRNA by 12 h, 54 f 4% by 24 h, and 93 sfi 1% by 72 h after treatment. The level of expression of a-MHC in age-matched euthyroid rats was 79 + 1% of total MHC mRNA. Northern blot analysis of SR calcium-ATPase

The level of expression of cardiac SR Ca-ATPase was examined by Northern blot analysis shown in Fig. 3. To determine the degree of expression, the Ca-ATPase mRNA within each sample was quantified by scanning densitometry and the value normalized for the amount of 18s rRNA within the same sample. The amount of SR Ca-ATPase mRNA in hypothyroid hearts decreased to 39% (P < 0.05) of the level expressed in euthyroid hearts. Administration of T4 to thyroidectomized rats caused a significant increase in expression to 75% of euthyroid levels by 12 h, 154% by 24 h, and 255% by 72 h. Expression of c-erb-A mRNA

Messenger RNA levels of both isoforms of the T3 receptor, c-erb-A a-1 and a-2, were measured by Northern blot analysis of poly(A)+ RNA from ventricular tissue (Fig. 4). The relative levels of c-erb-A a-l and (u-2 mRNA

were determined by densitometric scanning of the autoradiograms and corrected for gel loading variation by the amount of cy-tubulin mRNA expressed. Figure 5 shows

TABLE 1. Comparison of body wt, heart (Ht), and LV wt, heart rate (HR), and heart wt to body wt ratio [(H wt/B wt) x lo-'] of euthyroid rats to hypothyroid rats treated with T, for 12, 24, and 72 h

Euthyroid Hypothyroid TI administration 12 h 24 h 72 h

ND

Body wt (9) 376+17 157 f 13

2.59 f 0.16 2.33 3~0.28

967 + 22 362 + 12

812+200 311f 11

36Ok 19” 201+ 4

68.3 f 7.4 139.9 + 18.5 144.4 f 24.9

147 +5 146 + 3 142 + 2

2.35 f 0.06 2.65 + 0.21 3.26 f 0.35

344 + 20 386 + 30 461+ 43" ____---

289 zk 13 333 * 29 381f 41

2252 7 215 29 245 k ___-4"

T, h&l)

TI Wml)

4.0 + 0.4

59.9 + 8.6

NDb

37.1 + 2.3 23.1 + 0.8 19.3 zk 1.8

Hwt/Bwt

Ht wt (mg)

LV wt (mg)

HR (bpm)

* P < 0.05 compared with Hypo (n = 3 rats per group). b Not detectable.


2004

THYROID

Hypo

12h

24h

72h

HORMONE

AND

THE

Eu

HEART

Eu -----

Hypo

Endo. Voll30.

12h

24h

1992 No 4

72h

SR

*

l Ca”ATPase

p MHC-,

aMHC+

-18s

:,

FIG. 1. Sl nuclease protection analysis of cr- and fi-MHC mRNA in hearts of euthyroid (Eu), hypothyroid (Hypo), and T4 treated rats. The /3-MHC protected fragment is 46 bases in length, and o(-MHC is 38 bases.

FIG. 3. Northern blot analysis showing SR Ca-ATPase mRNA levels in ventricular tissue from euthyroid (Eu) rats, hypothyroid (Hypo) rats, and thyroidectomized rats treated with T, for 12, 24, and 72 h. Migration of the 18s and 28s ribosomal RNA subunits are indicated on the left. The lower panel shows hybridization of the same blot to an 18s rDNA probe.

H H 12 24 72 E 80 --

: alpha,

5 60-8 f5 a 40--

2.8 L

HOURS AFTER T4 TREATMENT

2. Time course of the effect of Tq treatment of thyroidectomized rats on the relative levels of (Y- and &MHC mRNA expressed in ventricular tissue shown as a percent of total MHC mRNA. Hypothyroid MHC mRNA levels are indicated by the 0 h time point; euthyroid levels indicated by Eu; closed symbols, percent P-MHC mRNA, open symbols, percent o(-MHC mRNA. Standard error bars not visible fall within the symbols. FIG.

the level of expression of c-erb-A a-l and a-2 mRNA normalized to cr-tubulin mRNA as a percent of the euthyroid values. Hypothyroid rats expressed levels of cerb-A which were 1.4- to 1.9-fold higher than euthyroid levels and which were progressively decreased by T4 treatment. 72 h after daily administration of T4 levels of c-erb-A-cu had fallen to 40 and 30% of the euthyroid values. Discussion In this study the time course of T4 responsiveness on cardiac growth, heart rate, and the changes in cardiacspecific gene expression in hypothyroid rats was investigated. Thyroid hormone is known to induce changes in

*

alpha,

FIG. 4. Northern blot analysis of poly(A)+ RNA from ventricular tissue of euthyroid (E), hypothyroid (H), and thyroidectomized rats treated with T4 for 12, 24, and 72 h. Each poly(A)+ RNA sample is derived from pooled total RNA samples purified from LVs of three hearts. mRNA species coding for c-erb-A a-1 and or-2 are indicated with their approximate molecular size (kilobase, kb) indicated on the left.

cardiac performance, including the speed of systolic contraction and diastolic relaxation which can be correlated to changes in the expression of specific cardiac proteins (2-8). In the present report we have investigated the coordinated changes in two thyroid hormone responsive genes. Since the direct cellular effects of thyroid hormone are thought to be mediated through its interactions with specific nuclear receptors (26, 27), temporal changes in the expression of T3 receptor mRNA were simultaneously examined. Characteristic of hypothyroidism, the thyroidectomized rats failed to grow, had lower heart weight/body weight ratios, and slower heart rates than age-matched euthyroid controls. In accordance with previously published data (3, 28, 29), ,&MHC mRNA was the only isoform detected in hypothyroid rat ventricles, whereas euthyroid rats expressed predominantly wMHC mRNA;


THYROID

HYPO

24

12 HOURS

72

HORMONE

EU

AFTER T4

E‘IG. 5. Relative levels of cardiac c-erb-A a-l (solid bars) and c-erb-A u-2 (lined bars) mRNA in hypothyroid (HYPO) and T, treated rats compared with euthyroid controls (EU). Values were derived from denaitometric ecanning of autoradiograms of Northern blots (Fig. 4) and corrected for loading variability using cY-tubulin mRNA as the invariant internal control.

SCadministration of T, to the thyroidectomized animals resulted in the appearance of cr-mRNA within 12 h and complete reversal to euthyroid levels by 72 h of treatment. Others have reported changes in MHC expression by 3 h after iv administration of T3 and within 6 h by SC injection (29, 30). The somewhat longer lag period observed in our studies may be attributable to the time required to convert the biologically less active form of the hormone T, to TB, whose binding affinity to nuclear receptors is lo-fold greater (15). However, recent data suggest that the biological activity of T, may be significant in cases of elevated serum free Tq in which nuclear thyroid receptor occupancy by Tq is increased (31). The expression of SR Ca-ATPase which was significantly decreased in hypothyroid rat ventricles was increased by T, administration. Rohrer and Dillmann (7) have reported that SR Ca-ATPase levels were significantly increased 2 h after T3 injection iv and that levels reached euthyroid values by 5 h. In the present studies, the time course of response of SR Ca-ATPase to T., was similar to that observed for MHC gene expression. To further investigate the in uiuo action of Tq on a-MHC gene transcription, we have in previous studies injected plasmid DNA containing a-MHC promoter sequences (-613 to +421) known to be Ts responsive (32) directly into the rat myocardium (33). The (Y-MHC gene-fusion construct was induced by Tq treatment indicating a direct role of the hormone on gene transcription. To determine whether the cellular effects of thyroid hormone could be modified by changes in the expression of myocardial thyroid hormone receptors, we examined the time course of expression of the c-erb-A-cY gene to T, treatment. We found that the c-erb-A mRNAs were

AND

THE

HEART

2005

elevated in the hypothyroid rats but declined within 12 h of Tq administration and continued to decrease at 24 and 72 h after treatment. Hodin et al. (14) reported a 65% and 86% decrease in c-erb A a-l and (~-2 mRNA, respectively, in hearts of hypothyroid rats after 6 days of TB treatment. In the present study, after 72 h of treatment c-erb-A (~-1 and (~-2 expression decreased to levels lower than that observed in euthyroid hearts. This is in contrast with the increase in levels of (r-MHC and CaATPase mRNAs observed over the same time course. Thus, as other authors have suggested it may be misleading to attribute cellular effects of thyroid hormone to total Ts receptor number since a change in the relative level of expression of the receptor isoforms could have profound effects on thyroid hormone responsiveness (14, 34, 35). This remains to be examined. We have previously reported that the effects of thyroid hormone on cardiac gene expression can be modified by alterations in cardiac work (23, 36). In the hemodynamically unloaded transplanted heart, we have shown that /3-MHC is expressed de nouo in animals with normal levels of thyroid hormone (23). Izumo et al. (37) have also shown that cardiac gene expression is modulated by the interactions of humoral and hemodynamic factors. In experimental pressure-overload hypertrophy of the adult myocardium several “fetal” isoforme of cardiac contractile proteins are reexpressed, including B-MHC (38). Administration of excess T, to these animals causes a rapid deinduction of fi-MHC expression despite the progression of hypertrophy (37, 39). The effect of thyroid hormone on cardiac growth does not occur in the absence of a hemodynamic workload on the heart since the unloaded heterotropically transplanted rat heart fails to grow in response to T4 treatment (36). Similarly, T, mediated cardiac hypertrophy observed in the normal working heart can be blocked by inhibiting the increase in heart rate and cardiac work associated with T, administration using the fi-adrenergic blocking agent, propranolol (17). In the present experiments, thyroid hormone administration to thyroidectomized rats significantly increased heart rate and heart size by 72 h. We suggest that these effects of thyroid hormone are the result of the effects of the hormone on parameters which alter cardiac work (2, 17, 36). We conclude from these studies that the expression of (Y-and /3-MHC, SR Ca-ATPase, and c-erb-A in the adult myocardium in response to thyroid hormone is indicative of a direct effect of the hormone on the myocyte. However, these results in conjunction with previous data in heterotropically transplanted nonworking heart (36) indicate that the direct cellular effects of TB may be modified by the hemodynamic alterations imposed on the heart as a consequence of thyroid hormone treatment.


THYROID

2006

HORMONE

Acknowledgment The authors wish to thank Ms. Linda Castelli for her expert secretarial assistance.

References 1. Klein I 1990 Thyroid hormone and the cardiovascular system. Am J Med m631-637 2. Klein I 1990 Thyroid hormone and blood pressure regulation. In: Laragh J, Brenner B (eds) Hypertension. Raven Press, New York, pp 1661-1674 3. Gust&on TA, Markham BE, Morkin E 1986 Effects of thyroid hormone on alpha-a&n and myosin heavy chain gene expression in cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic oligonucleotide probes. Circ Ree 59:194201 4. Izumo S, Nadal-Ginard B, Mahdavi V 1986 All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231:597-600 5. Morkin E, Flink IL, Goldman S 1983 Biochemical and physiologic effects of thvroid hormone on cardiac performance. Proa Cardiovast Dis 22435-464 6. Mintz G, Pizzarello R, Klein I 1991 Enhanced left diastolic function in hyperthyroidiam: noninvasive asaessment and response to treatment. J Clin Endocrinol Metab 73146-150 7. Rohrer D, Dii WH 1988 Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum calciumATPaae in the rat heart. J Biol Chem 2636941-6944 8. Arai M, Gtau K, MacLennan DH, Alpert NR, Periasamy M 1991 Effect of thvroid hormone on the expression of mRNA encoding aarcoplaemi~ reticulum proteins. Circ-Rea 69266-276 9. Evans R 1988 The steroid and thyroid hormone receptor superfamily. Science 240889-896 10. Samuels HH, Forman BM, Horowitz ZD, Ye ZS 1988 Regulation of gene expression by thyroid hormone. J Clin Invest 81:957-967 11. Weinberzer C. Thomnson CC. One ES. Lebo R. Gruol DJ, Evans RM 196 The c-erb-k gene encod% a .thyroid hormone receptor. Nature 324641-646 12. Lazar MA, Hodin RA, Darling D, Chin WW 1988 Identification of a rat c-erb-A alpha-related protein which binds deoxyribonucleic acid but doea not bind thyroid hormone. Mol Endocrinol 2:893901 13. Mitsuhashi T, Nikodem V 1989 Regulation of expression of the alternate mRNA’e of the rat alpha-thyroid hormone receptor gene. J Biol Chem 264:8900-8904 14. Hodin RA, Lazar MA, Chin WW 1990 Differential and tissuespecific regulation of the multiple rat c-erb A messenger RNA species by thyroid hormone. J Clin Invest B&101-105 15. Ladenson PW, Kieffer JD, Farwell AP, Ridgway EC 1986 Modulation of myocardial L-triiodothyronine receptors in normal, hypothyroid and hyperthyroid rats. Metabolism 355-12 16. Lazar MA, Chin WW 1990 Nuclear thyroid hormone receptors. J Clin Invest 86:1777-1782 cardiac hypertrophy: time course 17. Klein I 1988 Thyroxine-induced of development and inhibition by propranolol. Endocrinology 123:203-210 18. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162156169 19. Maniatis T, Fritsch EF, Sambrook J 1983 Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

AND

THE

HEART

Endo. Vol130.

1992 No 4

20. Klein I, Samarel AM, Welikson R, Hong C 1991 Heterotropic cardiac transplantation decreases the capacity for rat myocardial protein synthesis. Circ Res 68:1100-1107 21. Maciel LMZ, Polikar R, Rohrer D, Popovich BK, Dillmann WH 1990 Age-induced decreases in the messenger RNA coding for the sarcoplasmic reticulum calcium-ATPase of the rat heart. Circ Fl.es 67:230-234 22. Thompson C, Weinberger C, Lebo R, Evans RM 1987 Identification of a novel thyroid hormone receptor expressed in the mammalian central nervous system. Science 237:1610-1614 23. Klein I. Ojamaa K, Samarel AM, Welikson R, Hong C 1992 Hemodynamic regulation of myosin heavy chain gene expression: studies in the transnlanted rat heart. J Clin Invest 8968-73 24. Waape LE, Ordahl CP, Simpson PC 1990 The cardiac beta myoein heavy chain isogene is induced selectively in alpha adrenergic receptor-stimulated hvnertrophy of cultured rat heart myocytes. J ClinInvest 85:1206-iii4 - 25. Kraft R. Bravo-Zehnder M. Tavlor DA. Leinwand LA 1989 Complete nucleotide seouence of full length cDNA for rat alpha and beta cardiac myosin heavy chain. Nucleic Acids Res 17:75-29-7530 26. Oppenheimer JH 1985 Thyroid hormone action at the nuclear level. Ann Intern Med 102374-384 27. Koeniz RI. Wame RL. Brent GA. Harnev JW. Larsen PR. Moore DD 1988 Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. Proc Nat1 Acad S& USA 86:5031-5035 28. Green NK. Franklvn JA. Alhauist JAO. Gammaae MD, SheDDard MC 1989 Differeniial regulation by thyroid hormones-of myoein heavy chain alpha and beta mRNA’s in the rat ventricular myocardium. J Endocrinol122:193-200 29. Dillmann WH, Barrieux A, Shanker R 1989 Influence of thyroid hormone on myoein heavy chain mRNA and other messenger RNAs in the rat brain. Endocr Res 15:665-577 30. Everett AW, Sinha AM, Umeda PK, Jakovcic S, Rabinowitz M, Zak R 1984 Regulation of mvosin svntheeis by thyroid hormone: relative change-hi the alpha and beta myosin heavy chain mRNA levels in rabbit heart. Biochemistry 231596-1599 31. Halperin Y, Shapiro LE, Surks MI 1991 Role of L-thyroxine in nuclear thyroid hormone receptor occupancy and growth hormone production in cultured GC cells. J Clin Invest 88:1291-1299 32. Gustafson TA, Markham BE, Bahl JJ, Morkin E 1987 Thyroid hormone regulates expression of a transfected alpha-myosin heavy chain fusion gene in fetal heart cells. Proc Nat1 Acad Sci USA 84:3122-3126 33. Ojamaa K, Klein I 1991 Thyroid hormone regulation of alphamyosin heavy chain promoter activity by in uiuo DNA transfer in rat heart. Biochem Biophys Res Commun 1791269-1275 34. Mitsuhashi T, Tennyson G, Nikodem V 1988 Alternate splicing generates mesaages encoding rat c-erb-A proteins that do not bind thyroid hormone. Proc Nat1 Acad Sci USA 85:5840-5808 35. Izumo S, Mahdavi V 1988 Thyroid hormone receptor alpha isoforms generated by alternate splicing differentially activate myosin HC gene transcription. Nature 334539-542 36. Klein I, Hong C 1986 Effects of thyroid hormone on the cardiac size and myosin content of the heterotropically transplanted rat heart. J Clin Invest 77:1694-1698 37. Izumo S, Lompre A, Matsuoka R, Koren G, Schwartz K, NadalGinard B. Mahdavi V 1987 Mvosin heaw chain messenzer RNA and protein isoform transitions during cardiac hypertrophy. J Clin Invest 79:970-977 38. Nadal-Ginard B, Mahdavi V 1989 Molecular basis of cardiac performance: plasticity of the myocardium generated through protein isoform switches. J Clin Invest 84:1693-1700 39. Imamura S, Matsuoka R, Hiratsuka E 1990 Local response to cardiac overload on myosin heavy chain gene expression and isoenzyme transition. Circ Res 66:1067-1073


Thyroid hormone effects on cardiac gene expression independent of cardiac growth and protein synthesis.

Effects of thyroid hormone on sex hormone-binding globulin gene expression in human cells.

Effects of thyroid hormone deficiency and replacement on rat hypothalamic growth hormone (GH)-releasing hormone gene expression in vivo are mediated by GH.

Thyroid hormone regulation of gene expression.

Reduced effects of thyroid hormone on gene expression and metamorphosis in a paedomorphic plethodontid salamander.

In vivo effects of acute EtOH on rat alpha and beta luteinizing hormone gene expression.

Fetal and neonatal iron deficiency exacerbates mild thyroid hormone insufficiency effects on male thyroid hormone levels and brain thyroid hormone-responsive gene expression.

Comparison of the in vitro effects of TCDD, PCB 126 and PCB 153 on thyroid-restricted gene expression and thyroid hormone secretion by the chicken thyroid gland.

Differential effects of in vivo estrogen administration on hypothalamic growth hormone releasing hormone and somatostatin gene expression.

Thyroid - hormone effects on steroid - hormone metabolism.

Time-Course Gene Set Analysis for Longitudinal Gene Expression Data.

Translating thyroid hormone effects into clinical practice: the relevance of thyroid hormone receptor α1 in cardiac repair.

Decreased collagen gene expression and absence of fibrosis in thyroid hormone-induced myocardial hypertrophy. Response of cardiac fibroblasts to thyroid hormone in vitro.

Properties of sparse penalties on inferring gene regulatory networks from time-course gene expression data.

Regulation of growth hormone gene expression: synergistic effects of thyroid and glucocorticoid hormones.

Time-related effects of thyrotropin-releasing hormone (TRH) on the pituitary-thyroid axis and extrathyroidal targets.

Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo.

Thyroid hormone modulates apolipoprotein B gene expression in HepG2 cells.

Effects of perchlorate on BDE-47-induced alteration thyroid hormone and gene expression of in the hypothalamus-pituitary-thyroid axis in zebrafish larvae.

Effects of waterborne cadmium on thyroid hormone levels and related gene expression in Chinese rare minnow larvae.

The immediate and late effects of thyroid hormone (triiodothyronine) on murine coagulation gene transcription.

The time course of thyroid-hormone-induced changes in the isotonic and isometric properties of rat soleus muscle.

Peripheral thyroid hormone levels and hepatic thyroid hormone deiodinase gene expression in dairy heifers on the day of ovulation and during the early peri-implantation period.

Effects of selenium deficiency on thyroid hormone economy in rats.