TRs Have Common and Isoform-Specific Functions in Regulation of the Cardiac Myosin Heavy Chain Genes

Anethe Mansén, Fushun Yu, Douglas Forrest, Lars Larsson and Björn Vennström

Department of Cell and Molecular Biology (A.M., B.V.) and Department of Clinical Neuroscience (F.U., L.L.), Karolinska Institute, S-171 77 Stockholm, Sweden, Noll Physiological Research Center and Department of Cellular and Molecular Physiology (F.U., L.L.), Pennsylvania State University, University Park, Pennsylvania 16802; and Department of Human Genetics (D.F.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Dr. Bjorn Vennstrom, Department of Cell and Molecular Biology, Karolinska Institute, Room D427, Doktorsringen 2D, Soina, Sweden S-171 77. E-mail: bjorn.vennstrom{at}cmb.ki.se


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TR{alpha}1 and TRß mediate the regulatory effects of T3 and have profound effects on the cardiovascular system. We have analyzed the expression of the cardiac myosin heavy chain (MyHC) genes {alpha} and ß in mouse strains deficient for one or several TR genes to identify specific regulatory functions of TR{alpha}1 and TRß. The results show that TR{alpha}1 deficiency, which slows the heart rate, causes chronic overexpression of MyHCß. However, MyHCß was still suppressible by T3 in both TR{alpha}1- and TRß-deficient mice, indicating that either receptor can mediate repression of MyHCß. T3-dependent induction of the positively regulated MyHC{alpha} gene was similar in both TR{alpha}1- and TRß-deficient mice. The data identify a specific role for TR{alpha}1 in the negative regulation of MyHCß, whereas TR{alpha}1 and TRß appear interchangeable for hormone-dependent induction of MyHC{alpha}. This suggests that TR isoforms exhibit distinct specificities in the genes that they regulate within a given tissue type. Thus, dysregulation of MyHCß is likely to contribute to the critical role of TR{alpha}1 in cardiac function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ACTIVE FORM of thyroid hormone (TH), T3, plays a central role in the control of cardiac function by interacting with intracellular TRs. TRs are ligand- dependent transcription factors encoded by two different genes in mammals, each giving rise to several splice variants (1, 2, 3, 4, 5). The best characterized isoforms are TR{alpha}1, TR{alpha}2, TRß1, and TRß2, of which all but TR{alpha}2 bind T3 in their C-terminal domains. Deletion studies in mice have shown that loss of TR{alpha}1 slows heart rate and prolongs ventricular repolarization (6), whereas the loss of TRß slightly elevates heart rate, probably through the higher levels of T3 in these mice acting through TR{alpha}1 (7, 8). Deletion of both TR{alpha}1 and TRß results in a similar phenotype as deletion of TR{alpha}1 alone, thus indicating that TR{alpha}1 has the primary role in regulating heart rate (Refs. 9, 10, 11).

The cardiac muscle myosin heavy chain (MyHC) {alpha} and ß genes mediate contractility and are critical for normal heart function (12, 13, 14). The MyHC genes are known to be regulated by T3, suggesting that their regulation underlies some of the major actions of T3 in heart. The MyHC {alpha} and ß proteins form homo- or heterodimers: MyHC{alpha} homodimers form the high- ATPase, V1 isoform; MyHCß homodimers form the low-ATPase, V3 isoform; and MyHC{alpha}/ß heterodimers form the V2 isoform (for review see Ref. 15). Each of these native myosin isoforms contains the same complement of myosin light chain. The compositions of myosin reflect the mechanical performance of the heart. In adult rat with fast contracting ventricles, MyHC{alpha} (V1) dominates (80–90%), whereas, during embryonic and fetal development, MyHCß is the predominant isoform. The shift in expression of MyHC at birth is coincident with a perinatal increase in circulating TH levels, indicating that TH is an important mediator of this transition. Thyroid response elements (TREs) have been located in both MyHC{alpha} and MyHCß genes, with MyHC{alpha} being positively regulated and MyHCß negatively regulated (4, 16, 17).

TRs interact with specific TREs in target genes and thereby activate or repress transcription (18, 19). On a positive TRE, TR can activate gene expression in the presence of T3 and repress basal gene transcription in the absence of T3, thus indicating that TRs can also regulate transcription in a ligand-independent way. However, expression of certain target genes is down-regulated by the ligand-bound receptor via a negative TRE (19, 20).

Here we investigated the specific roles of the individual TR isoforms in regulation of the cardiac MyHC {alpha} and ß genes, using TR{alpha}1- and TRß-deficient strains. Our results show that the MyHC genes are regulated by TRs in an isoform- and target gene-specific manner and identify molecular defects that underlie the cardiac phenotype in TR{alpha}1-deficient mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Expression of Cardiac MyHCß RNA in TR-Deficient Mice
Since TH is known to regulate the expression of MyHC{alpha} positively and MyHCß negatively, we determined the ratio of MyHC{alpha} to MyHCß expression in different TR-deficient mouse strains. We prepared polyA+ RNA from the hearts of 4- to 6-month-old mice from three different TR-deficient strains and their corresponding wild-type (wt) animals. TR{alpha}1-/- mice produce all TR isoforms except TR{alpha}1, TRß-/- mice express none of the TRß isoforms, whereas TR{alpha}1-/-ß-/- animals express only TR{alpha}2 (Refs. 6 11A, 21, 22).

The Northern blot analyses (Fig. 1Go) showed that the expression of MyHCß was elevated 10- to 20-fold in TR{alpha}1-/- and 50-fold in TR{alpha}1-/-ß-/- mice, when comparing the different TR-deficient strains with their corresponding wt mice. This 50-fold induction of MyHCß in TR{alpha}1-/-ß-/- mice was observed in repeated experiments (Fig. 1Go and data not shown). A slight elevation of MyHCß was also seen in TRß-/- mice. In contrast, no major difference in MyHC{alpha} expression was detectable in the different mouse strains. The results suggest that the different TR isoforms have distinct functions in regulation of the MyHCß gene and indicate that TR{alpha}1 has the primary role in regulation of basal expression of the MyHCß gene. The exacerbated elevation of MyHCß in TR{alpha}1-/-ß-/- mice, however, reveals the same phenotype in the absence of all known TRs and indicates a minor contributory role for TRß isoforms.



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Figure 1. Elevated MyHCß Expression in TR{alpha}1 -/- and TR{alpha}1-/-ß-/- Mice

Autoradiograph of Northern blots containing polyA+ RNA (5 µg) from wt and TR{alpha}1-, TRß-, or TR{alpha}1/ß-deficient mice. Filters were hybridized with specific probes for MyHC{alpha}, MyHCß, and G3PDH. Levels of RNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt mice, which was set to 1.0. Each lane represents RNA pooled from two animals.

 
Hormonal Regulation of Cardiac MyHC RNA Expression by TR{alpha}1-/- and TRß-/- Mice
To assess the individual capacities of TR{alpha}1 and TRß to regulate MyHC expression in vivo, the response of hypothyroid TR{alpha}1-/- and TRß-/- mice to increasing doses of injected T3 was first determined. Animals (six mice per group) were first made hypothyroid by dietary treatment [iodine-deficient diet plus methimazole (LID + MMI)], after which the mice were injected for 5 d with either a low dose of T3 (0.05 µg T3/d; LID + MMI + lT3) or a higher dose (0.2 µg T3/d; LID + MMI + hT3). Table 1Go shows the resultant serum levels of free T3 (FT3) and T4 (FT4). The higher dose of injected T3, raised the level of FT3 to 19–24 pM in the TRß-/- and corresponding wt strains. It is suggested that these mice yielded higher levels of FT3 as compared with the TR{alpha}1 strain due to their different genetic backgrounds, which also results in distinct levels of 5'-deiodinase in peripheral organs (23).


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Table 1. FT4 (pmol/liter) and FT3 (pmol/liter) at Baseline (Normal Diet), During TH Deprivation (LID+MMI) and T3 Treatment (LID+MMI+T3)

 
PolyA+ RNA was prepared from hearts collected before treatment, after LID + MMI diet, and after T3 injections. Figure 2Go, A and B, shows that although untreated TR{alpha}1-/- mice expressed 13-fold higher levels of MyHCß RNA than the control animals, hypothyroidism resulted in similar, elevated levels in both animal groups. Injection of the low dose of T3 resulted in a 20-fold reduction in the wt animals, whereas only a 6-fold reduction was seen in TR{alpha}1-/- mice. The high dose of T3 reduced the levels in both animal groups to below that seen in untreated wt mice. This shows that the TRß isoforms, at normal or low hormone levels, are insufficient for normal suppression of the MyHCß gene, whereas higher hormone levels enable TRß to suppress fully.



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Figure 2. Effects of TH on MyHC{alpha} and MyHCß in TR{alpha}1-/- Mice

A, Representative autoradiograph of Northern blots containing mRNA (2 µg) from wt and TR{alpha}1-deficient mice on normal diet (ND), diet inducing hypothyroidism (LID + MMI), or injected with a low (LID + MMI + lT3) or high dose (LID + MMI + hT3) of T3. Levels of RNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt euthyroid mice, which was set to 1.0. Each lane represents RNA from one animal. B, Bar diagram showing relative expression levels (mean ± SEM) of MyHCß in wt and TR{alpha}1-/- mice. C, Bar diagram showing relative expression levels of MyHC{alpha} in wt and TR{alpha}1-/- mice.

 
In both TRß-/- and wt mice the induction of hypothyroidism caused an increase in MyHCß RNA (Fig. 3AGo). However, the increase was only 16-fold in the TRß-deficient mice as compared with 170-fold in wt controls. This reflects the higher TH levels observed in TRß -/- animals achieved after the LID + MMI treatment and indicates that the treatment was not sufficient to achieve severe hypothyroidism in these mice. After T3 injections the MyHCß gene was suppressed equally in both TRß-/- and wt mice (Table 1Go). We conclude from these experiments that TR{alpha}1 is needed to repress basal expression of MyHCß in the euthyroid state, and that both TR{alpha}1 and TRß isoforms can repress the MyHCß gene after T3 injections.



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Figure 3. Effects of TH on MyHC{alpha} and MyHCß in TRß-/- Mice

The bar diagrams show relative expression levels in wt and TRß-/- mice of MyHCß (A) and MyHC{alpha} (B). Results were obtained from Northern blots hybridization containing 2 µg RNA from wt and TRß-deficient mice on normal diet (ND), diet inducing hypothyroidism (LID + MMI), or injected with a low (LID + MMI + lT3) or high dose (LID + MMI + hT3) of T3. Levels of RNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt euthyroid mice, which was set to 1.0. Each bar represents mean ± SEM from three animals.

 
In contrast, there was no major difference in MyHC{alpha} expression levels, when comparing the TR{alpha}1-/- and TRß-/- mice with their respective wt controls (Figs. 2CGo and 3BGo). Upon TH deprivation, all groups of mice significantly reduced the MyHC{alpha} expression levels. Subsequent T3 injections resulted in elevated MyHC{alpha} levels in all groups, as compared with their hypothyroid expression levels. Next, we subjected mice devoid of all known T3 binding TRs [TR{alpha}1-/-ß-/- mice, Göthe et al. (21)] to hypo- and hyperthyroidism essentially as was done for the individual TR knockout strains. This allowed us to test whether hormonal regulation of MyHC{alpha} is mediated by nuclear TRs or through nongenomic pathways. Figure 4Go shows that MyHC{alpha} expression levels was unchanged in TR{alpha}1-/-ß-/- mice during hypo- and hyperthyroidism. In this experiment a 2.5-fold higher T3 dose was used, which could explain the higher induction of MyHC{alpha} seen in wt mice. We conclude that regulation of MyHC{alpha} requires nuclear TRs, but it is isoform independent (Figs. 2CGo, 3BGo, and 4Go, and Mansén, A., unpublished data).



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Figure 4. Effects of TH on MyHC{alpha} in TR{alpha} 1-/-ß-/- Mice

A, Representative autoradiograph of Northern blots containing mRNA (2 µg) from wt and TR{alpha}1/ß-deficient mice on normal diet (ND), diet inducing hypothyroidism (LID + MMI), or injected with T3 (0.5 µg T3/d)(LID + MMI + T3). Levels of RNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt euthyroid mice, which were set to 1.0. Each lane represents RNA from one animal. B, Bar diagram showing relative expression levels (mean ± SEM) of MyHC{alpha} in wt and TR{alpha}1-/-ß-/- mice. The LID + MMI-treated wt and TR{alpha}1-/-ß-/- mice had FT3 values of 0.2–0.5 and 2.1–2.4 pM, respectively. These levels were increased by T3 injections to 2.8–4.6 pM in wt and >360 pM in TR{alpha}1-/-ß-/- mice.

 
MyHC Protein Analysis
Next we determined whether the observed RNA levels reflected the amounts of protein. Mice from a separate animal experiment were used in this analysis. The protein analyses showed that the 10- to 20-fold elevation of MyHCß mRNA content in TR{alpha}1-/- mice and the 50-fold increase in TR{alpha}1-/-ß-/- mice caused a 16 ± 3% and 25 ± 4% increase (P < 0.001) in the relative content of the MyHCß protein isoform, respectively (Figs. 5Go, A and B, and 6). No MyHCß protein was detectable in untreated wt or TRß-/- mice.



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Figure 5. MyHC{alpha} and MyHCß Protein Levels in TR{alpha}1-/- and TRß-/- Mice

A, Representative silver-stained SDS separating gel, containing protein from the respective wt, TR{alpha}1-, and TRß-deficient mice on normal diet (ND), diet inducing hypothyroidism (LID + MMI), or injected with a high dose T3 (LID + MMI + hT3). B, Bar diagram showing relative expression levels (mean ± SEM) of MyHCß in wt and TR{alpha}1-/- mice. C, Bar diagram showing relative expression levels of MyHCß in wt and TRß-/- mice. Number of mice in each group analyzed individually is indicated below each bar. The FT3 and FT4 levels for the mice included in the protein analysis were similar to the levels presented in Table 1Go.

 
The relative amounts of MyHC{alpha} and MyHCß protein isoforms were not affected by changes in TH levels in the TR{alpha}1-deficient mice, i.e. the relative amount of the MyHCß protein was 16 ± 3%, 19 ± 5%, and 27 ± 3% in the untreated, LID + MMI-treated, and LID + MMI + T3-treated animals (Fig. 5Go, A and B). The wt mice, in contrast, showed an increase in the MyHCß protein isoform (35 ± 11%) expression as a response to LID + MMI treatment and a decrease in the MyHCß content (17 ± 4%) after the T3 injections.

The 2- to 3-fold elevation of MyHCß RNA seen in the TRß-/- mice did not result in detectable MyHC protein (Fig. 5Go). However, the MyHCß protein content increased in both TRß-/- and wt mice in response to the LID + MMI diet. The 16-fold increase in MyHCß mRNA content in TRß-/- and the 170-fold increase in the wt mice were paralleled by a significant change at the protein level, i.e. the MyHCß protein content was 15 ± 4% in TRß-/- and 28 ± 8% in wt animals after LID + MMI treatment as compared with 0% in the untreated TRß-/- and wt mice (Fig. 5Go, A and C). T3 injections did not induce any further change in MyHC isoform expression in either TRß-/- or wt mice.

The MyHCß contents were similar in untreated (25 ± 4%) and LID + MMI-treated (19%, n = 1) TR{alpha}1-/-ß-/- mice, and the MyHCß protein contents observed in the LID + MMI-treated (34 ± 18%) wt mice did not differ from that in TR{alpha}1/ß-deficient mice (Fig. 6).

The relatively high MyHCß protein content, in spite of the low mRNA level in the animals after T3 injections, is most probably related to the slow turnover rate of cardiac myosin protein [5–6 d half-life (24)] as compared with myosin mRNA [within hours (25)].


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The TR isoforms {alpha}1 and ß regulate distinct functions, e.g. TR{alpha}1 plays an important role in determining basal heart rate, whereas TRß is required for proper auditory functions and eye development, as well as for regulation of several liver enzymes (6, 21, 23, 26, 27, 28, 29). Here, we have studied the specific regulation by TR isoforms of the MyHC {alpha} and ß genes because they are differentially regulated by TH and are expressed in the same cells of the heart. The results show that the negatively regulated MyHCß gene can be regulated by both TR{alpha}1 and TRß. However, the receptors have distinct roles. MyHCß expression was elevated both at the mRNA and protein levels in untreated TR{alpha}1-/- and TR{alpha}1-/-ß-/-, but not in TRß-/- mice, suggesting that TR{alpha}1 is required for suppression of basal levels of MyHCß expression. This is further supported by Gloss et al. (10), who recently noted that TRß-/- mice that had been rendered euthyroid experimentally expressed levels of MyHCß similar to those of wt controls.

The TR{alpha}1-/-ß-/- mice, lacking all known T3-binding receptors, had a 50-fold increase in MyHCß mRNA (Fig. 1Go and data not shown). This increase is similar to that seen in the hypothyroid TR{alpha}1-/- and wt animals. These conclusions are reminiscent of previous observations with the negatively regulated TSHß gene: mice lacking both TR{alpha}1 and TRß (TR{alpha}1-/-ß-/-) have serum TSH and TSHß mRNA levels (Ref. 21 , and Mansén, A., unpublished data) that are similar to those found in severely hypothyroid mice. Thus, a mechanism of MyHCß gene activation by an aporeceptor may not be required.

TR{alpha}1 and TRß also exhibit differences in their ability to suppress MyHCß expression after injection of T3. A low dose of T3 allowed wt mice to down-regulate MyHCß RNA 20-fold, whereas only a 6-fold reduction was seen in the TR{alpha}1-/- mice. This suggests that TRß is less efficient than TR{alpha}1 or, alternatively, that the two receptors act in concert in suppressing MyHCß at low serum levels of T3. However, our data also show that TRß can, at higher serum T3 levels, efficiently suppress MyHCß. Taken together, our results indicate that TR{alpha}1 is more efficient than TRß in suppressing MyHCß. TR{alpha}2 appears to have, at most, a minor effect on MyHCß expression.

The MyHC{alpha} gene is, in contrast to MyHCß, induced by T3. No significant differences in MyHC{alpha} expression were observed in three independent experiments between untreated wt and the respective TR-deficient mice. Furthermore, the regimen used for inducing a hypothyroid state in the mice achieved a similar repression of MyHC{alpha} RNA levels in the TR{alpha}1-/-, TRß-/-, and control strains. Thus, we cannot ascribe a specific TR isoform a role in regulating the MyHC{alpha} gene. It is also noteworthy that the 5-d T3 injection regimen did not significantly increase the MyHC{alpha} levels above those seen in untreated mice, irrespective of the mouse strain studied. We have therefore examined the induction levels of MyHC{alpha} 2 h after injection of T3. This treatment did not result in an induction of MyHC{alpha} above that seen in untreated mice (data not shown). Therefore, the data indicate that in vivo, the major role of TRs in regulation of the MyHC{alpha} gene is to suppress expression during hypothyroidism and to relieve repression when ligand becomes available.

Gloss et al. (10) reported that TR{alpha}1 is expressed 3-fold higher than TRß on the RNA level in the mouse heart. It is therefore possible that the efficient suppression of MyHCß by TR{alpha}1 is due to its higher expression level. A further increase in TR{alpha}1 expression, as seen in the TR{alpha}2 mice (11A ), results in increased efficiency in hormone-dependent down-regulation of MyHCß (Mansén, A., unpublished data), thus supporting the hypothesis that receptor levels are important for gene regulation in vivo. However, we cannot exclude that possible differences between the receptors in binding to promoter-specific TREs or to corepressors also contribute to the relative efficacy of T3-dependent TR{alpha}1-mediated gene suppression. Nevertheless, MyHC{alpha} and MyHCß are expressed in the same cell type, indicating that TR{alpha}1 and TRß have distinct intrinsic gene-regulatory properties in vivo.

Adult mice have a high heart rate and accordingly express little or no MyHCß (15). Recently, Tardiff et al. (30) developed transgenic mice that significantly overexpress MyHCß (+12%) in the heart. These mice exhibit a decreased cardiac contractility, indicating that a shift in cardiac myosin composition, such as those we have described, markedly affects cardiac function. This is further supported by studies on transgenic mice expressing a dominant negative TRß (31, 32). Both mouse strains expressed highly elevated levels of MyHCß RNA, possibly as a consequence of an inability of the mutant TRß to down-regulate the MyHCß gene. Furthermore, a TR{alpha}-deficient mouse strain different from the one used by us showed a decreased MyHC{alpha} expression, which may be correlated to a shift in total myosin composition (10). The mouse strains referred to above have diminished contractile function, which was attributed to decreased MyHC{alpha} and sarcoplasmic reticulum Ca2+ ATPase levels (10, 31, 32). Since the mice analyzed by us carried genetically altered TRs similar to those studied by Gloss et al. (10), it is highly likely that the TR{alpha}1-deficient mice we have studied have similar deficiencies in contractile function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormone Determinations
Free T3 and T4 were measured using the Amerlex-MAB kit (Piscataway, NJ) as described previously (6).

Northern Blot Analyses
Polyadenylated RNA was prepared from frozen heart (ventricle and atrium) (33). Each preparation corresponds to one or two hearts as indicated in the figure legends and 2–5 µg mRNA were loaded per lane. Northern blots were hybridized with oligonucleotide probes specific for MyHC{alpha} or MyHCß (34). Hybridization with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (CLONTECH Laboratories, Inc., Palo Alto, CA) served as a control for equivalent loading of mRNA. Levels of MyHC{alpha} and MyHCß mRNA expression were normalized to that of G3PDH mRNA using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and BAS-2500 film (Fuji Photo Film Co., Ltd., Stamford, CT) for quantification. Data are presented as means ± SEM.

Animals and Experimental Setup
The Institutional Animal Care and Use Committee approved the studies. Genotyping was done by PCR or Southern blot analysis (6, 22). The genetic backgrounds of the mice differ among the different mouse strains (6, 21, 22). However, all wt control and TR-deficient mice used in this study were obtained from crosses between heterozygotes for the respective knockout strain. The control strains were bred in parallel form with the receptor-deficient strains for three to four generations, at which point new heterozygotes produced inbreeding, followed by renewed heterozygote breeding for homozygote wt and knockout strains. The control strains therefore had the same, mixed genetic background as their respective receptor-deficient strain. In summary, TRß mice hybrid of 129/Sv x C57Bl/6J, TR{alpha}1 mice hybrid of 129/OlaHsd x BALB/c, and the TR{alpha}1/ß mice were obtained from crosses of the above lines. The allele nomenclature suggested by the Mouse Nomenclature Guidelines & Locus Symbol Registry at The Jackson Laboratory (Bar Harbor, ME) is simplified in the text for readability. Thus Thratm1Ven/tm1Ven is denoted TR{alpha}1-/-, Thrbtm1Df/tm1Df is denoted TRß-/-, and finally Thratm1Ven/tm1VenThrbtm1Df/tm1Df is denoted TR{alpha}1-/-ß-/-.

Hypothyroidism was induced by an initial treatment of 2- to 5-month-old male mice with LID (R584, AnalyCen, Nordic AB, Lidköping, Sweden) for 14 d, followed by addition of 0.05% MMI and 1% potassium perchlorate to the drinking water) for an additional 21 d (LID + MMI). Thereafter, the mice were injected daily with either a low dose T3 (0.05 µg T3/d; LID + MMI + lT3) or a higher dose T3 (0.2 µg T3/d; LID + MMI+ hT3) for an additional 5 d to induce hyperthyroidism (Table 1Go) (27). The low dose of T3 was designed to raise the serum levels in hypothyroid mice to below normal. The treatment with LID + MMI significantly lowered the FT3/FT4 levels as compared with untreated mice. Since all animals given T3 injections were first made hypothyroid, we achieved maximal response to the T3 injections. The experimental setup for the TR{alpha}1-/-ß-/- and their wt controls was essentially as above; however, the LID treatment was extended to 21 d, followed by a period of 5 weeks on LID + MMI treatment, and finally 5 d of T3 injections (0.5 µg T3/d). Serum and organs were collected 1 d after the last T3 injection. A total of 24 mice of each TR{alpha}1-/- and TRß-/- plus an additional 24 of the respective wt animals was used in the experiments. The number of TR{alpha}1-/-ß-/- and wt mice used was 18 of each genotype. Six animals from each group were killed for analysis at each change of diet, although serum samples for hormone determinations were obtained from all mice. Animals that responded well to the dietary treatments, as judged by their FT3 and FT4 levels (Table 1Go), were selected for further Northern analyses.

The experiments in Fig. 1Go have been repeated three times; those in Figs. 2Go and 3Go were repeated four times, respectively, and that in Fig. 4Go was repeated once. The samples shown in Fig. 1Go represent a pool of RNA from two animals (females, 4 to 6 months old) of each genotype (12 mice total).

Electrophoretic Separation of Cardiac MyHC Isoform Composition
Frozen hearts (left and right ventricles) from a total of 88 mice of different TR strains were sectioned 10 µm thick with a cryotome (2800 Frigocut-E, Reichert-Jung GmbH, Heidelberg, Germany) at -20 C. Appropriate amounts of sample buffer (333 µl sample buffer/1 mg of wet tissue) were added according to the areas of each cross-section (3 x 105 µm2/1 µl sample buffer). The loading volumes of each sample were kept at 4 µl.

The MyHC composition was determined by SDS-PAGE (35). The total bis-acrylamide concentrations were 4% (wt/vol) in the stacking gel and 7% in the running gel (0.75 mm thick), with acrylamide: N,N'-methylene-bis-acrylamide in the ratio of 50:1. The gel matrix (both stacking and separating gels) included 5% glycerol as described previously (36). The gels were placed in the electrophoresis apparatus (SE 600 vertical slab gel unit, Hoefer Scientific, San Francisco, CA) connected to a power supply and a cooling unit. Electrophoresis was performed at 215 V for 22 h with a Tris-glycine electrode buffer (pH 8.3) at 10 C. Separating gels were silver stained and subsequently scanned in a soft laser densitometer (Molecular Dynamics, Inc.) with a high spatial resolution (50-µm pixel spacing) and 4,096 OD levels to determine the relative contents of the MyHC isoforms. The volume integration function was used to quantify the relative amount of protein (37), and background activity was subtracted from all pixel values (ImageQuant Software version 3.3, Molecular Dynamics, Inc.).

Means and SEM values were calculated from individual values by standard procedures. A two-way ANOVA was applied to test for the effect of TR deficiency and TH levels within each mouse strain. Differences were considered significant at P < 0.05.



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Figure 6. MyHC{alpha} and MyHCß Protein Levels in TR{alpha}1-/-ß-/- Mice

A, Representative silver-stained SDS separating gel, containing protein from the respective wt and TR{alpha}1/ß-deficient mice on normal diet (ND) and diet inducing hypothyroidism (LID + MMI). B, Graphic results of relative expression levels (mean ± SEM) of MyHCß in wt and TR{alpha}1-/-ß-/- mice. Number of mice in each group analyzed individually is indicated below each bar. The FT3 value in the LID + MMI-treated TR{alpha}1-/-ß-/- mouse was 2.1 pM.

 

    ACKNOWLEDGMENTS
 
We thank Kristina Nordström for animal setup, handling, and analyses.


    FOOTNOTES
 
This work was supported by grants from the Swedish Cancer Society, the Karolinska Institute, the Human Frontiers Science Program (to B.V.), the Muscular Dystrophy Association (to L.L.), the March of Dimes Birth Defects Foundation, National Institutes of Health, and a Hirschl Award (to D.F.).

Abbreviations: FT3, Free T3; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; hT3, high T3; LID, synthetic iodine-deficient diet; lT3, low T3; MMI, methimazole; MyHC, cardiac muscle myosin heavy chain; TH, thyroid hormone; TRE, thyroid response element; wt, wild type.

Received for publication May 18, 2001. Accepted for publication August 8, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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