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1Division of Endocrinology and Department of Medicine, North Shore University Hospital/New York University School of Medicine and North Shore-LIJ Research Institute, Manhasset; and 2Department of Cell Biology, New York University School of Medicine, New York, New York
Submitted 30 August 2004 ; accepted in final form 13 October 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-myosin heavy chain (MHC) transcription in hypothyroid hearts is rapid, exhibiting zero-order kinetics, whereas the repression of 
-MHC in these same hearts is much slower. To elucidate the mechanism for T3 transcriptional as well as posttranscriptional regulation of both MHC gene isoforms, we used an RT-PCR-based transcription assay and the RNA polymerase II inhibitor actinomycin D in an in vivo model to simultaneously measure specific - and -MHC heterogeneous nuclear RNA (hnRNA), mRNA kinetics, and MHC antisense RNA. In vivo actinomycin D treatment blocked -MHC transcription in euthyroid rats by >80% at 2 h and suggested a half-life of -MHC hnRNA of 
1 h, whereas actinomycin D inhibited -MHC transcription in hypothyroid rats by >75% at 6 h, suggesting a significantly longer hnRNA half-life of 4 h. The effect of actinomycin D on -MHC transcription was independent of T3. T3 treatment in hypothyroid animals caused -MHC mRNA to decline more rapidly than -MHC hnRNA, demonstrating, for the first time, a posttranscriptional mechanism(s). The measured change in -MHC mRNA half-life indicates a T3-mediated destabilization of -MHC mRNA. To understand the mechanism by which T3 destabilizes -MHC mRNA, we measured -MHC antisense RNA. -MHC antisense RNA is present in euthyroid myocytes, but levels are not significant in hypothyroid myocytes. This differential expression may explain some of the effects of T3 on MHC posttranscriptional regulation.
thyroid hormone; myocardium; gene transcription; actinomycin D; RNA stability; heterogeneous nuclear RNA
- and -MHC (4, 8, 23, 25). In euthyroid rats, -MHC is maximally expressed and -MHC is repressed. In thyroidectomized rats, the opposite occurs (8, 9, 23, 25). Our understanding of the regulation of the MHC genes is primarily derived from the T3-mediated positively regulated -MHC, whereas less is understood regarding the negative regulation of the -MHC gene (2, 3, 10, 25, 34, 41). -MHC is the predominant myofibrillar protein in the human heart, and it has been shown by others that, in the failing human heart, -MHC mRNA is decreased and -MHC mRNA is increased, possibly contributing to decreased contractility in the failing heart (21, 26, 31).
T3 mediates the transcription of -MHC as follows: T3 binds to nuclear receptor proteins (TRs), which in turn bind to TR response elements (TREs) in the promoter regions of positively regulated genes such as -MHC (4, 9, 14, 42). In the presence of T3, TRs are known to associate with coactivator complexes, (i.e., Trap220 and SRC-1), which possess histone acetyltransferase activity and activate genes (15, 31, 42). In the absence of T3, TRs recruit corepressor complexes (i.e., NCoR) and repress transcription (13, 42).
The mechanisms by which T3 mediates the transcription and repression of -MHC, a negatively regulated gene, are not fully understood. Several studies have identified the proximal -MHC promoter as an important region for transcriptional regulation, as it is for several other negatively regulated genes (10, 27, 41). However, it is not known whether TRs bind directly to the -MHC promoter for activation in hypothyroid animals or repression in the presence of T3.
To further study the T3-mediated regulation of MHC gene expression, we developed an RT-PCR assay to measure transcription in an in vivo model by quantitating specific - and -MHC heterogeneous nuclear RNA (hnRNA) from rat hearts. This technique permits, for the first time, the study of the temporal transcriptional response to various humoral and hemodynamic stimuli in vivo (8, 16, 17, 28, 33). In hypothyroid rats, induction of -MHC by T3 is rapid, but repression of -MHC transcription by T3 is not detectable until 6 h later, suggesting that separate molecular mechanisms and sites of action are involved in the coordinated regulation of these genes (8).
To explore the transcriptional and posttranscriptional activity of both genes, we used the RNA polymerase II inhibitor actinomycin D. We report here that -MHC hnRNA has a longer half-life than -MHC hnRNA and that -MHC mRNA is destabilized by T3. To explore a possible mechanism for the T3-mediated destabilization of -MHC mRNA, we measured -MHC antisense RNA in rat hearts. -MHC antisense RNA was demonstrated in euthyroid, but not hypothyroid, hearts and may serve as a site of posttranscriptional regulation for the effects of T3.
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MATERIALS AND METHODS |
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T3 treatment in hypothyroid rats. Tx rats (n = 3 in each group) were treated with a single injection of 1 µg of T3 (ICN Biomedicals, Aurora, OH) and killed at 0.5, 2, 6, 12, 24, or 36 h after treatment. Hearts were quickly excised and weighed, and left ventricles (LVs), including the septum, were rapidly frozen in liquid nitrogen and stored at –80°C until extracted for RNA. Three animals were used for each time point.
Actinomycin D and T3 treatment in hypothyroid and euthyroid rats. Tx and euthyroid rats were given actinomycin D (1 mg/kg; Sigma-Aldrich, St. Louis, MO) in PBS as a single intramuscular injection of 0.2 ml with or without a simultaneous intramuscular injection of 2 µg of T3 in 0.2 ml of PBS. Animals were killed at 2, 6, 12, 24, or 48 h after injection. Hearts were excised, LVs were weighed, and blood was collected for serum T3 analysis. Tissue was frozen as described above until used for RNA extraction. Three animals were used for each time point.
Total RNA isolation. Total RNA was extracted from frozen LV samples using the guanidinium thiocyanate method, as previously described (2).
Measurements of hnRNA.
We measured transcription using an RT-PCR-based transcription assay to quantitate hnRNA, as previously described (8). hnRNA is the primary transcript that is subsequently spliced and processed to form mature mRNA. Briefly, 50 µg of total RNA were treated with DNase I and subjected to the RNeasy miniprotocol for RNA cleanup (Qiagen, Valencia, CA). RT-PCR was performed as previously described with 2 µg of RNA using primers specific for - or -MHC hnRNA (8). Individual reactions were set up with the reverse primers for - or -MHC hnRNA, which annealed to sequences within the first intron of each gene: 5'-GACACAGAAAGAAAGGAAGGAT-3' for -MHC949R (GenBank accession no. AH002207) (23) and 5'-ACACACGCGCACACACTAGCA-3' for -MHC1456R (GenBank accession no. X16291).
PCR were performed as previously described (8) with the same reverse primers described for RT (-MHC949R and -MHC1456R) and forward primers that annealed to sequences within the first exon of each gene: 5'-ATTTCTCCATCCCAAGTAAG-3' for -MHC614F and 5'-TGAGCATTCTCCTGCTGTTTC-3' for -MHC1144F. The PCR product (10 µl) was run on a 2% agarose gel with ethidium bromide and quantitated by densitometry using Bio-Rad Quantity 4.2.2 software. All RT reactions were done in duplicate.
Measurements of fully processed mRNA.
RT-PCR of - and -MHC mRNA was performed as described above, but with 2 ng of total RNA and reverse primers that annealed to sequences at the 3'-untranslated end of the respective mRNAs [-MHC5892R (5'-GTGGGATAGCAACAGCGAGGC-3') and -MHC5869R (5'-CTCCAGGTCTCAGGGCTTCAC-3')], a region that differs between - and -MHC. PCR of mRNA was accomplished using the mRNA reverse primers described above and forward primers: -MHC5593F (5'-CTACCAGACAGAGGAAGACAAG-3') and -MHC5579F (5'-GACAGGAAGAACCTACTGCG-3').
Measurements of - and -MHC antisense RNA.
To quantitate - or -MHC antisense RNA in euthyroid and hypothyroid rat hearts, we performed RT on 2 µg of total RNA as described above but used the forward primers -MHC614F and -MHC1144F to synthesize the cDNA copy of the antisense hnRNA. Before amplification of - or -MHC antisense RNA by PCR, RT reactions were incubated with 1 µl of RNase A at 37°C for 20 min. PCR was carried out as described above with the same forward primers and reverse primers used for amplification of - or -MHC hnRNA.
Statistical analysis. Values are means ± SE. Statistical differences between values were evaluated by unpaired Student’s t-test with significant probability at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and -MHC gene transcription.
In response to T3 treatment (1 µg/animal) of hypothyroid animals, -MHC hnRNA, as a measure of transcription, appeared at 30 min and was 135 ± 16% of euthyroid levels at 6 h (Fig. 1). Simultaneous measures of -MHC hnRNA indicated no significant change in transcription rate until 6 h after treatment, when it was decreased by 14 ± 2%. Thus the initiation of transcription of -MHC and the suppression of transcription of -MHC can be temporally dissociated. To identify the mechanisms involved in these separate processes, we used actinomycin D to directly inhibit gene transcription.
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-MHC hnRNA.
To validate the effectiveness of in vivo actinomycin D treatment, we tested the ability of actinomycin D to block the T3-mediated induction of -MHC hnRNA in hypothyroid rats. Treatment with actinomycin D + T3 (2 µg/animal) prevented the increase in -MHC hnRNA completely at 2 h and by >80% at 6 h (Fig. 1). Analysis of serum T3 and LV weights performed when euthyroid and hypothyroid animals were killed indicates no differences as a result of treatment with actinomycin D or T3 on LV weights at that time point (Table 1) (7, 24).
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-MHC hnRNA and mRNA in euthyroid animals.
In the young adult euthyroid rat, where -MHC hnRNA and mRNA are expressed as the predominant MHC isoform, actinomycin D treatment significantly decreased the amount of -MHC hnRNA (transcription) by 77 ± 2% within 2 h and by 86 ± 3% at 6 h, with a half-life of 1 h (Fig. 2). In contrast, simultaneous measures of -MHC mRNA initially increased at 2 and 6 h and then declined steadily, with a half-life of 30 h (data not shown). This initial increase reflects stabilization of -MHC mRNA in the presence of a persistent low level of transcription.
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-MHC hnRNA and mRNA in response to T3.
Hypothyroid rats were treated with a single injection of T3 (1 µg) and then killed 0.5, 2, 6, 12, 24, or 36 h after treatment for analysis of cardiac -MHC hnRNA and mRNA (Fig. 3). -MHC hnRNA was unchanged at 2 h and first significantly declined by 14 ± 2% at 6 h after T3 treatment, with a nadir of 59% ± 2 at 36 h. In contrast, after T3 treatment, -MHC mRNA declined within 2 h by 29 ± 6% (P < 0.05 compared with hnRNA) and continued to decline to a nadir of 34 ± 1% of hypothyroid levels at 36 h. The half-lives of -MHC hnRNA and mRNA were calculated using the initial 12 h after T3 treatment and were 20 and 12 h, respectively.
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-MHC hnRNA and mRNA.
To understand the temporal disparity between the T3-mediated decline in -MHC hnRNA and mRNA, we treated hypothyroid rats with actinomycin D and focused on the responses observed at 2 and 6 h. At 2 h after actinomycin D treatment, -MHC hnRNA expression declined to 62 ± 4% of hypothyroid levels. At 6 h after actinomycin D treatment, -MHC hnRNA levels were 30 ± 2% of maximal, demonstrating a half-life of 4 h. The addition of T3 to actinomycin D did not further decrease the -MHC hnRNA content significantly, demonstrating a half-life of 3.5 h. -MHC hnRNA levels were significantly lower after treatment with actinomycin D or actinomycin D + T3 than after treatment with T3 alone (P < 0.01; Fig. 4A).
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-MHC mRNA in hypothyroid rats after actinomycin D treatment declined by 51 ± 1% at 2 h, as shown for -MHC hnRNA. Similar to -MHC hnRNA, the addition of T3 to actinomycin D had no significant effect on -MHC mRNA content at 2 or 6 h (Fig. 4B). The half-life of -MHC mRNA after treatment with actinomycin D and actinomycin D + T3 was 2 h.
Expression of - and -MHC antisense RNA.
Measurements of antisense RNA by PCR were performed using the same - and -MHC hnRNA primer sets. However, RT was performed using the specific forward primers. We were unable to detect any -MHC antisense RNA in hypothyroid or euthyroid rat hearts (12, 22). However, -MHC antisense RNA was present in euthyroid, and at substantially lower levels in hypothyroid, rat hearts. In euthyroid rat hearts, -MHC antisense RNA was 30% of the -MHC sense hnRNA expressed in the same samples (Table 2).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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and are regulated by T3 in a reciprocal fashion (3, 25). The observation that these two genes are located in tandem on chromosome 14 in humans and on chromosome 17 in the rat previously suggested that the activation of -MHC was temporally and mechanistically linked to the suppression of -MHC (11, 19, 24). However, as we recently showed, this regulation does not occur via a single coupled mechanism (8).
The T3-mediated (5 µg/kg) induction of -MHC transcription in hypothyroid rats occurs within minutes of T3 administration, whereas the repression of -MHC transcription in these same animals occurs more slowly over hours (8). In the present model for T3-mediated regulation of -MHC transcription, TRs constitutively occupy TREs and, in the presence and absence of T3, replace coactivator complexes for corepressor complexes, respectively (4, 7). This permits a fairly rapid response to changes in thyroid hormone levels (8). However, the mechanism by which T3 mediates the repression (with T3) and induction (without T3) of -MHC transcription is not known. There is no definitive evidence of a role for TRs at the -MHC promoter (27), and T3-mediated regulation may occur off the promoter, inasmuch as no traditional TREs that play a role in the TR-mediated regulation of this gene have been identified (38). It has been hypothesized that T3-regulated genes that do not possess TREs may be posttranscriptionally regulated. Cuadrado and colleagues (6) report that the neuron-specific neuronal HuD, an RNA-binding protein, is regulated by T3 and acts as an mRNA stability regulator under T3 control. However, no comparable proteins have been identified in cardiac myocytes.
To extend and further our understanding of the role of T3 in the expression of the cardiac-specific MHC genes, we studied - and -MHC mRNA stability simultaneously with its effects on transcription. T3 has been shown to regulate the stability of several other mRNAs. T3 decreased poly(A) tail length of the pituitary genes thyrotropin -subunit mRNA, a negatively regulated gene, and growth hormone mRNA, a positively regulated gene, decreasing the stability of both mRNAs (20, 37, 40). T3 increased the poly(A) tail length of hepatic NADPH-cytochrome P-450 reductase, but only transiently, inasmuch as cytoplasmic poly(A) tail length decreased 16 h later (36). However, overall stability of hepatic NADPH-cytochrome P-450 reductase mRNA was also decreased after the addition of T3. Sindhwani et al. (35) demonstrated that -MHC mRNA was polyadenylated at multiple sites, with an increase in the longest subspecies and a decrease in the shortest subspecies in the hypothyroid heart (35). They also identified two -MHC splice variants that were not affected by thyroid state. The significance of these findings as they relate to stability is not clear.
In the present study, 2 h after treatment with T3, when there was no detectable decline in -MHC transcription, -MHC mRNA content had already fallen by 30%, suggesting that T3 acts through a posttranscriptional mechanism to destabilize -MHC mRNA (12). After the initial destabilization of -MHC mRNA by T3, the further decline continued in parallel with the rate of repression of transcription (Fig. 3) (8). To better understand the potential transcriptional and posttranscriptional mechanisms by which T3 mediates the expression of these two genes, we undertook studies using actinomycin D, a potent inhibitor of DNA-dependent RNA synthesis (36). Animals were treated with actinomycin at 1 mg/kg (20), which was sufficient to block the in vivo induction of -MHC completely at 2 h and by >80% at 6 h. At 6 h after treatment of euthyroid rats with actinomycin D, -MHC transcription was reduced by 90%. -MHC hnRNA and mRNA content declined faster after treatment with actinomycin D than after treatment with T3 alone. The addition of T3 to actinomycin D treatment did not further decrease -MHC hnRNA or mRNA content, suggesting that the effect of actinomycin D (inhibition of RNA polymerase II) is faster and more complete than the T3-mediated inhibition of -MHC hnRNA and mRNA. These data suggest that a mechanism other than direct inhibition of transcription may be responsible for the T3-mediated repression. It should be noted that contractility, as well as T3, can affect MHC gene expression, both transcriptionally and posttranscriptionally (30). An independent effect of T3 and/or actinomycin D on contractility may contribute to the changes in MHC hnRNA and mRNA levels.
Haddad and colleagues (12) reported an antisense mechanism coordinating cardiac MHC ( and ) gene switching that involves the inhibition of -MHC hnRNA processing. Their findings demonstrate that -MHC transcription is coupled to -MHC antisense transcription and that antisense RNA serves to regulate the content of -MHC mRNA by targeting -MHC for degradation. This antisense RNA was determined to be full-length hnRNA. Therefore, using our -MHC hnRNA primers, we have also identified the presence of -MHC antisense RNA in euthyroid rat hearts but have not identified significant levels in hypothyroid rat hearts, supporting a role for -MHC antisense RNA in the regulation of -MHC repression in the presence of T3. To identify a mechanism for the stabilization of -MHC mRNA after treatment with actinomycin D, we hypothesized that the absence of a short-lived -MHC antisense RNA may account for the increased stability of -MHC mRNA. To validate the RT-PCR method for antisense RNA, we found it necessary to ensure the adequacy of the denaturation of the reverse transcriptase as described in MATERIALS AND METHODS (12, 22). Our findings, however, did not support this hypothesis, because we could detect no antisense -MHC in ventricular myocytes.
It is important to note that the COOH-terminal domain of RNA polymerase II directs splicing and polyadenylation factors to the hnRNA to facilitate processing, and it is possible that both of these coordinated processes may have been interrupted by treatment with actinomycin D (1). However, given that there were opposite effects of actinomycin D treatment on - and -MHC mRNA stability, this was not likely a factor.
In summary, we have demonstrated that there is a distinction between transcription of - and -MHC, both temporally and mechanistically, and that posttranscriptional mechanisms are involved in the regulation of mRNA content for both of these genes, destabilizing -MHC mRNA and stabilizing -MHC mRNA. The presence of -MHC antisense RNA may play a role in the T3-mediated negative regulation of -MHC expression in euthyroid cardiac myocytes.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and -myosin heavy chains and sarcomeric -actins are regulated through transcriptional mechanisms. Results from nuclear run-on assays in isolated rat cardiac nuclei. J Biol Chem 267: 12979–12985, 1992.-myosin heavy chain (MHC) expression. Biochem Biophys Res Commun 199: 1482–1488, 1994.[CrossRef][ISI][Medline]
- and -myosin heavy chain mRNA levels in rabbit heart. Biochemistry 23: 1596–1599, 1984.[CrossRef][Medline]
-myosin heavy chain in hypertrophied, failing ventricular myocardium. J Clin Invest 100: 2315–2324, 1997.[ISI][Medline]
- and -myosin heavy chain genes are organized in tandem. Proc Natl Acad Sci USA 81: 2626–2630, 1984.-myosin heavy chain promoter in the heart. Endocrinology 137: 802–808, 1996.[Abstract]
gene. Nucleic Acids Res 20: 1793–1799, 1992. cardiac myosin heavy chain gene expression. J Biol Chem 269: 3272–3276, 1994.-subunit messenger ribonucleic acid by thyroid hormone in murine thyrotrope tumor cells: a conserved mechanism across species. Endocrinology 139: 1093–1100, 1998.-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer. Am J Physiol Cell Physiol 276: C883–C891, 1999.This article has been cited by other articles:
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  J. Giger, A. X. Qin, P. W. Bodell, K. M. Baldwin, and F. Haddad Activity of the beta-myosin heavy chain antisense promoter responds to diabetes and hypothyroidism Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3065 - H3071. [Abstract] [Full Text] [PDF]
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K.-O. Larsen, I. Sjaastad, A. Svindland, K. A. Krobert, O. H. Skjonsberg, and G. Christensen Alveolar hypoxia induces left ventricular diastolic dysfunction and reduces phosphorylation of phospholamban in mice Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H507 - H516. [Abstract] [Full Text] [PDF]
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T. D. McClure, M. E. Young, H. Taegtmeyer, X.-H. Ning, N. E. Buroker, J. Lopez-Guisa, and M. A. Portman Thyroid hormone interacts with PPAR{alpha} and PGC-1 during mitochondrial maturation in sheep heart Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H2258 - H2264. [Abstract] [Full Text] [PDF]
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