Cardiac myosin heavy chain gene regulation by thyroid hormone involves altered histone modifications.

The antithetical regulation of cardiac α- and ß-myosin heavy chain (MHC) genes by thyroid hormone (T(3)) is not well understood but appears to involve thyroid hormone interaction with its nuclear receptor and MHC promoters as well as cis-acting noncoding regulatory RNA (ncRNA). Both of these phenomena involve epigenetic regulations. This study investigated the extent that altered thyroid state induces histone modifications in the chromatin associated with the cardiac MHC genes. We hypothesized that specific epigenetic events could be identified and linked to cardiac MHC gene switching in response to a hypothyroid or hyperthyroid state. A hypothyroid state was induced in rats by propylthiouracil treatment (PTU), whereas a hyperthyroid (T(3)) was induced by T(3) treatment. The left ventricle was analyzed after 7 days for MHC pre-mRNA expression, and the chromatin was assessed for enrichment in specific histone modifications using chromatin immunoprecipitation quantitative PCR assays. At both the α-MHC promoter and the intergenic region, the enrichment in acetyl histone H3 at K9/14 (H3K9/14ac) and trimethyl histone H3 at K4 (H3K4me3) changed in a similar fashion. They were both decreased with PTU treatment but did not change under T(3), except at a location situated 5' to the antisense intergenic transcription start site. These same marks varied differently on the ß-MHC promoter. For example, H3K4me3 enrichment correlated with the ß-promoter activity in PTU and T(3) groups, whereas H3K9/14ac was repressed in the T(3) group but did not change under PTU. Histone H3K9me was enriched in chromatin of both the intergenic and α-MHC promoters in the PTU group, whereas histone H4K20me1 was enriched in chromatin of ß-MHC promoter in the normal control and T(3) groups. Collectively, these findings provide evidence that specific epigenetic phenomena modulate MHC gene expression in altered thyroid states.

Regulation of antisense RNA expression during cardiac MHC gene switching in response to pressure overload.

Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster alpha- to slower beta-MHC isoform. The expression of the beta- and alpha-MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense beta-RNA were analyzed in three regions of the normal control (NC) and 12-day pressure-overloaded (AbCon) hearts: the left ventricle apex, left ventricle base, and the septum. The RNA analyses in the AbCon heart targeted both the 5' and the 3' ends of each RNA molecule. beta-MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5' or 3' end). In contrast, beta-MHC pre-mRNA expression in the AbCon heart depended on the site of the measurement (5' vs. 3' end). For example, whereas the pre-mRNA did not change when targeted at the 3' end (last intron), it increased significantly in the AbCon heart when measurement targeted the 5' end (2nd intron) of the 25-kb molecule. Analyses of the antisense beta-RNA revealed that its expression in the AbCon heart was significantly decreased relative to control regardless of its measurement site. A negative correlation was observed between the beta-mRNA expression and the antisense beta-RNA (P < 0.05), suggesting an inhibitory role of antisense RNA on the sense beta-MHC gene expression. In contrast, a positive correlation was observed between the antisense beta-RNA and the alpha-MHC pre-mRNA (P < 0.05). This latter observation along with the alpha-MHC gene position relative to that of the beta-antisense suggest that the alpha-MHC sense and beta-antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased beta-MHC expression in the AbCon heart not only is the result of increased beta-MHC transcription but also involves an antisense beta-RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the beta-MHC gene and posttranscriptional processing.

Transcriptional regulation of the type I myosin heavy chain gene in denervated rat soleus.

Denervation (DEN) of rat soleus is associated with a decreased expression of slow type I myosin heavy chain (MHC) and an increased expression of the faster MHC isoforms. The molecular mechanisms behind these shifts remain unclear. We first investigated endogenous transcriptional activity of the type I MHC gene in normal and denervated soleus muscles via pre-mRNA analysis. Our results suggest that the type I MHC gene is regulated via transcriptional processes in the denervated soleus. Deletion and mutational analysis of the rat type I MHC promoter was then used to identify cis elements or regions of the promoter involved in this response. DEN significantly decreased in vivo activity of the -3,500, -2,500, -914, -408, -299, and -215 bp type I MHC promoters, relative to the alpha-skeletal actin promoter. In contrast, normalized -171 promoter activity was unchanged. Mutation of the betae3 element (-214/-190) in the -215 promoter and deletion of this element (-171 promoter) blunted type I downregulation with DEN. In contrast, betae3 mutation in the -408 promoters was not effective in attenuating the DEN response, suggesting the existence of additional DEN-responsive sites between -408 and -215. Western blotting and gel mobility supershift assays demonstrated decreased expression and DNA binding of transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this decrease may contribute to type I MHC downregulation in denervated muscle.

In vivo regulation of the beta-myosin heavy chain gene in hypertensive rodent heart.

The main goal of this study was to examine the transcriptional activity of different-length beta-myosin heavy chain (beta-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by approximately 45% relative to control. Results show that beta-MHC promoter activity of all tested wild-type constructs, i.e., -3500, -408, -299, -215, -171, and -71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the -71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting betae1, betae2, GATA, and betae3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the beta-MHC gene. Collectively, these results indicate that three separate regions on the beta-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between -408 and -3500 bp, 2) a proximal region between -299 and -215 bp, and 3) a basal region within -71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate beta-MHC transcriptional regulation in response to pressure overload.

In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats.

In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

Effects of spaceflight and thyroid deficiency on rat hindlimb development. II. Expression of MHC isoforms.

Both slow-twitch and fast-twitch muscles are undifferentiated after birth as to their contractile protein phenotype. Thus we examined the separate and combined effects of spaceflight (SF) and thyroid deficiency (TD) on myosin heavy chain (MHC) gene expression (protein and mRNA) in muscles of neonatal rats (7 and 14 days of age at launch) exposed to SF for 16 days. Spaceflight markedly reduced expression of the slow, type I MHC gene by approximately 55%, whereas it augmented expression of the fast IIx and IIb MHCs in antigravity skeletal muscles. In fast muscles, SF caused subtle increases in the fast IIb MHC relative to the other adult MHCs. In contrast, TD prevented the normal expression of the fast MHC phenotype, particularly the IIb MHC, whereas TD maintained expression of the embryonic/neonatal MHC isoforms; this response occurred independently of gravity. Collectively, these results suggest that normal expression of the type I MHC gene requires signals associated with weight-bearing activity, whereas normal expression of the IIb MHC requires an intact thyroid state acting independently of the weight-bearing activities typically encountered during neonatal development of laboratory rodents. Finally, MHC expression in developing muscles is chiefly regulated by pretranslational processes based on the tight relationship between the MHC protein and mRNA data.

In vivo regulation of beta-MHC gene in rodent heart: role of T3 and evidence for an upstream enhancer.

Cardiac beta-myosin heavy chain (beta-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length beta-MHC promoter fragments in normal control and in altered thyroid states. The test beta-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control alpha-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that beta- and alpha-MHC genes were individually and reciprocally regulated by thyroid hormone. The beta-to-alpha ratios of reporter gene expression demonstrated an almost threefold larger beta-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the -408-bp beta-MHC promoter construct caused transcriptional activity to drop to null. When studied in the -3, 500-bp beta-MHC promoter, construct activity was reduced ( approximately 100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the beta-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression.

We examined the novel interaction of hyperthyroidism and hindlimb suspension on the pattern of myosin heavy chain (MHC) expression (mRNA and protein) in skeletal muscles. Female Sprague-Dawley rats were assigned to four groups: 1) normal control (Con); 2) thyroid hormone treated [150 micrograms 3,5,3'-triiodothyronine (T3). kg-1. day-1] (T3); 3) hindlimb suspension (HS); or 4) T3-treated and HS (T3 + HS). Results show for the first time the novel observation that the combination T3 + HS induces a rapid and sustained, marked (80-90%) downregulation of type I MHC gene expression that is mirrored temporally by concomitant marked upregulation of type IIb MHC gene expression, as evidenced by the de novo synthesis of type IIb MHC protein in the soleus. The fast type IIx MHC isoform showed a differential response among the experimental groups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing in the plantaris muscles. The fast type IIa MHC was the least responsive to suspension of the MHCs and reflected its greatest responsiveness to T3 treatment while also undergoing differential adaptations in slow vs. fast muscle (increases vs. decreases, respectively). These results confirm previous findings that all four adult MHC genes are sensitive to T3 and suspension in a muscle-specific manner. In addition, we show that T3 + HS can interact synergistically to create novel adaptations in MHC expression that could not be observed when each factor was imposed separately.

Thyroid receptor plasticity in striated muscle types: effects of altered thyroid state.

This study examined nuclear thyroid receptor (TR) maximum binding capacity (Bmax), dissociation constant (Kd), and TR isoform (alpha1, alpha2, beta1) mRNA expression in rodent cardiac, "fast-twitch white," "fast-twitch red," and "slow-twitch red" muscle types as a function of thyroid state. These analyses were performed in the context of slow-twitch type I myosin heavy-chain (MHC) expression, a 3,5,3'-triiodothyronine (T3)-regulated gene that displays varying responsiveness to T3 in the above tissues. Nuclear T3 binding analyses show that the skeletal muscle types express more TRs per unit DNA than cardiac muscle, whereas the latter has a lower Kd than the former. Altered thyroid state had little effect on either cardiac Bmax or Kd, whereas hypothyroidism increased Bmax in the skeletal muscle types without affecting its Kd. Cardiac muscle demonstrated the greatest mRNA signal of TR-beta1 compared with the other muscle types, whereas the TR-alpha1 mRNA signals were more abundant in the skeletal muscle types, especially fast-twitch red. Hyperthyroidism increased the ratio of beta1 to alpha1 and decreased the ratio of alpha2- to alpha1+beta1-mRNA signal across the muscle types, whereas hypothyroidism caused the opposite effects. The nuclear T3 affinity correlated significantly with the TR-beta1 mRNA expression but not with TR-alpha1 mRNA expression. Collectively, these findings suggest that, despite a divergent pattern of TR mRNA expression in the different muscle types, these patterns follow similar qualitative changes under altered thyroid state. Furthermore, TR expression pattern cannot account for the quantitative and qualitative changes in type I MHC expression that occur in the different muscle types.

Effects of isometric training on skeletal myosin heavy chain expression.

This study tested the hypothesis that an isometric resistance-training program induces upregulation of slow myosin heavy chain (MHC) expression in a fast-twitch skeletal muscle. Thus we studied the effects of two resistance-training programs on rodent medial gastrocnemius (MG) muscle that were designed to elicit repetitive isometric contractions (10-12 per set; 4 sets per session) of different duration (8 vs. 5 s) and activation frequency (100 vs. 60 Hz) per contraction during each training session (total of 6 and 12 sessions). Results showed that both training paradigms produced significant increases in muscle weight ( approximately 11-13%) after completion of training (P < 0.05). Significant transformations in MHC expression occurred and involved specifically a decrease in the relative expression of the fast type IIb MHC and concomitant increased expression of the fast type IIx MHC. These adaptations were observed in both the "white" and "red" regions of the MG, and they occurred at both the mRNA and protein levels. These adaptations were detected after only six training sessions. Neither of the training programs produced any change in the relative expression of either the slow type I MHC or the moderately fast type IIa MHC, which can be upregulated in the red MG by chronic functional overload. These findings show that the isometric protocols used in this investigation were not sufficient to induce the hypothesized changes in the myosin heavy chain isoform expression in rodent skeletal muscle.

Analysis of myosin heavy chain mRNA expression by RT-PCR.

An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.