Regulation Of Myosin Heavy Chain Genes Research Articles

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

The antithetical regulation of cardiac α- and β-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 β-MHC promoter. For example, H3K4me3 enrichment correlated with the β-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 β-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.

Novel epigenetic regulation of skeletal muscle myosin heavy chain genes. Focus on “Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading”

skeletal muscle is a highly plastic tissue, capable of responding and adapting to a variety of physiological stimuli, e.g., muscle loading and unloading. Multiple functional phenotypes of skeletal muscle exist throughout the body at the same time. For example, muscles required for antigravity support of the skeleton or sustained locomotion are generally slow-contracting, low force-generating, and relatively resistant to fatigue, while other muscles required for quick, powerful movements are generally fast-contracting, high force-generating, and easily fatigued. In fact, skeletal muscle phenotypes have been characterized using combinations of muscle properties, including metabolic, fatigability, color, innervation, or predominant contractile protein expression. Myosin heavy chain (MHC) is the most abundant protein in skeletal muscle, comprising ∼25% of the total protein content, and is a major component of the complex responsible for generating contractile force in skeletal muscle. At least nine MHC isoforms, each transcribed from their own gene, exist in mammalian striated muscle; but only slow type I MHC and various isoforms of fast type II (IIa, IIx/d, and IIb MHC isoforms) are present in adult limb skeletal muscle that is not undergoing regeneration (16). It is well established that skeletal muscle phenotype, particularly MHC gene expression, can be altered by different states of muscular activity or inactivity, as well as various hormonal and metabolic factors (1).

Dynamics of Myosin Heavy Chain Gene Regulation in Slow Skeletal Muscle

The evolutionarily conserved order of the skeletal muscle myosin heavy chain (MHC) genes and their close tandem proximity on the same chromosome are intriguing and may be important for their coordinated regulation. We investigated type II MHC gene regulation in slow-type muscle fibers undergoing a slow to fast MHC transformation in response to inactivity, 7 days after spinal cord isolation (SI) in rats. We examined the transcriptional products of both the sense and antisense strands across the IIa-IIx-IIb MHC gene locus. A strand-specific reverse transcription (RT)-PCR approach was utilized to study the expression of the mRNA, the primary transcript (pre-mRNA), the antisense RNA overlapping the MHC genes, and both the intergenic sense and antisense RNAs. Results showed that the mRNA and pre-mRNA of each MHC had a similar response to SI, suggesting regulation of these genes at the transcriptional level. In addition, we detected previously unknown antisense strand transcription that produced natural antisense transcripts (NATs). RT-PCR mapping of the RNA products revealed that the antisense activity resulted in the formation of three major products: aII, xII, and bII NATs (antisense products of the IIa, IIx, and IIb genes, respectively). The aII NAT begins in the IIa-IIx intergenic region in close proximity to the IIx promoter, extends across the 27-kb IIa MHC gene, and continues to the IIa MHC gene promoter. The expression of the aII NAT was significantly up-regulated in muscles after SI, was negatively correlated with IIa MHC gene expression, and was positively correlated with IIx MHC gene expression. The exact role of the aII NAT is not clear; however, it is consistent with the inhibition of IIa MHC gene transcription. In conclusion, NATs may mediate cross-talk between adjacent genes, which may be essential to the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts.

Effect of unloading on type I myosin heavy chain gene regulation in rat soleus muscle

Slow-twitch soleus, a weight-bearing hindlimb muscle, predominantly expresses the type I myosin heavy chain (MHC) isoform. However, under unloading conditions, a transition in MHC expression occurs from slow type I toward the fast-type isoforms. Transcriptional processes are believed to be involved in this adaptation. To test the hypothesis that the downregulation of MHC1 in soleus muscle following unloading is controlled through cis element(s) in the proximal region of the promoter, the MHC1 promoter was injected into soleus muscles of control rats and those subjected to 7 days of hindlimb suspension. Mutation analyses of six putative regulatory elements within the -408-bp region demonstrated that three elements, an A/T-rich, the proximal muscle-type CAT (betae3), and an E-box (-63 bp), play an important role in the basal level of MHC1 gene activity in the control soleus and function as unloading-responsive elements. Gel mobility shift assays revealed a diminished level of complex formation of the betae3 and E-box probes with nuclear extract from hindlimb suspension soleus compared with control soleus. Supershift assays indicated that transcriptional enhancer factor 1 and myogenin factors bind the betae3 and E-box elements, respectively, in the control soleus. Western blots showed that the relative concentrations of the transcriptional enhancer factor 1 and myogenin factors were significantly attenuated in the unloaded soleus compared with the control muscle. We conclude that the downregulation of MHC1 in response to unloading is due, in part, to a significant decrease in the concentration of these transcription factors available for binding the positive regulatory elements.

Effect of endurance exercise on myosin heavy chain gene regulation in human skeletal muscle.

The purpose of this study was to determine the effect of endurance-oriented exercise on myosin heavy chain (MHC) isoform regulation in human skeletal muscle. Exercise consisted of 1 h of cycle ergometer work per day at 75% maximal oxygen consumption for seven consecutive days. Muscle was obtained before the first bout of exercise, 3 h after the first bout of exercise, and before and 3 h after the final exercise bout on day 7 (n = 9 subjects). No changes in MHC mRNA (I, IIa, IIx) were evident after the first exercise period. There was, however, a significant (P < 0.05) decline (-30%) in MHC IIx mRNA 3 h after the final training bout. An interesting finding was that a higher pretraining level of MHC IIx mRNA was associated with a greater decline in the transcript before (r = 0.68, P < 0.05) and 3 h after (r = 0.82, P < 0.05) the final exercise bout. These findings suggest that MHC IIx mRNA is downregulated during the early phase of endurance-oriented exercise training in human skeletal muscle but only after repeated contractile activity. Pretraining MHC IIx mRNA content may influence the magnitude of this response.

Local response to cardiac overload on myosin heavy chain gene expression and isozyme transition.

It is uncertain whether the shift of cardiac myosin heavy chain (MHC) during pressure overload can be induced by some intrinsic factors or by the stress imposed directly on the individual myocytes. To study whether the changes in cardiac MHC gene expression produced by one-sided overload are limited to the involved ventricle or extend to the other ventricle, we examined MHC gene expression and isozyme transition in the left and right ventricles in aortic coarctated and pulmonary artery-banded rats. It has been confirmed that the pressure overload is indeed limited to the loaded ventricle. The results showed that, compared with sham-operated rats, there was no significant induction of the beta-MHC messenger RNA and corresponding protein in the unloaded ventricle, whereas significant induction was observed in the overloaded ventricle. These results demonstrated that the changes in MHC gene expression and isozyme produced by one-sided ventricular overload are limited to the involved ventricle. We conclude that the MHC gene regulation during hemodynamic overload may not be induced by intrinsic factors, such as hormones, catecholamine, or atrial natriuretic peptide, but is induced by direct local response to increased load.

Differential regulation by thyroid hormones of myosin heavy chain alpha and beta mRNAs in the rat ventricular myocardium.

The effect of tri-iodothyronine (T3) treatment on myocardial levels of alpha and beta myosin heavy chain (MHC) mRNAs in the rat was defined in vivo and in vitro. Dose-response experiments were performed in intact hypothyroid and euthyroid rats; in addition, studies in vitro examined the effect of T3 on MHC mRNAs in neonatal cardiac myocytes in primary culture. Specific alpha and beta MHC mRNAs were determined by Northern blot and dot hybridization to oligonucleotide probes complementary to the 3' untranslated regions of the MHC genes. An increase in myocardial beta MHC mRNA was demonstrated in hypothyroidism, accompanied by a reduction in alpha MHC mRNA. Marked differences in the sensitivity of alpha and beta MHC mRNAs to T3 replacement were found; a dose-dependent increase in alpha mRNA was evident at 6 h after T3 treatment, in the absence of consistent effects on beta mRNA, whereas 72 h after T3 replacement was commenced, stimulatory effects of T3 on alpha MHC mRNA, evident at all doses, were accompanied by a dose-dependent inhibition of beta MHC mRNA. No effect of thyroid status on actin mRNA was found, indicating the specificity of MHC gene regulation. T3 treatment of cardiac myocytes in vitro exerted similar actions on MHC mRNAs to those found in vivo, with a more marked influence on alpha than beta MHC mRNA. These studies of the action of T3 in vivo and in vitro have thus demonstrated specific effects of T3 on pretranslational regulation of the alpha and beta MHC genes, influences which differ not only in terms of stimulation or inhibition, but also in magnitude of effect.

Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature.

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.

Characterization of a developmentally regulated perinatal myosin heavy-chain gene expressed in skeletal muscle.

A cDNA clone, labeled pFOD5, isolated from a fetal-rat skeletal-muscle cDNA library, has been characterized and found to contain sequences corresponding to a perinatal-specific skeletal myosin heavy-chain (MHC) mRNA. This MHC cDNA demonstrates a high degree of nucleotide- and amino acid-sequence conservation with other MHC genes, but its carboxyl-terminal peptide and 3'-untranslated region are highly divergent and specific for this gene. S1 nuclease mapping experiments have shown that the perinatal MHC gene represented by this cDNA clone is only transiently expressed during skeletal-muscle development. Perinatal MHC mRNA is first detected late in fetal life, reaches maximal levels of expression at the end of the first postnatal week, and is de-induced thereafter. Its levels are almost undetectable at 28 days of postnatal life. During fetal and early postnatal life, the expression of this perinatal gene in skeletal muscle overlaps with the expression of the embryonic MHC gene. After the first week of extrauterine life, this gene is coexpressed with two adult MHC genes. The transient expression of this perinatal MHC gene raises interesting questions about the physiological significance of the MHC transitions and offers an interesting model for the study of MHC gene regulation.