A novel mechanism of regulation of cardiac ␣ and ␤ myosin heavy chain gene by naturally occurring antisense transcription was elucidated via pre-mRNA analysis. Herein, we report the expression of an antisense ␤ myosin heavy chain RNA in the normal rodent myocardium. The pattern of expression of the antisense ␤MHC RNA (␤ RNA) under altered thyroid state and in diabetes directly correlates with that of the ␣ pre-mRNA/mRNA, whereas it negatively correlates with the ␤ mRNA expression. Rapid amplification of the 5 end shows that this antisense transcript originates 2 kb downstream of the ␤ gene, and it is transcribed across the entire ␤ gene from the opposite strand. Our results demonstrate that the ␤-␣ myosin heavy chain intergenic DNA possesses a bidirectional transcriptional activity, one direction transcribing the ␣ gene, and the opposite direction transcribing the antisense ␤ RNA. This process turns on the ␣ expression, and it simultaneously turns off that of the ␤ and thus coordinates ␣ and ␤ expression in an opposite fashion. Comparative analyses of the intergenic DNA sequence across five mammalian species revealed a conserved region that is proposed to be a common regulatory region for the ␣ and antisense ␤ promoter. This finding unravels the mechanism of cardiac ␣-␤ gene switching and implicates the role of cardiac myosin gene organization with their function.Cardiac muscle expresses two myosin heavy chain (MHC) 1 isoforms designated as ␣ (high ATPase) and ␤ (low ATPase) that are encoded by two distinct genes located in close proximity on the same chromosome (1-4). The MHC is the molecular motor driving muscle contraction, and its phenotypic composition regulates the intrinsic contraction properties of the heart (5, 6). Cardiac MHC isoform expression is developmentally regulated (5), and it can change totally in either direction under certain pathophysiological states (7-9). For example during the first 3 weeks of postnatal life of rodents there is a complete switch from a predominant ␤MHC expression at birth (Ͼ90%) to a predominant ␣MHC expression at 3 weeks of age (Ͼ95%). Throughout adult life, the ␣MHC expression predominates in a normal rodent heart, with ␤MHC expression gradually increasing as the animal gets older. At any time during life, the pattern of MHC expression can be altered. Hypothyroidism and diabetes are associated with a switch in the cardiac MHC gene expression from a predominant ␣MHC to a predominant ␤MHC. In contrast, thyroid hormones treatment increases the ␣MHC expression while down-regulating the ␤MHC expression. The exact molecular mechanisms causing this tightly coordinated regulation of these two genes remains unclear. Thyroid hormone has been shown to be a major regulator of MHC gene expression, and its regulation is thought to occur mainly via transcriptional processes regulating each gene independently in a well coordinated fashion (8). Several thyroid responsive elements have been located on the promoter of the ␣MHC gene, whereas the localized action of thyroid hormone on the ...

Background: The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primerindependent cDNA synthesis using a popular RNase H -RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.

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.

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.

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.