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

Department of Physiology and Biophysics, University of California, Irvine, California

Submitted 20 October 2005 ; accepted in final form 9 January 2006

	ABSTRACT

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Hypertension has been shown to cause cardiac hypertrophy and a shift in myosin heavy chain (MHC) gene expression from the faster - to slower -MHC isoform. The expression of the - and -MHC pre-mRNAs, mRNAs, as well as the newly discovered antisense -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. -MHC mRNA expression significantly increased relative to control in all three regions, regardless of the target site (5' or 3' end). In contrast, -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 -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 -mRNA expression and the antisense -RNA (P < 0.05), suggesting an inhibitory role of antisense RNA on the sense -MHC gene expression. In contrast, a positive correlation was observed between the antisense -RNA and the -MHC pre-mRNA (P < 0.05). This latter observation along with the -MHC gene position relative to that of the -antisense suggest that the -MHC sense and -antisense transcription are coregulated likely via common intergenic regulatory sequences. Our results suggest that the increased -MHC expression in the AbCon heart not only is the result of increased -MHC transcription but also involves an antisense -RNA regulation scheme. Although the exact mechanism concerning antisense regulation is not clear, it could involve modulation of both transcriptional activity of the -MHC gene and posttranscriptional processing.

premRNA; gene transcription; posttranscription; hypertension; RT-PCR; Sprague-Dawley rats

In the myocardium, the MHC isoform composition is of great physiological significance to cardiac performance, and it affects the physiodynamics and energetic properties of the working heart. The -MHC isoform is characterized by a higher ATPase activity and faster shortening speed than the -MHC isoform (1, 12). Thus hearts rich in the -isoform have a high intrinsic contractility, whereas those rich in the -MHC have a lower contractility but a higher economy of tension development (1). In the adult rodent heart, the -MHC is the predominant isoform expressed in the ventricles, accounting for 85–90% of the total MHC protein pool, whereas the -MHC accounts for the remaining 10–15% (9). In cardiac myocytes of different mammalian species, the expression of the two MHCs is developmentally regulated (17), and it can be altered by a variety of pathophysiological conditions, including abnormal thyroid status (6, 14, 17, 29), diabetes (4), and hemodynamic overload (13, 18).

One of the unique features of the cardiac MHCs is that the - and -MHC genes are located in tandem on chromosome 15 in the rat, and each gene is 25 kb separated by 4.5 kb of intergenic region. They are in the order of -gene followed by the -gene. Each gene consists of 40 exons and 39 introns and yields a 6-kb mature mRNA. Their organization and order are well conserved in mammalian species through millions of years of evolution, which suggests that these genes’ structure is of functional significance. In a recent study (8), we have shown that a naturally occurring antisense RNA is transcribed from the DNA strand that is opposite to the MHC genes, with an initiation site located in the intergenic region between the - and -genes. Transcription occurs in the direction of the -MHC gene, thus creating a -antisense RNA. This antisense -transcript is associated with the MHC isoform gene switching in the heart in response to diabetes and hypothyroidism (8). Interestingly, in the normal control heart we showed that both the - and -MHC genes are actively transcribed based on primary transcript (pre-mRNA) analyses; however, the antisense -RNA is also highly expressed and appears to be involved in the repression of the MHC gene expression into mRNA via a complex, less understood mechanism (8). Diabetes and hypothyroidism are associated with a decrease in the antisense expression and a concomitant increase in the -MHC mRNA. Furthermore, we have shown a significant positive correlation between -MHC gene transcription and that of the antisense -transcript, which suggests that the - and antisense -transcriptions are coregulated likely via some common regulatory elements located on the intergenic region between the - and -MHC genes (8).

Given this proposed novel mode of coordinated regulation of cardiac MHC gene expression, the primary objective of the present study was to address the question whether this mode of regulation observed in diabetes and hypothyroidism, also applies to MHC regulation in the hypertensive heart. Cardiac pressure overload is associated with an increase in muscle mass, as well as a significant phenotype shift in MHC mRNA and protein composition (7). The end result is that the hypertensive heart becomes larger, and the -MHC mRNA and protein composition is enhanced beyond that of a normal control heart.

Previous studies suggest that the increase in -MHC protein expression in pressure-overloaded hearts is partly the result of a pretranslational mechanism based on significant correlations between -MHC protein and -MHC mRNA expression (7). Furthermore, based on promoter-reporter studies reporting significant increase in -MHC promoter activity in pressure-overloaded hearts (32) and the fact that this occurs without any obvious change in the -MHC promoter activity, we hypothesized that the increase in -MHC expression is due to transcriptional activation of the -MHC gene independently of the involvement of the newly discovered antisense -MHC mRNA, which appears to play a critical role in the MHC shift occurring in thyroid deficiency and diabetes. Our results support the idea that the observed increase in -MHC gene expression in the AbCon heart is the result of not only transcriptional activation of the -MHC gene but that it also involves regulation by the antisense -MHC transcript via a complex mechanism that might influence transcriptional and posttranscriptional processes within the cardiac MHC gene locus. The exact mechanism of the antisense -RNA regulation has not been determined at the molecular level of analyses, but it could be implicated in a universal mode of coordinated regulation between two adjacent genes of the same family.

	METHODS

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Animal model. Twelve young adult female Sprague-Dawley rats (150 g body wt, purchased from Taconic Farms, Germantown, NY) were used as subjects for these experiments. Animals were assigned to one of two groups: the normal control group (NC; n = 6) and the hypertensive group (n = 6). Hypertension was induced via the abdominal aortic constriction model (AbCon). Rats were anesthetized with a mixture of ketamine, acepromazine, and xylazine (40, 1, and 4 mg/kg, respectively). The abdominal aorta was surgically isolated just rostral to the left renal artery. A 2-0 silk suture was tied tightly around a blunt 22-gauge needle placed along the side of the aorta. The needle was removed, leaving the vessel constricted. The abdomen was closed with sterile surgical sutures, and animals were allowed to recover before they were returned to their vivarium cages. In previous studies, our laboratory has observed that this procedure causes a significant elevations in mean arterial blood pressure as measured at 6 and 12 days (32). The experimental procedure was carried out for 12 days. In a previous study, the -MHC promoter activity and MHC mRNA expression were significantly altered in 12-day AbCon hearts (32); therefore, a 12-day period appears to be suitable in examining altered expression of the MHC pre-mRNA and the newly discovered antisense -RNA in the early phase of AbCon induced MHC remodeling. At the end of 12 days, the animals were euthanized with Nembutal overdose administered via intraperitoneal injection (100 mg/kg body wt). Once the animals were dead, the heart was quickly removed and rinsed in ice-cold saline to remove excess blood. The atria were separated, and the ventricles were weighed. The ventricles were divided into right, left, and septum pieces; in addition, the left ventricle was cut in transverse into base and apex. The left ventricle apex, base, and the septum pieces were stored at –80 until analyzed for cardiac MHC RNA expression. Animal procedures described in this study were approved by our institutional animal care and use committee and comply with National Institutes of Health animal use and care guidelines.

RNA extraction. Total RNA was extracted from frozen cardiac muscle pieces (septum, apex, and base of the left ventricles) using the TRI Reagent protocol (Molecular Research Center). The extracted RNA was DNase treated using 1 unit of RQ1 RNase-free DNase (Promega) per microgram of total RNA at 37°C for 30 min. The DNase treatment was immediately followed by a second RNA extraction using the TRI Reagent LS for liquid samples (Molecular Research Center). At the end of this extraction, the RNA was dissolved in nuclease-free water, and its concentration was determined by optical density at 260 nm using a factor of 40, i.e., one unit absorbance equivalent to 40 µg/ml. RNA quality was checked by gel electrophoresis whereby 0.5 µg of the total RNA was separated by electrophoresis on 1% agarose gel-1x Tris acetate buffer, and it was stained with ethidium bromide. All the samples used in this study showed the 28S and 18S ribosomal RNA bands, which is consistent with a good quality, e.g., not degraded total RNA.

Determination of MHC mRNA percent distribution. Cardiac MHC distribution in different regions of the heart muscle was evaluated by reverse transcription (RT)-polymerase chain reaction (PCR) using random primers for the reverse transcription reaction followed by PCR with primers targeting - and -MHC mRNAs, as described previously (32). This method allows one to determine the composition of cardiac MHC mRNA in each region of the myocardium and confirms that the AbCon procedure was associated with MHC phenotype remodeling consisting of a previously known - to -shift in MHC. However, these data will not be able to determine how the shifts occurred. Is it via change in the -MHC mRNA, or change in the -MHC mRNA expression, or both? One must analyze MHC mRNA expression for each isoform in a constant amount of total RNA to answer this question.

Specific MHC RNA analyses. Complete DNA sequence data on the rat cardiac MHC genes and the intergenic region is available in Genbank (chromosome 15, contig NW_047454). Specific RT primers were designed to target the exonic sequences from the 5' and 3' ends of the - and -MHC genes. RT reactions using these exonic RT primers generate cDNA from both the pre-mRNA and the mRNA. These cDNAs were amplified by PCR using primers specific for the mRNA (exonic primers) or the pre-mRNA (which includes at least 1 intronic primer). In addition, RT primers targeting the antisense RNA were designed based on the positive-strand sequence of the MHC gene locus and they targeted different sites such as the 5' end, the 3' end of the -MHC gene, and the intergenic region between the - and -MHC genes. The cDNA product complementary to the antisense RNA was amplified using PCR primers targeting intergenic sequences. In all these reported RT-PCR amplification results, the PCR primers’ target was located within a short distance from the specific RT primer to ensure high amplification efficiency. DNA sequence and other details on the primers used can be found in Table 1. Specific RT reactions used 2 µg of total RNA in 20 µl of total volume in presence of 5 pmol of specific primers using Superscript II reverse transcriptase (Invitrogen). PCR amplifications used Biolase DNA polymerase (Bioline) in the presence of magnesium (at an optimized concentration ranging from 1.5 to 2 mM), 0.2 mM deoxynucleotide triphosphates, and 0.6 µM PCR primers and were carried out on a Robocycler (Stratagene). The annealing temperature was adjusted based on the PCR primers’ optimal annealing temperature. The amount of cDNA and PCR cycles was adjusted so that the accumulated product was in the linear range of the exponential curve of the PCR amplifications. PCR products were separated by electrophoresis on agarose gels and stained with ethidium bromide. The ultraviolet light-induced fluorescence of stained DNA was captured by a digital camera, and the band intensities were quantified by densitometry with ImageQuant software (Molecular Dynamics) on digitized images and were reported as arbitrary scan units. In addition, all PCR reactions were performed with two negative control reactions. 1) The total RNA was subjected to reverse transcription whereby the RT enzyme was omitted from the reaction, whereas all other ingredients were kept intact. Detection of a PCR product from these first negative-control reactions may be attributed to either reagent contamination with PCR products or RNA contamination with genomic DNA. This latter component is important for unspliced RNA target, which amplifies the same fragment size as the genomic DNA. The absence of product ensures the lack of contaminants as well as the effectiveness of DNase I treatment of the total RNA. All the utilized RNA in this study was DNase treated and no PCR signal could be detected from these negative control reactions, thus demonstrating that the DNase I treatment was fully effective. 2) The total RNA was subjected to the RT reactions in the absence of any RT primers, but all other ingredients in the reaction were kept intact. The second negative control is necessary because in some cases a fairly robust PCR signal can be detected despite the absence of RT primers in the RT reaction. This signal can account for up to 30% of the signal obtained from cDNA made with certain specific primers. It is not clear why RT without primers followed by PCR with primers to the cDNA region of interest would give, in some cases, a measurable signal. This signal is highly reproducible from different samples done at separate times as well as from different cDNAs of the same sample, and it can also be formed using different primer sets targeting the same RNA/cDNA region. We speculate that some form of self-priming during the RT reaction occurs (of an antisense fragment to sense strand and vice versa, or involving the RNA secondary structure), which allows the reverse transcription to proceed, and thus the PCR amplification. Based on having a PCR signal without targeting the RNA with RT primer, it becomes important to account for this product when the PCR amplifies a targeted cDNA. The most logical way to account for this "background" signal was to subtract it from the PCR signal. Thus the RT-PCR data reported herein reflect subtraction of this second negative control RT-PCR from the corresponding specifically primed RT-PCR signal.

Statistical analyses. Data are reported as means ± SE. The effect of AbCon was studied for each region of the heart independently of the other. Differences between the two experimental groups (NC vs. AbCon) were analyzed using an unpaired t-test. For multiple comparisons, a one-way analysis of variance with Newman-Keuls post hoc test was utilized. Relationships between two variables were assessed using linear regression and correlation analyses (GraphPad Software). Statistical significance was set at P < 0.05.

	RESULTS

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Body weight and heart weight. Body weight was not different between the NC and the AbCon group [190 ± 3 vs. 183 ± 3 g, respectively]. However, absolute heart weight and the relative heart weight (heart weight-to-body weight ratio) were significantly elevated by 39% (P < 0.05) and 44% (P < 0.05), respectively, in the AbCon group relative to the NC group. This is a signature response in this model whereby the increased cardiac mass is considered to be a compensatory adaptation of the heart muscle to increased work demand. This hypertrophic response is due to the increased mean blood pressure and increased afterload that is imposed, and the heart undergoes remodeling in an effort to reduce the wall stress (30).

MHC mRNA analyses. Throughout this paper we report the various results based on analyses from the apex, base, and septum regions of the heart to ensure that the AbCon hypertension affects the cardiac MHC shifts in a global way. To initially assess the shift between the - and -isoform, cardiac MHC mRNA composition was analyzed using the standard competitive MHC PCR approach that was developed by our laboratory (32). These analyses show that after 12 days of AbCon, the MHC isoform composition is altered to be significantly enriched in the -MHC mRNA by 104% in the base, by 91% in the apex, and by 68% in the septum relative to control (Fig. 1). Comparisons of the -MHC mRNA induction between different regions of the hearts revealed that the septum response was slightly lower compared with the base response, while the apex response was not different from either the base or the septum. The reason for this difference in the magnitude of septum response at this time is unclear and could be the result of one of three possibilities. 1) The -MHC percent composition in the control septum was slightly higher than the control base and apex (27 vs. 21%; P = 0.052, 1-way ANOVA); 2) it is possible that -MHC mRNA response has to do with the regional effect of the afterload on the left ventricle such that the stimulus is less at the septum level compared with base and apex; or 3) it is possible that in a time course of 12 days, the heart has not reached a steady state, and this response might be identical in all three regions of the heart if analyzed at a later time point.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Myosin heavy chain (MHC) mRNA analyses by reverse transcription (RT)-polymerase chain reaction (PCR) using competitive PCR as described in Wright et al. (32). A: representative gel showing PCR products. The MHC refers to cDNA amplification products resulting from using - or -MHC-specific primer pairs; the control refers to the amplification product of the control fragment that was added externally to each cDNA to be coamplified with the same PCR primers yielding PCR products of 250 or 224 bp, using the - and -primer pairs, respectively, and allowing to correct for PCR efficiency. B: %-MHC mRNA in the 12-day pressure-overloaded (AbCon) heart relative to normal control (NC) in 3 regions of the heart, the apex, the base, and the septum. Values are the ratio of %-MHC mRNA in AbCon to %-MHC mRNA in NC. *P < 0.05 septum vs. base. C: %-MHC mRNA in the NC heart regions. Values are means ± SE; n = 6 animals/group. *P = 0.052 (1-way ANOVA).

Expression of -MHC transcripts in the AbCon heart. MHC gene expression was analyzed at the level of pre-mRNA and mRNA. The -MHC pre-mRNA was studied as a marker for transcriptional activity of the -MHC gene. The analyses of the pre-mRNA expression used RT-PCR techniques in which the pre-mRNA was targeted using gene-specific antisense RT primers to generate a cDNA followed by amplification using PCR primers. The RT primers were designed to target the pre-mRNA at both the 5' end of the gene (close to the transcription start site) as well as at the 3' end of the gene (close to the termination site). Because of the long MHC pre-mRNA molecule (25 kb), these distant analyses are important to ensure that any observed change in the AbCon heart can be equally detected at the 5' end as well as at the 3' end of the long target RNA. At the same time, these analyses may be more complex and difficult to interpret for such long transcripts whereby processing may be occurring along with transcription. For example, enhanced intron removal during splicing of the primer targeted region would result in a decrease RT-PCR product, even though the actual number of transcripts is the same as that analyzed at different sites. Also, as more transcripts are being initiated at the 5' end, for example, in response to activation, fewer will be detected at the 3' end because transcription has not been completed yet. Results from the -MHC pre-mRNA analyses are reported in Fig. 2, and they show that whereas the pre-mRNA is increased at the 5' end of the gene in response to AbCon, it was not significantly induced at the 3' end. This discrepancy between the 5' end and 3' end response of the pre-mRNA was found in all three regions of the left ventricles that were analyzed (apex, base, septum). The increased MHC pre-mRNA at the 5' end is consistent with an increased transcription of the gene in response to AbCon. It was not expected that the pre-mRNA as measured at the level of the last intron (3' end) was not increased in the AbCon heart as observed for the 5' end measurement. The reason for this difference is not clear but could involve limited elongation of the transcript as it is synthesized from the 5' end to the 3' end. That is, whereas transcription initiation is increased in the AbCon state, there is a limit concerning how many transcripts can be completed as they are being extended to the 3' end of the gene. On the other hand, it is possible that the splicing of the introns at the 3' end of the gene is enhanced in the AbCon heart so that the pre-mRNA may appear as not changing when studied at this level because it was spliced (loss of introns) at a higher rate.

View larger version (15K): [in this window] [in a new window]   Fig. 2. -MHC pre-mRNA expression in NC and AbCon heart. A: RT-PCR targeting the 5' end of the -MHC pre-mRNA in 3 cardiac regions. Gel images of PCR products obtained from both -specific RT reactions (RT-PCR) and RT reactions from RNA without primers (ØRT). Presented data are the net signal (difference in signal between both reactions). PCR was carried out on 1 µl cDNA that was diluted 15-fold for 28 cycles. B: RT-PCR targeting the 3' end of the -MHC pre-mRNA. -pre-mRNA expression in NC and AbCon heart, analyses in 3 cardiac regions. Also shown are representative gel images of PCR products obtained from both -specific RT reactions (RT-PCR) and RT reactions from RNA without primers (ØRT). Presented data are the net signal [difference in signal between both reactions in arbitrary scan units (ASU)]. PCR was carried out using 1 µl cDNA that was diluted 5-fold for 29 cycles. Values are means ± SE; n = 6 animals/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test).

View larger version (14K): [in this window] [in a new window]   Fig. 3. -MHC mRNA expression in NC and AbCon heart. RT-PCR targeting the 5' end (A) and the 3' end (B) of the -MHC mRNA in 3 cardiac regions. Gel images of PCR products obtained from -specific RT reactions. PCR from RT reactions on RNA without primers gave no detectable product at the dilutions used for these amplification reactions. PCR was carried out on 1 µl cDNA that was diluted 40-fold for 24 cycles. Values are means ± SE; n = 6/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test).

Expression of -MHC antisense RNA.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Antisense -MHC RNA expression in NC and AbCon heart in 3 regions of the myocardium. RT-PCR targeting the antisense overlapping the 3' end of the -MHC gene (A) or the intergenic (IG) region between and (B). Gel images of PCR products obtained from both DNA strand-specific RT reactions (RT-PCR) and RT reactions from RNA without primers (ØRT). Presented data are the net signal (difference in signal between both reactions in ASU). PCR was carried out on 1 µl cDNA that was diluted 5-fold for 30 cycles. Values are means ± SE; n = 6 animals/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test).

Quantitative PCR using SYBRgreen real-time PCR to analyze -MHC RNA expression.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Quantitative -MHC RNA analyses in the apex region using real-time PCR and SYBRgreen fluorescence. A: -MHC pre-mRNA expression targeted at the 5' end and 3' end. B: antisense -RNA expression targeted at the 5' end and 3' end. C: -MHC mRNA expression in NC and AbCon apex as analyzed using real-time PCR [quantitative PCR (QPCR)] and using the ethidium bromide gel (EtBr) end-point PCR method. In A, B, and C, values are reported as ratio of copy number to NC average, which makes the NC expression equivalent to 1 and the AbCon expression relative to NC. Values are means ± SE; n = 6 animals/group. *Mean is significantly different from 1; P < 0.05.

Expression of -MHC transcripts in the AbCon heart.

View larger version (19K): [in this window] [in a new window]   Fig. 6. -pre-mRNA expression in NC and AbCon heart. A: RT-PCR targeting the 5' end of the -MHC pre-mRNA in 3 cardiac regions. Gel images of PCR products obtained from both -specific RT reactions (RT-PCR) and RT reactions from RNA without primers (ØRT). Presented data are the net signal (difference in signal between both reactions). PCR was carried out on 1 µl cDNA that was diluted 15-fold for 28 cycles. Values are means ± SE; n = 6 animals/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test). B: RT-PCR targeting the 3' end of the -MHC pre-mRNA. -pre-mRNA expression in NC and AbCon heart, analyses in 3 cardiac regions. Also shown are representative gel images of PCR products obtained from both -specific RT reactions (RT-PCR) and RT reactions from RNA without primers (ØRT). Presented data are the net signal (difference in signal between both reactions) in ASU. PCR was carried out using 1 µl cDNA that was diluted 5-fold for 29 cycles. Values are means ± SE; n = 6 animals/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test).

View larger version (16K): [in this window] [in a new window]   Fig. 7. -MHC mRNA expression in NC and AbCon hearts. RT-PCR targeting the 5' end (A) and the 3' end (B) of the -MHC mRNA in 3 cardiac regions. Gel images of PCR products obtained from -specific RT reactions. PCR from RT reactions on RNA without primers gave no detectable product at the dilutions used for these amplification reactions. PCR was carried out on 1 µl cDNA that was diluted 40-fold for 23 cycles. Values are means ± SE; n = 6 animals/group. *P < 0.05, NC vs. AbCon (Ab) (unpaired t-test).

	DISCUSSION

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Relationship between the -and -MHC genes in response to pressure overload. Based on the discovery of the -antisense RNA, the observed relationships between cardiac MHC pre-mRNA, mRNA, and antisense RNA, and in conjunction with the evolutionary conserved cardiac MHC gene organization, a model of coordinated regulation of cardiac MHC gene expression was proposed (8). This model, shown in Fig. 8A, basically proposes that the transcribed -antisense RNA interferes with -MHC gene expression 1) via interference with the normal transcription of the sense -MHC gene and/or 2) via inhibiting the processing of the primary transcript (pre-mRNA) into mature mRNA. In addition, based on positive correlation between the antisense -RNA and the -MHC pre-mRNA, in conjunction with the proximity of the two promoter regions, we proposed that the antisense -RNA transcriptional activity is linked to that of the -MHC gene via some common regulatory elements in the intergenic region. The tight coordinated regulation between the - and -MHC gene, which is demonstrated by the rapid, simultaneous, and reversible shifts between - and -MHC gene expression to correspond to the functional demands of the myocardium, further corroborate this model.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Coordinated regulation of cardiac MHC gene via antisense transcription. A: schematic of cardiac MHC gene locus and proposed model of gene interaction. Cardiac MHC genes are located in tandem on the same chromosome. Each gene is 25 kb, and they are separated by 4 kb of intergenic region. P is the promoter of the MHC gene transcribing the sense -MHC pre-mRNA. The P is the promoter of the MHC gene transcribing the sense -MHC pre-mRNA. The (as)P is an intergenic promoter transcribing the antisense -RNA, which is proposed to interfere with the P activity and inhibit the processing of the sense -pre-mRNA into mature -mRNA. The data suggest that both sense - and antisense -transcriptions are in part regulated by a common promoter region (CPR). B: relationship between -MHC mRNA and antisense -RNA. C: relationship between -MHC pre-mRNA and antisense -RNA. The line (in B and C) was generated by linear regression analysis; R is the correlation coefficient. Data points are from individual samples representing NC and AbCon groups for each heart region. AC and AH, apex control and apex AbCon (hypertensive); BC and BH, base control and base hypertensive; SC and SH, septum control and septum hypertensive, respectively.

The idea of negative regulation of gene expression by an overlapping antisense transcript is not new nor unique to the -MHC gene, because it has been observed for several other genes, including the endothelial nitric oxide synthase (28), Hox A11 (2), atrial essential myosin light chain 1 (27), and collagen I ₁-gene (5), and in the regulation of X inactivation (19). The proposed coordinated regulation between two adjacent genes via an antisense RNA is novel, and to date it has not been yet proposed for other genes. However, this may be an important mechanism for gene cross talk within gene clusters in order for the cell to simultaneously activate one gene while turning off another related gene. This idea is supported by the fact that the gene order, orientation, and spacing are conserved through evolution, suggesting functional significance.

In the context of these responses involving the heart, we have also noted that there is a similar phenomenon occurring in skeletal muscle involving fast MHC gene expression. In studying the fast MHC gene RNA expression in different models, there is an antisense IIa RNA expression associated with the IIa IIx gene switching in slow muscle (vastus intermedius and soleus) undergoing slow-to-fast MHC transition such as during unloading (26), and an antisense IIx RNA expression associated with the IIb IIx gene switching in fast muscles (white medial gastrocnemius) undergoing fast-to-slow transition such as occurring in an overload state (F. Haddad and K. M. Baldwin, unpublished observations). As in the case of the heart MHC genes, the skeletal fast type MHC IIa, IIx, and IIb genes are aligned in tandem on the same chromosome with much conserved gene order, orientation, and spacing throughout evolution. Taken together, it would appear that the classical gene switching that occurs among MHC isoforms involves a common phenomenon that implicates antisense RNA expression.

Validation of the antisense -RNA results in the AbCon heart. The antisense -MHC RNA expression was analyzed using specific RT-PCR methods and was found to be significantly decreased in the AbCon heart in all three separate regions (apex, base, and septum). The antisense RNA was analyzed in four separate regions across the MHC gene locus: in the intergenic region (Fig. 4B), in the region overlapping the 3' end of the -MHC gene (Fig. 4A), in the region overlapping the 5' end of the -MHC gene (Fig. 5B), and in the promoter region upstream of the -MHC gene (data not shown). For all these analyses, the antisense was expressed in the NC heart, and the AbCon heart consistently expressed significantly less antisense -RNA product. Real-time PCR analyses were performed to validate the quantitative comparisons between control and AbCon, and these results were well in correspondence to the end-point PCR as reported in the results above. In addition to quantitative validation of the results on the response of the antisense to AbCon, having detected the expression of the -antisense RNA when targeted with specific primers at different sites in the MHC gene locus suggests that this antisense RNA is overlapping the entire -MHC gene and extending into the promoter region as shown in the proposed model in Fig. 8A. In addition to the reported PCR results that were carried within short distance from the RT primers (for quantitative purpose), the antisense RNA extent was qualitatively assessed. The antisense RNA was targeted near the initiation start site of the -MHC gene, and the PCR amplification targeted a region that is in the intergenic region, 26 kb from the RT primer target sequence, and we got a positive net response between a negative RT and the specific RT. These results were not used for quantitative assessment because the PCR amplification target is far away from the RT primer (25 kb), making the reaction less efficient to occur. However, they confirm that the antisense exists as a long RNA, which overlaps the entire -MHC gene.

Validation of the -MHC pre-mRNA results. In this study, the pre-mRNA levels were used as a measure of transcriptional activation because it is the primary product of the RNA polymerase II activity, although these measurements may depend on the stability of targeted intronic sequence considering the fact that the pre-mRNA is processed quickly into mRNA as it is synthesized. It is intriguing that the -MHC pre-mRNA expression in response to AbCon depended on the site of analyses. At the 5' end, 3 kb from the transcription start site, AbCon hearts expressed significantly more -pre-mRNA. This response is consistent with a higher transcriptional activity of the gene. Analyses of the pre-mRNA targeted at the 3' end of the -MHC gene, e.g., at the site of the last intron, show that the -MHC pre-mRNA expression was not appreciably altered in response to AbCon. We speculate that the pre-mRNA is rapidly processed to keep up with the high transcription rate, and this is consistent with the associated decrease in the antisense -RNA, which we postulate to be an inhibitor of pre-mRNA processing. Thus, in the AbCon heart, intron splicing may be occurring at a rate faster than in the normal heart, which is consistent with the observed increase in the -MHC m-RNA in the AbCon heart that exceeds the increase in the corresponding pre-mRNA. This difference in the stoichiometry between pre-mRNA increase (100%) vs. the mRNA increase (225%) in the AbCon heart relative to NC was validated using quantitative PCR analyses (Fig. 5). This latter observation is also consistent with the model illustrated in Fig. 8A.

How does -MHC gene regulation in the AbCon heart fit the model? In the model of hypertension, previous reports on the -MHC gene regulation are equivocal. It has been reported to either decrease (22, 23) or not change (11, 13, 24). In the normal control heart in rodents, the -MHC expression is predominant. In response to pressure overload, the heart hypertrophies and its MHC profile becomes richer in -MHC expression. This can be possible by an increased -MHC expression regardless of whether the -MHC expression was maintained at control level or decreased. In this study, the results on the -MHC were consistent with the literature, and they ranged between either no change or being decreased. For example, when analyzed at the 5' end of the -MHC gene, the pre-mRNA was not changed in the AbCon apex and septum, but it was significantly reduced in the AbCon base (Fig. 6A). The corresponding mRNA was significantly reduced in the AbCon apex and base but not in the septum (Fig. 7A). When analyzed at the 3' end, the -MHC pre-mRNA expression was reduced in the apex and base but not in the septum (Fig. 6B), whereas the corresponding mRNA expression was not changed in all three regions of the hearts (Fig. 7B). These results on the -MHC suggest that the -MHC expression is altered in response to AbCon, but it follows a different time course from that of the -MHC and the antisense RNA regulation, which occurs very early after induction of pressure overload. We speculate that at a later time point after the pressure overload, the -MHC expression will be significantly reduced in all regions of the hearts. Despite this varying response in -MHC expression, the overall response is a reduction in its pre-mRNA and mRNA level. Statistical analyses examining the relationship between the antisense -MHC RNA (at 3' end of the -MHC gene) and the 5' end -MHC pre-mRNA revealed a positive correlation that was statistically significant (P < 0.0001) (Fig. 8B). The Pearson correlation coefficient was 0.62, which translates into a coefficient of determination of 0.4, which leads us to the following interpretation. In the AbCon heart, the -MHC pre-mRNA and antisense -RNA regulation is likely coordinated, and such coordination is postulated to occur, at least in part, via a common regulatory mechanism (Fig. 8A). But there are other independent regulatory factors that are affecting the -antisense without altering the . The nature of these factors remain to be determined in future studies.

Role of naturally occurring antisense RNA. It is important to note that the phenomenon of naturally occurring antisense RNA expression is not unique to the MHC gene family but appears to be prevalent in the mammalian genome. Recent reports have revealed a surprisingly large number of antisense transcripts (15, 34), but their function and mechanism of action are complex and remain largely speculative. The large number of bidirectionally transcribed genes is intriguing and substantiates the physiological importance of antisense regulation. Antisense gene products can exert their influence at any gene expression stage, including transcription initiation, elongation, RNA processing, RNA stability, and translation (21). Based on the literature, long naturally occurring antisense RNAs have been implicated in the regulation of gene expression via at least four different mechanisms as reviewed by Lavorgna et al. (16). 1) The first mechanism is transcriptional interference, whereby transcription of a gene encoded on the sense strand of the DNA inhibits concomitant transcription of the overlapping gene encoded on the opposite strand. When transcription occurs on both strands in opposite direction, there is attenuation of the polymerase II activity in one or both directions due to collision of large transcription complexes. 2) The second mechanism is RNA masking, whereby the formation of double stranded RNA may mask regulatory signals necessary for splicing, probably by blocking the accessibility of cis-regulatory elements. This mechanism may play a role in the alternative splicing between two gene variants, such as described for the thyroid receptor -gene (10). 3) The third mechanism include double-stranded RNA-dependent mechanisms, whereby the double-stranded RNA in the nucleus triggers RNA editing [involving selective deamination of adenosine (A) to inosine (I) in the pre-mRNA] leading to nuclear RNA retention or RNA interference-dependent gene silencing. 4) The fourth mechanism is antisense-induced methylation, whereby the expression of the antisense transcript induces the methylation of the CpG island upstream of the sense gene, thus inhibiting its transcription. This latter mechanism is particularly true for antisense transcripts involved in gene imprinting (25, 31, 33) and thus is unlikely occurring in the cardiac MHC gene regulation because the effect is not total inhibition of the -MHC mRNA expression.

Although our results do not allow definition of the precise mechanism of action of this MHC RNA antisense, they suggest negative regulation of -MHC gene expression by the antisense -RNA, and this is supported by the negative correlation between the -MHC mRNA and the antisense -RNA. Direct sequencing of cDNA product failed to detect any deamination (A to I conversion) on the overlapping -MHC RNA sequence (data not shown), which suggests that the third mechanism is unlikely to be occurring. This makes either the first and/or the second mechanism as possibly occurring in cardiac MHC gene regulation, and the presented data support either one. The first mechanism of transcription interference may be possible and can be occurring as follows: when the antisense transcription is attenuated in the AbCon heart, the sense -MHC gene transcription is activated by having less interference from the opposite strand transcription. The second mechanism could involve the antisense RNA making double-stranded RNA with the sense pre-mRNA, thus masking the splicing recognition sites, and allowing less mRNA to be made. If this inhibition is occurring in the NC heart, processing may be activated in the AbCon heart expressing less antisense RNA. Two pieces of data support this notion: 1) the increase in mRNA in response to AbCon exceeds the increase in the corresponding pre-mRNA (difference validated with quantitative PCR), and 2) the pre-mRNA measurement at the last intron showed no change in the AbCon heart, suggesting a higher rate of intron removal at this site. While these ideas remain speculative in nature, clearly, a better understanding of the mode of action of the antisense RNA is needed especially in the context of maintaining tight coordinated regulation between two adjacent genes of related function. Future studies are needed to address this issue. For example, the consequences of targeted disruption of the antisense transcript on cardiac MHC gene expression should be examined to gain insight into its role in the cardiac MHC gene regulation. Disruption of the antisense can be achieved at the transcriptional level by altering its basal promoter located in the --intergenic region, or posttranscriptionally via the use of RNA interference system.

In summary, in this paper we reinvestigated a long-known phenomenon that involves MHC phenotype shifts in the pressure-overloaded heart. We investigated the mechanism of cardiac MHC shifts in view of the novel finding reported recently that describes the involvement of antisense RNA in the antithetical regulation of the cardiac MHC gene expression in thyroid deficiency and diabetes (8). Our results demonstrate that the increased -MHC expression in the AbCon heart not only is the result of increased -MHC transcription but also involves an increased accumulation of the -MHC mRNA via a mechanism involving a reduced inhibitory effect of the antisense -RNA via a complex mechanism of gene cross talk. Thus these findings add to an expanding database to suggest that the antithetical regulation of /-MHC gene switching is mediated by an intergenic mechanism involving naturally occurring -antisense RNA transcription. The exact details of this mechanism at the molecular level remain to be elucidated.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was funded by National Heart, Lung, and Blood Institute Grant HL-73473-01 (to K. M. Baldwin).

	ACKNOWLEDGMENTS

 
The authors thank Adrienne Ma, Danny Cheng, Timothy Dah Jong Leetrakul, Cori Kobayashi, and Toni Garma for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: F. Haddad, Physiology and Biophysics Dept., Univ. of California, Irvine, CA 92697-4560 (e-mail: fhaddad{at}uci.edu)

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.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Alpert NR and Mulieri LA. Functional consequences of altered cardiac myosin isoenzymes. Med Sci Sports Exerc 18: 309–313, 1986.[CrossRef][ISI][Medline]
2. Chau YM, Pando S, and Taylor HS. HOXA11 silencing and endogenous HOXA11 antisense ribonucleic acid in the uterine endometrium. J Clin Endocrinol Metab 87: 2674–2680, 2002.[Abstract/Free Full Text]
3. Dahary D, Elroy-Stein O, and Sorek R. Naturally occurring antisense: transcriptional leakage or real overlap? Genome Res 15: 364–368, 2005.[Abstract/Free Full Text]
4. Dillmann WH. Diabetes and thyroid-hormone-induced changes in cardiac function and their molecular basis. Annu Rev Med 40: 373–394, 1989.[CrossRef][ISI][Medline]
5. Farrell CM and Lukens LN. Naturally occurring antisense transcripts are present in chick embryo chondrocytes simultaneously with the down-regulation of the alpha1(I) collagen gene. J Biol Chem 270: 3400–3408, 1995.[Abstract/Free Full Text]
6. Gustafson TA, Markham BE, and Morkin E. Analysis of thyroid hormone effects on myosin heavy chain gene expression in cardiac and soleus muscles using a novel dot-blot mRNA assay. Biochem Biophys Res Commun 130: 1161–1167, 1985.[CrossRef][ISI][Medline]
7. Haddad F, Bodell PW, and Baldwin KM. Pressure-induced regulation of myosin expression in rodent heart. J Appl Physiol 78: 1489–1495, 1995.[Abstract/Free Full Text]
8. Haddad F, Bodell PW, Qin AX, Giger JM, and Baldwin KM. Role of antisense RNA in coordinating cardiac myosin heavy chain gene switching. J Biol Chem 278: 37132–37138, 2003.[Abstract/Free Full Text]
9. Haddad F, Masatsugu M, Bodell PW, Qin A, McCue SA, and Baldwin KM. Role of thyroid hormone and insulin in control of cardiac isomyosin expression. J Mol Cell Cardiol 29: 559–569, 1997.[CrossRef][ISI][Medline]
10. Hastings ML, Ingle HA, Lazar MA, and Munroe SH. Post-transcriptional regulation of thyroid hormone receptor expression by cis-acting sequences and a naturally occurring antisense RNA. J Biol Chem 275: 11507–11513, 2000.[Abstract/Free Full Text]
11. Herzig TC, Jobe SM, Aoki H, Molkentin JD, Cowley AW Jr, Izumo S, and Markham BE. Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload. Proc Natl Acad Sci USA 94: 7543–7548, 1997.[Abstract/Free Full Text]
12. Holubarsch C, Goulette RP, Litten RZ, Martin BJ, Mulieri LA, and Alpert NR. The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium. Circ Res 56: 78–86, 1985.[Abstract/Free Full Text]
13. Imamura S, Matsuoka R, Hiratsuka E, Kimura M, Nakanishi T, Nishikawa T, Furutani Y, and Takao A. Adaptational changes of MHC gene expression and isozyme transition in cardiac overloading. Am J Physiol Heart Circ Physiol 260: H73–H79, 1991.[Abstract/Free Full Text]
14. Izumo S, Nadal-Ginard B, and Mahdavi V. All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231: 597–600, 1986.[Abstract/Free Full Text]
15. Kiyosawa H, Yamanaka I, Osato N, Kondo S, and Hayashizaki Y. Antisense transcripts with FANTOM2 clone set and their implications for gene regulation. Genome Res 13: 1324–1334, 2003.[Abstract/Free Full Text]
16. Lavorgna G, Dahary D, Lehner B, Sorek R, Sanderson CM, and Casari G. In search of antisense. Trends Biochem Sci 29: 88–94, 2004.[CrossRef][ISI][Medline]
17. Lompre AM, Nadal-Ginard B, and Mahdavi V. Expression of the cardiac ventricular alpha- and beta-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem 259: 6437–6446, 1984.[Abstract/Free Full Text]
18. Lompre AM, Schwartz K, d'Albis A, Lacombe G, Van Thiem N, and Swynghedauw B. Myosin isoenzyme redistribution in chronic heart overload. Nature 282: 105–107, 1979.[CrossRef][Medline]
19. Luikenhuis S, Wutz A, and Jaenisch R. Antisense transcription through the Xist locus mediates Tsix function in embryonic stem cells. Mol Cell Biol 21: 8512–8520, 2001.[Abstract/Free