the 259, no. 10, 25, pp. 6437-6446 1984 of in s. a. 0 ... · expression of the cardiac ventricular...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biologid Chemists, Inc.

Vol. 259, No. 10, Issue of May 25, pp. 6437-6446 1984 Printed in d. S. A.

Expression of the Cardiac Ventricular CY- and &Myosin Heavy Chain Genes Is Developmentally and Hormonally Regulated*

(Received for publication, December 15,1983)

Anne-Marie Lompre:$, Bernard0 Nadal-Ginard, and Vijak Mahdavit From the Department of Pedintrics, Haruard Medical School and the Department of Cardhbgy, Children’s Hospital, Boston, Massachusetts 021 15

The cardiac ventricular myosin phenotype is developmentally and hormonally regulated. The genes coding for the two myosin heavy chains (MHCs), a and @, have been recently isolated and characterized. In this study, we establish the precise temporal expression of these MHC genes in correlation with the myosin phenotype both during cardiac development and in response to different thyroid hormone levels and also document their expression in other muscle tissues. The close correlation observed between the relative abundance of the a- and &MHC mRNAs and corresponding isozymes demonstrates that the MHC phenotype is produced by the expression of the a- and &MHC genes and is regulated by changes in the level of their respective mRNAs. The opposite effect of thyroid hormone on the expression of the a- and @-MHC genes in the ventricular myocardium indicates that these genes are regulated in an antithetic fashion. Finally, the MHC mRNAs encoded by the a- and @-MHC genes are also present in the atrial myocardium and in the soleus, respectiveIy.

Myosin, a multisubunit protein composed of two heavy chains (M, = 200,000), two phosphorylatable light chains (M, = 18,000-22,000), and two nonphosphorylatable light chains (Mr = 16,000-27,000), is the major structural component of the contractile apparatus. The role of myosin as an enzyme that converts chemical energy in the form of ATP into mechanical work of muscle contraction is well established.

Different myosin isozymes are found in cardiac, skeletal, smooth, and nonmuscle tissues (reference cited in Refs. 1 and 2). In the cardiac ventricles of several mammalian species, three myosin isozymes have been identified (VI, V2, and V3) on the basis of their electrophoretic mobility in nondenaturing gels (3-5). However, these three myosins are composed of only two distinct types of MHCs’ referred to as a and 8. V1 and V3 are composed of aa and &3 homodimers, respectively, while V2 has been postulated to be an a@ heterodimer (6, 7). The two types of MHCs differ in their peptide maps, immu- nological properties (5-lo), and enzymatic activities (3,4, 11,

* This work was supported by grants from the National Institutes of Health and The American Heart Association and by Grant 13- 522-812 from The American Heart Association, Central Massachu- setts Division. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Supported by the Institut National de la Santk et de la Recherche Medicale, Paris, France. Present address, INSERM U127, Hopital Lariboisiere 41, bd de la Chapelle, 75010 Paris, France.

8 To whom correspondence and reprint requests should be sent. The abbreviations used are: MHC, myosin heavy chain; bp, base

pair; PIPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

12), suggesting that they are products of different MHC genes. Indeed, two ventricular MHC mRNA sequences (13,141 and their corresponding genes (15) have been isolated and characterized. In the rat, these ventricular MHC genes are linked (15) and are part of the highly conserved sarcomeric MHC multigene family, which is composed in vertebrates by 8-10 members (16-20).

The myosin composition of the myocardium is of physiological significance since the relative distribution of the two major ventricular MHCs, a and @, is in direct correlation with the contractile performance of the heart (21, 22). The a- MHC, which confers the high Ca2+-ATPase activity to the cardiac myosin VI, is associated with increased shortening velocity of the cardiac fibers. In contrast, the ,9-MHC, which confers the low Ca*’-ATPase activity to the cardiac myosin V3, is associated with hypocontractile states.

The ratio of the different cardiac myosin isozymes is developmentally regulated. In all the species studied so far, V3 is the most abundant myosin in late fetal life. In rat and mouse, V1 increases at birth and becomes the predominant form throughout perinatal and adult life. In contrast, in larger mammals, V1 is transiently predominant after birth with V3 becoming the most abundant myosin in the adult animal (3- 5, 10).

The distribution of the cardiac myosin isozymes can be modified in certain pathological and experimental conditions such as mechanical overload (8,23-26), diabetes (27,28), and changes in thyroid hormone levels (3, 7, 10, 12, 14, 21, 29- 31). Hypothyroidism is associated with a shift from a- to 8- MHC. Protein data analysis suggests that the 8-MHC reap- pearing in the heart of hypothyroid rats is the same as the one expressed in fetal life (32). This redistribution of MHC isozyme ratio can be reversed by the administration of thyroid hormone (3, 7, 10, 29, 31).

Atrial and ventricular myosins differ from each other in their enzymatic activity and structure of their light chains (3). In addition, two different atrial myosins have been detected by nondenaturing gel electrophoresis in the rat (3) and the rabbit (5). One of the atrial MHCs is structurally and immunologically closely related to the a-MHC present in the ventricles (5, 31, 33-35). However, it has not yet been determined whether ventricular a- and atrial MHCs share a limited number of antigenic determinants or are encoded by the same gene.

The relevance of these observations to cardiac physiology and pathology makes it imperative to determine the molecular basis of the temporal, hormonal, and tissue-specific regulation of the cardiac MHC genes. First, the precise genetic basis of the cardiac MHC phenotype has to be established. Second, in order to understand the mechanisms responsible for the regulation of the MHC isozymic transitions, it is essential to

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6438 Expression of Cardiac MHC Genes

determine whether modulation of expression of the cardiac MHC genes occurs at the transcriptional, post-transcriptional, or translational levels.

Using a- and B-MHC gene-specific sequences and the S1 nuclease mapping procedure, we show that the MHC isozymic transitions in the ventricular myocardium are determined by the expression of the a- and B-MHC genes and can be entirely accounted for by changes in the accumulation rates of their respective mRNAs. Expression of the a- and B-MHC genes is not limited to the ventricular myocardium; the a-MHC gene is also expressed in the atrial myocardium, while the 8-MHC gene is expressed in slow twitch skeletal muscle fibers. Fur- thermore, our results suggest that thyroid hormone has opposite effects on the transcription of the a- and P-MHC genes.

EXPERIMENTAL PROCEDURES

Animuls-Ten-week-old male Wistar rats (Charles River Breeding Laboratories, Inc.) were thyroidectomized and kept for 6 weeks before starting thyroxin treatment by daily intraperitoneal injections of 5 pg of L-thyroxin (Sigma) dissolved in 1 ml of 0.01 N NaOH, 0.9% NaCl. 10 mM CaClz was added to the drinking water of all rats. In the development study, male Wistar rats of various ages were used.

Levels of Thyroid Hormone in the Serum-Serum concentration of total T4 was measured by a radioimmunoassay (Clinical Assays Inc., Cambridge, MA). Average values obtained per 100 ml of serum were 3.74 f 0.33 ng in normal animals, 0.23 f 0.13 ng in thyroidectomized animals, and 6.12 & 0.78 ng in thyroxin-treated animals.

Separation of Myosin Isozymes-Part of the left ventricle wall of the hearts used for MHC RNA determination was kept for analysis of the myosin isozymic pattern. The myosin isoforms were separated by 4% polyacrylamide gel electrophoresis in the presence of 2 mM sodium pyrophosphate, 2 mM EDTA, 10% (v/v) glycerol, 0.01% (v/ v) 2-mercaptoethanol, pH 8.5 (3, 36).

RNA Preparation-Total cytoplasmic RNA was isolated from various muscle tissues by using the hot phenol procedure (37).

Si Nuclease Mapping Analysis Procedure-The cDNA clones pCMHC5 and pCMHC21/26 were cleaved with the restriction endonuclease PstI, 3' end-labeled with deoxyadenosine 5'-triphosphate, 3'-[~x-~~P] (3000 ci/mmol), and terminal transferase (New England Nuclear). After size separation by electrophoresis, the desired frag-

described (38). ments were isolated and strand-separated on 5% acrylamide gel as

The probes were hybridized in DNA excess to 30 pg of total RNA extracted from different muscle tissues, as indicated. Hybridization was in 25 pl of 80% deionized formamide, 10 mM PIPES buffered at pH 6.4, 1 mM EDTA, 0.05% sodium dodecyl sulfate for 20 h at 42 "c. S1 nuclease digestion was in 300 pl for 1 h at 25 "C with 150 units of enzyme (New England Nuclear) in 300 mM NaC1,30 mM Na acetate, pH 4.5, 3 mM ZnSO,. At the end of the reaction, the samples were made 10 mM in EDTA, ethanol-precipitated, resuspended in 85% formamide, and run on 6% polyacrylamide, 8.3 M urea sequencing gels (38).

RESULTS

Procedure for the Determination and Quantitatwn of the MHC mRNAs-Previous characterization of cDNA recombi- nant clones representing cardiac MHC mRNA sequences demonstrated that two closely related MHC genes are expressed in the ventricular myocardium of the rat (13). The MHC cDNA clones named pCMHC5 and pCMHC21/26 are represented schematically in Fig. 1. The PstI restriction fragments containing the 3'-terminal portion of these clones (pCMHC5 and pCMHC21 probes) comprise a 180-base pair common sequence and the complete untranslated 3' end of two cardiac MHC mRNAs (13). These two 3"untranslated sequences are different in length and nucleotide sequence, unique in the rat genome, and therefore specific for each of the two cardiac MHC genes they represent. The remaining coding portion in both MHC cDNA clones is 95% identical except in an internal PstI fragment, pCMHC26 probe in clone

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restriction endonuclease sites and number of nucleotides are indicated in clones pCMHC5 and pCMHC21/26 (13). The fil&d and open boxes in the pCMHC5, -21, and -26 probes represent, respectively, regions of identity and divergence between the nucleotide sequence of the two MHC cDNA clones. The expected length of fully and partially S1 nuclease-protected fragments are represented by arrows. Note that the pCMHC5 and pCMHC21 probes contain poly(A) and synthetic poly(G) oligomers that are not protected from S1 nuclease digestion by the respective MHC mRNAs.

pCMHC21/26, where several regions of sequence divergence were observed (13).

These PstI restriction fragments were used as hybridization probes in S1 nuclease mapping experiments (39,40) in order to follow the expression of their corresponding MHC genes in the heart during development and in response to different thyroid hormone levels. In this procedure, three types of double-stranded RNA-DNA hybrids can be produced (see Fig. 1). Each of them will generate an S1 nuclease-protected fragment of different length; hybridization of the pCMHC5, pCMHC21, and pCMHC26 probes to their homologous RNA will generate a fully protected fragment of 304, 260, and 252 base pairs, respectively. Hybridization of the pCMHC5 probe to the mRNA represented by the cDNA clone pCMHC21/26 or hybridization of the pCMHC21 probe to the mRNA corresponding to the cDNA clone pCMHC5 will give a partially protected fragment of 180 bp corresponding to the portion common to both probes. A 102-bp fragment will be produced after complete S1 nuclease digestion of the hybrids formed by the pCMHC26 probe and the mRNA corresponding to the cDNA clone pCMHC5. Other MHC mRNAs (cardiac and skeletal) might give intermediate size or no protected fragments, depending on the location and degree of sequence homology with the probe. It is important to emphasize that both cardiac MHC mRNAs are detected by each probe, thus allowing one to follow simultaneously the expression of the two genes with any single probe. The intensity of the radioactivity in the fully and partially protected fragments will reflect the amount of each cardiac MHC mRNA present in a particular tissue.

Developmental Expression of the Cardiac a- and B-MHCs Is Regulated at the Level of mRNA Availability-The relative amount of the cardiac ventricular myosin isozymes is developmentally regulated. A significant decrease in the proportion of the V3 isozyme, which is the most abundant myosin in the late fetal life, occurs after birth and during the first 3-4 weeks of life. Conversely, the V1 isozyme which is low in fetus increases dramatically after birth and is practically the only myosin present in 28-day-old rats. Later, low amounts of V3 myosin can be detected again in adult rats (3,4). To establish at what level the regulation of these myosin transitions occurs,

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Expression of Cardiac MHC Genes 6439

the pattern of accumulation of the different ventricular MHC mRNAs was determined during development using the S1 nuclease mapping technique and the three MHC cDNA probes described above. In parallel, on samples obtained from the same hearts, the relative amount of the different myosins isozymes was determined by nondenaturing gel electrophoresis.

At least two MHC mRNAs are detected in the ventricles at almost all stages of development studied (from 18-day-old fetuses to 6-month-old adults) as shown by the presence of two SI-resistant fragments of the expected sizes, generated by using pCMHC5 (Fig. 2A), pCMHC21 (Fig. 2B), or pCMHC26 (Fig. 2C) probes. As shown in Fig. 2A, the fully protected pCMHC5 probe is only more intense than its partially protected counterpart in the ventricular RNAs of 18- and 20-day-old fetuses. After birth, the absolute level of MHC mRNA homologous to the pCMHC5 probe decreases slightly, as visualized by the amount of radioactivity in the 304-bp fragment. This MHC mRNA is barely detectable in the 28- day-old ventricle, but reappears in the 3- and 6-month-old animals. The relative abundance of this mRNA correlates very well with the relative levels of V3 myosin and 8-MHC in the same samples (see Fig. 3, C and D). Consequently, these results demonstrate that the cardiac MHC mRNA represented by pCMHC5 in fact codes for the P-MHC that is expressed both in fetal and adult life. Therefore, this mRNA

ing the untranslated 3‘ terminus, to that of pCMHC5 (data not shown).

The 180-bp long partially protected fragment shown in Fig. 2A is produced by the hybridization of the portion of the probe common to both cardiac MHC mRNAs and presumably corresponds to the a-MHC mRNA represented by the pCMHC21 probe. This mRNA is present in low amounts in the fetus, increases rapidly during the first 3 weeks of postnatal life, and is apparently the only species present at 4 weeks of age. Its pattern of expression correlates very well with the amount of a-MHC mRNA predicted by the accumulation pattern of V1 isozyme and a-MHC in the same hearts (see Fig. 3, C and D). Consequently, the S1 nuclease protection pattern using the pCMHC21 probe should be the converse image of that shown in Fig. 2A.

As expected, when the pCMHC21 probe is used (Fig. 2B), the faint fully protected 260-bp band observed with fetal ventricular RNA indicates that the RNA corresponding to pCMHC21 is a minor component of the fetal MHC mRNA. However, although the intensity of this band increases signif- icantly at birth, it remains almost constant throughout postnatal life and never becomes stronger than i ts partially protected counterpart, as would be predicted from the results shown in Fig. 2A. Furthermore, in the RNA of the 28-day-old animal, two bands are observed, one fully protected (260 bp) and one partially protected (180 bp), indicating the presence of two MHC mRNAs at this stage of development. Yet. as

is not preferentially adult-specific, as previously reported shown in Fig. 2A, the &MHe mRNA represented ’ by based on RNA blot analysis data (13). This conclusion is pCMHC5 is not detected in the RNA isolated from the same further reinforced by the fact that MHC cDNA clones isolated heart. from a cDNA library constructed from ventricular RNA of The difference between relative intensities of the label in 20-day-old fetuses have identical nucleotide sequence, includ- the fully and partially protected fragments obtained with the

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FIG. 2. Detection of cardiac ventricular “IC mRNAs during normal development by S1 nuclease mapping. Ventricular RNAs were from fetuses a t 18 or 20 days of gestation period and from 1-day to 6-month- old postpartum animals. A single animal was used for each time point a t or after 5 days of age. The probes used were pCMHC5 (A), pCMHC21 ( B ) , and pCMHC26 (C). In A, undigested pCMHC5 probe containing poly(A) and poly(G) oligomers was used as reference marker for S1 nuclease digestion. Molecular weight markers in bases are from selected PstI-digested fragments of the cDNA clones pCMHC5, -21, and -26 (13).

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FIG. 3. Relative levels of MHC mRNAs and isozymes during development. The S1 nuclease-protected bands obtained by hybridization of the different RNA samples with the pCMHC5 (A), pCMHC21 (W), and pCMHC26 (0) probes (Fig. 2) were excised from the gels, the autoradiograms of which are shown in Fig. 2. A, for each sample, the radioactivity in the fully and partially protected bands was determined in a scintillation counter and represents the total amount of MHC mRNA detected by either probe. B, the radioactivity in the fully protected bands, calculated as percentage of the total MHC mRNA, represents the percentage of each MHC mRNA homologous to the different probes. C, myosin isozyme distribution was determined by polyacrylamide gel electrophoresis in presence of pyrophosphate. D, relative proportion of a (0) and 8 (A) MHC isozymes was calculated by densitometric scanning of the gels presented in C, assuming that V1 is an aa homodimer, V2 an ab heterodimer, and V3 a 88 homodimer.

pCMHC21 and pCMHC5 probes suggests the existence of two types of a-MHC mRNAs: one represented by the cDNA clone pCMHC21/26, the other having a sequence identical to the two probes for 180 bp, but diverging at the 3' terminus. Alternatively, this result could also be explained by a specific conformation of the RNA-DNA hybrid that generates single- stranded regions. "Breathing" in the pCMHC21 RNA-DNA hybrid due to A + T rich regions would also allow digestion by the S1 nuclease. These two later possibilities are unlikely, however, given the primary sequence of the probe (13) and the fact that the ratio between the label in fully and partially protected fragments does not change with increasing S1 nuclease concentrations or the stringency of the digestion (data not shown). Furthermore, it is improbable that artifactual cleavage of the hybrid would occur consistently at the precise point of divergence between the 3' end sequence of pCMHC5 and pCMHC21.

Consequently, the existence of a second mRNA coding for an a-type MHC cannot be ruled out. In order to determine whether the sequence divergence between the two putative a- MHC mRNA species is limited to the 3' end or can be observed in other portions of the molecule, the same RNA samples used in Fig. 2, A and B, were hybridized to the pCMHC26 probe (Fig. 1) that contains the 5"upstream ye- quences of the pCMHC21/26 cDNA clone.

As shown in Fig. 2C, a band of faint intensity corresponding to the fully protected probe is detected in the RNA samples from the fetuses. The intensity of the label in this fragment increases immediately after birth, reaches a maximum at 4 weeks, and remains the strongest during the adult life, indicating that the pCMHC26 fragment represents all the a- MHC mRNA sequences expressed in the ventricles at different developmental stages. This observation is further con-

firmed by the fact that throughout development, the accumulation pattern of the MHC mRNAs detected by the pCMHC26 probe (Fig. 2C) is the exact converse image of the accumulation pattern of the MHC mRNAs detected by the pCMHC5 probe (Fig. 2A) . As expected, only the fully protected form of the pCMHC26 probe is detected in the 28-day- old ventricle.

The partially protected fragments ranging from 102 to 120 bp (Fig. 2C) correspond to the 8-MHC mRNA represented by clone pCMHC5. Indeed, scattered nucleotide sequence divergence in that region of clones pCMHC5 and pCMHC21/26 (see Fig. 1 and, for complete sequence, Ref. 13) produces unstable duplex regions in the heterologous MHC mRNA- DNA hybrids and explains the presence of several S1 nuclease-partially protected fragments. The ratio of these fragments changes with increasing concentration of S1 nuclease; the top bands disappear progressively, while the intensity of the lower one increases (data not shown).

The results shown in Fig. 2, A-C, as well as experiments performed with other portions of the cDNA clones ( d a t a not shown) demonstrate that a minimum of two different ventricular MHC mRNAs are expressed during development: one coding for P-MHC and represented by the cDNA clone pCMHC5, the other coding for a-MHC and represented by the cDNA clone pCMHC21/26. These results also suggest, but do not conclusively prove, the existence of a second a- MHC mRNA identical to the known a-MHC sequence in the entire light meromyosin-coding portion but different in the last COOH-terminal codons and the untranslated 3' end.

In addition to the changes in relative amounts of a- and 8- MHC mRNAs occurring during development, there are significant changes in the absolute level of MHC mRNA as a proportion of the total cellular RNA. Determination of the

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radioactivity in the RNA-DNA hybrids produced with each of the three probes (Fig. 3A) clearly shows that the level of total MHC mRNA is not constant during development but increases approximately 3-fold in the first 4 weeks of life. This change is mainly produced by a 10-fold increase in the level of a-MHC mRNA and a slight decrease in the level of P-MHC mRNA (see Fig. 2, A and C). Later in adult life, the level of MHC mRNA drops slowly, mainly due to the progressive decrease in a-MHC mRNA. Thus, both absolute and relative levels of a- and P-MHC mRNAs change continuously throughout the life span of the organism.

In order to determine whether the changing levels of a- and (3-MHC mRNAs during development are directly reflected in the MHC protein phenotype, the relative amounts of each MHC mRNA and isozyme, isolated from the same heart, were quantitated (Fig. 3, B and C). The relative level of P-MHC mRNA detected by the fully protected pCMHC5 probe (Fig. 3B) equals that detected by the partially protected pCMHC26 probe. Conversely, the a-MHC mRNA is detected either by the fully protected pCMHC26 probe or by the partially protected pCMHC5 probe. These observations assess the internal consistency of the data shown in Fig. 2 and demonstrate that within the technical limitation of the experimental protocol, these two probes hybridize probably to the total amount of a- and 0-MHC mRNAs present in the ventricles. However, approximately 50% of the a-MHC mRNA detected throughout development by the pCMHC26 probe is not represented by the pCMHC21 probe (Fig. 3B). This MHC mRNA, which is different from either pCMHC5 or pCMHC21/26 only at the very 3’ end, could be the product of a different gene or the result of differential splicing of the transcipts encoded by a single a-MHC gene.

The relative abundance of a- and P-MHC isozymes during development was determined from the myosin isozyme pattern shown in Fig. 3C, assuming that V2 is an ab heterodimer. These measurements (Fig. 30) clearly illustrate that the a- and @-isozymes follow a pattern of accumulation which is almost identical to that of the corresponding MHC mRNAs. Therefore, the qualitative and quantitative changes in the MHC phenotype observed during development can be entirely accounted for by changes in the level of the respective MHC mRNAs. Translational and post-translational mechanisms, if present, do not appear to play a major role in the production of the MHC phenotype during normal cardiac development.

Thyroid Hormone Modulates the Expression of the a- and P-MHC Genes-Ablation of the thyroid gland in adult rats leads to a shift of the cardiac myosin phenotype from V1 to V3. This isozyme shift can be reversed by daily injections of L-thyroxin (3). In order to gain a better understanding of the regulation of the cardiac MHC switches, it was of interest to determine the level at which thyroid hormone affects the MHC phenotype and also to establish whether reappearance of V3 in the hypothyroid animals represents re-expression and high level of accumulation of the same P-MHC mRNA present in late fetal life. For that reason, the ventricular MHC mRNA and protein phenotypes were determined in animals taken at different time intervals after thyroidectomy and hormone replacement therapy. The levels of a- and P-MHC mRNAs were measured by hybridization to the pCMHC5 (Fig. 4A) and pCMHC26 (Fig. 4B) probes.

Thyroid gland removal results in a decrease of the measured T4 serum levels in the operated animals (see “Experimental Procedures”). Three to nine weeks after thyroid ablation, the P-MHC mRNA is the only ventricular MHC transcript detected by either probe (Fig. 4, A and B). In contrast, a-MHC mRNA is predominant in the age-matched control (Fig. a,

Adult). Thyroid hormone treatment of the operated animals raises their serum T4 to equal or higher levels than normal (see “Experimental Procedures”). The hormone replacement therapy results in an immediate and progressive reversion of the MHC mRNA pattern toward the normal adult phenotype (compare first and last lanes in Fig. 4A). This reversion is produced simultaneously by a decrease in P-MHC mRNA (Fig. 4A) and an increase in a-MHC mRNA (Fig. 4B). It merits mention that the p-MHC mRNA never disappears in the thyroidectomized or normal animals that have been treated daily with 5-20 pg of L-thyroxin but remains at levels comparable to the normal age-matched controls. It should also be noted that despite the dramatic switch in the type of MHC mRNA present, no significant variability in the total amount of MHC mRNA as a fraction of the total cellular RNA was observed in the different hormonal states.

To establish the correlation between a- and P-MHC mRNA levels and the protein phenotype, the myosin isozyme pattern was determined in a tissue sample taken from the same ventricles used for the MHC mRNA determination. As shown in Fig. 4C, the V1 and V3 myosin isozyme distribution follows very closely that of a- and P-MHC mRNAs in the different thyroid hormonal states. Quantitation of these results, dis- played graphically in Fig. 5, A and B, clearly illustrates that changes in the MHC isozyme pattern produced by thyroid hormone are the result of corresponding changes in the MHC mRNA levels. The time lag observed between the mRNA and protein decay and accumulation curves can be accounted for by the respective apparent half-life of the MHC mRNAs, which is -3 days, and that of the proteins, which is -7 days. It is remarkable that the tlh values of the MHC proteins and mRNAs are very similar during the induction and de-induction process, strongly suggesting that changes in stability do not play a significant role in the production of the MHC isozyme switches.

The experiments presented above demonstrate that thyroid ablation stimulates the reexpression of the “fetal” cardiac P- MHC mRNA which then constitutes the only MHC mRNA present in the ventricular myocardium and, at the same time, represses the expression of the “adult” a-MHC mRNA to the extent that it completely disappears from the ventricles. Con- versely, thyroid hormone replacement induces synthesis and accumulation of a-MHC mRNA while it reduces, but does not completely suppress, the synthesis and accumulation of 6-MHC mRNA. Because these two mRNAs are the product of different but closely related MHC genes, (13, 15), these results demonstrate that thyroid hormone has opposite effects on the expression of the cardiac a- and P-MHC genes.

The Genes Coding for a- and &MHC mRNAs Are Also Expressed in Tissues Other Than the Ventricular Myocar- dium-In order to understand the significance and possible regulatory mechanisms involved in the production of the ventricular MHC switches shown above, the tissue-specific pattern of expression of the a- and 8-MHC genes needs to be defined. It has been shown that slow twitch (soleus) skeletal muscle and V3 myosins (35, 41, 42) and one atrial and ventricular a-MHCs (5, 31, 33-35) are structurally and immunologically related. These observations suggested the possibility that MHCs present in atrial and soleus muscle might by encoded by the cardiac a- and P-MHC genes, respectively. To test this possibility, S1 nuclease mapping experiments using the gene-specific pCMHC5, pCMHC21, and pCMHC26 probes and atrial, slow and fast skeletal muscle RNAs were performed.

AS shown in Fig. 6A, the fully protected fragment, produced by the hybridization of soleus mRNA with the pCMHC5

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FIG. 4. Effect of thyroid hormone on the levels of a- and /3-MHC mRNAs and V1 and V3 myosins. The left ventricle from a single animal was used for each sample: 10-week-old adult control; adults 3-9 weeks after thyroidectomy (7"); adults 6 weeks after thyroidectomy and treated with L-thyroxin for 4-26 days (+T). S1 mapping analysis of ventricular RNAs hybridized to the pCMHC5 probe ( A ) and pCMHC26 probe (B) . Distribution pattern of ventricular myosins, in the same samples, obtained by polyacrylamide gel electrophoresis (C).

FIG. 5. Effect of thyroid hormone on the relative levels of ventricular a- and &MHC mRNAs and isozymes. The relative amounts of &MHC mRNA (A) and isozyme (A) ( A ) and a- MHC mRNA (0) and isozyme (0) ( B ) were determined as described in the leg- end to Fig. 3. Solid line, thyroidectomized animals; dashed line, thyroidectomized animals injected daily with L-thyroxin.

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probe, demonstrates that a MHC mRNA expressed in this muscle is identical to the cardiac p-MHC mRNA and is therefore encoded by the cardiac 0-MHC gene. A low level of expression of the p-MHC in fast twitch skeletal muscle (extensor digitorum longus), seen only upon long exposure of the autoradiogram, can be accounted for by the presence of slow fibers in this muscle (43,44). Similarly, trace amounts of fully protected probe can be also detected in RNAs from right and left atria, suggesting a very low level of expression of the p- MHC gene in this tissue as well.

No full protection of the pCMHC21 probe was detected with RNAs from soleus or extensor digitorum longus (Fig. 6B), indicating that the cardiac a-MHC gene is probably not expressed in these muscle types. The 180-bp fragment observed with soleus RNA can be accounted for by hybridization of the P-MHC mRNA to its homologous portion in the pCMHC21 probe. In contrast, this probe is completely protected by the atrial mRNAs, indicating that a MHC mRNA present in the left and right atria is encoded by the a-MHC

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gene. The 180-bp fragment protected by atrial RNA is not due to the presence of high levels of p-MHC mRNA as shown by the protection pattern of the pCMHC5 probe (Fig. 6A). This partially protected fragment could be produced by hybridization to another atrial MHC mRNA which shares common sequences to both probes, as was shown to be the case in the ventricles (see above). The fact that the pCMHC26 probe is completely protected by atrial mRNA (Fig. 6C) strongly indicates that the same a-MHC sequences are expressed in both atrial and ventricular cells of the heart. Similar S1 nuclease protection pattern, obtained by using the genomic sequences encoding the NHj-terminal portion of a- MHC as hybridization probe (15), corroborates this conclusion. With the pCMHC26 probe, a faint partially protected fragment is also observed after long exposure of the autoradiogram. This result, consistent with that obtained with the pCMHC5 probe, suggests the expression a t a very low level of the p-MHC gene in the atria. No qualitative differences between left and right ventricular and atrial RNAs were

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FIG. 6. Expression of a- and &MHC mRNAs in different tissues. S1 mapping analysis was performed using RNAs extracted from soleus muscle (SOL), extensor digitorum longus ( E D L ) , left (LV), or right (RV) ventricle, and left ( L A ) or right ( R A ) atrium. The probes used were pCMHC5 (A ) , pCMHC21 ( B ) , and pCMHC26 ( 0 .

observed in the hybridization patterns to each probe. The fact that the a-MHC gene is expressed in both ven-

tricular and atrial myocardium and that the 0-MHC gene is expressed in both ventricular myocardium and slow skeletal muscle fibers reveals a new aspect of the tissue-specific expression of the MHC genes and raises important questions related to their developmental stage- and hormone-mediated regulation.

DISCUSSION

The Ventricular a- and p-MHC Phenotype Is Produced by Modulation of the Expression of the MHC Genes-The results presented here demonstrate that the ontogenic changes of the ventricular MHC phenotype leading to the replacement of a MHC with low ATPase activity (P-MHC) predominant in late fetal life by a MHC of higher ATPase activity (a-MHC) predominant in adult life are produced, a t least in the rat, by closely parallel changes in the levels of 8- and a-MHC mRNAs. The same correlation was also found during experimental manipulation of the ventricular MHC phenotype by thyroid hormone and was also observed in the rabbit (7, 14, 31). The P-MHC mRNA and protein expressed in fetal and adult life as well as in the hypothyroid state are identical and therefore the products of the same gene. These results confirm and extend previous protein data in which the MHC from fetal hearts could not be distinguished from MHC obtained

from hypothyroid animals (32). Although only one a-MHC protein has been identified so far, our results suggest that there might be two different a-MHC mRNAs that are identical for most of their length but diverge at the region coding for the last four or five carboxyl-terminal amino acids.

The two types of MHC mRNAs, a and 0, account for the complete ventricular MHC phenotype. No evidence for a V2- specific MHC mRNA was found. The fact that the level of V2 myosin is high only when both a- and D-MHC mRNAs are present in significant amounts in the same tissue supports the conclusion of other investigators (6, 7) that this MHC is an a@ heterodimer. The lack of binomiality in the distribution of this myosin isozyme can easily be accounted for by the heterogeneous distribution of a- and p-MHC mRNAs among different ventricular cells, as has been demonstrated at the protein level (10, 42).

We have recently identified and characterized the genes coding for the P-MHC mRNA, represented by the cDNA clone pCMHC5, and for the a-MHC mRNA, represented by the cDNA clone pCMHC21/26 (15). These two genes are organized in tandem, 4 kilobase pairs apart, and oriented 5‘ to 3‘ in the order of their developmental expression. The origin of the putative second a-MHC mRNA is unclear. This transcript could be the product of another MHC gene that has not been identified with the probes used or the result of an alternative splicing pathway of the a-MHC gene primary

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transcript. Although not yet proven, the latter possibility is favored by the fact that the two genes isolated so far appear to contain all the common sequences of the a- and @"HC mRNAs that are present in the genome. Moreover, in these two genes, the 3' end-specific sequences are encoded by sep- arate exons (15). Thus, the existence of a second 3' end exon in the a-MHC gene could produce the second a-MHC mRNA. In this respect, it is interesting that differential splicing pathways of the 3' end of the Drosophila MHC gene transcript have been demonstrated (45,46). Thus, although the existence of a third MHC gene expressed in the myocardium has not been conclusively ruled out, the data presently available strongly suggest that the ventricular MHC phenotype can be entirely accounted for by the modulation of the expression of two MHC genes.

The developmentally and physiologically regulated MHC phenotype, produced by the presence of a minimum of two MHC isozymes in the same tissue, seems to be a general feature of all striated muscles. Transitions between a minimum of three temporally regulated MHC isozymes have been observed during the development of individual muscles in the rat (47), chicken (2, 48), and Drosophila (45, 46). In rat (49) and chicken (50,51), the different MHC isozymes are encoded by different genes, whereas in Drosophila (45, 46), they are produced through three different splicing pathways of the same MHC mRNA transcript.

The MHC Switches during Development and in Response to Thyroid Hormone Levels Are Regulated by the Level of MHC mRNA Availability-The close correlation between the levels of a- and @-MHC mRNAs and the corresponding proteins, both during development and in response to thyroid hormone, strongly suggests that the MHC phenotype is regulated by change in the level of cytoplasmic a- and @-MHC mRNAs. Control mechanisms occurring at the translational or post- translational level do not need to be evoked to produce the observedphenotype. A similar correlation between the pattern of contractile protein accumulation and mRNA levels has been observed during in vitro myogenesis (52-59) and development in Drosophila (60).

The experiments presented here do not allow the determination of whether induction and de-induction of a- and @- MHC mRNAs during development and in response to thyroid hormone are produced by changes in the rate of transcription, changes in the stability of the transcripts, or a combination of both. However, the kinetics of disappearance of the @-MHC mRNA and appearance of the a-MHC mRNA after thyroxin treatment is practically identical, with the apparent half-life of both mRNAs being approximately 3 days. This value is in good agreement with the tlhvalue reported previously for MHC mRNA (61) and strongly suggests that induction and de- induction of the ventricular MHC mRNAs are regulated by the same mechanism. The modulation in the mRNA levels can be best explained by changes of the transcriptional rate of the corresponding genes. This postulate is in agreement with the demonstration that the main regulatory event in MHC expression during L6E, cell myogenesis occurs at the transcriptional level (61). In addition, as a result of the appropriate hormonal stimulus, enhanced transcription of genes such as growth hormone (62,63), rat a - 2 ~ globulin (641, mouse mammary tumor virus (65), ovalbumin, and conalbumin (66) has been observed. However, hormones may also affect mRNA metabolism by changing its stability, as has been shown for ovalbumin and conalbumin (67, 68), and casein genes (69). Although, from the results presented here, it is not possible to rule out such post-transcriptional mechanisms in the accumulation of a- and @-MHC mRNAs during

development and in response to thyroid hormone, their role seems limited at best, given the similar kinetics of induction and de-induction of the two mRNAs.

The a- and P-MHC Genes Are Also Expressed in Atrial and Slow Twitch Skeletal Muscle, Respectively-As discussed above, the a- and 0-MHC genes account for the complete ventricular MHC phenotype. However, these two genes are not only expressed in the ventricular myocardium. The data presented here and elsewhere (15) unambiguously demonstrate that the a-MHC gene is also expressed in the atria, while the B-MHC gene is expressed in slow twitch skeletal muscle fibers. Consistent with these results, previous protein data analysis has shown close structural similarities between the ventricular a-MHC and one MHC present in the atrium (5, 31, 33) and between ventricular @-MHC and the slow twitch muscle MHC (35, 41, 42). However, this is the first demonstration that at least one of the MHCs in atrial and slow twitch skeletal muscles is encoded by the ventricular a- and @-MHC genes, respectively. Although another MHC gene is also expressed in soleus muscle: only the a-MHC and low levels of (3-MHC mRNAs have so far been detected in the atria with the probes presently available.

Co-expression in skeletal and cardiac muscle has also been demonstrated for other contractile protein genes. For in- stance, the fetal skeletal muscle light chain is also found in the fetal ventricles (70) and adult atria (1); skeletal and cardiac a-actin genes are co-expressed in skeletal muscle and in fetal and adult hearts in the mouse (71), rat: and human (72). Thus, it is becoming apparent that skeletal and cardiac muscles, although quite distinct at the cellular and physiological levels, share a significant number of contractile protein genes. The ability to analyze the expression of a given gene in two different tissues offers new insights into its regulation (see below) and into the plasticity and complexity of the sarcomeric contractile apparatus. While the a- and p-MHCs associate with the same light chains in the ventricle to form the V1, V2, and V3 myosin molecules, they combine with different light chains both in the atria and in slow twitch muscle (73) to constitute different myosins, as was also shown for skeletal MHCs during muscle development (47). Although the physiological significance of the multiplicity of myofibers generated by the assembly of a limited number of constituents is not yet clear, these observations demonstrate the necessity to biochemically define these different myofibers before meaningful physiological comparisons can be established.

Is Thyroid Hormone the Regulator of the a- and P-MHC Genes?-The results presented here clearly demonstrate that modulation in the MHC phenotype in response to thyroid hormone depletion/replacement is produced by the corresponding changes in MHC mRNA accumulation, strongly pointing toward changes in transcription rates as a main response to the thyroxin levels. The identification of chro- matin-associated T3 receptors (74) supports the concept of a direct effect of this hormone on a- and P-MHC gene transcription. Indeed, thyroxin has been shown to directly en- hance transcription of the growth hormone gene (63). Alter- natively, thyroid hormone may act indirectly on the MHC phenotype through, for example, changes in the circulating levels of growth hormone. However, the fact that thyroid hormone administration to hypothyroid animals results in the appearance of newly synthesized a-MHC mRNA after a lag

D. Bois and B. Nadal-Ginard, manuscript in preparation. R. Grebenau, V. Mahdavi, and B. Nadal-Ginard, unpublished

observation.

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of only a few hours' together with the ability of this hormone to induce changes in the MHC protein pattern in vitro (75) points toward a direct effect of thyroid hormone in the regulation of the a- and p-MHC genes.

Despite the ability of thyroid hormone to directly or indirectly modulate the expression of the a- and P-MHC genes, its role as the sole or main modulator of these genes during development has not yet been established. In rat, the increase of a-MHC and decrease of P-MHC after birth closely relate with increased levels of circulating thyroid hormone (76, 77), thereby suggesting a direct role of the hormone in the production of the adult phenotype. However, the ontogenic changes are mainly the result of the modulation of a-MHC gene expression, whereas thyroid hormone in the adult ap- pears to modulate both a- and p-MHC genes. To evaluate the relevance of these observations, it should be noted that during fetal and early postnatal development, the increase in cardiac cell mass is produced both by cell growth and cell division (78). It is possible that at these early stages, the heart is constituted by a heterogeneous population of cardiocytes that are at different points in the maturation pathway and might not respond simultaneously to the factors responsible for the change in the MHC phenotype. Heterogeneity among the cardiac cells, in which either a-, p-, or both MHC isozymes are detected, has been observed particularly during the periods of transition from one phenotype to the other (10,42). On the other hand, the cardiac MHC switches in the adult and those produced in response to thyroid hormone occur in stationary cells that are presumably more synchronous in their response to external stimuli. Whether or not these differences at the cellular level play a role in the regulation of the MHC phenotype is not yet known.

There is no evidence that the significant increases in p- MHC in response to work overload (8, 23, 25, 26), aging (31, diabetes (27, 28), and castration (79) are produced through changes in thyroid hormone levels. Moreover, the a- and p- MHC genes respond differently to thyroid hormone in ventricular myocardium and in extraventricular tissues. First, high levels of a-MHC mRNA are found in the atria of long term hypothyroid animals,' whereas this mRNA is not detectable in the ventricles. Second, in adult euthyroid animals, the p-MHC mRNA is present in high amounts in soleus muscle, whereas very low amounts are detected in the ventricles. Ontogenic and tissue-specific differences in thyroid hormone receptors, either quantitative or qualitative, could be the underlying basis of this differential expression of the MHC genes.

Despite these unresolved questions, the striking effect of thyroid hormone, and presumably of the pathological conditions that have been shown to alter the MHC phenotype in the ventricular myocardium, is that it can have an opposite effect on two closely related genes. The a- and @-MHC genes are tightly linked and have a high degree of sequence conservation throughout their entire length (15), indicating that they have originated by a relatively recent duplication of a common ancestor. I t is obvious that despite the high degree of conservation in the coding sequences, the two genes have evolved different regulatory sequences that permit them to respond, directly or indirectly, in an antithetic manner to the same stimulus. Interestingly, the modulation of the expression of the MHC genes occurs in the absence of DNA replication. This fact, observed as well in a myogenic cell line (59, 80), is relevant because it has been postulated that changes in chro- matin conformation and DNA methylation, associated with " .

' A.-M. Lomprb, B. Nadal-Ginard, and V. Mahdavi, unpublished observation.

changes in gene expression, only occur during DNA replication (reviewed in Ref. 81).

Obviously, the biochemical parameters involved in the developmental, hormonal, and tissue-specific regulation of the a- and P-MHC genes need to be analyzed in further detail. The recent isolation and characterization of these genes (15) should facilitate progress in this analysis.

Acknowledgments-We would like to thank C. Wisnewsky for her excellent technical assistance in performing the protein analysis and Dr. K. Schwartz (both from the Institut National de la Santi et de la Recherche Medicale U127) for helpful discussions and Drs. H. T. Nguyen, E. E. Strehler, and D. Wieczorek for critical reading of the manuscript. The expert secretarial assistance of M. Hager is gratefully appreciated.

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A M Lompré, B Nadal-Ginard and V Mahdavidevelopmentally and hormonally regulated.

Expression of the cardiac ventricular alpha- and beta-myosin heavy chain genes is

1984, 259:6437-6446.J. Biol. Chem.

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