Catecholamines and cardiac growth.

The present knowledge concerning the alpha- and beta-adrenergic systems in the regulation of cardiac growth and gene expression is reviewed. To investigate the mechanism by which cAMP regulates the expression of cardiac genes we have used cultured myocytes derived from fetal rat hearts. We have shown previously that the addition of Br cAMP to the culture medium produced an increase in alpha-myosin heavy chain (alpha-MHC) mRNA level, in its rate of transcription as well as in the amount of V1 isomyosin. To characterize the promoter element(s) involved in cAMP responsive regulation of alpha-MHC expression we performed transient transfection analysis with a series of alpha-MHC gene promoter-CAT constructs. We have identified a 13 bp E-box/M-CAT hybrid motif (EM element) which conferred a basal muscle specific and cAMP inducible expression of the alpha-MHC gene. Using mobility shift assay we have documented that one of the EM element binding protein is TEF-1. Moreover, by incubating cardiac nuclear extracts with the catalytic subunit of PK-A we have found that factor(s) binding to the EM element is a substrate for cAMP dependent phosphorylation.

Developmental shift of myosin heavy chain mRNA expression due to neural factor(s) and muscle activity.

The adult ventricular isoform of chicken myosin heavy chain (MHC-V) is transiently expressed in all skeletal muscle primordia analyzed and is completely repressed around embryonic days 10-12, when functional innervation is established. By ribonuclease protection assay, we demonstrated that denervation of the adult anterior latissimus dorsi muscle resulted in reexpression of MHC-V mRNA. In contrast, treatment of primary cultures of fetal breast or leg muscles with embryonic brain extract or conditioned media from glial or neuroblastoma cell lines, but not from a myogenic cell line or primary muscle cell cultures, led to inhibition of MHC-V expression. This inhibitory activity was abolished by heating and increased with protein concentration. The acquisition of both brain inhibitory activity and the competence of myogenic cells to downregulate MHC-V mRNA expression were age dependent. Furthermore, either paralysis of muscle in ovo by curare or contraction arrest of cultured myotubes resulted in persistent expression of MHC-V mRNA. Thus a putative soluble factor(s) of nerve origin as well as muscle activity are involved in the developmental downregulation of MHC-V expression in muscle primordia.

Cardiac phenotypic markers expressed in early stages of both cardiac and skeletal muscle development.

We have examined changes in the expression of chicken myosin heavy chain (MHC) mRNAs in the heart and skeletal muscles during normal development and in regenerating adult muscles. cDNA clones isolated from adult heart and regenerating skeletal muscle libraries revealed more than 98% sequence homology in the 3' untranslated regions. Using specific cDNA probes we have detected ventricular MHC transcripts in the heart and in early developmental stages of fast as well as slow skeletal muscles. The expression of ventricular MHC mRNA in skeletal muscles is especially significant since, in contrast to mammals, the avian ventricular and slow MHC mRNAs are encoded by different genes.

Differential expression of ventricular-like myosin heavy chain mRNA in developing and regenerating avian skeletal muscles.

Based on previous immunological data, cross-reactivity of myosin heavy chain (MHC) with the ventricular (V) isoform was observed in primordia of avian skeletal muscles and in regenerating adult anterior latissimus dorsi (ALD) muscle. To determine whether this primordial (P) MHC is identical to adult V-MHC gene product, we have cloned and characterized the 3' portion of MHC cDNA that is expressed in ALD muscle at 3 d of regeneration. Comparison of nucleotide sequences between adult V-MHC and P-MHC cDNAs revealed more than 98% homology in the 3'-untranslated (UT) portions of these genes. The expression pattern of P-MHC was analyzed in adult regenerating muscles using total RNA from two fast muscles, posterior latissimus dorsi (PLD) and pectoralis major (PM), as well as from slow ALD and mixed fast/slow gastrocnemius muscles at 0, 1, 3, 4, 6, 9, and 14 d after cold injury. Identical results were obtained by RNase protection assays using either a probe specifying the coding region of adult V-MHC or a P-MHC probe encoding the carboxy end plus the 3'-UT region. The expected protected fragments were detected early from day 2 up to day 6 in ALD muscle. Similar rate of appearance, reaching the highest level at day 3, was observed in PLD, PM, and gastrocnemius muscles. However, the amount and the kinetics of disappearance differed among the various muscles analyzed. In contrast, during development, steady-state levels and kinetics of V-MHC mRNA expression were found to be alike in axial and appendicular muscles. These data strongly suggest the identity of P-MHC as the ventricular isoform and support the concept that expression of P-MHC mRNA is a common feature of developing as well as of all regenerating adult skeletal muscles. Interestingly, no expression of cardiac specific myosin light chain (MLC) 2A was observed after cold injury, suggesting independent regulatory pathways for the two kinds of myosin subunits.

Structural and phylogenetic analysis of the chicken ventricular myosin heavy chain rod.

We have isolated and characterized five overlapping clones that encompass 3.2 kb and encode a part of the short subfragment 2, the hinge, and the light meromyosin regions of the myosin heavy chain rod as well as 143 bp of the 3' untranslated portion of the mRNA. Northern blot analysis showed expression of this mRNA mainly in ventricular muscle of the adult chicken heart, with trace levels detected in the atrium. Transient expression was seen in skeletal muscle during development and in regenerating skeletal muscle following freeze injury. To our knowledge, this is the first report of an avian ventricular myosin heavy chain sequence. Phylogenetic analysis indicated that this isoform is a distant homolog of other ventricular and skeletal muscle myosin heavy chains and represents a distinct member of the multigene family of sarcomeric myosin heavy chains. The ventricular myosin heavy chain of the chicken is either paralogous to its counterpart in other vertebrates or has diverged at a significantly higher rate.

Muscle-specific regulation of a transfected rabbit myosin heavy chain beta gene promoter.

We have examined the transcriptional regulation of the rabbit myosin heavy chain (HC) beta gene by using DNA-mediated transfection experiments. To analyze the activity of the myosin HC beta promoter in a myogenic background, cultured myoblasts from 12-day-old chick embryonic breast muscle were transfected with a chimeric gene containing 781 base pairs of the promoter region fused to the gene for chloramphenicol acetyltransferase (CAT). As indicated by the transient expression of chloramphenicol acetyltransferase, the activity of the promoter in myoblast cultures increased at least 32-fold following differentiation and was selectively inhibited when myogenesis was blocked with 5-bromodeoxyuridine. Furthermore, RNase protection experiments showed that the in vivo myosin HC beta transcriptional initiation (or cap) site was utilized in the transfected skeletal muscle cells and also that the regulation of the exogenous promoter was similar to the induction of the endogenous skeletal alpha-actin gene. The results indicated that the exogenous promoter is regulated in a tissue- and stage-specific manner. By creating progressive 5' deletions of the promoter, we showed that only the region extending -294 base pairs upstream from the cap site is necessary for the muscle-specific expression. Linker-scanner mutagenesis of this region indicated that the positive regulation in differentiated skeletal muscle is mediated by at least two distinct elements within the 5'-flanking region of the myosin HC beta gene.

Control of myosin heavy chain expression in cardiac hypertrophy.

The control of myosin expression by thyroid hormone is analyzed as an example of compensatory mechanisms of the heart. Two topics are discussed in detail: polymorphism of cardiac myosin heavy chains in the mammalian heart, and effect of thyroid hormone on myosin heavy chain expression by thyroid hormone. Our current knowledge about the identity of heavy chains and their corresponding isomyosins myosins is summarized and the dynamic nature of the myosin phenotype of the heart is discussed. The data on the thyroid hormone's role include studies in which the synthesis rate of the 2 classes of heavy chains (alpha and beta) was compared with their respective messenger RNA levels. A close correlation was observed and is consistent with pretranslational control. Transcription of myosin heavy chain genes was examined using isolated nuclei in a run-off experiment The rate of gene transcription was found to be the principal determinant of the cytoplasmic level of messenger RNA and of the isomyosin composition of the heart.

Characterization of genomic clones specifying rabbit alpha- and beta-ventricular myosin heavy chains.

We have isolated gene sequences coding for the alpha- and beta-myosin heavy chains (HC) of rabbit ventricular muscle. A rabbit genomic library was screened with previously characterized cDNA clones specifying part of the light meromyosin and the entire subfragment 2 portion of alpha- and beta-myosin HCs, as well as with a clone containing the 3' nontranslated sequences of the alpha-myosin HC mRNA. Seven strongly hybridizing clones were analyzed in detail. One genomic clone encoded all of the 3' nontranslated sequences of an alpha-cDNA clone and, therefore, contained the 3' end of the alpha-myosin HC gene. Electron microscopic heteroduplex analysis and DNA sequence analysis showed that this clone overlapped a second genomic clone providing more than 25 kilobase pairs of the alpha-myosin HC gene. The exons within this region corresponded to approximately equal to 85% of the mRNA and were separated by at least 28 introns. A clone for the beta-myosin HC gene was also identified by Southern blot hybridization, by heteroduplex mapping, and by comparing the DNA sequence of a subfragment 2 exon to sequences of the alpha- and beta-cDNA clones. The introns of the alpha- and beta-myosin HC genes were in the same position but showed marked variation in length. These results conclusively showed that the alpha- and beta-myosin HCs are products of separate genes.

Expression of rabbit ventricular alpha-myosin heavy chain messenger RNA sequences in atrial muscle.

We have constructed and isolated a cardiac myosin heavy chain (HC) cDNA clone, pMHC alpha 81, with mRNA from ventricular heart muscle of hyperthyroid rabbits. The clone encodes approximately 480 amino acids of the COOH terminus of light meromyosin and all of the 3' nontranslated region of the corresponding mRNA. Nuclease S1 analyses indicated that the clone is transcribed in hyperthyroid, but not in hypothyroid ventricles and, therefore, corresponds to ventricular alpha-HC mRNA. With probes from the more divergent 3' non-translated region of pMHC alpha 81 and also from selected portions of two previously characterized rabbit cDNA clones ( pMHC alpha 252 and pMHC beta 174), we analyzed the myosin HC mRNAs of atrial, fast skeletal, and slow skeletal muscles by nuclease S1 mapping. In atrial muscle, only one major transcript was detected. The sequence of this transcript was indistinguishable from ventricular alpha-HC mRNA in the 3' nontranslated region and in two coding segments. In contrast, the sequence divergence between the ventricular alpha-HC mRNA and the mRNAs of ventricular beta, fast skeletal, and slow skeletal myosin HCs was clearly detected. There appeared to be, however, considerable homology between coding sequences of ventricular beta and slow skeletal myosin HC mRNAs. The results strongly suggest that rabbit atrial and ventricular alpha-HCs are encoded by the same gene.

Regulation of myosin synthesis by thyroid hormone: relative change in the alpha- and beta-myosin heavy chain mRNA levels in rabbit heart.

The expression of mRNAs for two cardiac myosins has been examined in the ventricles of hypo- and hyperthyroid rabbits by means of cloned cDNA sequences corresponding to the mRNAs of the alpha- and beta-myosin heavy chains (HCs). The temporal change in the relative levels of the alpha- and beta-HC mRNAs after 3,5,3'-triiodothyronine (T3) treatment of hypothyroid rabbits was determined by nuclease S1 mapping. In the hypothyroid state, only HC beta-mRNA was expressed in the ventricles. The HC alpha-mRNA was first detectable 4 h after administration of T3 (200 micrograms/kg) to hypothyroid animals. By 12 h, HC alpha-mRNA represented 20% of total myosin mRNA, increasing to 50% by 24 h and to about 90% by 72 h. The relationship between the relative mRNA levels and relative synthesis rates of the myosin HCs was evaluated in 5-6-week-old normal and thyrotoxic rabbits. Myosin synthesis rates were determined by labeling of protein in vivo with [3H]leucine. The V1 (HC alpha) and V3 (HC beta) isomyosins were separated by affinity chromatography with monoclonal antibodies, and the HCs were isolated electrophoretically. In a normal euthyroid group of animals and in animals 12 and 24 h after administration of 200 micrograms of 3,5,3',5'-tetraiodothyronine/kg, the relative mRNA levels and relative synthesis rates of the alpha- and beta-HCs were not significantly different. Our results show that, first, thyroid hormone causes a rapid accumulation of HC alpha-mRNA and loss of HC beta-mRNA and, second, in normal and thyrotoxic rabbits, the relative synthesis rates of HC alpha and HC beta reflect the relative abundance of the alpha- and beta-HC mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of cloned mRNA sequences encoding subfragment 2 and part of subfragment 1 of alpha- and beta-myosin heavy chains of rabbit heart.

Two cardiac myosin heavy chain cDNA clones, pMHC alpha 252 and pMHC beta 174, were constructed using rabbit ventricular mRNA isolated from adult thyrotoxic and normal hearts, respectively. The complete DNA sequences of the 2.2- and 1.4-kilobase inserts of pMHC beta 174 and pMHC alpha 252, respectively, were obtained. The 736 amino acids specified by pMHC beta 174 begin 439 (1.3 kilobases) residues from the heavy chain NH2 terminus and include a 400-amino acid segment of subfragment 1 and the entire subfragment 2 region. Clone pMHC alpha 252 encodes 465 amino acids encompassing all of subfragment 2 and a portion of light meromyosin. Comparison of these two clones revealed extensive sequence overlap which included 1107 nucleotides specifying a 369-amino acid segment corresponding to subfragment 2. Within this region 78 (7%) base and 32 (8.7%) amino acid mismatches were noted. These differences were clustered within discrete regions, with the subfragment 1/subfragment 2 junctional region being particularly divergent. Structural differences between pMHC alpha 252 and pMHC beta 174 indicate that these two clones represent two similar but distinct myosin heavy chain genes whose expression is responsible for ventricular myosin heavy chain isoforms alpha and beta, respectively. The derived amino acid sequences of both clones exhibit extensive homology (greater than 81%) with sequences obtained by direct analysis of adult rabbit skeletal muscle myosin heavy chain protein. The sequences corresponding to the subfragment 2 region are consistent with an alpha-helical conformation with a characteristic 7-residue periodicity in the linear distribution of nonpolar amino acids. Conversely, subfragment 1 sequences specified by pMHC beta 174 suggest a folded highly irregular structure.

Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. I. DNA and derived amino acid sequence of light meromyosin.

Two myosin heavy chain cDNA clones (251 and 110), constructed from chick embryonic skeletal muscle mRNA, were subjected to extensive DNA sequence analysis. A complete description of the DNA sequence of clone 251 was obtained. This 1.5-kilobase pair cDNA sequence specified the COOH-terminal 439 amino acids of the myosin heavy chain, and included the entire 3' nontranslated region. The translated and 3' nontranslated sequences were purine- (64%) and AT-(71%) rich, respectively. The derived amino acid sequence of clone 251 correlated well with sequences obtained by direct amino acid sequencing of adult rabbit back muscle myosin heavy chain protein (87% homology), as well as with cloned myosin heavy chain sequences from other species. Comparison of clone 251 with a partial DNA sequence of clone 110 revealed significant structural differences both in the translated, and 3' nontranslated regions. This data indicates that these two clones represent two distinct myosin heavy chain genes. The protein sequence specified by clone 251 corresponds to the light meromyosin portion of the myosin heavy chain rod. These sequences, like other myosin heavy chain rod sequences, are alpha-helical and exhibit 7- and 28-residue periodicities in the linear distribution of nonpolar, and basic and acidic amino acids, respectively.

Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. II. Expression during development using S1 nuclease mapping.

We have examined the expression of two embryonic myosin HC mRNAs using two cDNA clones (110 and 251) which we have previously constructed from RNA isolated from 14-day-old embryonic chick skeletal muscle. Sequence divergence in the 3' nontranslated regions enabled us to analyze the differential expression of the mRNAs corresponding to the two clones using the S1 nuclease mapping procedure. Clone 251 mRNA is expressed primarily in embryonic fast muscle, where its transcripts appear to be the predominant species. This mRNA is minimally expressed in the posthatching period, but it is not detected in adult leg and breast muscle. Messenger RNA for clone 110 is also primarily expressed in embryonic fast muscle. However, in the posthatching and adult stages of development, it continues to be expressed at a low level in leg muscle but not in breast muscle. The differential expression of these mRNAs during development strongly indicates that they correspond to two different genes coding for embryonic myosin HCs. Other myosin HC mRNAs which were partially homologous to the clone 110 or 251 mRNAs were also identified by S1 nuclease mapping. Using the probes from these two clones, a minimum of four other developmentally expressed forms were detected. Two of these correspond to "neonatal" myosin HCs, while the other two code for different adult myosin HCs present in leg and in breast muscle, respectively. The results therefore suggest a much greater diversity of myosin HC mRNAs expressed during development than previously reported.

Molecular cloning of mRNA sequences for cardiac alpha- and beta-form myosin heavy chains: expression in ventricles of normal, hypothyroid, and thyrotoxic rabbits.

We have isolated cDNA clones from thyrotoxic (pMHC alpha) and normal (pMHC beta) adult rabbit hearts. Restriction map analysis and DNA sequence analyses show that, although there is strong homology between overlapping regions of the two clones, they are distinctly different. The two clones exhibited 78-83% homology between the derived amino acid sequences and those determined by direct amino acid sequence analysis of rabbit fast skeletal muscle myosin heavy chains. The clones specify a segment of the myosin heavy chain corresponding to subfragment 2 and the COOH-terminal portions of subfragment 1. Nuclease S1 mapping was used to compare transcription of the two clones with expression of the alpha and beta forms of myosin heavy chains in the ventricles of thyrotoxic, hypothyroid (propylthiouracil-treated), and normal rabbits. Thyrotoxic ventricles contained only pMHC alpha transcripts whereas hypothyroid ventricles contained exclusively pMHC beta transcripts. These data correlate well with the presence of alpha- and beta-form myosin heavy chains. In the normal young adult rabbit, pMHC beta transcripts predominate, agreeing with the known beta form/alpha form ratio of 4:1. We therefore conclude that pMHC alpha and pMHC beta contain sequences of the alpha- and beta-form myosin heavy chain genes, respectively.

Isolation and characterization of two molecular variants of myosin heavy chain from rabbit ventricle. Change in their content during normal growth and after treatment with thyroid hormone.

We have prepared monoclonal antibodies specific for cardiac myosin heavy chain. These antibodies were used for the separation and characterization of the molecular variants of myosin heavy chain present in the rabbit heart. Two molecular forms of myosin heavy chain, HC alpha and HC beta, were isolated from the euthyroid rabbit heart by affinity chromatography. Their reactivity with our antibodies indicated that the primary structures of HC alpha and HC beta differ in at least four and share at least two antigenic determinants. Differences in the primary structure of HC alpha and HC beta were confirmed by analysis of the peptides produced by limited chymotryptic digestion of the two heavy chains. Thirteen peptide differences were consistently found. The HC alpha and HC beta variants are shown by immunologic analysis and in chymotryptic peptide profiles to be identical with the predominant forms of myosin heavy chain synthesized in the hearts of hyperthyroid and adult euthyroid rabbits, respectively. During development and maturation of the euthyroid rabbit heart, HC alpha comprises approximately 50% of the ventricular myosin between birth and 4 weeks of age; it diminishes to 20-30% by 8 weeks and to 10-20% by 12 weeks of age. Cardiac myosin from a 1-year-old rabbit is composed almost entirely of HC beta. Cardiac myosin from embryonic animals at 20 days gestation contained 20% HC alpha. These results show that HC alpha occurs normally in the euthyroid rabbit heart and that the relative proportions of HC alpha and HC beta depend on both the developmental stage and the thyroid state of the animal.

Molecular cloning of two fast myosin heavy chain cDNAs from chicken embryo skeletal muscle.

Recombinant DNA clones containing sequences for two different types of myosin heavy chain (HC) genes from chicken embryonic skeletal muscle were constructed and analyzed. Specificity of the clones for myosin HC was demonstrated by hybrid-arrested translation, by hybridization to a 7.0-kb mRNA, and by comparison of DNA sequences with known amino acid sequences of rabbit skeletal muscle myosin HC. Restriction enzyme and electron-microscopic heteroduplex analysis showed the presence of two distinct but homologous cDNA sequences. Hybrid melting curves indicated that both types of sequences represent fast myosin HC sequences.

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states.

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.