Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle.

In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.     

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

Molecular cloning and characterization of human atrial and ventricular myosin alkali light chain cDNA clones

We have isolated essentially full-length cDNA clones for atrial (ALC1) and ventricular (VLC1) myosin alkali light chains from a human fetal heart cDNA library. Comparison of overall nucleotide sequences of ALC1 and VLC1 cDNA clones has revealed that, while these two inserts show significant DNA sequence homology (78.4%) with respect to their coding regions, the 5'- and 3'-untranslated regions are highly divergent. Our statistical analysis suggests that human ALC1 and VLC1 diverged approximately 300 million years ago, during the time of separation of birds and mammals. RNA blot analysis shows that ALC1 mRNA is expressed in fetal ventricular and fetal skeletal muscles as well as fetal and adult atrial muscles and VLC1 mRNA is expressed in adult slow skeletal muscle as well as fetal and adult ventricular muscles. Southern blot analysis indicates that each protein is encoded by a single gene. Finally, we show that VLC1 mRNA is induced in pressure-overloaded human atrium.     

Cloning and characterization of cDNA sequences corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin

A library of cDNA clones was constructed from adult rat skeletal muscle mRNA, from which a set of contractile protein clones was selected. These clones were identified by sequencing the cDNA inserts and comparing the derived amino acid sequences with published sequences of rabbit contractile proteins. In this manner, clones corresponding to myosin light chains 1, 2, and 3, troponin-C, troponin-T, alpha-tropomyosin, and alpha-actin were identified. A high degree of amino acid sequence conservation was found upon comparison of the rat and rabbit proteins. Using the cDNA clone panel, we analyzed the expression of abundant rat muscle mRNAs. We show that abundant rat muscle mRNAs can be classified into four developmentally regulated groups, based upon their expression at different stages of myogenesis. One class of mRNAs is expressed during all stages of muscle development. Since these mRNAs are also present in nonmuscle tissues, we conclude that they code for housekeeping proteins. The second class of mRNAs is present in both embryonic and adult muscle, while a third class of mRNAs is expressed only in adult muscle. A small number of mRNAs, which are present at greater levels in undifferentiated myoblasts than in adult muscle, comprise a fourth class. These results suggest the existence of at least four modes of gene control during myogenesis.     

Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain

We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers.     

Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Two different forms of beta myosin heavy chain are expressed in human striated muscle

Summary We have found evidence for two beta-like myosin heavy chains in humans, one cardiac and one skeletal. The cDNA sequences of the cardiac beta myosin heavy chain cDNA clone pHMC3 and the skeletal beta-like myosin heavy chain cDNA clone pSMHCZ, were compared to each other. It was found that the 3 untranslated regions as well as 482 nucleotides specifying the carboxyl coding region, were 100% homologous. Further examination revealed that the skeletal clone pSMHCZ diverges from the human cardiac beta myosin heavy chain cDNA clone pHMC3 at the 5 end. We present evidence in this report which indicates that the cardiac beta myosin heavy chain mRNA is expressed in skeletal muscle tissues. The human cardiac beta myosin heavy chain cDNA clone, pHMC3, which codes for a portion of the light meromyosin section of the myosin heavy chain, was used as a probe for S1 nuclease mapping studies with RNA derived from cardiac tissue, smooth muscle and skeletal muscle tissues consisting of fast-twitch, slow-twitch and mixed fast- and slow-twitch muscle fibres. Two probes were used to examine the expression of the mRNA. One probe (406 nucleotides) constitutes the 3 untranslated region and a portion of the coding region of the beta cardiac myosin heavy chain cDNA clone, which is 100% homologous to pSMHCZ, the skeletal cDNA clone. The other constitutes the majority of the coding region (1017 nucleotides) of the cardiac clone pHMC3 in which the first 216 nucleotides from the labelled end are 100% homologous to the skeletal clone pSMHCZ. In the soleus muscle, which is rich in slow-twitch type I muscle fibres, the expression of the cardiac beta myosin heavy chain mRNA was very prominent. In gastrocnemius muscle, a mixed fibre muscle, the expression of this mRNA was detected to a lesser degree than that for the soleus muscle. In vastus lateralis and vastus medialis, which consist of predominantly type II, fast-twitch fibres, there were trace amounts of the cardiac beta myosin heavy chain mRNA. When expression of this mRNA was tested in smooth muscle tissue none could be detected.     

Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC1 and MLC3; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLCemb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC1, MLC3 and MLCemb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC1 and MLC3 is located on human chromosome 2.     

Construction of a human ventricular cDNA library and characterization of a beta myosin heavy chain cDNA clone

Summary We have constructed and characterized for the first time a complementary DNA (cDNA) clone, pHMC3, which codes for a cardiac myosin heavy chain mRNA from human heart. This clone contains a 1.7 kb DNA segment and specifies 543 amino acids of the carboxyl portion of the myosin heavy chain. The DNA sequence and encoded amino acid sequence were compared to the hamster alpha (pVHC1) and beta (pVHC2/pVHC3) cardiac myosin heavy chain cDNA and amino acid sequences and the rat cardiac myosin heavy chain sequences as well. The myosin heavy chain mRNAs are highly conserved and this is reflected in our cDNA clone. The pHMC3 clone is 87.9% homologous to the hamster alpha cDNA and 92.2% homologous to the hamster beta cDNA clones. The 3 untranslated region of pHMC3 is 64.1% homologous to the hamster beta clone while the hamster alpha myosin heavy chain shows only 25% homology to pHMC3 and exhibits extensive diversity. Similar results rere obtained when pHMC3 was compared to the rat cardiac myosin heavy chain cDNA sequences. The comparisons showed that pHMC3 is a beta cardiac myosin heavy chain cDNA clone.     

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.     

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

Summary 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.Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL60637, USA     

Partial characterization of the human beta-myosin heavy-chain gene which is expressed in heart and skeletal muscle

A human myosin heavy-chain gene, cloned in gamma Charon 4A phage (and as a clone designated lambda gMHC-1), was shown to code for a cardiac myosin heavy chain of the beta-type. The 5' end of the 14,200-base-pair genomic DNA clone is located in the head region of the myosin chain. The 3' end was shown to extent to the COOH terminus and includes the 3'-nontranslated sequence of the corresponding mRNA. The identification of lambda gMHC-1 as coding for a cardiac beta-myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy-chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the clones DNA including the 3'-nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the beta-myosin heavy-chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle.     

The analysis of a chicken myosin heavy chain cDNA clone

A cDNA library has been constructed in the plasmid pBR322 using a large size class of RNA derived from chicken embryonic leg muscle as the template material. A clone containing a 2350-base pair insert was selected and identified as coding for the myosin heavy chain sequence, based upon its ability to hybridize to genomic myosin heavy chain clones, and by direct nucleotide sequencing. Cross-hybridization experiments with myosin heavy chain genomic clones, and mRNAs derived from different muscle types were used to explore the heterogeneity of the various myosin heavy chain isoforms at the level of the coding sequences. Although extensive sequence homology with the other isoforms was observed, a fast white isoform-specific subclone was constructed, and used to demonstrate that different genes code for the adult and embryonic fast white myosin heavy chain proteins.     

Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma. A monoclonal antibody study

Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue.