Cloning and characterization of mammalian myosin regulatory light chain (RLC) cDNA: the RLC gene is expressed in smooth, sarcomeric, and nonmuscle tissues.

The 20-kD regulatory light chain (RLC) plays a central role in the regulation of smooth muscle contraction. Little is known about the structure or expression of smooth muscle myosin light chain (MLC) genes. A cDNA library was constructed in the expression vector, lambda gt-11, with mRNA derived from cultured rat aortic smooth muscle cells. Using antibody generated against tracheal smooth muscle myosin, three cDNA clones encoding a RLC were isolated, one of which, SmRLC-2, represents a full-length transcript of the RLC mRNA. The derived amino acid sequence shows 94.2% homology with the chicken gizzard RLC, and 70 and 52% homology with the rat skeletal and cardiac muscle MLC-2 proteins, respectively. Thus, the gene encoding the putative smooth muscle RLC appears to have originated by duplication of the same ancestor that gave rise to the sarcomeric MLC-2 genes. Contrary to the stringent tissue-specific expression of sarcomeric MLC-2 genes, RNA blot hybridization and S1 nuclease mapping demonstrates that the putative smooth muscle RLC gene is expressed in smooth, sarcomeric, and nonmuscle tissues at significant levels. Primer extension analysis suggests that the same promoter region is used in these different tissues. Thus the putative smooth muscle RLC gene appears to be a gene that is constitutively expressed in a large variety of cells and has a differentiated function in smooth muscle.

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications.

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature.

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.

The expression of sarcomeric muscle-specific contractile protein genes in BC3H1 cells: BC3H1 cells resemble skeletal myoblasts that are defective for commitment to terminal differentiation.

The BC3H1 cell line has been used widely as a model for studying regulation of muscle-related proteins, such as the acetylcholine receptor, myokinase, creatine kinase, and actin. These cells, derived from a nitrosourea-induced mouse brain neoplasm, have some of the morphological characteristics of smooth muscle and have been shown to express the vascular smooth muscle isoform of alpha-actin. To provide further information about the contractile protein phenotype of BC3H1 and to gain additional insights into the possible tissue of origin of these cells, we have examined the expression of a battery of contractile protein genes. During rapid growth, subconfluent BC3H1 cells express the nonmuscle isoform of alpha-tropomyosin (alpha-Tm) and the nonsarcomeric isoforms of myosin heavy and light chains (MHCs and MLCs, respectively), but do not express troponin T(TnT). However, when BC3H1 cells differentiate in response to incubation in serum-deprived medium or upon approaching confluence, they express TnT as well as sarcomeric muscle isoforms of MHC, MLC 2 and 3, alpha-Tm, and alpha-actin. These results suggest that BC3H1 is a skeletal muscle cell line of ectodermal origin that is defective for commitment to terminal differentiation.

Mammalian nonsarcomeric myosin regulatory light chains are encoded by two differentially regulated and linked genes.

The myosin 20,000-D regulatory light chain (RLC) has a central role in smooth muscle contraction. Previous work has suggested either the presence of two RLC isoforms, one specific for nonmuscle and one specific for smooth muscle, or the absence of a true smooth muscle-specific isoform, in which instance smooth muscle cells would use nonmuscle isoforms. To address this issue directly, we have isolated rat RLC cDNAs and corresponding genomic sequences of two smooth muscle RLC based on homology to the amino acid sequence of the chicken gizzard RLC. These cDNAs are highly homologous in their amino acid coding regions and contain unique 3'-untranslated regions. RNA analyses of rat tissue using these unique 3'-untranslated regions revealed that their expression is differentially regulated. However, one cDNA (RLC-B), predominantly a nonmuscle isoform, based on abundant expression in nonmuscle tissues including brain, spleen, and lung, is easily detected in smooth muscle tissues. The other cDNA (RLC-A; see Taubman, M., J. W. Grant, and B. Nadal-Ginard. 1987. J. Cell Biol. 104:1505-1513) was detected in a variety of nonmuscle, smooth muscle, and sarcomeric tissues. RNA analyses comparing expression of both RLC genes with the actin gene family and smooth muscle specific alpha-tropomyosin demonstrated that neither RLC gene was strictly smooth muscle specific. RNA analyses of cell lines demonstrated that both of the RLC genes are expressed in a variety of cell types. The complete genomic structure of RLC-A and close linkage to RLC-B is described.

Slow myosin in developing rat skeletal muscle.

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo.

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.

All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner.

In mammals different isoforms of myosin heavy chain are encoded by the members of a multigene family. The expression of each gene of this family is regulated in a tissue- and developmental stage-specific manner as well as by hormonal and various pathological stimuli. In this study the molecular basis of isoform switches induced in myosin heavy chain by thyroid hormone was investigated. The expression of the myosin heavy chain gene family was analyzed in seven different muscles of adult rats subjected to hypo- or hyperthyroidism with complementary DNA probes specific for six different myosin heavy chain genes. The results demonstrate that all six genes are responsive to thyroid hormone. More interestingly, the same myosin heavy chain gene can be regulated by thyroid hormone in highly different modes, even in opposite directions, depending on the tissue in which it is expressed. Furthermore, the skeletal embryonic and neonatal myosin heavy chain genes, so far considered specific to these two developmental stages, can be reinduced by hypothyroidism in specific adult muscles.

Catalytic activity of an RNA domain derived from the U6-U4 RNA complex.

U6 RNA contains two regions that are essential for proper splicing of nuclear precursor messenger RNA (pre-mRNA). A comparison of putative secondary structures of the U6-U4 RNA complexes from different phyla revealed a conserved domain that is similar to the catalytic hammerhead RNA motif. Although no catalytic activity was detected in the mammalian U6-U4 RNA complexes, two nucleotide changes in U6 RNA and one in U4 RNA conferred cleavage activity to the complex. Furthermore, the highly conserved domain of the wild-type complex, without the accompanying flanking regions, cleaved an RNA substrate and exhibited other characteristics of the hammerhead ribozyme. The possible involvement of this structure in pre-mRNA splicing is also discussed.

Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD.

Muscle enhancer factor-2A (MEF2A), a member of the MADS family, induced myogenic development when ectopically expressed in clones of nonmuscle cells of human clones, a function previously limited to the muscle basic helix-loop-helix (bHLH) proteins. During myogenesis, MEF2A and bHLH proteins cooperatively activate skeletal muscle genes and physically interact through the MADS domain of MEF2A and the three myogenic amino acids of the muscle bHLH proteins. Thus, skeletal myogenesis is mediated by two distinct families of mutually inducible and interactive muscle transcription factors, either of which can initiate the developmental cascade.

Reversal of terminal differentiation mediated by p107 in Rb-/- muscle cells.

The terminal differentiation of mammalian muscle cells requires the tumor suppressor retinoblastoma protein (Rb). Unlike their wild-type counterparts, multinucleated myotubes from mouse cells deficient in Rb (Rb-/-) were induced by serum to re-enter the cell cycle. Development of the myogenic phenotype in Rb-/- cells correlated with increased expression of p107, which interacted with myogenic transcription factors. Serum-induced cell cycle reentry, on the other hand, correlated with decreased p107 expression. Thus, although p107 could substitute for Rb as a cofactor for differentiation, it could not maintain the terminally differentiated state in Rb-/- myotubes.

Multigene family for sarcomeric myosin heavy chain in mouse and human DNA: localization on a single chromosome.

Cloned myosin heavy chain DNA probes from rat and human were hybridized to restriction endonuclease digests of genomic DNA from somatic cell hybrids and their parental cells. The mouse myosin heavy chain genes detectable by this assay were located on chromosome 11, and three different human sarcomeric myosin heavy chain genes were mapped to the short arm of chromosome 17. A synteny between myosin heavy chain and two unrelated markers, thymidine kinase and galactokinase, was found to be preserved in the rodent and human genomes.

Gating charge differences between two voltage-gated K+ channels are due to the specific charge content of their respective S4 regions.

Voltage-gated ion channels that differ in their primary amino acid sequence in the putative voltage sensor, the S4 region, show distinct voltage-sensing characteristics. In this study, we directly compared two voltage-gated K+ channels, the mammalian RCK1 with the Drosophila Shab11, and correlated the specific amino acid content of their respective S4 regions with the distinct voltage-sensing properties they exhibit. We find that specific differences in the charge content of the S4 region are sufficient to account for the distinct gating valence of each channel. However, differences in residues inside the S4 region are not sufficient to account for each channel's characteristic voltage range of activation.

Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells.

We have cloned a cDNA coding for a delayed rectifier K+ channel from rat brain (RCK1) and rat muscle (RMK1) and expressed it in Xenopus oocytes and in a myoblast cell line (Sol-8). Stably transfected Sol-8 cells exhibited large outward K+ currents, which were indistinguishable from the K+ currents induced in Xenopus oocytes by injection of mRNA transcribed in vitro. RCK1 encodes a K+ channel with a unitary conductance of approximately 14 pS. The steep voltage dependence of channel opening resides in transitions between closed states, whereas the direct transitions into and out of the open state are very rapid and not markedly voltage-dependent. Channel inactivation is very slow, voltage-independent, and occurs from the open state only. We present a simple model that incorporates our findings and is consistent with the presumed structural symmetry of a functional K+ channel.

Incremental reductions of positive charge within the S4 region of a voltage-gated K+ channel result in corresponding decreases in gating charge.

The S4 region of voltage-dependent ion channels is involved in the voltage-sensing mechanism of channel activation. Previous studies in fast inactivating channels have used non-steady-state measurements and thus have not allowed the quantitative assessment of activation parameters. Using site-directed mutagenesis and voltage-clamp recordings in a noninactivating channel (RCK1), we demonstrate that stepwise reductions of positive charge within the S4 region correlate with a progressive decrease in the channel's overall gating valence. In addition to testing for electrostatic behavior of individual charged residues, our study was designed to probe nonelectrostatic influences on charge movement. We provide evidence that individual charged residues behave differentially in response to the electric field, so that purely electrostatic influences cannot fully account for the gating movement of certain charges.

Age-related differences in the expression of proto-oncogene and contractile protein genes in response to pressure overload in the rat myocardium.

Cardiac adaptation to hemodynamic stress involves both quantitative (hypertrophy) and qualitative (pattern of gene expression) changes. Our previous studies have shown that advancing age in the rat is associated with diminished capacity to develop left ventricular hypertrophy in response to either ascending aortic constriction (AoC). In this study, we examined whether the expression of protooncogenes and contractile protein genes in response to AoC differs between adult (9-mo-old) and old (18-mo-old) rats. RNA was isolated from the left ventricles of AoC animals of both age groups subjected to a similar hemodynamic stress. Immediately after AoC, the levels of the ventricular expression of c-fos and c-jun protooncogenes were markedly lower in the old rats than in the adult animals. 5 d after the operation, the ratio of beta- to alpha-myosin heavy chain mRNAs increased significantly after AoC in both age groups. In contrast, AoC was associated with a marked reduction in the levels of mRNAs encoding sarcoplasmic reticulum Ca(2+)-ATPase (by 69%) and cardiac calsequestrin (by 49%) in the old rats but not in the adults. The mRNAs encoding atrial natriuretic factor and skeletal alpha-actin increased in response to AoC only in the adult rats. There were no significant differences in expression of the cardiac alpha-actin mRNA among the experimental groups. These data suggest that (a) the expression of protooncogenes in response to acute pressure overload is significantly reduced in the aged rats and (b) the pattern of expression of the contractile protein gene in response to AoC in the old rats differs qualitatively as well as quantitatively from that in younger animals. These age-related differences may play a role in the higher frequency of heart failure in the aged during hemodynamic stress.

Myosin heavy chain messenger RNA and protein isoform transitions during cardiac hypertrophy. Interaction between hemodynamic and thyroid hormone-induced signals.

Expression of the cardiac myosin isozymes is regulated during development, by hormonal stimuli and hemodynamic load. In this study, the levels of expression of the two isoforms (alpha and beta) of myosin heavy chain (MHC) during cardiac hypertrophy were investigated at the messenger RNA (mRNA) and protein levels. In normal control and sham-operated rats, the alpha-MHC mRNA predominated in the ventricular myocardium. In response to aortic coarctation, there was a rapid induction of the beta-MHC mRNA followed by the appearance of comparable levels of the beta-MHC protein in parallel to an increase in the left ventricular weight. Administration of thyroxine to coarctated animals caused a rapid deinduction of beta-MHC and induction of alpha-MHC, both at the mRNA and protein levels, despite progression of left ventricular hypertrophy. These results suggest that the MHC isozyme transition during hemodynamic overload is mainly regulated by pretranslational mechanisms, and that a complex interplay exists between hemodynamic and hormonal stimuli in MHC gene expression.

Identification of a cis-acting regulatory element conferring inducibility of the atrial natriuretic factor gene in acute pressure overload.

To identify the cis-acting regulatory element(s) which control the induction of the atrial natriuretic factor (ANF) gene in acute pressure overload, DNA constructs consisting of promoter elements linked to a reporter gene were injected into the myocardium of dogs, which underwent aortic banding or were sham-operated. Expression of a reporter gene construct harboring the ANF promoter (-3400ANF) was induced 6-12-fold after 7 d of pressure overload. An internal deletion of 556 bp (nucleotide sequence -693 to -137) completely abrogated the inducibility of the ANF reporter gene construct. An activator protein-1 (AP1)-like site (-496 to -489) and a cAMP regulatory element (CRE) (-602 to -596) are located within the deleted sequence. Site-directed mutagenesis of the AP1-like site but not the CRE completely prevented the induction of this construct to acute pressure overload. Further, the AP1-like site was able to confer inducibility of a heterologous promoter (beta-myosin heavy chain) to higher values than controls. Gel mobility shift assay (GMSA) supershift analysis was performed using a radiolabeled probe of the ANF promoter (-506/-483) that included the AP1-like site (ATGAATCA) sequence, as well as a probe converted to contain an AP1 consensus sequence (ATGACTCA). GMSA analysis demonstrated that the ANF AP1-like element could bind both a constitutively expressed factor and the AP1 proteins, and conversion to a true AP1 site increased its affinity for AP1. However, 7 d after the onset of pressure overload, the AP1 proteins were present only at low levels, and the major complex formed by the ANF AP1-like probe was not supershifted by a jun antibody. Using a large animal model of pressure overload, we have demonstrated that a unique cis-acting element was primarily responsible for the overload induction of the ANF gene.

Overexpression of Gs alpha protein in the hearts of transgenic mice.

Alterations in beta-adrenergic receptor-Gs-adenylyl cyclase coupling underlie the reduced catecholamine responsiveness that is a hallmark of human and animal models of heart failure. To study the effect of altered expression of Gs alpha, we overexpressed the short isoform of Gs alpha in the hearts of transgenic mice, using a rat alpha-myosin heavy chain promoter. Gs alpha mRNA levels were increased selectively in the hearts of transgenic mice, with a level 38 times the control. Despite this marked increase in mRNA, Western blotting identified only a 2.8-fold increase in the content of the Gs alpha short isoform, whereas Gs activity was increased by 88%. The discrepancy between Gs alpha mRNA and Gs alpha protein levels suggests that the membrane content of Gs alpha is posttranscriptionally regulated. The steady-state adenylyl cyclase catalytic activity was not altered under either basal or stimulated conditions (GTP + isoproterenol, GTP gamma S, NaF, or forskolin). However, progress curve studies did show a significant decrease in the lag period necessary for GppNHp to stimulate adenylyl cyclase activity. Furthermore, the relative number of beta-adrenergic receptors binding agonist with high affinity was significantly increased. Our data demonstrate that a relatively small increase in the amount of the coupling protein Gs alpha can modify the rate of catalyst activation and the formation of agonist high affinity receptors.

Transcriptional and posttranscriptional control of c-myc during myogenesis: its mRNA remains inducible in differentiated cells and does not suppress the differentiated phenotype.

It is widely accepted that the cellular oncogene c-myc plays an important role in the control of cell proliferation and that its expression diminishes in differentiated cells. We examined whether there is a correlation between c-myc expression and cell proliferation or differentiation by using a subclone of a rat skeletal muscle cell line L6E9. Myoblasts irreversibly withdraw from the cell cycle, fuse to form multinucleated myotubes, and express muscle-specific genes (terminal differentiation). Muscle-specific genes can also be expressed in the absence of fusion (biochemical differentiation). Such mononucleated but biochemically differentiated cells can be stimulated to reenter the cell cycle. c-myc was induced by insulin, insulin-like growth factor, or serum factors in G0-arrested cells, whereas induction by protein synthesis inhibitors or superinduction by protein synthesis inhibitors in combination with serum factors occurred in all physiological states tested. We found that c-myc expression was reduced in biochemically and terminally differentiated cells as well as in quiescent undifferentiated cells but that it remained inducible by growth factors in all three physiological states. Results of nuclear runoff transcription assays suggested that the induction of c-myc mRNA by growth factors and its deinduction in these physiological states were regulated mainly at the transcriptional level. In contrast, induction and superinduction of c-myc mRNA by protein synthesis inhibitors alone and in combination with growth factors, respectively, were regulated posttranscriptionally mainly by stabilization of c-myc mRNA. Moreover, c-myc and muscle-specific genes could be simultaneously transcribed in both biochemically and terminally differentiated cells. These results indicate that irreversible repression of c-myc is not required for terminal myogenic differentiation and that its expression is insufficient by itself to suppress the differentiated phenotype.