Biomedical subjects

E Hardeman

Publications and source records attributed to E Hardeman.

14 recordsLinked to original sources

PubMed1998

Creating intracellular structural domains: spatial segregation of actin and tropomyosin isoforms in neurons.

Actin microfilaments play a direct role in a variety of cell processes. Distinct populations of microfilaments are associated with different cellular compartments, such as growth cones, filipodia, stress fibers, and lamellipodia. It is becoming clear that these different populations are often composed of different isoforms of the two core microfilament components, actin and tropomyosin. This is particularly true in neurons, where actin and tropomyosin isoforms are segregated into different intracellular compartments which correspond to functionally distinct regions of the neuron. Developmental regulation of this isoform sorting suggests a specific role for some isoforms in growth and for others in stabilization of neuronal structure. This provides a mechanism by which a neuron can create and independently regulate intracellular domains composed of microfilaments with different functional properties.

Actin Cytoskeleton↗

PubMed1998

Isoform sorting and the creation of intracellular compartments.

The generation of isoforms via gene duplication and alternative splicing has been a valuable evolutionary tool for the creation of biological diversity. In addition to the formation of molecules with related but different functional characteristics, it is now apparent that isoforms can be segregated into different intracellular sites within the same cell. Sorting has been observed in a wide range of genes, including those encoding structural molecules, receptors, channels, enzymes, and signaling molecules. This results in the creation of intracellular compartments that (a) can be independently controlled and (b) have different functional properties. The sorting mechanisms are likely to operate at the level of both proteins and mRNAs. Isoform sorting may be an important consequence of the evolution of isoforms and is likely to have contributed to the diversity of functional properties within groups of isoforms.

Actin Cytoskeleton↗

PubMed1997

Variations in the relative mRNA levels of actins and myosin heavy chains do not produce corresponding differences in their proteins in the adult human heart.

This paper examines the quantitative relationship between the expression of myosin heavy chain (MHC) and actin at both the levels of their mRNAs and their proteins. Explanted human left ventricle tissues were obtained from non-diseased (ND) individuals and from dilated cardiomyopathy (DCM) patients with terminally failing hearts who underwent heart transplantation. We found: (1) there are substantial differences in the stoichiometry of sarcomeric MHC and actin transcripts in hearts of DCM patients as well as in ND individuals; (2) there are substantial differences between levels of total sarcomeric actin transcripts from different individual patients; (3) by and large variations in transcript levels between samples from the same heart are much less than between samples from different hearts; and (4) the ratio of MHC to sarcomeric actin proteins expressed by different ND and DCM hearts remains essentially constant. We conclude that the human ventricle can accommodate a substantial imbalance between sarcomeric MHC and actin mRNA levels while maintaining a constant ratio of their corresponding proteins.

Actins↗

PubMed1997

Impact of alpha-skeletal actin but not alpha-cardiac actin on myoblast morphology.

Muscle differentiation involves a profound change in cell cytoarchitecture. This is accompanied by extensive isoform replacement in which the major non-muscle isoforms of the actin filament system are replaced by their muscle isoform counterparts. We have tested whether the sequential expression of the actin isoforms is functionally significant by precociously expressing the two striated muscle actins (alpha-skeletal and alpha-cardiac) in mouse myoblasts. The human alpha-skeletal and alpha-cardiac actin genes were transfected into mouse C2 myoblasts and clones expressing the human genes at the highest level were identified. Expression of the human alpha-skeletal actin gene was low with the highest mRNA level found to be 4% of that in adult human skeletal muscle. Clones expressing alpha-cardiac actin accumulated the mRNA up to 13% of the level of alpha-skeletal actin in adult human skeletal muscle. Despite the low level of alpha-skeletal actin expression, myoblasts transfected with this gene displayed a profound decrease in cell spreading. In contrast, alpha-cardiac actin had no impact on cell spreading. Neither alpha-skeletal actin nor alpha-cardiac actin had any impact on the total actin protein pool nor on the levels of the high molecular weight tropomyosins. The organisation of actin and tropomyosin into stress fibres was similar between transfected and control cells. We conclude that precocious expression of alpha-skeletal actin, but not alpha-cardiac actin, compromises myoblast morphology but not the ability of the cell to assemble stress-fibre-like structures.

Actins↗

PubMed1996

Different electrophoretic techniques produce conflicting data in the analysis of myocardial samples from dilated cardiomyopathy patients: protein levels do not necessarily reflect mRNA levels.

A variety of electrophoretic techniques were used to search for potential causes of human dilated cardiomyopathy (DCM). Northern blots were used to quantify alpha-cardiac and alpha-skeletal muscle actins, and beta-myosin heavy chain mRNAs which are the predominant expressed isoform species. We found a wide range of mRNA levels expressed in both DCM and nondiseased (ND) samples of left ventricles. However, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of the same heart samples revealed a stable and constant ratio of actin and myosin. Dystrophin deficiency might account for the DCM symptoms and so dystrophin levels of DCM and ND samples were evaluated using Western blots probed with monoclonal antibodies for the N-, C- and mid-rod portions of this protein. We found that dystrophin levels were constant in all 29 DCM and 5 ND samples suggesting that dystrophin deficiency is probably not a contributing cause. We explored the possibility that terminal failure may be due to an apoptotic-like event in the cardiomyocytes. Zymograms of DCM and ND samples revealed a significant increase in DNase I activity in the DCM group compared to the ND samples. These data raise the possibility that end-stage failure may be associated with apoptosis.

Actins↗

PubMed1993

Nerve-dependent and -independent patterns of mRNA expression in regenerating skeletal muscle.

Muscle regeneration was used to assess the roles of innervation and intrinsic genetic information in the acquisition of predominant slow and fast contractile protein mRNA profiles in adult skeletal muscle. Rat soleus (slow) and EDL (fast) muscles were allowed to regenerate in the presence and absence of their respective slow and fast nerves. Levels of mRNAs encoding fast, slow, and cardiac contractile protein isoforms were quantitatively determined at specific times during regeneration. All muscles initially expressed a heterogeneous pattern of fast, slow, and cardiac isoform mRNAs during myotube formation. Subsequently, the EDL muscle made a transition to a predominantly fast profile of mRNAs which was independent of innervation. The mRNA profile for the noninnervated regenerating soleus muscle was similar to both the innervated and the noninnervated EDL muscle profile. Thus, the decision to express predominantly fast isoform mRNAs is intrinsic to in vivo muscle regeneration with the fast nerve not appearing to be informative. In contrast, acquisition of a slow mRNA profile is dependent on the presence of a slow nerve. The mRNAs encoding slow isoforms from all of the contractile protein gene families are upregulated during the period of reestablishment of neuromuscular transmission. Additionally, there is no concomitant down-regulation of the fast isoform mRNAs upon reinnervation in the soleus regenerate. We propose that information both intrinsic to the muscle and supplied by the in vivo environment acts to provide potential isoform mRNA options to the regenerating muscle.

Animals↗

PubMed1992

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.

Adult↗

PubMed1991

Multiple mechanisms regulate muscle fiber diversity.

Adult skeletal muscles are composed of clusters of multinucleated muscle cells called myofibers. At least three different types of myofibers can be detected within mammals based on their physiological properties and their expression of different contractile protein isoforms. Different skeletal muscles display a wide range of combinations of myofibers. Recent work has demonstrated that multiple mechanisms are responsible for the generation of these myofiber types during development. Muscle progenitor cells have been dissected into two categories on the basis of which isoforms of myosin heavy chain (MHC) they express when they differentiate. Neural and other environmental influences act to modify decisions concerning the type of contractile protein a myofiber may express, and this is most apparent for MHC. The other contractile protein gene families are initially regulated independent of the MHC gene family. One or more events late in development are responsible for coordinating isoform expression between the gene families to generate the adult phenotype. Studies of muscle gene expression have revealed that regulation can occur at the levels of transcription, alternative splicing of primary transcripts, mRNA stability, and translation. The current challenge is to decipher how environmental and functional information is interpreted in terms of the activity of the regulators of muscle gene expression.

Animals↗

PubMed1990

Coordinate reciprocal trends in glycolytic and mitochondrial transcript accumulations during the in vitro differentiation of human myoblasts.

Changes in the mRNA levels during mammalian myogenesis were compared for seven polypeptides of mitochondrial respiration (the mitochondrial DNA-encoded cytochrome oxidase subunit III, ATP synthase subunit 6, NADH dehydrogenase subunits 1 and 2, and 16S ribosomal RNA; the nuclear encoded ATP synthase beta subunit and the adenine nucleotide translocase) and three polypeptides of glycolysis (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and triose-phosphate isomerase). Progressive changes during the conversion from myoblasts to myotubes were monitored under both atmospheric oxygen (normoxic) and hypoxic environments. Northern analyses revealed coordinate, biphasic, and reciprocal expression of the respiratory and glycolytic mRNAs during myogenesis. In normoxic cells the mitochondrial respiratory enzymes were highest in myoblasts, declined 3- to 5-fold during commitment and exist from the cell cycle, and increased progressively as the myotubes matured. By contrast, the glycolytic enzyme mRNAs rose 3- to 6-fold on commitment and then progressively declined. When partially differentiated myotubes were switched to hypoxic conditions, the glycolytic enzyme mRNAs increased and the respiratory mRNAs declined. Hence, the developmental regulation of muscle bioenergetic metabolism appears to be regulated at the pretranslational level and is modulated by oxygen tension.

Cell Differentiation↗

PubMed1990

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.

Animals↗

PubMed1990

Regulation of contractile protein gene family mRNA pool sizes during myogenesis.

During myogenesis, muscle contractile protein gene expression is induced and the products are used to assemble the contractile apparatus characteristic of striated muscle. The different muscle proteins are accumulated in a fixed stoichiometric ratio related to their organization in the contractile apparatus. We have examined the relationship between contractile protein gene expression and the maintenance of stoichiometry at different stages of human myogenesis. Essentially all of the known components of adult human skeletal muscle thick and thin filaments have been cloned in the form of cDNAs and used to generate isoform-specific DNA probes. The expression of fast, slow, and cardiac isoforms was measured in human myogenic primary culture and in fetal and adult human skeletal muscle. We observed that neither fast nor slow nor cardiac isoforms are coordinately regulated at the level of comparative transcript accumulation throughout myogenesis. Thus, the stoichiometry of contractile protein levels cannot be explained by coordination of expression in each of these isoform classes. However, we find that the stoichiometry of mRNA accumulation of each gene family is very similar among three developmental stages: myotubes, fetal skeletal muscle, and adult skeletal muscle. This is consistent with the possibility that the maintenance of stoichiometry between the contractile proteins could be largely regulated by the total accumulation of mRNA from each of these gene families.

Actins↗

PubMed1987

Differential patterns of transcript accumulation during human myogenesis.

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

Adult↗

PubMed1985

Isolation of full-length cDNAs encoding abundant adult human skeletal muscle mRNAs.

We have used a method [Gunning et al., Mol. Cell. Biol. 3 (1983) 787-795] of cDNA clone isolation from a cDNA library that selects for clones corresponding to abundant mRNAs and simultaneously yields a large number of different cDNA clones containing a high fraction of nearly full-length inserts. We screened an adult human skeletal muscle (skm) cDNA library and have isolated 46 cDNA clones which correspond to different mRNAs expressed at significant levels in adult skm. Of these cDNA clones 17 appear to be muscle-specific. Eleven are expressed in both cardiac muscle and skm but six are expressed primarily in skm. The remainder are expressed in muscle as well as in human fibroblasts. Comparison of cDNA insert size with mRNA size for the 17 clones expressed only in skeletal plus cardiac muscle revealed that eight are full-length, five are not and four recognize multiple transcripts which prevent a definitive conclusion. These cDNA clones will greatly facilitate the characterization of genes which are regulated during human muscle development.

Cloning, Molecular↗

PubMed1983

Targeted selection of recombinant clones through gene dosage effects.

The availability of multicopy plasmid vectors for the yeast Saccharomyces cerevisiae allows the selective amplification of individual segments of the genome. Increased dosage of particular genes results in overproduction of specific gene products and thereby confers resistance to certain metabolic inhibitors. Advantage was taken of this fact to isolate recombinant clones that increase the activities of the enzymes UDP-N-acetylglucosamine-1-P transferase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Cloning, Molecular↗