Human Fetal Muscle Research Articles

Expression of the low‐affinity NGF receptor during human muscle development, regeneration, and in tissue culture

The expression of the low-affinity NGF receptor (LNGFR) during human muscle development, regeneration, and in tissue culture was analyzed using a murine monoclonal antibody to human LNGFR (MAb ME 20.4). Muscle cells from 12-22-week fetuses stained strongly for LNGFR. In adult normal muscle, only intramuscular nerve endings showed immunoreactivity with MAb ME 20.4, but no staining was detected in muscle fibers. In Duchenne muscular dystrophy, immunohistologically demonstrable LNGFR was present in regenerating muscle fibers. In these fibers, LNGFR gene expression was also demonstrated at the transcriptional level using in situ hybridization with riboprobes coding with human LNGFR. Cultures from human fetal and adult muscle were studied by double label indirect immunofluorescence microscopy with MAb ME 20.4 and antisera against human fetal myosin. Most myosin-positive cells, both at the myoblast and myotube stages, displayed surface LNGFR immunostaining. In cells from fetal muscle, LNGFR was detected during the first 2 weeks in vitro, whereas in cells from adult muscle the expression of LNGFR was observed for up to 7 weeks. These findings suggest a potential involvement of LNGFR in human muscle development and regeneration.

Splicing of the Muscle‐Specific Plasma Membrane Ca2+‐ATPase Isoform PMCA1c Is Associated with Cell Fusion in C2 Myocytes

The regulation of intracellular calcium is essential for proper muscle function. Muscle cells have several mechanisms for dealing with the rapid and large changes in cytosolic calcium level that occur during contraction. Among these is the plasma membrane Ca(2+)-ATPase (PMCA), which pumps calcium from the cytosol to the extracellular space. We have previously shown that in human fetal muscle the PMCA1 isoforms present are PMCA1a-d, with PMCA1b and c predominating. Alternative splicing of mRNAs encoding proteins involved in muscle contraction is common in developing muscle. Therefore, we examined the expression of muscle-specific PMCA mRNAs in pre- and postfusion mouse C2 myoblasts. The housekeeping form of the CA(2+)-ATPase, PMCA1b, was found at all times and under all conditions. However, the other predominating isoform found in muscle, PMCA1c, was expressed on myotube formation. Simple cell-cell contact was not sufficient to induce PMCA1c expression, as cells plated at confluence but harvested before myotubule formation did not express PMCA1c. The induction of this muscle-specific Ca(2+)-ATPase at myotube formation suggests that it may play an important role in muscle function.

IGF-II in the pathogenesis of rhabdomyosarcoma: a prototype of IGFs involvement in human tumorigenesis.

In recent years there has been a growing interest in the role of peptide growth factors in the regulation of the biologic behavior of several human malignancies. Among those peptides that might have importance in neoplastic pathogenesis are the insulin-like growth factors I and II (IGF-I and IGF-II). These proteins have been shown to stimulate myoblast proliferation and differentiation (1), to promote nutrient uptake and to inhibit proteolysis (2, 3). IGF-II mRNA is highly expressed in human skeletal fetal muscle tissue, but it is not detectable in normal adult skeletal muscle by standard Northern analysis and in situ hybridization (4). In mouse myoblasts, IGF-I and IGF-II mRNA levels increase transiently (IGF-II > > IGF-I) within 48-72 hours of the beginning of the myogenic differentiation process. The expression of mRNA is accompanied by secretion of the peptides in the culture medium that peaks at hour 92. IGF-I receptors increase transiently, doubling by 48 hours after the onset of differentiation, while IGF-II receptors increase and remain at a higher number throughout the differentiated state (5, 6). The production of IGF-binding proteins of Mr 29,000 to 32,000 is also induced throughout differentiation (7).

Expression of myosin heavy chain isoforms in developing human muscle spindles.

We studied serial sections of human fetal limb muscles (10-25 weeks of gestation) by light microscopic (LM) immunocytochemistry, using specific antibodies against slow-tonic, slow-twitch, fetal, embryonic, and alpha-cardiac myosin heavy chain (MHC) isoforms, neurofilament protein, laminin, and myomesin. One set of the first-generation myotubes expressed slow-tonic MHCs, and slow-twitch, fetal, and embryonic MHCs from the tenth week of gestation. These primary myotubes were identified as developing nuclear bag fibers. Second-generation myotubes in close apposition to the primary nuclear bag myotubes initially expressed only fetal and embryonic MHCs. One or more of these secondary myotubes acquired expression of slow-tonic and slow-twitch MHCs and gave rise to nuclear bag fibers. Most of the nuclear bag precursors expressed alpha-cardiac MHC. The secondary myotubes that expressed fetal and embryonic MHC but not slow-tonic, slow-twitch, or alpha-cardiac MHCs gave rise to the nuclear chain fibers. This study shows that different populations of fiber precursors, each with a unique sequence of MHC expression, gave rise to the nuclear bag and chain fibers, despite the presence of a common afferent nerve, from the early stages.

Complete sequence of human fast‐type and slow‐type muscle myosin‐binding‐protein C (MyBP‐C)

Myosin-binding-protein C (MyBP-C) is a myosin-associated protein of unknown function found in the cross-bridge-bearing zone (C region) of A bands in striated muscle. Using a cDNA clone encoding the fast-type isoform of chicken MyBP-C, we screened a human fetal muscle cDNA library and isolated clones encoding the full-length human fast-type isoform of MyBP-C. cDNA clones encoding the slow-type isoform of human MyBP-C, were also isolated and fully sequenced. Northern-blot analysis demonstrated skeletal muscle-specific expression of these gene products. Using human/hamster somatic-cell hybrids, we were able to map the slow-type MyBP-C to human chromosome 12, and the fast-type MyBP-C to chromosome 19. The cDNA for human fast-type MyBP-C encodes a polypeptide of 1142 amino acids with an expected molecular mass of 128.1 kDa. Comparison of this cDNA with other members of the MyBP family reveals extensive primary-sequence conservation. Each MyBP-C contains seven immunoglobulin C2 motifs and three fibronectin type-III repeats in the arrangement C2-C2-C2-C2-C2-III-III-C2-III-C2. Regions of high identity shared by the chicken and the two human proteins are not restricted to the immunoglobulin and fibronectin motifs. Sequence comparison of all three proteins has allowed us to map a highly conserved region between the first and second C2 motifs, the only large spacer sequence present between motifs in these proteins.

Neurite outgrowth of spinal neurons on tissue sections of embryonic muscle is largely integrin dependent

Embryonic chick spinal neurons have been cultured over sections of human foetal muscle to determine which cell adhesion molecules present in embryonic muscle are important in promoting neurite outgrowth. Using blocking antibodies against the major cell adhesion molecules, neural cell adhesion molecule (NCAM), N-cadherin and the β 1 subunit of the integrins, neurite outgrowth was significantly blocked only by anti-integrin antibodies. In addition other agents that block neurite outgrowth stimulated by NCAM, N-cadherin and L1, such as the calcium channel antagonists verapamil and ω-conotoxin and pertussis toxin which inactivates G-proteins also had no effect. This suggests that in this culture system integrins are able to promote neurite outgrowth whereas NCAM and N-cadherin are not.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9-26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9-20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17-18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle

Spontaneous Contractility of Human Fetal Airway Smooth Muscle

Physical forces such as fetal breathing and fluid secretion may influence lung development, perhaps by their ability to distend the fetal lung. We found that airway smooth muscle cells in first-trimester human lung tissue and cultured lung tissue explants spontaneously contract. The contractions caused visible movement of intraluminal fluid and distended the distal ends of the epithelial tubules, suggesting that they produced significant changes in intraluminal pressure. Smooth muscle contractility and responses to pharmacologic manipulations were recorded with video microscopy. The interval between contractions ranged from 8 to 135 s (mean +/- SEM, 54 +/- 5 s; n = 20). Smooth muscle contractility was not inhibited by tetrodotoxin or atropine, implying a myogenic rather than neurogenic origin. Because cholinergic nerves modulate adult airway smooth muscle contractility, we asked whether fetal airway smooth muscle cells were regulated by cholinergic agents. The cholinergic agonists acetylcholine and carbachol both increased fetal airway smooth muscle contractility. Contractions were inhibited by the calcium channel blockers CdCl2 and nifedipine, suggesting that an influx of extracellular Ca2+ accompanied airway smooth muscle contractions. Isoproterenol dilated the contractile regions of the epithelial tubules and stopped contractions. Lemakalim, an activator of smooth muscle ATP-sensitive K+ channels, also arrested contractions and relaxed fetal airway smooth muscle. In conclusion, human fetal airway smooth muscle contacts spontaneously and exhibits pharmacologic responsiveness similar to adult airway smooth muscle. We speculate that fetal airway smooth muscle contractions, possibly exerting effects through phasic changes in intraluminal pressure, may be an important physical force contributing to lung development.

Molecular characterization of the gene encoding the gamma subunit of the human skeletal muscle 1,4-dihydropyridine-sensitive Ca2+ channel (CACNLG), cDNA sequence, gene structure, and chromosomal location

cDNA clones of the gamma subunit of the skeletal muscle 1,4-dihydropyridine-sensitive voltage-dependent Ca2+ channel were isolated from a human fetal skeletal muscle cDNA library using the rabbit gamma cDNA as a probe. The DNA sequence of the entire human cDNA was determined. Cosmids that contained the human gamma gene were isolated and used to determine the genomic organization of the coding sequences. Four exons were identified, spanning 12.5 kilobases of DNA. Reverse-transcribed polymerase chain reaction analysis detected the gamma transcript in human and mouse skeletal muscle RNAs, but not in RNA from human brain or cardiac muscle or from mouse brain, cardiac muscle, spleen, kidney, liver, or stomach. A polymorphic dinucleotide repeat within the gamma gene was identified. This repeat was used to type a subset of the Centre d'Etude du Polymorphisme Humain families. Linkage analysis indicates that the gamma gene is tightly linked (Z = 12.94, theta = 0.001) to growth hormone at chromosome 17q23, a region that also contains the adult skeletal muscle Na+ channel.

Localization of the growth hormone receptor, identified by immunocytochemistry, in second trimester human fetal tissues and in placenta throughout gestation.

Pituitary GH secretion appears largely unnecessary for the attainment of normal birth size in many species, including man. This is believed to be due to an immaturity and/or an absence of GH receptors in many fetal tissues. However, in vitro studies using late first trimester human fetal tissues have demonstrated mitogenic actions of GH on liver and stimulation of insulin biosynthesis in pancreas. To resolve this discrepancy, we have employed immunocytochemistry to identify the presence and distribution of GH receptors in various human fetal tissues. Fetuses of 14-16 weeks gestation were obtained after therapeutic abortion, tissues were fixed, and immunocytochemistry was performed using monoclonal antibodies against purified rat or rabbit GH receptor. The specificity of staining was confirmed by preabsorption of the antibodies with 1) adult rat liver membranes or 2) human fetal liver membranes, both of which possess specific GH-binding sites, or 3) human fetal skeletal muscle membranes, which do not specifically bind GH. Positive staining was seen in a subpopulation of liver parenchymal cells, in the ductal and endocrine tissue of pancreas, in the germinal layer of the epidermis and the deeper dermal layers of skin, and in the tubular epithelium of kidney. No immunopositive staining was seen in skeletal or cardiac muscle, epiphyseal growth plate, lung, intestine, or adrenal. Positive staining was present in the neuronal cell bodies of the cerebral cortex. GH receptor was also detectable as early as 8 weeks gestation in syncytial layers of the placenta and was maintained until term. Results demonstrate the presence of immunoreactive GH receptor/binding protein in some human fetal tissues early in development. In particular, these results would support a role for GH in the growth and function of liver and pancreas.

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

Immunohistochemical and biochemical characterisation of the expression of a human embryonal carcinoma cell proteoglycan antigen in human germ cell tumours and other tissues

In the embryonal carcinoma (EC) cell line GCT 27, monoclonal antibody GCTM-2 recognises an epitope on a 200 kD pericellular matrix keratan sulphate proteoglycan. Immunohistochemical analyses demonstrated staining of tissue sections from 21 out of 22 human non-seminomatous germ cell tumours, and from 22 out of 28 sections of seminomas. In normal human fetal tissues gut epithelium and muscle stained strongly, and certain other epithelia stained moderately. In adult tissues, the distribution of the epitope was similar, but staining intensity was weaker. Neoplastic tissues showed reactivity with embryonal rhabdomyosarcoma and colorectal carcinoma, but no other non-germ cell tumours. Immunofluorescence microscopy showed that GCTM-2 also stained cell lines from human colorectal carcinoma, embryonal rhabdomyosarcoma and choriocarcinoma. In contrast to EC cells the epitope in these other cell types required permeabilisation of the cells to be visualised, and the protein bands in immunoblots lacked extensive modification with keratan sulphate and were smaller. Thus, GCTM-2 reacts with an epitope which has a previously unrecognised tissue distribution; its expression as a pericellular matrix proteoglycan is predominantly a characteristic of human EC cells.

Dystrophin in Fetal Muscle

Dystrophin, the product of the Duchenne muscular dystrophy gene, was studied in human fetal skeletal muscle from 9 to 26 weeks of gestation at the Jerry Lewis Muscle Research Centre, Hammersmith Hospital, London.

Immunoreactive arginine vasopressin in human fetal and neonatal skeletal muscle

We provide evidence for the presence of arginine vasopressin (AVP) in human fetal and neonatal skeletal muscle using a combination of specific RIA, tangential flow ultrafiltration and reverse-phase HPLC separation. The IR-AVP concentrations are negatively correlated with gestational age ( r = −0.75, P < 0.0001) and range from 1 to 10 pmol/g wet wt at 15 weeks gestation to 0.04 pmol/g wet wt at term. This IR-AVP substance is of low molecular weight (< 3000 mol. wt), elutes in the same position as standard AVP after HPLC separation and is detected by four different anti-AVP antisera.

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes.

Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14-15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin and alpha-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycnic centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells may provide a useful experimental system for the study of human cardiac muscle cell biology.

Characterisation of dystrophin during development of human skeletal muscle.

Dystrophin, the 427 x 10(3) Mr product of the Duchenne muscular dystrophy (DMD) gene, was studied in human foetal skeletal muscle from 9 to 26 weeks of gestation. Dystrophin could be detected from at least 9 weeks of gestation at the sarcolemmal membrane of most myotubes, though there was differential staining with antibodies raised to various regions of the protein. Dystrophin immunostaining increased and became more uniform with age and by 26 weeks of gestation there was intense sarcolemmal staining of all myotubes. On a Western blot, a doublet of smaller relative molecular mass than that seen in adult tissue was detected in all foetuses studied. There was a gradual increase in abundance of the upper band from 9 to 26 weeks, and the lower band, although present in low amounts in young foetuses, increased significantly between 20 and 26 weeks of gestation. These data indicate that there are several specific isoforms of dystrophin present in developing skeletal muscle, though the role of these is unknown.

Identification of a tissue-specific regulatory element within the human muscle glycogen phosphorylase gene.

Northern blot analysis showed that human muscle glycogen phosphorylase is developmentally regulated in human adult and fetal skeletal muscle. Furthermore, muscle phosphorylase mRNA expression is temporally regulated in the C2C12 mouse muscle cell line. To define regulatory elements that control expression of the human muscle glycogen phosphorylase gene, the structure of the 5' end of the gene was determined, and 1,129 base pairs of the 5'-flanking region were subcloned and sequenced. Primer extension, RNase protection, and S1 nuclease protection experiments mapped the transcription start site to 76 base pairs upstream from the starting methionine. Sequential deletions of the 5'-flanking region were tested for the ability to activate chloramphenicol acetyltransferase (CAT) expression in fused or proliferating C2C12 cells. Inclusion of the 43 base pairs between -612 and -570 led to a 9-fold increase in CAT activity in fused myotubes. No increase was observed in proliferating myoblasts. This region contains a 10-base pair sequence, CTCCAAAAGG, at -592, which is also repeated at -252. Mutation of the sequence at -592 results in a 50% decrease in CAT activity compared with the amount of CAT activity observed with the normal or a control mutation. These results indicate that a regulatory element is found within -612 to -570 of the 5'-flanking DNA of the human muscle glycogen phosphorylase gene which activates transcription only in differentiated muscle cells.

Identification and pattern of transitions of some developmental and adult isoforms of fast troponin T in some human and rat skeletal muscles

Using a monoclonal antibody (F24) in an immunoblotting procedure, the composition of fast troponin T in several adult and developing skeletal muscles of rat and human was studied. With the exception of diaphragm, four isoforms of fast troponin T (HF1-HF4) were detected in all the adult human skeletal muscles investigated. Another isoform of fast troponin T undetectable in the adult human skeletal muscles, designated the fetal isoform (HFF1), was found to be present in all fetal skeletal muscles at 20 weeks of gestation except the diaphragm. Unlike isoform HF4 that was undetectable in all the fetal skeletal muscles, isoforms HF1-HF3 were present in all the human fetal skeletal muscles including the diaphragm. At least five isoforms of fast troponin T (AF1-AF5) could be detected in adult rat skeletal muscles. An additional isoform designated (D) appeared to be present in the rat diaphragm. In some muscles one of the isoforms, AF1, could be further resolved into two to three variants. The proportions and the level of expression of AF1-AF5 isoforms varied not only in different muscles but in some cases also in different parts of the same muscle. In addition to the adult isoforms, four other developmental isoforms termed fetal (FF1 and FF2) and neonatal (NF1 and NF2), were detected during the early development in the rat skeletal muscles. Their presence was first detected during the late fetal to early neonatal period and these isoforms were generally undetectable in a majority of the muscles after 1-2 months of age although their low level of expression persisted in a small number of muscles.

Insulin-Like Growth Factor (IGF) Binding to Cell Monolayers Is Directly Modulated by the Addition of IGF-Binding Proteins*

Insulin-like growth factor-I (IGF-I) binds to specific receptors and IGF-binding proteins (IGFBPs) that are present on cell surfaces. The analysis of [125I]IGF-I binding to human fibroblasts is complicated by IGFBPs on the cell surface and their release into the medium during the binding assay. This release alters the distribution of [125I]IGF-I between type I IGF receptors and both soluble as well as cell surface-associated IGFBPs. In the present study we have determined the effects of three different forms of IGFBPs on [125I]IGF-I binding to cell surface binding sites of human fetal fibroblasts (GM10 cells) and porcine smooth muscle cells. Human 29,000 mol wt (Mr; IGFBP-1), bovine 34,000 Mr (IGFBP-2), and bovine 46,000 Mr (IGFBP-3) forms of IGFBP were compared. Each of the three IGFBPs inhibited [125I]IGF-I binding to the cell surface of both cell types. This effect was due to increased binding of [125I]IGF-I by the IGFBPs in the assay buffer. At equimolar concentrations, IGFBP-3 was more effective than either IGFBP-1 or IGFBP-2 in blocking cell surface binding. The addition of increasing concentrations of unlabeled IGF-I in the presence of each IGFBP showed that either IGFBP-1 or IGFBP-3, but not IGFBP-2, resulted in a paradoxical increase in [125I]IGF-I binding to the cell surface. The paradoxical increase occurred in the presence of excess insulin, indicating that unsaturated type I IGF receptors are not required to demonstrate this phenomenon. In a physiological salt solution, the order of affinity of the IGFBPs for IGF-I was IGFBP-3 greater than IGFBP-1 greater than IGFBP-2. These differences in affinity appear to account for the differences in IGF-I competition for binding that are seen when each of the three proteins is added. Thus, IGFBPs have the potential to alter the partitioning of IGF-I between cell surface-associated IGFBPs, membrane receptors, and the IGFBPs in extracellular fluids. The various forms of IGFBP affect IGF cell surface binding differently, and therefore, each may have distinct effects on IGF target cell actions.