Growth of human muscle in tissue culture

Summary A method is described for the culture of normal and diseased human muscle cells. Cell outgrowth was obtained from 63/63 biopsies, and cells differentiated to form myotubes in 57/63 biopsies. The culture technique used readily permitted the growth of both normal and diseased human muscle cells. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and the Medical Research Council.     

DISEASED MUSCLE CELLS IN CULTURE

(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster. (2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells. (3) Myoblasts obtained from dy^2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy^2J spinal cord neurones on the differentiation of normal, dy^2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder. (4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non-fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue-culture studies suggest that the dy mouse mutation is a myopathic disorder. (5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well-differentiated myotubes with cross-striations. mdg myotubes have normal electro-physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation-contraction coupling. (6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti-proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi-directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy. (7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue-culture studies have not been made of hamster skeletal muscle. (8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl-cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid-maltase deficiency but phosphorylase activity re-appeared in myotubes cultured from biopsies of phosphorylase-deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes. (9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

Skeletal muscle carnitine metabolism in patients with unilateral peripheral arterial disease.

Patients with peripheral arterial disease (PAD) have abnormalities of carnitine metabolism that may contribute to their functional impairment. To test the hypothesis that muscle acylcarnitine generation (intermediates in oxidative metabolism) in patients with PAD provides a marker of the muscle dysfunction, 10 patients with unilateral PAD and 6 age-matched control subjects were studied at rest, and the patients were studied during exercise. At rest, biopsies of the gastrocnemius muscle in the patients' nonsymptomatic leg revealed a normal carnitine pool and lactate content compared with control subjects. In contrast, the patients' diseased leg had higher contents of lactate and long-chain acylcarnitines than controls. The muscle short-chain acylcarnitine content in the patients' diseased leg at rest was inversely correlated with peak exercise performance (r = -0.75, P less than 0.05). With graded treadmill exercise, only patients who exceeded their individual lactate threshold had an increase in muscle short-chain acylcarnitine content in the nonsymptomatic leg, which was identical to the muscle carnitine response in normal subjects. In the patients' diseased leg, muscle short-chain acylcarnitine content increased with exercise from 440 +/- 130 to 900 +/- 200 (SE) nmol/g (P less than 0.05). In contrast to the nonsymptomatic leg, there was no increase in muscle lactate content in the diseased leg with exercise, and the change in muscle carnitine metabolism was correlated with exercise duration (r = 0.82, P less than 0.01) and not with the lactate threshold. We conclude that energy metabolism in ischemic muscle of patients with PAD is characterized by the accumulation of acylcarnitines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency.

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells greater than more mature muscle cells greater than muscle. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control subjects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme of adenylate deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.     

Two-dimensional proton magnetic resonance of human muscle extracts

In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha-Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts.     

Serine proteinase inhibitors in human skeletal muscle: Expression of β-amyloid protein precursor and α1-antichymotrypsin in vivo and during myogenesis in vitro

The balance of serine proteases and inhibitors in nerve and muscle is altered during programmed- and injury-induced remodeling. A serpin, α₁-antichymotrypsin (α₁-ACT), and Kunitz-inhibitor containing forms of the β-amyloid precursor protein (βAPP) may be important components of this balance. In the present study, we analyzed their expression in primary cultures of human myogenic (satellite) cells that mimic myogenic differentiation using Western blotting and immunocytochemistry. In vitro results were compared to in vivo results from normal adult human skeletal muscle biopsies. Using an anti-α₁-ACT polyclonal antibody, we detected a 62 kDa immunoreactive band both in cultured human myogenic cells (mononucleated myoblasts as well as multi-nucleated myotubes) and in extracts of human muscle biopsies. With a polyclonal anti-βAPP antibody we found two bands (105 and 120 kDa) in myoblasts and myotubes in culture. However, the same antibody recognized only a single band at 92 kDa in biopsies. By immunocytochemistry, both α₁-ACT and βAPP were indistinctly present on localized to the surface of myoblasts in culture. In contrast, these inhibitors were dense on myotube surfaces, where they often formed distinct aggregates and frequently co-localized. In permeabilized muscle cells, α₁-ACT and βAPP appeared to be localized to the perikarya of both myoblasts and myotubes. Confirming previous results, both α₁-ACT and βAPP were present at the neuromuscular junction in human muscle sections. These developmental changes found during in vitro myogenesis for α₁-ACT and βAPP, both serine protease inhibitors, reinforce the hypothesis that regulation of the serine proteases and serine protease inhibitors plays an important role in neuromuscular differentiation. © 1995 Wiley-Liss Inc.     

Expression of fetal antigens by normal human skin cells grown in tissue culture.

Normal human sera are capable of causing complement-mediated lysis of normal human skin cells grown in tissue culture. This lytic reactivity can be completely removed by absorption with first trimester fetal tissue. Absorption with a variety of normal adult human tissues including lymphocytes, decidua, skin, and muscle are incapable of absorbing reactivity. Absorption of reactivity by fetal tissue is specific and not due to the introduction of anti-complementary or other nonspecific factors, as evidenced by the inability of simultaneous fetal absorption to remove reactivity from antisera with specificity for HLA antigens. Similarly, absorption of lytic sera with fetal calf serum proteins was incapable of removing reactivity against normal cells in tissue culture. It thus appears that normal human cells in tissue culture express antigens shared by the first trimester human fetus, but not present on a variety of adult human tissues. This "neoantigen" present on normal human cells when grown in tissue culture is a potential source of confusion and must be accounted for in searching for human tumor-specific antigens utilizing tissue culture cells.     

A human skeletal muscle cell line obtained from an adult donor.

A cell line (RCMH) in permanent culture was established from surgically removed adult normal human skeletal muscle by exposure to conditioned media obtained from thyroid cells. Cells proliferated indefinitely but displayed density inhibition of growth while maintaining some differentiated markers. Under certain incubation conditions, cells fused into myotube-like structures, with a concomitant increase in muscle specific proteins, such as human myoglobin, skeletal muscle myosin, desmin and dystrophin, as identified using immunocytochemical procedures. In addition, RCMH cells displayed high affinity receptors for alpha-bungarotoxin (Bmax = 0.7 pmol/mg protein, Kd = 1.5 nM) and dihydropyridines (Bmax = 0.3 pmol/mg protein, Kd = 0.5 nM for [3H]PN200-110); these values are comparable to those reported for muscle cells in primary culture. Patch-clamp studies showed the presence of 42 pS carbachol gated channels and of 5 pS calcium channels (current carried by barium); chloride and potassium channels were also seen. This new cell line appears to be a convenient model system to study skeletal muscle function.     

Ultrastructural studies of human basophils and mast cells.

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.     

Aspects of normal and dystrophic chicken muscle grown in vitro.

Pectoral muscle from normal and dystrophic New Hampshire chicken embryos was dissociated and grown in vitro. Marked differences between the two types of cell cultures were observed with the light and electron microscopes during early myogenic stages. The diseased myoblasts assumed a polarized affect and fused into smaller and fewer myotubes. Pseudostraps rather than true muscle straps were often seen in diseased cultures. There was also a delay in the appearance of myosin containing thick myofilaments in differentiating dystrophic muscle cells.     

Contractile and cytoskeletal proteins of smooth muscle cells in rat, rabbit, and human arteries

The aim of this study was to determine whether similar populations of smooth muscle cells, in relation to contractile and cytoskeletal proteins, are present in normal and diseased human coronary arteries and normal and injured rat and rabbit arteries. Rat aortae and rabbit carotid arteries were de-endothelialised and the resulting neointimal thickening examined at set time points 2-24 weeks later. Immunohistochemistry revealed that arteries had three distinct populations of cells in respect to alpha-smooth muscle actin, smooth muscle myosin heavy chain and vimentin (staining intensities '-', '+' or '++' for each protein), but only two populations in respect to desmin ('-' and '+'). The different populations of cells were found in the neointima at all times after injury, in human atherosclerotic plaque and in the media of diseased, injured and uninjured vessels, although in different proportions. It was concluded that arteries of the human, rat and rabbit have cells with a wide spectrum of contractile and cytoskeletal proteins. Expression of the different proteins did not reflect the state of the artery after injury or during the disease process, and was not associated with the expansion of a subset of cells within the artery wall.     

Insulin resistance and type 2 diabetes are not related to resistin expression in human fat cells or skeletal muscle.

Resistin is secreted by rodent fat cells and was recently postulated to be an important link between obesity and insulin resistance. We examined Resistin gene expression with real-time RT-PCR in human isolated fat cells, adipose tissue, and muscle from 42 individuals of varying degrees of overweight and who had normal insulin sensitivity or were insulin-resistant or Type 2 diabetic. Resistin was not expressed in human muscle nor was it expressed in most human isolated fat cells or intact biopsies. No difference was found between normal, insulin-resistant, or Type 2 diabetic samples. However, a very low but specific Resistin expression could be demonstrated in isolated fat cells and intact adipose tissue from some individuals (n = 3 and n = 4, respectively). There was no evidence for the expression of splice variants in the human samples. Thus, Resistin does not seem to be an important link to insulin resistance and Type 2 diabetes in human.     

Isolation of single atrial and ventricular cells from the human heart.

The single isolated heart cell has recently emerged as a model for the study of the structure and function of cardiac cells. Heart muscle cells of adult animals of various species have been successfully isolated by enzymatic digestion of intact cardiac tissue. In this paper a dissociation method that yields living cells from atrial and ventricular tissue of young and adult humans is detailed. The cells retain the morphologic features of cells in intact cardiac tissue, and they generate action potentials and contractions in response to electrical stimulation. The study of isolated human heart cells should make a valuable contribution to knowledge of the normal and diseased heart.     

ApoTome to visualize E-cadherin and p63 expression in oral pre-cancer

Precise histopathological localization of E-cadherin and p63 is of immense importance in understanding the integrity of oral mucosal stratified epithelium in normal and diseased conditions. Necessarily immunohistochemical imaging should have minimum bleaching impact on the dyes and ability to produce clear and crisp images. Here ApoTome provides an alternative with metal halide light source and structured illumination under the assistance of grids, along with integrated image processing modality to generate crisp images with digital interface. The current study demonstrates the applicability of such microscopic system in capturing fluorescence images of immunohistochemical sections of normal and precancerous biopsies in respect to the expression of p63 and E-cadherin in the epithelial cells. The ApoTome images localize the nuclear and membranous expressions of p63 and E-cadherin, respectively, with remarkable specificity. The findings on E-cadherin expression have enormous diagnostic significance as these images clearly differentiate the early and advanced stages of oral submucous fibrosis based on their cytoplasmic and membranous location. Thus, this study clearly depicts a remarkable performance of ApoTome with diagnostic significance.     

Long-term culture of human aortas. Development of atherosclerotic-like plaques in serum-supplemented medium

Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures.     

Intact individual heart cells isolated from human ventricular tissue.

The intricate architecture of heart muscle, comprising irregularly shaped cells which interdigitate in a complex three-dimensional array, has often compromised clear interpretation of experimental data obtained from the whole organ. One approach to minimise some of the difficulties is to use individual muscle cells in suspension, and data have already been reported using myocytes isolated from mammalian ventricles. It is difficult, however, to extrapolate results obtained from animal tissues to situations of medical relevance in man. Intact isolated muscle cells were obtained from human ventricular tissue by modifications of methods used for isolating smooth muscle, atrial, and ventricular tissue from animals. Electrical studies showed that these myocytes had functional characteristics similar to those observed in the whole heart. Such cells will prove a useful preparation for studies on both the mechanisms underlying myocardial performance in normal and diseased states and the response of heart tissue at the cellular level to conditions found during cardiac surgery.     

Myosin and actin containing cells in the human postnatal thymus. Ultrastructural and immunohistochemical findings in normal thymus and in myasthenia gravis.

Samples of normal human thymus of different ages (4-63 years old) were studied by immunofluorescence microscopy (using antibodies to smooth muscle myosin, to actin from the chicken gizzard, and antibodies to myosin from human striated muscle) as well as by routine electron microscopy. Thymus tissue from myasthenia gravis patients was also investigated for comparative reasons. Epithelial cells reacted with anti-smooth, but not with anti-striated muscle myosin, whereas myoid cells reacted with antibodies to striated, but not to smooth muscle myosin. Both epithelial and myoid cells displayed a strong immunoreactivity with antiactin. Corresponding to this immunoreactivity, both cell types contained bundles of thin, actin-like filaments. Myoid cells occurred in the rounded and elongated variety, and they were a normal constituent of all thymuses investigated in this study. Ultrastructurally, this non-innervated, striated muscle-like cell type possessed bundles of thin and thick filaments as well as Z lines in a rather disorganized arrangement, resembling striated muscle after denervation or various other pathologic conditions. There were no overt differences in the number and structure of myoid cells between healthy and myasthenic patients.     

Expression of urotensin-II in human coronary atherosclerosis

The vasoactive peptide urotensin-II (U-II) is best known for its ability to regulate peripheral vascular and cardiac contractile function in vivo, and recent in vitro studies have suggested a role for the peptide in the control of vascular remodeling by inducing smooth muscle proliferation and fibroblast-mediated collagen deposition. Therefore, U-II may play a role in the etiology of atherosclerosis. In the present study we sought to determine the expression of U-II in coronary arteries from patients with coronary atherosclerosis and from normal control subjects, using immunohistochemistry and in situ hybridization. In normal coronary arteries, there was little expression of U-II in all types of cells. In contrast, in patients with coronary atherosclerosis, endothelial expression of U-II was significantly increased in all diseased segments (P < 0.05). Greater expression of U-II was noted in endothelial cells of lesions with subendothelial inflammation or fibrofatty lesion compared with that of endothelial cells underlined by dense fibrosis or minimal intimal thickening. Myointimal cells and foam cells also expressed U-II. In most diseased segments, medial smooth muscle cells exhibited moderate expression of U-II. These findings demonstrate upregulation of U-II in endothelial, myointimal and medial smooth muscle cells of atherosclerotic human coronary arteries, and suggest a possible role for U-II in the pathogenesis of coronary atherosclerosis.