• View in gallery
    Figure 1—

    Amino acid sequences of horse and human MyoD. Differences between sequences (bold letters) and conserved regulatory domains (underlined) are indicated. The proteins from each species consist of 319 amino acids and contain high degrees of homology.

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    Figure 2—

    Diagrammatic representation of the plasmid vector for transient transfection of pIRES2-eqMyoD-EGFP in equine skin–derived fibroblasts and 3T3-NIH cells (A), photomicrographs of transiently transfected skin fibroblasts (B), and western immunoblots of control and transfected cells (C). In panel A, notice that the vector is 6,284-bp long and contains the CMV promoter that drives expression of bicistronic eqMyoD and EGFP by virtue of the IRES. Images in panel B were acquired at different wavelengths and are of equine skin–derived fibroblasts transiently transfected with pIRES2-eqMyoD-EGFP. Cells were immunostained with an antibody against MyoD (red). Notice the EGFP expression (green) throughout the same cell stained with an antibody against MyoD (arrows). The eqMyoD labeling is mainly in the nucleus as confirmed by the DAPI staining (blue). Nontransfected nuclei (blue) in the combined image serve as internal control cells. Bar = 20 μm. Panel C is a western immunoblot for MyoD in control (−) and transfected (+) 3T3-NIH cell extracts. The scale on the right represents the number of kilodaltons. Notice that the transfected cells contain a band at approximately 45 kd. An additional nonspecific band at approximately 33 kd in both lanes confirms equal loading of the lanes.

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    Figure 3—

    Diagrammatic representation of the lentiviral vector for transduction of eqMyoD into equine skin–derived fibroblasts (A) and photomicrographs of immunocytochemical analysis of equine skin–derived fibroblasts on day 7 of culture (B and C). In panel A, the vector is 9,136-bp long and contains the CMV promoter that drives expression of bicistronic eqMyoD and EGFP by virtue of the IRES. The 5′ and the 3′ long terminal repeats (LTR) are depicted. The vector also confers resistance to zeomycin (Zeo; not used). Panel B provides high-resolution images of individual fluorescence channels and combined channels of lentiviral-transducted (+) and control (−) equine skin–derived fibroblasts. Notice that the entire syncytial cell contains EGFP (green) and also expresses desmin (red). The DAPI staining (blue) labels nuclear chromatin. One cell, likely a myofibroblast in the control fibroblasts, expressed low amounts of desmin (arrow). Panel C shows the immunocytochemical analysis for the muscle-specific protein desmin (red) combined with fluorescent channels for EGFP (green) and DAPI (blue) in equine skin–derived fibroblasts. Notice the alignment of multiple multinucleated myotubes. In panels B and C, bars = 10 μm.

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    Figure 4—

    Photomicrographs of equine skin–derived fibroblasts or extracts 15 days after lentiviral transduction (stained with PAS; A and B) and results of western immunoblotting for those cells (C). Notice that in contrast to the control (nontransduced) fibroblasts in panel A, the treated fibroblasts in panel B have abundant stores of glycogen (purple) as well as tubular structure and multiple aligned nuclei. Bars in panels A andB=30 μm. In panel C, results are provided for western immunoblots of lentiviral transduced (+) and control (−) equine skin–derived fibroblast extracts for the muscle-specific proteins desmin (53 kd), sarcomeric myosin (200 kd), and troponin-T (51 kd). An unspecified band (approx 33 kd) is evident in the treated fibroblasts for the muscle-specific protein troponin-T. Coomassie staining of the original gel confirmed equivalent loading of control and treated lanes. The scale on the left side represents the number of kilodaltons.

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Assessment of the transformation of equine skin–derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1

Marta Fernandez-FuenteComparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Ln, Hertfordshire AL9 7TA, England
Dubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England

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Elizabeth G. AmesDepartment of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Michelle L. WagnerDepartment of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Haiyan ZhouDubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England

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Molly StromDivision of Molecular Neuroendocrinology, National Institute for Medical Research, Mill Hill, London NW7 1AA, England

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Peter S. ZammitRandall Division of Cell and Molecular Biophysics, King's College London, Strand, London WC2 2LS, England

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James R. MickelsonDepartment of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108

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Francesco MuntoniDubowitz Neuromuscular Centre, Hammersmith Hospital, Imperial College London, DuCane Rd, London W12 0NN, England

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Richard J. PiercyComparative Neuromuscular Diseases Laboratory, Department of Veterinary Clinical Sciences, Royal Veterinary College, Hawkshead Ln, Hertfordshire AL9 7TA, England
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Abstract

Objective—To develop a reliable method for converting cultured equine skin–derived fibroblasts into muscle cells.

Sample Population—Equine skin–derived fibroblasts.

Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.

Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.

Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Abstract

Objective—To develop a reliable method for converting cultured equine skin–derived fibroblasts into muscle cells.

Sample Population—Equine skin–derived fibroblasts.

Procedures—The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and western immunoblots in equine fibroblasts and fibroblasts from National Institutes of Health Swiss mouse embryos, prior to generation of a lentiviral vector containing the same coding sequences. Transformation of equine skin–derived cells into skeletal myotubes was examined by use of immunohistochemical analysis, western immunoblotting, and periodic acid–Schiff staining.

Results—eqMyoD mRNA consists of 960 bp and shares high homology with myogenic differentiation 1 from other mammals. Transfection confirmed the expression of a 53-kd protein with mainly nuclear localization. Lentiviral transduction was efficient, with approximately 80% of EGFP-positive cells transformed into multinucleated myotubes during 15 days, as determined by expression of the muscle-specific proteins desmin, troponin-T, and sarcomeric myosin and by cytoplasmic storage of glycogen.

Conclusions and Clinical Relevance—Equine primary fibroblasts were transformed by lentiviral transduction of eqMyoD into fusion-competent myoblasts. This may offer a preferable alternative to primary myoblast cultures for the investigation of cellular defects associated with muscle diseases of horses, such as recurrent exertional rhabdomyolysis and polysaccharide storage myopathy.

Scientists and veterinarians have identified several inherited skeletal muscle diseases of horses. Some of these diseases are rare, but others, particularly the exertional myopathies, are common. For example, recurrent exertional rhabdomyolysis of Thoroughbreds is believed to affect up to 7% of racehorses throughout the world,1–3 and polysaccharide storage myopathy affects between 6% and 12% of Quarter Horses4 and 36% of certain draft breeds.5 Understanding the pathophysiologic processes of these conditions is essential for provision of better management and application of treatments. Furthermore, in the diseases for which the precise genetic cause is unknown, improved understanding of the underlying disease process may improve accurate diagnosis and suggest possible candidate genes, thereby expediting and facilitating genetic investigation.

Study of the pathophysiologic processes of disease at the cellular level offers many advantages over whole-animal experiments, not the least of which is a reduction in costs. Additionally, studying the specific cell type that is involved in the disorder enables a more directed approach and allows hypothesis testing that is not feasible in live animals because of experimental constraints or welfare concerns. The cellular investigation of muscle disease is no exception, and experiments involving muscle cell cultures substantially compliment other approaches to disease investigation.6 For example, muscle cell culture has helped confirm that an abnormality in calcium regulation exists in Thoroughbreds with recurrent exertional rhabdomyolysis.7

Culture of mammalian muscle cells is technically demanding and involves certain problems not encountered with some other cell types. Skeletal muscle is a postmitotic tissue, hence cultured muscle cells (myoblasts) are derived from the quiescent population of stem cell–like satellite cells that occupy a position between a muscle fiber's cell membrane (sarcolemma) and basement membrane.8 Satellite cells enable regeneration in damaged or diseased muscle. When a skeletal muscle sample is cultured in vitro, the activated satellite cells divide, and in appropriate conditions, the resulting myoblasts then can be induced to fuse to form more mature and differentiated multinucleated cells, known as myotubes.8 However, there is general consensus that in older and diseased muscle, the satellite cell population is depleted. Therefore, culturing myotubes from diseased adult patients can be difficult because relatively few satellite cells remain.9 Furthermore, in certain diseases associated with a major inflammatory component, muscle cell culture results in contamination with other cell types (eg, fibroblasts) that are not immediately relevant to the primary abnormality being investigated.10 Finally, after several passages, primary myoblasts typically undergo senescence11 (severely limiting their potential); thus, in our experience, additional primary cells have to be derived regularly from fresh muscle samples.

Myogenesis involves the coordinated expression of a series of muscle-specific transcription factors. Of these, transcription factor MyoD is regarded as a key regulator in the myogenic pathways, where it activates the expression and maintenance of many muscle-specific genes. When ectopically expressed in certain nonmuscle cells, MyoD activates positive feedback mechanisms that result in myogenic conversion.12,13 Hence, forced exogenous expression of MyoD in cultured fibroblasts from mice and humans converts them into fusion competent myotubes.14,15 This myogenic conversion has been found to accurately reproduce the characteristics of myotubes derived from primary muscle cells with respect to the expression and assembly of myofibrillar proteins.12,13 Importantly, transformed fibroblasts from human patients have enabled phenotypic classification of patients with mutations in proteins that cause primary muscle diseases, thereby avoiding the need for more invasive muscle biopsy.16,17

Introduction of exogenous transgenes into cells may be accomplished in several ways, but efficient transduction is often accomplished by inserting the transgene into a viral vector. Lentiviral vectors offer advantages over certain other viral systems (such as adenoviruses) because they cause permanent integration of the exogenous transgene within the host cell's genome. In preliminary experiments, we forced the expression of human MyoD into equine fibroblasts, but this did not result in cell transformation and myotube formation, despite expression of the human protein, which suggested that equine fibroblasts are refractory to myogenic conversion or, more likely, that the effects of the transcription factor may be a species-specific event (ie, transformation of equine fibroblasts into muscle would require expression of eqMyoD). For the study reported here, we hypothesized that fibroblasts derived from equine skin could be induced to differentiate into skeletal myoblasts and then into myotubes in culture after exogenous viral expression of the eqMyoD gene. Thus, we sought to clone the eqMyoD gene and create a lentiviral vector capable of efficient conversion of equine skin–derived fibroblasts to muscle cells. This would provide a simple method for obtaining large numbers of muscle cells for the investigation of muscle diseases in horses without the need for invasive muscle biopsy.

Materials and Methods

Sequencing of the eqMyoD gene—Three techniques were used to generate the genomic sequence of the eqMyoD gene. Equine bacterial artificial chromosome clonesa 140N18 and 178B12 containing the eqMyoD gene were obtained, purified, and used for direct sequencing. The PCR primers were designed from human sequences for amplification of the eqMyoD gene from horse genomic DNA. The PCR conditions were as follows: 1.5mM MgSO4, 1.4X PCR buffer solution,b 67μM deoxynucleoside triphosphates, 1μM of each primer, 25 ng of horse genomic DNA, and 0.3 units of Taq polymerase.c The PCR assay was performed in a thermal cyclerd and consisted of 20 minutes at 95°C; 40 cycles of 30 seconds at 94°C, 30 seconds at 56°C, and 30 seconds at 72°C; and a final extension of 15 minutes at 72°C. The PCR products were purified on gels by use of a gel-extraction kitc and submitted for sequencing.e The PCR primers were also designed from the horse genome trace files containing sequences for the MyoD gene. Each PCR assay (performed as previously described) contained 25 ng of DNA, 1.5mM MgCl2, 0.2μM of each primer, 20μM deoxynucleoside triphosphates, and 0.3 units of Taq polymerase.b Products were assembled into a consensus sequence by use of specialized software.f Sequences were submitted online for analysisg and confirmed as eqMyoD.

Cloning of eqMyoD—A sample of semimembranosus muscle (1 cm3) was obtained from a 3-day-old male Thoroughbred foal immediately after it was euthanized for reasons unrelated to our study. The sample was snap-frozen in liquid nitrogen and stored at −80°C. After crushing approximately 20 mg of this muscle sample in liquid nitrogen with a mortar and pestle, total RNA was isolated by use of a commercially available kit,h and cDNA was generated with reverse transcriptasei in accordance with the manufacturer's directions to synthesize first-strand cDNA from purified poly(A)+ or total RNA. The cDNA coding sequence of eqMyoD was amplified in a PCR assay by use of Taq DNA polymerasej and a primer pair (forward, 5′–CTA CGA TGG CAC CTA CTA CAG–3′; reverse, 5′–CTC AGA GCA CCT GGT AGA TG–3′). The program for the thermal cycler was initial denaturation at 94°C for 2 minutes, 31 cycles of 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 68°C for 1 minute. The 960-bp amplified product was cloned into a plasmid vectork that self-catalyzed a ligation reaction of PCR fragments via topoisomerase I (in accordance with the manufacturer's directions) to generate pCR-eqMyoD.

The pCR-eqMyoD was transformed into competent bacterial cellsl by heat shock in accordance with the manufacturer's directions and incubated overnight at 37°C on bacterial agarm plates containing kanamycinn (100 μg/mL). Single colonies were subsequently cultured overnight in 5 mL of bacterial growth culture mediao containing ampicillinn (100 μg/mL) in sterile universal containers.p Plasmid preparations were created by use of commercially available kitsc in accordance with the manufacturer's directions, and plasmids that contained the appropriate eqMyoD insert were confirmed by restriction digest with EcoRI.q Clones with positive results were sequenced with M13 and T7 primersb by the sequencing service at the Imperial College Clinical Sciences Centre and were assessed by use of sequencing software.r

The eqMyoD EcoRI insert was then subcloned into EcoRI-digestedq pIRES2-EGFP by use of T4 DNA ligases to create pIRES2-EGFP-eqMyoD. This new vector was then transformed and prepared as previously indicated and used in transient transfection experiments.

A 2,467-bp fragment from pIRES2-EGFP-eqMyoD was removed by double digest with NheIq and MfeIq and ligated with T4 DNA ligase into the lentiviral vector pCMV-GINs that had been digested with NheI and EcoRI to create the lentiviral vector pCMV-eqMyoD-IRESEGFP. The plasmid was then transformed and prepared as previously described.

Cell lines—The cell lines, human embryonic kidney cell line 293T and 3T3-NIH, were cultured in normal growth medium consisting of Dulbecco modified Eagle medium,n 10% fetal bovine serum,n penicillinstreptomycinb (1 U/mL and 1 μg/mL, respectively), and 2mM L-glutamine.n

Primary equine skin–derived cells—Primary equine skin–derived fibroblasts were obtained from skin samples collected from two 2-year-old male ponies and a 4-year-old Thoroughbred mare that were euthanized for reasons unrelated to our study. Immediately after the animals were euthanized, the skin was aseptically prepared and 4-mm skin samples were obtained with a punch biopsy.t The skin samples were maintained in normal growth medium for up to 48 hours at 4°C until further processing.

Briefly, biopsy specimens were cut into 1-mm cubes with a sterile scalpel blade, placed into the center of 3.5-cm tissue culture dishes, and maintained in an incubator (37°C and 5% carbon dioxide) within a single drop of growth medium. After 6 hours, another 2 mL of medium was added, and the culture dishes were returned to the incubator for 2 to 3 days, by which time, fibroblast-like cells were readily visible migrating outward from the tissue. Fresh medium was then added, and cultures were maintained for an additional 7 to 10 days until a confluent bed of fibroblast-like cells had been obtained. Cultures were split with routine trypsin digestion (0.05% trypsin and 0.53mM EDTA • 4 Na)b and expanded for 2 passages. Cells were frozen in freezing medium (5% dimethyl sulfoxiden in fetal bovine serum) and maintained in cryovials in liquid nitrogen for future use.

Transient transfection experiments—Six-well tissue culture platesu containing 13-mm glass coverslipsv were coated with basement membrane matrixw (0.1 mg/mL), incubated for 30 minutes at 37°C, and then air-dried in a tissue culture flow cabinet. The day before transfection, 2 × 105 of both primary equine fibroblast–like cells and 3T3-NIH cells were plated in 2 mL of normal growth medium in separate wells of 6-well plates (1 plate/cell type). The next day, transfection complexes were formed by adding 8 μL of transfection reagentx to 2 μg of pIRES2-EGFP-MyoD, diluted in 100 μL of serum free medium.y After incubation for 15 minutes at 21°C, the complexes were added in a dropwise manner to the plates. Forty-eight hours after transfection, equine fibroblasts were fixed and processed to confirm the expression of eqMyoD (12 coverslips were examined) by immunocytochemical analysis, and 3T3 cell extracts were collected for western immunoblot analysis.

Virus production—The human embryonic kidney cell line 293T cells were cotransfected by use of a mammalian transfection systemz with the required transfer vector plasmid pCMV-eqMyoD-IRES-EGFP, which was generated as described previously, the packaging plasmid (p8.91),18 and the vesicular stomatitis virus envelope glycoprotein–encoding plasmid.19 The following day, cells were incubated with 10mM sodium butyraten for 4 hours, and medium was replaced with fresh growth medium and incubated for an additional 24 hours. Medium containing virus particles was then collected, filtered through a 0.45-μm filter, and stored at −80°C. To calculate the titer of the viral stock solution, 1 × 104 3T3-NIH cells were plated in 6 wells of a 24-well plate.u Twenty-four hours later, cells were transduced with serial dilutions of the viral stock solution (1 to 100 μL in a final volume of 500 μL of normal growth medium). Four hours later, 1 mL of growth medium was added, and cells were incubated for 5 days before the number of EGFP-positive cells per well were counted. Titer was calculated as the number of transducing units (EGFP-positive cells) per milliliter. In our experiments, the virus titer was 5 × 103 EGFP-positive cells/mL.

Lentiviral transduction of equine fibroblasts—Basement membrane matrixw–coated coverslips in 6-well tissue culture plates were seeded with 1 × 105 equine skin–derived fibroblast–like cells/well and cultured overnight in normal growth medium at 37°C and 5% carbon dioxide. After 24 hours, the cells were transduced with 1 mL (titer of 5 × 103 EGFP-positive cells/mL) of the thawed medium containing the viral particles. After incubation for 4 hours, 2 mL of prewarmed growth medium was added. Control cells received no viral treatment. After incubation for an additional 24 hours, medium was replaced with differentiation medium (Dulbecco modified Eagle medium containing 2% horse serum,s penicillin-streptomycin [1 U/mL and 1 μg/mL, respectively], and 2mM L-glutamine). Medium was changed approximately every 4 days, and cells were examined daily. Coverslips were subsequently processed 7 (to confirm the expression of desmin by immunohistochemical analysis) or 15 (western immunoblotting with desmin and sarcomeric myosin antibodies and PAS staining) days later.

Immunocytochemical analysis—All experiments were performed at least in triplicate, and at least 6 coverslips for each condition were examined within each experiment. Transduced and control cells on coverslips were rinsed in PBS solutionn and then fixed-permeabilized in 4% paraformaldehydet in 250mM HEPESn (pH, 7.4), containing 0.1% triton X-100n at 4°C for 20 minutes, which was followed by incubation in 8% paraformaldehyde in 250mM HEPES for 15 minutes at 21°C. After copious washing in PBS solution, cells were incubated in 20mM glycinen in PBS solution for 15 minutes and then further permeabilized in 0.5% triton X-100 in PBS solution for 30 minutes.

Following incubation for 30 minutes in blocking solution (1% bovine serum albumin,n 0.2% gelatin,n and 0.05% caseinaa in PBS solution), coverslips were incubated for 2 hours with the primary antibody diluted in blocking solution. Antibodies used were a rabbit polyclonal anti-MyoD antibodybb (1:100) for transiently transfected cells and a mouse monoclonal antibody against desmincc (1:100) for virally treated cells. After several washes in PBS solution, coverslips were incubated for 1 hour with secondary antibody (goat antimousedd or goat anti-rabbit IgG,ee as appropriate [diluted 1:500]), washed in PBS solution, and mounted in reagent containing DAPI.ff Cells were examined by use of a fluorescence microscopegg equipped with appropriate wavelength filters (350 nm, 488 nm, and 594 nm), and images were obtained by use of a cooled camerahh and processed by use of commercially available software.ii

PAS staining—Fifteen days after transduction, treated and untreated control equine skin–derived fibroblasts on coverslips (3 in each well of a 6-well plate for each condition) were washed twice with PBS solution and then fixed for 15 minutes in formyl-alcohol (9:1 mixture of 95% ethanol:formaldehyde). Slides were then rinsed with 95% ethanol, air-dried, and rinsed twice with tap water. After incubation for 15 minutes in 0.5% periodic acid,n cells were incubated for 20 minutes with Schiff's reagentu and then counterstained with Harris hematoxylin.jj

Western immunoblotting—Two days after transfection (for confirmation of MyoD expression) or 15 days after transduction with the lentiviral vector (to study expression of muscle-specific proteins after differentiation), cells were rinsed twice with PBS solution, scraped from the plates with a sterile rubber policeman,n and then sedimented by centrifugation (800 × g for 5 minutes). Cell pellets were resuspended in 100 μL of loading buffer (75mM Tris-HCln [pH, 6.8], 15% SDS, 5% β-mercaptoethanol,n 2% glycerol,n 0.02% bromophenol blue,n and protease inhibitorskk; 1 tablet/50 mL of cell extract). Protein concentration was measured by use of a bicinchoninic acid kit,ll in accordance with the manufacturer's directions.

Cell extracts were denatured at 95°C for 5 minutes and centrifuged (14,250 × g for 5 minutes at 4°C). Fifty micrograms of each protein sample and a protein marker laddermm were then loaded into wells on a bis-tris gradient gelnn and electrophoresed for 2 hours at 200 V in running buffer containing 0.05% antioxidant.oo Proteins were then transferred to a nitrocellulose membranepp by incubation for 1 hour at 30 V. Membranes were blocked in 10% nonfat dried milkqq in TBS-T (20mM Tris-HCl, 150mM NaCl, and 0.05% Tween 20n [pH, 7.5]) overnight at 4°C and then incubated for 1 hour at 21°C with the appropriate antibody (anti-MyoD antibodybb [diluted 1:100], antibody against desmincc [diluted 1:100], anti-sarcomeric myosinrr [diluted 1:50], or anti-troponin-Tss [diluted 1:200]); antibodies were diluted in TBS-T. After 4 washes in TBS-T during a 1-hour period, membranes were incubated with a polyclonal rabbit anti-mouse antibody conjugated to HRPtt [diluted 1:200]) or a polyclonal goat anti-rabbit antibody conjugated to HRPtt (for western immunoblotting for MyoD [diluted 1:2,000]), which was followed by the luminal-HRP-chemiluminescence reaction.uu Protein bands were assessed visually.

Gels were stained after protein transfer in accordance with a standard Coomassie protocol to confirm equivalent protein loading for each lane. Briefly, gels were fixed with 40% methanoln and 10% acetic acidn in water for 1 hour, then incubated for 1 additional hour with constant stirring in Coomassie solution.n Finally, gels were destained in 7.5% methanoln and 10% acetic acid until the background between the protein bands had cleared.

Results

The total genomic sequence of eqMyoD is 4,349 bp. Homology of this sequence with Homo sapiens chromosome 11 (ENSG00000129152) is 79%. The coding sequence for eqMyoD consists of 960 bp, and it has homology of 90% with human MyoD mRNA (NM_ 002478). Translation of the coding sequence yielded 1 likely reading frame that yielded a 319-amino acid residue protein that contained a myogenic basic domain and a helix-loop-helix domain (Figure 1).

Figure 1—
Figure 1—

Amino acid sequences of horse and human MyoD. Differences between sequences (bold letters) and conserved regulatory domains (underlined) are indicated. The proteins from each species consist of 319 amino acids and contain high degrees of homology.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1637

Equine myogenic differentiation 1 has 91% homology with the amino acid sequence of human MyoD (NP_002469; Figure 1), and it has similar high homology with the protein in other mammals (dogs, 95% [XP_853636]; pigs, 94% [NP_001002824]; cattle, 93% [NP_001035568]; sheep, 92% [P29331]; and rats and mice, 89% [Q02346 and P10085, respectively]). Comparisons with nonmammalian species revealed that eqMyoD was 65% and 62% identical to the homologous proteins of chickens (P16075) and zebra fish (Q90477), respectively.

Reverse-transcriptase PCR assay of equine muscle mRNA generated the expected 960-bp eqMyoD cDNA amplicon, which was cloned to generate pIRES2-eqMyoD-EGFP (Figure 2). Transient transfection of this plasmid vector into equine skin–derived cells resulted in coexpression of eqMyoD and EGFP under control of the CMV promoter in approximately 10% of cells (the IRES within the vector-created bicistronic mRNA). No cells were detected with eqMyoD expression or EGFP expression alone. Expression of eqMyoD was mostly within the nucleus; however, there was low expression within the cytoplasm. Expression of EGFP was evident throughout each cell. Western immunoblots of 3T3-NIH cultures containing transfected cells confirmed the expression of a protein with an approximate molecular weight of 45 kd (as expected), in comparison with results for nontransfected cells.

Figure 2—
Figure 2—

Diagrammatic representation of the plasmid vector for transient transfection of pIRES2-eqMyoD-EGFP in equine skin–derived fibroblasts and 3T3-NIH cells (A), photomicrographs of transiently transfected skin fibroblasts (B), and western immunoblots of control and transfected cells (C). In panel A, notice that the vector is 6,284-bp long and contains the CMV promoter that drives expression of bicistronic eqMyoD and EGFP by virtue of the IRES. Images in panel B were acquired at different wavelengths and are of equine skin–derived fibroblasts transiently transfected with pIRES2-eqMyoD-EGFP. Cells were immunostained with an antibody against MyoD (red). Notice the EGFP expression (green) throughout the same cell stained with an antibody against MyoD (arrows). The eqMyoD labeling is mainly in the nucleus as confirmed by the DAPI staining (blue). Nontransfected nuclei (blue) in the combined image serve as internal control cells. Bar = 20 μm. Panel C is a western immunoblot for MyoD in control (−) and transfected (+) 3T3-NIH cell extracts. The scale on the right represents the number of kilodaltons. Notice that the transfected cells contain a band at approximately 45 kd. An additional nonspecific band at approximately 33 kd in both lanes confirms equal loading of the lanes.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1637

Because we believed that the aforementioned information established that our construct resulted in expression of eqMyoD after transfection, we next sought to evaluate a means for increasing the efficiency of delivery. The cassette containing the sequences for eqMyoD, IRES, and EGFP was subcloned into the lentiviral transfer vector, and this was used to generate viral particles coding for eqMyoD and EGFP (Figure 3). Twenty-four hours after infection with viral particles, the serum concentration in the media was reduced, which is a standard technique used to induce cell fusion.

Figure 3—
Figure 3—

Diagrammatic representation of the lentiviral vector for transduction of eqMyoD into equine skin–derived fibroblasts (A) and photomicrographs of immunocytochemical analysis of equine skin–derived fibroblasts on day 7 of culture (B and C). In panel A, the vector is 9,136-bp long and contains the CMV promoter that drives expression of bicistronic eqMyoD and EGFP by virtue of the IRES. The 5′ and the 3′ long terminal repeats (LTR) are depicted. The vector also confers resistance to zeomycin (Zeo; not used). Panel B provides high-resolution images of individual fluorescence channels and combined channels of lentiviral-transducted (+) and control (−) equine skin–derived fibroblasts. Notice that the entire syncytial cell contains EGFP (green) and also expresses desmin (red). The DAPI staining (blue) labels nuclear chromatin. One cell, likely a myofibroblast in the control fibroblasts, expressed low amounts of desmin (arrow). Panel C shows the immunocytochemical analysis for the muscle-specific protein desmin (red) combined with fluorescent channels for EGFP (green) and DAPI (blue) in equine skin–derived fibroblasts. Notice the alignment of multiple multinucleated myotubes. In panels B and C, bars = 10 μm.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1637

Cells derived from the various animals and cells that had been frozen behaved identically. Within 4 days after lentiviral transduction, approximately 5% of cells had fused, and by 7 days, cell morphology had changed dramatically, with alignment and fusion of cells to form multinucleated myotubes. In contrast to nontransduced cells, approximately 80% of transduced cells expressed the muscle-specific protein desmin (Figure 3). Within the nontransduced control cells, a few (< 1%) cells were identified that were mildly positive for desmin expression.

After an additional week of incubation in differentiation medium, myotubes had differentiated further. They appeared more mature phenotypically, with long tubular processes, squared cellular attachments, and multiple nuclei; they were often raised from the culture bed. There was intense glycogen staining within these cultures, in comparison with staining in nontransduced control cells (as evident after PAS staining; Figure 4). Western immunoblotting of protein extracts from these cultures confirmed expression of desmin and also revealed expression of the muscle-specific proteins, sarcomeric myosin and troponin-T, in contrast with results for control cells.

Figure 4—
Figure 4—

Photomicrographs of equine skin–derived fibroblasts or extracts 15 days after lentiviral transduction (stained with PAS; A and B) and results of western immunoblotting for those cells (C). Notice that in contrast to the control (nontransduced) fibroblasts in panel A, the treated fibroblasts in panel B have abundant stores of glycogen (purple) as well as tubular structure and multiple aligned nuclei. Bars in panels A andB=30 μm. In panel C, results are provided for western immunoblots of lentiviral transduced (+) and control (−) equine skin–derived fibroblast extracts for the muscle-specific proteins desmin (53 kd), sarcomeric myosin (200 kd), and troponin-T (51 kd). An unspecified band (approx 33 kd) is evident in the treated fibroblasts for the muscle-specific protein troponin-T. Coomassie staining of the original gel confirmed equivalent loading of control and treated lanes. The scale on the left side represents the number of kilodaltons.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1637

Discussion

In the study reported here, we sought to develop a practical system that could reliably generate muscle cells in culture from a skin sample. Skin biopsy is likely to be more acceptable than muscle biopsy to owners with valuable horses in training, and primary muscle cells are hard to culture and maintain. We initially derived the eqMyoD genomic sequence and then used species-specific primers to amplify the appropriate cDNA coding sequence, which was then expressed as a transgene by use of a lentiviral vector. The system we used resulted in the reproducible generation of abundant differentiated myotubes that express proteins present in mature adult muscle and that, similar to mature muscle, store glycogen. Furthermore, these cells were stable in culture for up to 15 days and could be derived from skin samples stored for 2 days after collection during biopsy. In addition, myotubes can be derived from fibroblast-like cells that have been frozen, stored, and subsequently thawed. Coexpression of EGFP and eqMyoD by use of the IRES element within our vector allowed us to confirm the fusion of living transduced cells to form myotubes.

We tested the muscle-specific transduction of equine skin–derived cells in comparison with control cells by examining the expression of muscle-specific proteins by both immunocytochemical analysis (for desmin) and western immunoblotting (for desmin, troponin-T, and sarcomeric myosin). In control cells (ie, nonvirally transduced cells), there were extremely low numbers of cells mildly positive for desmin detected by use of immunocytochemical analysis that were considered likely to have been myofibroblasts derived from the original primary cultures. In contrast to the treated cells, however, these cells were mononucleated. Western immunoblotting is particularly helpful in protein identification when investigators use antibodies in new species, and in the experiments reported here, bands of the expected molecular weight were detected in treated cells for each of the proteins examined. However, additional unexpected protein bands were identified when we used 2 of the antibodies (anti-MyoD and anti-troponin-T). The anti-MyoD antibody that we used was a polyclonal antibody that appeared to recognize an additional protein, which accounted for the smaller band at 33 kd. An additional faint unexpected band was also detected when the troponin-T antibody was used, although the band was only evident for the treated cells. It is conceivable that this monoclonal antibody recognized an epitope on a smaller protein that was only expressed in the muscle-transformed cells. Alternatively, it is possible that the protein band was a cleavage product of full-size troponin-T.

Myogenic differentiation 1 is a muscle-specific transcription factor in the MRF family.20 Other members include myogenin, Myf5, and MRF4.21 These transcription factors are basic helix-loop-helix proteins that play key roles in the differentiation of the skeletal muscle lineage. Myogenic differentiation 1 transforms fibroblasts from a variety of species12,14,15 into myotubes, similar to the results for other factors, such as Myf5.22 Some factors, (eg, myogenin, which has been detected in equine satellite cells23) transform fibroblasts less efficiently24 probably because they act farther downstream in the myogenic pathways.25 For the study reported here, we chose to concentrate on MyoD because experiments conducted in other species have revealed that myotubes derived from fibroblasts transformed with this transcription factor are morphologically indistinguishable from their muscle-derived counterparts.12,13 Members of our research group have examined the phenotypic properties of human skin cells that have been converted to myotubes by human MyoD–induced conversion,16 but our preliminary experiments revealed that human MyoD did not induce equine fibroblasts to transform into myotubes. In the set of experiments described here, we detected efficient myogenic conversion of equine skin–derived cells following transduction with eqMyoD, which suggests that the sequence differences between eqMyoD and human MyoD that we reported here may have been responsible. Although analysis of the amino acid sequences of human MyoD and eqMyoD revealed a high degree of homology (as expected), there were several differences in amino acids in the key regulatory domains of the proteins. We believe it is likely that these differences confer the species-specific nature of the proteins' myogenic functions.

Although members of our research group have used an adenoviral system in human cells to initiate myogenic conversion,16 adenoviruses are somewhat selective in their relative infection of different cell types.26 Adenoviruses can infect cultured equine cells of mesenchymal origin27; however, fibroblasts in other species have relatively low expression of the receptor for virus entry,26,28 and high viral titers or other strategies must sometimes be used to increase infection rates.29 For the study reported here, we chose a lentiviral vector, rather than an adenoviral vector, to deliver the exogenous transgenes. To our knowledge, this is the first time that a lentiviral vector has been used to introduce transgenic DNA into equine cells. However, a lentiviral vector has been described that can induce myogenic conversion of human chorionic villus and fibroblast cultures.17 In contrast to adenoviral vectors, which result in transient expression of exogenous DNA, lentiviral vectors result in permanent integration of exogenous DNA within the host cell's genome. Although we did not compare the relative efficacy of adenoviral and lentiviral systems for fibroblast conversion to myotubes, the persistent expression of eqMyoD may better mimic myogenesis in vivo because MyoD is expressed into adult life.30

We believe that myotubes derived from equine skin samples may be a useful in vitro method for investigating muscle diseases of horses. Additional studies will be required to validate the reproducibility of specific functional pathways within equine skin–derived cells. The lentiviral approach that we used likely results in a heterogeneous population of cells with integration of the CMV-MyoD-IRES-EGFP element at various locations within the nuclear DNA. As such, observed differences that could result from the interruption or misexpression of genes in cis or trans to the inserted transgene can be avoided by measuring or recording from a population of cells within the same culture. Our intent is to focus on separate systems of relevance to exertional myopathies in horses, such as glycogen metabolism (with regard to polysaccharide storage myopathy31) and calcium homeostasis (with regard to recurrent exertional rhabdomyolysis),32,33 and the effect of relative states of differentiation on these systems.

The study reported here confirmed that a lentiviral vector expressing eqMyoD induced myogenic conversion of equine skin–derived cells. Additional studies are being conducted to determine whether this system offers a feasible and practical alternative to the use of cultured primary muscle cells for the investigation of the pathophysiologic processes of specific muscle diseases of horses and for the accurate diagnosis of certain inherited myopathies in which the genetic cause has not been identified.

ABBREVIATIONS

CMV

Cytomegalovirus

DAPI

4′,6-diamidino-2-phenylindole

EGFP

Enhanced green fluorescent protein

eqMyoD

Equine myogenic differentiation 1

HRP

Horseradish peroxidase

IRES

Internal ribosome entry site

MRF

Myogenic regulatory factor

Myf5

Muscle-specific transcription factor in the myogenic regulatory factor family

MyoD

Myogenic differentiation 1

PAS

Periodic acid–Schiff

TBS-T

Tris-buffered saline–Tween-20

3T3-NIH

Fibroblasts from National Institutes of Health Swiss mouse embryos

a.

Children's Hospital Oakland Research Institute, Oakland, Calif.

b.

Invitrogen Corp, Carlsbad, Calif.

c.

Qiagen Inc, Valencia, Calif.

d.

MJ Research, Reno, Nev.

e.

BioMedical Genomics Center, DNA Sequencing and Analysis Facility, University of Minnesota, Saint Paul, Minn.

f.

Sequencher, version 4.7, Gene Codes Corp, Ann Arbor, Mich.

g.

Basic local alignment search tool (BLAST), NCBI, Bethesda, Md.

h.

Qiagen RNeasy mini kit, Qiagen Ltd, Crawley, West Sussex, England.

i.

Superscript III, Invitrogen Ltd, Paisley, England.

j.

Platinum Taq DNA polymerase high fidelity, Invitrogen Ltd, Paisley, England.

k.

pCRII-TOPO, Invitrogen Ltd, Paisley, England.

l.

Top10, Invitrogen Ltd, Paisley, England.

m.

Lennox L agar, Invitrogen Ltd, Paisley, England.

n.

Sigma-Aldrich Co, Gillingham, Dorset, England.

o.

Lennox L broth, Invitrogen Ltd, Paisley, England.

p.

Barloworld-Scientific, Maidenhead, Berkshire, England.

q.

New England Biolabs (UK) Ltd, Hitchin, Hertfordshire, England.

r.

Vector NTI, Invitrogen Ltd, Paisley, England.

s.

Autogen Bioclear UK Ltd, Calne, England.

t.

Stiefel Laboratorium GmbH, Offenbach am Main, Germany.

u.

Becton Dickinson UK Ltd, Oxford, England.

v.

VWR International Ltd, Lutterworth, Leicestershire, England.

w.

Matrigel, Becton Dickinson UK Ltd, Oxford, England.

x.

FuGene HD transfection reagent, Roche Diagnostics Ltd, Burgess Hill, West Sussex, England.

y.

Opti-MEM, British Biocell International Ltd, Cardiff, England.

z.

Profection mammalian transfection system, Promega, Southampton, England.

aa.

British Biocell International Ltd, Cardiff, England.

bb.

Clone M-318, Santa Cruz Biotechnology, Santa Cruz, Calif.

cc.

Clone D33, Dako UK Ltd, Ely, Cambridgeshire, England.

dd.

Alexa-fluor 488 goat anti-mouse, Invitrogen Ltd, Paisley, England.

ee.

Alexa-fluor 594 goat anti-mouse, Invitrogen Ltd, Paisley, England.

ff.

Prolong Gold antifade reagent containing DAPI, Invitrogen Ltd, Paisley, England.

gg.

Leica DMR fluorescence microscope, Leica Microsystems AG, Wetzlar, Germany.

hh.

CDD Photometrics Coolsnap, Roper Scientific, Tucson, Ariz.

ii.

Metamorph, version 7.5, Universal Imaging, West Chester, Pa.

jj.

Raymond A Lamb Ltd, Eastbourne, East Sussex, England.

kk.

Protease inhibitor cocktail tablets, Roche Diagnostics Ltd, Burgess Hill, West Sussex, England.

ll.

Pierce Biotechnology Inc, Rockford, Ill.

mm.

SeeBlue Plus2 prestained standard, Invitrogen Ltd, Paisley, England.

nn.

NUPAGE 4-12% bis-tris gradient gel, Qiagen Ltd, Crawley, West Sussex, England.

oo.

NUPAGE MOPS SDS, Invitrogen Ltd, Paisley, England.

pp.

Hybond ECL, Amersham PLC, Little Chalfont, Buckinghamshire, England.

qq.

Néstle UK Ltd, Croydon, Surrey, England.

rr.

Clone MF20, Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, Iowa City, Iowa.

ss.

Clone JLT-12, Sigma-Aldrich Co, Gillingham, Dorset, England.

tt.

Dako UK Ltd, Ely, Cambridgeshire, England.

uu.

ECL Plus western blotting detection system, Amersham PLC, Little Chalfont, Buckinghamshire, England.

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Contributor Notes

Supported by the Horserace Betting Levy Board.

Address correspondence to Dr. Piercy.