- Email: [email protected]
Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells
Available online at www.sciencedirect.com
Veterinary Immunology and Immunopathology 125 (2008) 225–233 www.elsevier.com/locate/vetimm
Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells Ste´phanie Gofflot, Philippe Kischel, Caroline Thielen, Vincent Radermacher, Jacques Boniver, Laurence de Leval * Department of Pathology, University of Lie`ge, Pathology Building B23+1, CHU Sart Tilman, B-4000 Lie`ge, Belgium Received 30 October 2007; received in revised form 8 May 2008; accepted 27 May 2008
Abstract Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology. # 2008 Elsevier B.V. All rights reserved. Keywords: Muscle markers; Immunohistochemistry; Canine muscle
1. Introduction Over the past years, cell-based therapies have revolutionized the field of regenerative medicine. There is indeed increasing interest for generating functional contractile muscle and cardiac cells by transplantation of undifferentiated stem cells or myoblasts (Grounds et al., 2002). Cellular therapies are presently under investigation for the treatment of numerous muscular disorders, such as ischemic heart diseases, cardiomyopathies and hereditary muscular dystrophies. Relevant
* Corresponding author at: Department of Pathology, University of Lie`ge, Pathology Building B23+1, CHU Sart Tilman, B-4000 Lie`ge, Belgium. Tel.: +32 43662410; fax: +32 43662919. E-mail address: [email protected] (L. de Leval). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.05.029
animal models are mandatory in this type of research and dogs have been widely used as experimental models for Duchenne’s muscular dystrophy (Cooper et al., 1988; Valentine et al., 1988; Cooper, 1989) and in preclinical studies investigating the use of stem cell transplantation after induced heart damage (Vulliet et al., 2004; He et al., 2005; Linke et al., 2005; Silva et al., 2005). In addition to functional studies, evaluation of these experimental strategies requires specific methods of cell characterization, in order to evaluate the degree of colonization of the diseased tissue by the grafted cells and to assess lineage-specific differentiation of these cells. Immunohistochemistry is an invaluable tool for targeting and identifying differentiation markers. Yet, to our knowledge, the specificity of antibodies for muscular markers has not been extensively characterized in canine tissues. In a recent report, anti-human antibodies directed
226
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Table 1 Specific markers expressed by the different muscle types
There are two main types of muscle cells, smooth and striated, the latter including voluntary skeletal muscle and involuntary cardiac muscle.
against Troponin I and Troponin T were shown to react with dog cardiomyocytes (Fishbein et al., 2003). The current study was undertaken to extend the panel of antibodies useful for immunohistochemical characterization of muscular differentiation in canine tissues. A broad panel of antibodies raised against human, rat or chicken epitopes of muscle-specific proteins, validated for diagnostic or experimental use on human tissues, were evaluated for their reactivity on canine tissues. The markers tested in this study are summarized in Table 1. Desmin is an intermediate filament protein that is a structural cytoplasmic component of all muscle cells, i.e. cardiomyocytes, skeletal and smooth muscle cells (Costa et al., 2004). Alpha Smooth Muscle Actin (SMA) is an actin isoform that contributes to cell-generated mechanical tension. Its expression is normally restricted to smooth muscle cells, myoepithelial cells and myofibroblasts (Skalli et al., 1986). h-Caldesmon, a cytoplasmic protein that regulates cellular contraction through its interaction with actin and myosin is another specific marker of smooth muscle differentiation (Sobue and Sellers, 1991). HDAC8, a class I histone deacetylase was recently characterized as a marker of smooth muscle differentiation as it is detected in the cytoplasm of visceral and vascular smooth muscle cells, myoepithelial cells and myofibroblasts (Waltregny et al., 2004). Conversely, Myosin Heavy Chain (MHC), the most abundant protein in the striated muscle where it acts as the molecular motor of muscle contraction, is not detected in smooth muscle cells. Troponin (Tn), a protein complex of three subunits (TnC, TnI, and TnT), is found in both skeletal and cardiac muscles, and plays the role of conferring calcium sensitivity to muscle cells (Farah and Reinach, 1995).
Several nuclear proteins functioning as transcription factors are known to be specifically expressed in cardiomyocytes or in skeletal myocytes (Dias et al., 1994; Bruneau, 2002). GATA-4 transcription factor, which binds a DNA consensus sequence WGATAR in the promoter region of many cardiac and gut-specific genes, is expressed in various tissues of endodermal or mesodermal derivation such as heart, liver, lung, gonad, and gut where it plays critical roles in regulating tissuespecific gene expression (Molkentin, 2000). GATA-4 is present in adult cardiac tissue as well as in the developing heart and is essential for proper cardiac morphogenesis (Pikkarainen et al., 2004). MEF-2C, a member of the myocyte-specific enhancer factor 2 (MEF-2) family is an essential regulator of cardiac myogenesis and right ventricular development (Lin et al., 1997). The homologue of the tinman gene in Drosophila melanogaster called Nkx-2.5 in vertebrates, encodes for a homeodomain-containing transcription factor, which is expressed at high levels in the developing and adult heart and plays a critical role in the myocardial development (Lints et al., 1993; Shiojima et al., 1996). MyoD1 and myogenin are myogenic regulatory factors restricted to skeletal muscle cells; MyoD1 plays a critical role in initiating the onset of muscle-specific gene expression whereas myogenin is involved in the later steps of skeletal muscle differentiation (Weintraub et al., 1991). 2. Materials and methods 2.1. Tissues Normal tissues were obtained from 10 to 15 monthsold Beagle dogs (15–45 kg) of either sex, purchased from Harlan (The Netherlands). They were used in an experimental protocol of cardiac injury and repair by injection of stem cells (Bartunek et al., 2007). The study was approved by the Ethical Committee for the animal research of the Louvain Medical School, Brussels and the Faculty of Medicine of the Free University of Brussels, Belgium. At the end of the treatment, the dogs were euthanized by intravenous barbiturate overdosage administration (200 mg/kg). Control animals with no cardiac injury or with no stem cell injection, were selected for harvesting of normal tissue samples from heart (ventricular wall) or skeletal muscle (thigh) and organs containing a muscularis layer (uterus, intestines and bladder). Tissues were either fixed in 10% buffered formalin and embedded in paraffin blocks for immunohistochemistry, or snapfrozen for western blot analysis.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
227
Table 2 Summary of antibodies and their conditions of use Antibody
Origin-immunogen
Cytoplasmic markers h-Caldesmon Monoclonal mouse anti-human
Designation
Source
Pre-treatment for IHC
Dilution IHC a
h-CD
DAKO
Water-bath at 37 8C 20 min in 0,25% trypsin/PBS + water-bath at 95 8C 40 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Water-bath at 95 8C for 40 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8)
1:100
Microwave 2 5 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Microwave 2 5 min in 10 mM citrate buffer (pH 6) Microwave 2 5 min in 10 mM citrate buffer (pH 6) Water-bath at 95 8C 40 min in EDTA buffer (pH 8)
1:50
Desmin
Monoclonal mouse anti-human
D33
DAKO
HDAC8
Polyclonal goat anti-human
N-20
Santa Cruz
MHC
Monoclonal mouse anti-chicken
F59
Santa Cruz
SMA
Monoclonal mouse anti-human
1A4
DAKO
Troponin I
Polyclonal goat anti-human
C19
Santa Cruz
Troponin T
Monoclonal mouse anti-chicken
T1/61
Novocastra
Nuclear markers GATA-4
Monoclonal mouse anti-human
G4
Santa Cruz
MEF-2C
Polyclonal goat anti-human
C-17
Santa Cruz
MyoD1
Monoclonal mouse anti-human
5.8A
DAKO
Myogenin
Monoclonal mouse anti-rat
F5D
DAKO
Nkx-2.5
Polyclonal goat anti-human
AF2444
R&D
a
Dilution WB
1:200
1:500
1:40
1:600
1:200
1:2500
1:200
1:5000
1:50
1:1000
1:10
1:250
1:25 1:100
1:100
1:25
The same dilution was applied to human and canine tissues.
2.2. Antibodies The antibodies tested in this study were: hcaldesmon, desmin, HDAC8, MHC, a-SMA, Troponin I, Troponin T, GATA-4, MEF-2C, MyoD1, myogenin, Nkx-2.5. The clone designations, sources of antibodies and their conditions of use established based on their expected reactivity on normal adult human tissues are summarized in Table 2. 2.3. Immunohistochemistry Briefly, four micrometers paraffin sections were dewaxed in xylol and rehydrated in methanol. Endogenous peroxidase activity was inhibited with 3%
hydrogen peroxide in methanol for 5 min. After washing in distilled water and antigen retrieval, the sections were blocked with ChemMateTM Antibody Diluent (S2022, Dako, Glostrup, Denmark) and incubated at room temperature for 1 h with the working dilution of primary antibody, excepted for MyoD1 antibody which required overnight incubation at 4 8C. For mouse primary antibodies, the slides were subsequently incubated for 30 min with the biotinylated link (LSAB2 system-HRP, Dako), washed and incubated for 30 min with streptavidin–peroxidase (HRP) (Dako). ENVISION developing system (Dako) was required for MyoD1 antibody. Visualization of goat primary antibodies was performed using HRP-coupled rabbit anti-goat immunoglobulin secondary antibody. Washes were performed with PBS
228
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
after each incubation step. Antibody binding was revealed by using H2O2 as a substrate and 3,30 diaminobenzidine as chromogen (Dako). Sections were counterstained with hematoxylin and mounted with a Tissue-Tek SCA film coverslipper (Sakura Finetek, Zoeterwoude, The Netherlands). Negative controls by omission of the primary antibody and positive controls consisting of adequate human tissues were included in each staining batch. 2.4. Western blotting Cardiac, skeletal and smooth muscles from human and canine species were pulverized frozen using a MikroDismembrator U (Braun Biotech) and tissue powders were lysed in a PBS buffer containing 1% NP40, 0.5% DOC, 1% SDS and a protease inhibitor cocktail (Halt, Pierce-Thermo Scientific). The lysates were subjected to SDS-PAGE in 4–12% gels (NuPAGE, Invitrogen) under reducing conditions. Separated proteins were electrotransferred to nitrocellulose membranes overnight at constant voltage (35 V), except for MHC proteins that were transferred at constant current (120 mA). Membranes were blocked in TBS (Tris Buffer Saline) containing 5% non-fat dried milk, 0.05% Tween-20, and incubated with antibodies overnight at 4 8C. Bound antibodies were visualized using ECL chemiluminescent substrate (GE Healthcare) and exposure to X-ray films (Fuji).
3. Results 3.1. Immunohistochemistry: cytoplasmic markers Anti-desmin antibody induced a strong and diffuse cytoplasmic positivity in all three kinds of canine muscle cells (not shown). Smooth muscle cells of the myometrium, bladder and intestinal walls were strongly desmin-positive, as were cardiomyocytes and skeletal muscle cells. Arteriolar smooth muscle cells in all tissues also stained positively. The staining was specific, since reactivity was observed neither in the epithelial compartments, nor in nerves. Only scattered spindled cells stained positively in mucosal or interstitial connective tissue. Anti-SMA antibodies produced a strong cytoplasmic staining in most muscle cells in the myometrium, in the muscularis mucosae and muscularis propria layers of the bowel and bladder (Fig. 1A). A strong staining was also identified in arteriolar smooth muscle cells (Fig. 1A–C). Conversely, in the endometrium and mucosal layers of the intestine and bladder, only scattered spindled cells showed positivity for this marker (Fig. 1A). A similar pattern was observed in the corresponding human viscera. In the skeletal muscle and heart, the staining for SMA was restricted to smooth muscle cells of the vascular walls (internal positive controls), whereas the myocytes were clearly negative (Fig. 1B–C).
Fig. 1. IHC stains of canine tissues with smooth muscle specific markers. Expression of SMA (A–C) and HDAC8 (D–F) in canine muscle tissues (uterus: A and D; skeletal muscle: B and E; myocardium: C and F). Positive staining for both markers is observed in the cytoplasm of smooth muscle cells in the myometrium and the positivity is restricted to the vascular endothelial cells of skeletal muscle and in myocardium (magnification 100).
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
229
Fig. 2. IHC stains of canine tissues with striated muscle markers. The antibodies MHC (A–C), Troponin I (D–F) and Troponin T (G–I) positively stain cytoplasm of cardiac myocytes (C, F, and I-100) and skeletal muscle cells (B, E, and H-100) whereas smooth muscle cells in the intestines (A-100) and bladder (D-100, G-200) are negative.
The pattern of staining observed with anti-HDAC8 antibody was similar to that described above for SMA (Fig. 1D–F). In canine uterus, HDAC8 showed positivity exclusively in the myometrium and vessel walls (Fig. 1D). In the intestine tissue, HDAC8 produced cytoplasmic staining in the muscularis layer and in vessel walls, in contrast to the mucosa that remained completely negative. The muscularis layer of bladder was also positively stained with HDAC8 antibody. h-Caldesmon did not produce any reactivity on canine tissue while a positivity was observed in human tissues. MHC was uniformly negative in non-muscle cells of striated muscles and in smooth muscle cells of viscera and arterioles (Fig. 2A). Conversely, the cytoplasm of skeletal muscle cells and of cardiomyocytes was strongly positive (Fig. 2B–C). No reactivity was observed for TnI within muscular or mucosal layers of intestine, bladder and uterus (Fig. 2D). Anti-TnI antibody produced a cytoplasmic staining in striated muscle cells, including skeletal and
cardiac myocytes (Fig. 2E–F). Virtually all striated muscle cells were positively stained, with a staining pattern distinctively very heterogeneous within each cell, that tended to be more intense towards one pole of the cytoplasm. Vascular walls in all tissues were negative for this marker. No specific immunoreactivity with anti-TnT antibody was observed in the canine tissues containing smooth muscle (Fig. 2G). TnT displayed moderate to strong cytoplasmic staining in the majority of skeletal muscle cells. A small subset of skeletal striated cells remained negative, likely corresponding to slow muscle cells, since the antibody is reported to be specific for fast ones (Fig. 2H). Conversely, virtually all-cardiac myocytes were strongly and homogeneously positive for TnT (Fig. 2I). 3.2. Immunohistochemistry: nuclear markers Among the cardiac-specific markers tested in the present study, Nkx-2.5 was strongly positive in canine
230
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Fig. 3. IHC stains of canine tissues with cardiac-specific transcription factors. Nkx-2.5 stains nuclei of dog myocardium cells (A-200) and is negative in intestinal cells (B-200) and skeletal muscle cells (C-200). GATA-4 is positive in cardiomyocytes (D-200) and crypts of the intestines (E-100) but is negative in skeletal muscle cells (F-200).
myocytes (Fig. 3A). No staining was seen for this marker in smooth or skeletal muscle cells (Fig. 3B–C) in all other tissues studied. Anti-GATA-4 antibody produced a nuclear staining in canine cardiomyocytes (Fig. 3D). No staining was seen in the vessels and in the muscularis of bladder and uterus. However, in the intestine, GATA-4 displayed a nuclear staining of smooth muscle cells in the muscularis propria and in epithelial cells lining crypts, similar to the staining observed in the corresponding human viscera (Fig. 3E). No staining was seen for this marker in skeletal muscle cells (Fig. 3F). The antibodies against MEF-2C, MyoD1 and myogenin did not produce any cross-reactivity on canine tissues in the conditions used for the staining of human tissues.
was detected in all human and canine samples, with human cardiac muscle showing the lowest reactivity and human smooth muscle showing the strongest reactivity. Myosin heavy chains (cardiac cMHC and fast fMHC)
3.3. Validation of antibody cross-reactivity Even though the immunohistochemistry staining observed with the different antibodies was similar in human and canine tissues, we sought to confirm the specificity of these antibodies. By sequence analysis (http://www.expasy.org), we were able to compare the sequence of five protein markers, and found a good consensus (over 80%) between both species for desmin, MHC, Nkx-2.5 and Troponin I whereas less then 60% identity was found for Troponin T. Then, we carried out a western blot analysis on human and canine muscle protein homogenates (Fig. 4). Desmin
Fig. 4. Protein lysates from human and canine cardiac, skeletal and smooth muscles were subjected to immunoblot analysis of desmin, MHC, SMA, HDAC8, TnT and TnI expression, as described in Section 2.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
231
Table 3 Summary of immunohistochemistry staining on canine muscle tissues and specificity on human muscle cells Canine muscle tissues
Human specificity
Antibody
Smooth muscle
Striated cardiac muscle
Striated skeletal muscle
Cytoplasmic markers Desmin
+
+
+
+ +
+ + +
+ + +
Smooth, skeletal and cardiac muscles Smooth muscle Smooth muscle Smooth muscle Striated muscle Striated muscle Striated muscle
a
+ +
Cardiac muscle Cardiac muscle Skeletal muscle Skeletal muscle Cardiac muscle
h-Caldesmon HDAC8 SMA MHC Troponin I Troponin T Nuclear markers GATA-4 MEF-2C MyoD1 Myogenin Nkx-2.5 a
Nuclear staining of smooth muscle cells in the muscularis propria and epithelial cells lining crypts.
were detected in cardiac and skeletal muscles from both human and canine origin, as expected from our IHC data and the previously described reactivity of the F59 antibody (Miller et al., 1989). SMA and HDAC8 were specifically detected in smooth muscles of both human and canine species. Cardiac muscle troponin T (cTnT) was recognized in cardiac muscles from both species, and several fast muscle troponin T isoforms (fTnT) were recognized in skeletal muscles from human and canine origin. Fast TnI was expressed in skeletal muscles from both human and canine species. Moreover, in canine cardiac muscles, two TnI isoforms were recognized: cardiac TnI (cTnI) appeared clearly as the main isoform expressed, while a lower expression of slow TnI (sTnI) was also detected. In human cardiac muscle, only a faint band of cTnI could be discerned. Unfortunately, antibodies against nuclear markers GATA-4 and Nkx2.5 did not produce any specific results in western blotting. 4. Discussion Muscular disorders represent a large field for potential application of cell-based therapies and various protocols are currently tested in different animal models, in particular dogs. Immunohistochemical analyses of animal models of cell-based therapies for the treatment of hereditary or acquired organ dysfunctions are becoming an important aspect of the evaluation of these experimental trials. This technique allows to assess the differentiation stage of the grafted cells in situ and at the cellular level.
The aim of the present study was to characterize an antibody panel useful for the analysis of canine muscle cells in routinely processed formalin-fixed paraffinembedded tissues. Twelve antibodies raised against cytoplasmic or nuclear proteins expressed in muscle cells, validated for use in human tissues for diagnostic or research purposes, were tested for staining quality and specificity in canine normal tissues. Normal heart, skeletal muscle and various viscera containing a muscularis layer made up of smooth muscle cells (bladder, intestine, and uterus) were selected for evaluation. Eight antibodies (five mouse monoclonal, three goat polyclonal summarized in Table 3) demonstrated a staining of good quality in canine tissues, with a specificity identical to that achieved in human tissues. The specificity of the cross-reactivity of these markers was definitely confirmed by western blot analysis. Valuable antibodies for dog tissues include a ‘‘panmuscle cell’’ marker (desmin) as well as markers with restricted specificity towards the three main subtypes – smooth, skeletal and cardiac – of muscle cells. SMA and HDAC8 appeared equally valuable for the specific delineation of smooth muscle cells whereas h-Caldesmon, a smooth muscle marker well validated for its use in human diagnostic pathology, produced no cross-reactivity in canine tissues. The three markers specific of striated muscle in humans, tested in this study (MHC, TnI, TnT) were equally specific for identification of striated skeletal and cardiac muscle cells in canine tissues. Thus, our findings confirm and expand the observations previously reported by Fishbein et al. (2003).
232
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Among the transcription factors tested for their use in dog tissues, none of the two markers specific in human for skeletal muscles (MyoD1 and myogenin) produced reactivity in homologous canine tissues. Conversely, two of the three cardiac-specific transcription factors produced a similar reactivity in canine and human hearts. In summary we have characterized an antibody panel valuable for immunohistochemical analysis of canine muscle cells in formalin-fixed paraffinembedded tissues. The panel comprise a pan-muscle cell marker (desmin), two markers of smooth muscle cells (SMA and HDAC8), three markers reacting with both skeletal and cardiac striated muscle cells (MHC, TnI, TnT) and two nuclear markers specific for cardiac transcription factors (GATA-4, Nkx-2.5). We believe that this panel of antibodies will be useful in experimental pathology for the evaluation of protocols using cell-based strategies for these treatment of muscular disorders in dogs. Moreover, the antibodies may represent a valuable tool for veterinary pathologists involved in the diagnostic interpretation of soft tissue neoplasms in dogs. Acknowledgements The authors wish to thank Ste´phanie Maquet and Pascale Heneaux for their skilled technical assistance.L. de Leval is a Senior Research Associate of the FNRS, S. Gofflot, P. Kischel and C. Thielen are Scientific Research Workers of the FNRS. References Bartunek, J., Croissant, J.D., Wijns, W., Gofflot, S., de Lavareille, A., Vanderheyden, M., Kaluzhny, Y., Mazouz, N., Willemsen, P., Penicka, M., Mathieu, M., Homsy, C., De Bruyne, B., McEntee, K., Lee, I.W., Heyndrickx, G.R., 2007. Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. Am. J. Physiol. Heart Circ. Physiol. 292, 1095–1104. Bruneau, B.G., 2002. Transcriptional regulation of vertebrate cardiac morphogenesis. Circ. Res. 90, 509–519. Cooper, B.J., Winand, N.J., Stedman, H., Valentine, B.A., Hoffman, E.P., Kunkel, L.M., Scott, M.O., Fischbeck, K.H., Kornegay, J.N., Avery, R.J., Williams, J.R., Schmickel, R.D., Sylvester, J.E., 1988. The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature 334, 154–156. Cooper, B.J., 1989. Animal models of Duchenne and Becker muscular dystrophy. Br. Med. Bull. 45, 703–718. Costa, M.L., Escaleira, R., Cataldo, A., Oliveira, F., Mermelstein, C.S., 2004. Desmin: molecular interactions and putative functions of the muscle intermediate filament protein. Braz. J. Med. Biol. Res. 37, 1819–1830.
Dias, P., Dilling, M., Houghton, P., 1994. The molecular basis of skeletal muscle differentiation. Semin. Diagn. Pathol. 11, 3–14. Farah, C.S., Reinach, F.C., 1995. The troponin complex and regulation of muscle contraction. FASEB J. 9, 755–767. Fishbein, M.C., Wang, T., Matijasevic, M., Hong, L., Apple, F.S., 2003. Myocardial tissue troponins T and I. An immunohistochemical study in experimental models of myocardial ischemia. Cardiovasc. Pathol. 12, 65–71. Grounds, M.D., White, J.D., Rosenthal, N., Bogoyevitch, M.A., 2002. The role of stem cells in skeletal and cardiac muscle repair. J. Histochem. Cytochem. 50, 589–610. He, K.L., Yi, G.H., Sherman, W., Zhou, H., Zhang, G.P., Gu, A., Kao, R., Haimes, H.B., Harvey, J., Roos, E., White, D., Taylor, D.A., Wang, J., Burkhoff, D., 2005. Autologous skeletal myoblast transplantation improved hemodynamics and left ventricular function in chronic heart failure dogs. J. Heart Lung Transpl. 24, 1940–1949. Lin, Q., Schwarz, J., Bucana, C., Olson, E.N., 1997. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF-2C. Science 276, 1404–1407. Linke, A., Muller, P., Nurzynska, D., Casarsa, C., Torella, D., Nascimbene, A., Castaldo, C., Cascapera, S., Bohm, M., Quaini, F., Urbanek, K., Leri, A., Hintze, T.H., Kajstura, J., Anversa, P., 2005. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proc. Natl. Acad. Sci. U.S.A. 102, 8966–8971. Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I., Harvey, R.P., 1993. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431. Miller, J.B., Teal, S.B., Stockdale, F.E., 1989. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression. J. Biol. Chem. 264, 13122–13130. Molkentin, J.D., 2000. The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissuespecific gene expression. J. Biol. Chem. 275, 38949–38952. Pikkarainen, S., Tokola, H., Kerkela, R., Ruskoaho, H., 2004. GATA transcription factors in the developing and adult heart. Cardiovasc. Res. 63, 196–207. Shiojima, I., Komuro, I., Mizuno, T., Aikawa, R., Akazawa, H., Oka, T., Yamazaki, T., Yazaki, Y., 1996. Molecular cloning and characterization of human cardiac homeobox gene CSX1. Circ. Res. 79, 920–929. Silva, G.V., Litovsky, S., Assad, J.A., Sousa, A.L., Martin, B.J., Vela, D., Coulter, S.C., Lin, J., Ober, J., Vaughn, W.K., Branco, R.V., Oliveira, E.M., He, R., Geng, Y.J., Willerson, J.T., Perin, E.C., 2005. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111, 150–156. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., Gabbiani, G., 1986. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell. Biol. 103, 2787–2796. Sobue, K., Sellers, J.R., 1991. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J. Biol. Chem. 266, 12115–12118. Valentine, B.A., Cooper, B.J., de Lahunta, A., O’Quinn, R., Blue, J.T., 1988. Canine X-linked muscular dystrophy. An animal model of Duchenne muscular dystrophy: clinical studies. J. Neurol. Sci. 88, 69–81. Vulliet, P.R., Greeley, M., Halloran, S.M., MacDonald, K.A., Kittleson, M.D., 2004. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet 363, 783–784.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233 Waltregny, D., de Leval, L., Glenisson, W., Ly Tran, S., North, B.J., Bellahcene, A., Weidle, U., Verdin, E., Castronovo, V., 2004. Expression of histone deacetylase 8, a class I histone deacetylase, is restricted to cells showing smooth muscle differentiation in normal human tissues. Am. J. Pathol. 165, 553–564.
233
Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., Lassar, A., 1991. The myoD gene family: nodal point during specification of the muscle cell lineage. Science 251, 761–766.
Veterinary Immunology and Immunopathology 125 (2008) 225–233 www.elsevier.com/locate/vetimm
Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells Ste´phanie Gofflot, Philippe Kischel, Caroline Thielen, Vincent Radermacher, Jacques Boniver, Laurence de Leval * Department of Pathology, University of Lie`ge, Pathology Building B23+1, CHU Sart Tilman, B-4000 Lie`ge, Belgium Received 30 October 2007; received in revised form 8 May 2008; accepted 27 May 2008
Abstract Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology. # 2008 Elsevier B.V. All rights reserved. Keywords: Muscle markers; Immunohistochemistry; Canine muscle
1. Introduction Over the past years, cell-based therapies have revolutionized the field of regenerative medicine. There is indeed increasing interest for generating functional contractile muscle and cardiac cells by transplantation of undifferentiated stem cells or myoblasts (Grounds et al., 2002). Cellular therapies are presently under investigation for the treatment of numerous muscular disorders, such as ischemic heart diseases, cardiomyopathies and hereditary muscular dystrophies. Relevant
* Corresponding author at: Department of Pathology, University of Lie`ge, Pathology Building B23+1, CHU Sart Tilman, B-4000 Lie`ge, Belgium. Tel.: +32 43662410; fax: +32 43662919. E-mail address: [email protected] (L. de Leval). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.05.029
animal models are mandatory in this type of research and dogs have been widely used as experimental models for Duchenne’s muscular dystrophy (Cooper et al., 1988; Valentine et al., 1988; Cooper, 1989) and in preclinical studies investigating the use of stem cell transplantation after induced heart damage (Vulliet et al., 2004; He et al., 2005; Linke et al., 2005; Silva et al., 2005). In addition to functional studies, evaluation of these experimental strategies requires specific methods of cell characterization, in order to evaluate the degree of colonization of the diseased tissue by the grafted cells and to assess lineage-specific differentiation of these cells. Immunohistochemistry is an invaluable tool for targeting and identifying differentiation markers. Yet, to our knowledge, the specificity of antibodies for muscular markers has not been extensively characterized in canine tissues. In a recent report, anti-human antibodies directed
226
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Table 1 Specific markers expressed by the different muscle types
There are two main types of muscle cells, smooth and striated, the latter including voluntary skeletal muscle and involuntary cardiac muscle.
against Troponin I and Troponin T were shown to react with dog cardiomyocytes (Fishbein et al., 2003). The current study was undertaken to extend the panel of antibodies useful for immunohistochemical characterization of muscular differentiation in canine tissues. A broad panel of antibodies raised against human, rat or chicken epitopes of muscle-specific proteins, validated for diagnostic or experimental use on human tissues, were evaluated for their reactivity on canine tissues. The markers tested in this study are summarized in Table 1. Desmin is an intermediate filament protein that is a structural cytoplasmic component of all muscle cells, i.e. cardiomyocytes, skeletal and smooth muscle cells (Costa et al., 2004). Alpha Smooth Muscle Actin (SMA) is an actin isoform that contributes to cell-generated mechanical tension. Its expression is normally restricted to smooth muscle cells, myoepithelial cells and myofibroblasts (Skalli et al., 1986). h-Caldesmon, a cytoplasmic protein that regulates cellular contraction through its interaction with actin and myosin is another specific marker of smooth muscle differentiation (Sobue and Sellers, 1991). HDAC8, a class I histone deacetylase was recently characterized as a marker of smooth muscle differentiation as it is detected in the cytoplasm of visceral and vascular smooth muscle cells, myoepithelial cells and myofibroblasts (Waltregny et al., 2004). Conversely, Myosin Heavy Chain (MHC), the most abundant protein in the striated muscle where it acts as the molecular motor of muscle contraction, is not detected in smooth muscle cells. Troponin (Tn), a protein complex of three subunits (TnC, TnI, and TnT), is found in both skeletal and cardiac muscles, and plays the role of conferring calcium sensitivity to muscle cells (Farah and Reinach, 1995).
Several nuclear proteins functioning as transcription factors are known to be specifically expressed in cardiomyocytes or in skeletal myocytes (Dias et al., 1994; Bruneau, 2002). GATA-4 transcription factor, which binds a DNA consensus sequence WGATAR in the promoter region of many cardiac and gut-specific genes, is expressed in various tissues of endodermal or mesodermal derivation such as heart, liver, lung, gonad, and gut where it plays critical roles in regulating tissuespecific gene expression (Molkentin, 2000). GATA-4 is present in adult cardiac tissue as well as in the developing heart and is essential for proper cardiac morphogenesis (Pikkarainen et al., 2004). MEF-2C, a member of the myocyte-specific enhancer factor 2 (MEF-2) family is an essential regulator of cardiac myogenesis and right ventricular development (Lin et al., 1997). The homologue of the tinman gene in Drosophila melanogaster called Nkx-2.5 in vertebrates, encodes for a homeodomain-containing transcription factor, which is expressed at high levels in the developing and adult heart and plays a critical role in the myocardial development (Lints et al., 1993; Shiojima et al., 1996). MyoD1 and myogenin are myogenic regulatory factors restricted to skeletal muscle cells; MyoD1 plays a critical role in initiating the onset of muscle-specific gene expression whereas myogenin is involved in the later steps of skeletal muscle differentiation (Weintraub et al., 1991). 2. Materials and methods 2.1. Tissues Normal tissues were obtained from 10 to 15 monthsold Beagle dogs (15–45 kg) of either sex, purchased from Harlan (The Netherlands). They were used in an experimental protocol of cardiac injury and repair by injection of stem cells (Bartunek et al., 2007). The study was approved by the Ethical Committee for the animal research of the Louvain Medical School, Brussels and the Faculty of Medicine of the Free University of Brussels, Belgium. At the end of the treatment, the dogs were euthanized by intravenous barbiturate overdosage administration (200 mg/kg). Control animals with no cardiac injury or with no stem cell injection, were selected for harvesting of normal tissue samples from heart (ventricular wall) or skeletal muscle (thigh) and organs containing a muscularis layer (uterus, intestines and bladder). Tissues were either fixed in 10% buffered formalin and embedded in paraffin blocks for immunohistochemistry, or snapfrozen for western blot analysis.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
227
Table 2 Summary of antibodies and their conditions of use Antibody
Origin-immunogen
Cytoplasmic markers h-Caldesmon Monoclonal mouse anti-human
Designation
Source
Pre-treatment for IHC
Dilution IHC a
h-CD
DAKO
Water-bath at 37 8C 20 min in 0,25% trypsin/PBS + water-bath at 95 8C 40 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Water-bath at 95 8C for 40 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8) Pressure cooker 5 min in EDTA buffer (pH 8)
1:100
Microwave 2 5 min in 10 mM citrate buffer (pH 6) Pressure cooker 5 min in EDTA buffer (pH 8) Microwave 2 5 min in 10 mM citrate buffer (pH 6) Microwave 2 5 min in 10 mM citrate buffer (pH 6) Water-bath at 95 8C 40 min in EDTA buffer (pH 8)
1:50
Desmin
Monoclonal mouse anti-human
D33
DAKO
HDAC8
Polyclonal goat anti-human
N-20
Santa Cruz
MHC
Monoclonal mouse anti-chicken
F59
Santa Cruz
SMA
Monoclonal mouse anti-human
1A4
DAKO
Troponin I
Polyclonal goat anti-human
C19
Santa Cruz
Troponin T
Monoclonal mouse anti-chicken
T1/61
Novocastra
Nuclear markers GATA-4
Monoclonal mouse anti-human
G4
Santa Cruz
MEF-2C
Polyclonal goat anti-human
C-17
Santa Cruz
MyoD1
Monoclonal mouse anti-human
5.8A
DAKO
Myogenin
Monoclonal mouse anti-rat
F5D
DAKO
Nkx-2.5
Polyclonal goat anti-human
AF2444
R&D
a
Dilution WB
1:200
1:500
1:40
1:600
1:200
1:2500
1:200
1:5000
1:50
1:1000
1:10
1:250
1:25 1:100
1:100
1:25
The same dilution was applied to human and canine tissues.
2.2. Antibodies The antibodies tested in this study were: hcaldesmon, desmin, HDAC8, MHC, a-SMA, Troponin I, Troponin T, GATA-4, MEF-2C, MyoD1, myogenin, Nkx-2.5. The clone designations, sources of antibodies and their conditions of use established based on their expected reactivity on normal adult human tissues are summarized in Table 2. 2.3. Immunohistochemistry Briefly, four micrometers paraffin sections were dewaxed in xylol and rehydrated in methanol. Endogenous peroxidase activity was inhibited with 3%
hydrogen peroxide in methanol for 5 min. After washing in distilled water and antigen retrieval, the sections were blocked with ChemMateTM Antibody Diluent (S2022, Dako, Glostrup, Denmark) and incubated at room temperature for 1 h with the working dilution of primary antibody, excepted for MyoD1 antibody which required overnight incubation at 4 8C. For mouse primary antibodies, the slides were subsequently incubated for 30 min with the biotinylated link (LSAB2 system-HRP, Dako), washed and incubated for 30 min with streptavidin–peroxidase (HRP) (Dako). ENVISION developing system (Dako) was required for MyoD1 antibody. Visualization of goat primary antibodies was performed using HRP-coupled rabbit anti-goat immunoglobulin secondary antibody. Washes were performed with PBS
228
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
after each incubation step. Antibody binding was revealed by using H2O2 as a substrate and 3,30 diaminobenzidine as chromogen (Dako). Sections were counterstained with hematoxylin and mounted with a Tissue-Tek SCA film coverslipper (Sakura Finetek, Zoeterwoude, The Netherlands). Negative controls by omission of the primary antibody and positive controls consisting of adequate human tissues were included in each staining batch. 2.4. Western blotting Cardiac, skeletal and smooth muscles from human and canine species were pulverized frozen using a MikroDismembrator U (Braun Biotech) and tissue powders were lysed in a PBS buffer containing 1% NP40, 0.5% DOC, 1% SDS and a protease inhibitor cocktail (Halt, Pierce-Thermo Scientific). The lysates were subjected to SDS-PAGE in 4–12% gels (NuPAGE, Invitrogen) under reducing conditions. Separated proteins were electrotransferred to nitrocellulose membranes overnight at constant voltage (35 V), except for MHC proteins that were transferred at constant current (120 mA). Membranes were blocked in TBS (Tris Buffer Saline) containing 5% non-fat dried milk, 0.05% Tween-20, and incubated with antibodies overnight at 4 8C. Bound antibodies were visualized using ECL chemiluminescent substrate (GE Healthcare) and exposure to X-ray films (Fuji).
3. Results 3.1. Immunohistochemistry: cytoplasmic markers Anti-desmin antibody induced a strong and diffuse cytoplasmic positivity in all three kinds of canine muscle cells (not shown). Smooth muscle cells of the myometrium, bladder and intestinal walls were strongly desmin-positive, as were cardiomyocytes and skeletal muscle cells. Arteriolar smooth muscle cells in all tissues also stained positively. The staining was specific, since reactivity was observed neither in the epithelial compartments, nor in nerves. Only scattered spindled cells stained positively in mucosal or interstitial connective tissue. Anti-SMA antibodies produced a strong cytoplasmic staining in most muscle cells in the myometrium, in the muscularis mucosae and muscularis propria layers of the bowel and bladder (Fig. 1A). A strong staining was also identified in arteriolar smooth muscle cells (Fig. 1A–C). Conversely, in the endometrium and mucosal layers of the intestine and bladder, only scattered spindled cells showed positivity for this marker (Fig. 1A). A similar pattern was observed in the corresponding human viscera. In the skeletal muscle and heart, the staining for SMA was restricted to smooth muscle cells of the vascular walls (internal positive controls), whereas the myocytes were clearly negative (Fig. 1B–C).
Fig. 1. IHC stains of canine tissues with smooth muscle specific markers. Expression of SMA (A–C) and HDAC8 (D–F) in canine muscle tissues (uterus: A and D; skeletal muscle: B and E; myocardium: C and F). Positive staining for both markers is observed in the cytoplasm of smooth muscle cells in the myometrium and the positivity is restricted to the vascular endothelial cells of skeletal muscle and in myocardium (magnification 100).
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
229
Fig. 2. IHC stains of canine tissues with striated muscle markers. The antibodies MHC (A–C), Troponin I (D–F) and Troponin T (G–I) positively stain cytoplasm of cardiac myocytes (C, F, and I-100) and skeletal muscle cells (B, E, and H-100) whereas smooth muscle cells in the intestines (A-100) and bladder (D-100, G-200) are negative.
The pattern of staining observed with anti-HDAC8 antibody was similar to that described above for SMA (Fig. 1D–F). In canine uterus, HDAC8 showed positivity exclusively in the myometrium and vessel walls (Fig. 1D). In the intestine tissue, HDAC8 produced cytoplasmic staining in the muscularis layer and in vessel walls, in contrast to the mucosa that remained completely negative. The muscularis layer of bladder was also positively stained with HDAC8 antibody. h-Caldesmon did not produce any reactivity on canine tissue while a positivity was observed in human tissues. MHC was uniformly negative in non-muscle cells of striated muscles and in smooth muscle cells of viscera and arterioles (Fig. 2A). Conversely, the cytoplasm of skeletal muscle cells and of cardiomyocytes was strongly positive (Fig. 2B–C). No reactivity was observed for TnI within muscular or mucosal layers of intestine, bladder and uterus (Fig. 2D). Anti-TnI antibody produced a cytoplasmic staining in striated muscle cells, including skeletal and
cardiac myocytes (Fig. 2E–F). Virtually all striated muscle cells were positively stained, with a staining pattern distinctively very heterogeneous within each cell, that tended to be more intense towards one pole of the cytoplasm. Vascular walls in all tissues were negative for this marker. No specific immunoreactivity with anti-TnT antibody was observed in the canine tissues containing smooth muscle (Fig. 2G). TnT displayed moderate to strong cytoplasmic staining in the majority of skeletal muscle cells. A small subset of skeletal striated cells remained negative, likely corresponding to slow muscle cells, since the antibody is reported to be specific for fast ones (Fig. 2H). Conversely, virtually all-cardiac myocytes were strongly and homogeneously positive for TnT (Fig. 2I). 3.2. Immunohistochemistry: nuclear markers Among the cardiac-specific markers tested in the present study, Nkx-2.5 was strongly positive in canine
230
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Fig. 3. IHC stains of canine tissues with cardiac-specific transcription factors. Nkx-2.5 stains nuclei of dog myocardium cells (A-200) and is negative in intestinal cells (B-200) and skeletal muscle cells (C-200). GATA-4 is positive in cardiomyocytes (D-200) and crypts of the intestines (E-100) but is negative in skeletal muscle cells (F-200).
myocytes (Fig. 3A). No staining was seen for this marker in smooth or skeletal muscle cells (Fig. 3B–C) in all other tissues studied. Anti-GATA-4 antibody produced a nuclear staining in canine cardiomyocytes (Fig. 3D). No staining was seen in the vessels and in the muscularis of bladder and uterus. However, in the intestine, GATA-4 displayed a nuclear staining of smooth muscle cells in the muscularis propria and in epithelial cells lining crypts, similar to the staining observed in the corresponding human viscera (Fig. 3E). No staining was seen for this marker in skeletal muscle cells (Fig. 3F). The antibodies against MEF-2C, MyoD1 and myogenin did not produce any cross-reactivity on canine tissues in the conditions used for the staining of human tissues.
was detected in all human and canine samples, with human cardiac muscle showing the lowest reactivity and human smooth muscle showing the strongest reactivity. Myosin heavy chains (cardiac cMHC and fast fMHC)
3.3. Validation of antibody cross-reactivity Even though the immunohistochemistry staining observed with the different antibodies was similar in human and canine tissues, we sought to confirm the specificity of these antibodies. By sequence analysis (http://www.expasy.org), we were able to compare the sequence of five protein markers, and found a good consensus (over 80%) between both species for desmin, MHC, Nkx-2.5 and Troponin I whereas less then 60% identity was found for Troponin T. Then, we carried out a western blot analysis on human and canine muscle protein homogenates (Fig. 4). Desmin
Fig. 4. Protein lysates from human and canine cardiac, skeletal and smooth muscles were subjected to immunoblot analysis of desmin, MHC, SMA, HDAC8, TnT and TnI expression, as described in Section 2.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
231
Table 3 Summary of immunohistochemistry staining on canine muscle tissues and specificity on human muscle cells Canine muscle tissues
Human specificity
Antibody
Smooth muscle
Striated cardiac muscle
Striated skeletal muscle
Cytoplasmic markers Desmin
+
+
+
+ +
+ + +
+ + +
Smooth, skeletal and cardiac muscles Smooth muscle Smooth muscle Smooth muscle Striated muscle Striated muscle Striated muscle
a
+ +
Cardiac muscle Cardiac muscle Skeletal muscle Skeletal muscle Cardiac muscle
h-Caldesmon HDAC8 SMA MHC Troponin I Troponin T Nuclear markers GATA-4 MEF-2C MyoD1 Myogenin Nkx-2.5 a
Nuclear staining of smooth muscle cells in the muscularis propria and epithelial cells lining crypts.
were detected in cardiac and skeletal muscles from both human and canine origin, as expected from our IHC data and the previously described reactivity of the F59 antibody (Miller et al., 1989). SMA and HDAC8 were specifically detected in smooth muscles of both human and canine species. Cardiac muscle troponin T (cTnT) was recognized in cardiac muscles from both species, and several fast muscle troponin T isoforms (fTnT) were recognized in skeletal muscles from human and canine origin. Fast TnI was expressed in skeletal muscles from both human and canine species. Moreover, in canine cardiac muscles, two TnI isoforms were recognized: cardiac TnI (cTnI) appeared clearly as the main isoform expressed, while a lower expression of slow TnI (sTnI) was also detected. In human cardiac muscle, only a faint band of cTnI could be discerned. Unfortunately, antibodies against nuclear markers GATA-4 and Nkx2.5 did not produce any specific results in western blotting. 4. Discussion Muscular disorders represent a large field for potential application of cell-based therapies and various protocols are currently tested in different animal models, in particular dogs. Immunohistochemical analyses of animal models of cell-based therapies for the treatment of hereditary or acquired organ dysfunctions are becoming an important aspect of the evaluation of these experimental trials. This technique allows to assess the differentiation stage of the grafted cells in situ and at the cellular level.
The aim of the present study was to characterize an antibody panel useful for the analysis of canine muscle cells in routinely processed formalin-fixed paraffinembedded tissues. Twelve antibodies raised against cytoplasmic or nuclear proteins expressed in muscle cells, validated for use in human tissues for diagnostic or research purposes, were tested for staining quality and specificity in canine normal tissues. Normal heart, skeletal muscle and various viscera containing a muscularis layer made up of smooth muscle cells (bladder, intestine, and uterus) were selected for evaluation. Eight antibodies (five mouse monoclonal, three goat polyclonal summarized in Table 3) demonstrated a staining of good quality in canine tissues, with a specificity identical to that achieved in human tissues. The specificity of the cross-reactivity of these markers was definitely confirmed by western blot analysis. Valuable antibodies for dog tissues include a ‘‘panmuscle cell’’ marker (desmin) as well as markers with restricted specificity towards the three main subtypes – smooth, skeletal and cardiac – of muscle cells. SMA and HDAC8 appeared equally valuable for the specific delineation of smooth muscle cells whereas h-Caldesmon, a smooth muscle marker well validated for its use in human diagnostic pathology, produced no cross-reactivity in canine tissues. The three markers specific of striated muscle in humans, tested in this study (MHC, TnI, TnT) were equally specific for identification of striated skeletal and cardiac muscle cells in canine tissues. Thus, our findings confirm and expand the observations previously reported by Fishbein et al. (2003).
232
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233
Among the transcription factors tested for their use in dog tissues, none of the two markers specific in human for skeletal muscles (MyoD1 and myogenin) produced reactivity in homologous canine tissues. Conversely, two of the three cardiac-specific transcription factors produced a similar reactivity in canine and human hearts. In summary we have characterized an antibody panel valuable for immunohistochemical analysis of canine muscle cells in formalin-fixed paraffinembedded tissues. The panel comprise a pan-muscle cell marker (desmin), two markers of smooth muscle cells (SMA and HDAC8), three markers reacting with both skeletal and cardiac striated muscle cells (MHC, TnI, TnT) and two nuclear markers specific for cardiac transcription factors (GATA-4, Nkx-2.5). We believe that this panel of antibodies will be useful in experimental pathology for the evaluation of protocols using cell-based strategies for these treatment of muscular disorders in dogs. Moreover, the antibodies may represent a valuable tool for veterinary pathologists involved in the diagnostic interpretation of soft tissue neoplasms in dogs. Acknowledgements The authors wish to thank Ste´phanie Maquet and Pascale Heneaux for their skilled technical assistance.L. de Leval is a Senior Research Associate of the FNRS, S. Gofflot, P. Kischel and C. Thielen are Scientific Research Workers of the FNRS. References Bartunek, J., Croissant, J.D., Wijns, W., Gofflot, S., de Lavareille, A., Vanderheyden, M., Kaluzhny, Y., Mazouz, N., Willemsen, P., Penicka, M., Mathieu, M., Homsy, C., De Bruyne, B., McEntee, K., Lee, I.W., Heyndrickx, G.R., 2007. Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. Am. J. Physiol. Heart Circ. Physiol. 292, 1095–1104. Bruneau, B.G., 2002. Transcriptional regulation of vertebrate cardiac morphogenesis. Circ. Res. 90, 509–519. Cooper, B.J., Winand, N.J., Stedman, H., Valentine, B.A., Hoffman, E.P., Kunkel, L.M., Scott, M.O., Fischbeck, K.H., Kornegay, J.N., Avery, R.J., Williams, J.R., Schmickel, R.D., Sylvester, J.E., 1988. The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature 334, 154–156. Cooper, B.J., 1989. Animal models of Duchenne and Becker muscular dystrophy. Br. Med. Bull. 45, 703–718. Costa, M.L., Escaleira, R., Cataldo, A., Oliveira, F., Mermelstein, C.S., 2004. Desmin: molecular interactions and putative functions of the muscle intermediate filament protein. Braz. J. Med. Biol. Res. 37, 1819–1830.
Dias, P., Dilling, M., Houghton, P., 1994. The molecular basis of skeletal muscle differentiation. Semin. Diagn. Pathol. 11, 3–14. Farah, C.S., Reinach, F.C., 1995. The troponin complex and regulation of muscle contraction. FASEB J. 9, 755–767. Fishbein, M.C., Wang, T., Matijasevic, M., Hong, L., Apple, F.S., 2003. Myocardial tissue troponins T and I. An immunohistochemical study in experimental models of myocardial ischemia. Cardiovasc. Pathol. 12, 65–71. Grounds, M.D., White, J.D., Rosenthal, N., Bogoyevitch, M.A., 2002. The role of stem cells in skeletal and cardiac muscle repair. J. Histochem. Cytochem. 50, 589–610. He, K.L., Yi, G.H., Sherman, W., Zhou, H., Zhang, G.P., Gu, A., Kao, R., Haimes, H.B., Harvey, J., Roos, E., White, D., Taylor, D.A., Wang, J., Burkhoff, D., 2005. Autologous skeletal myoblast transplantation improved hemodynamics and left ventricular function in chronic heart failure dogs. J. Heart Lung Transpl. 24, 1940–1949. Lin, Q., Schwarz, J., Bucana, C., Olson, E.N., 1997. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF-2C. Science 276, 1404–1407. Linke, A., Muller, P., Nurzynska, D., Casarsa, C., Torella, D., Nascimbene, A., Castaldo, C., Cascapera, S., Bohm, M., Quaini, F., Urbanek, K., Leri, A., Hintze, T.H., Kajstura, J., Anversa, P., 2005. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proc. Natl. Acad. Sci. U.S.A. 102, 8966–8971. Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I., Harvey, R.P., 1993. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431. Miller, J.B., Teal, S.B., Stockdale, F.E., 1989. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression. J. Biol. Chem. 264, 13122–13130. Molkentin, J.D., 2000. The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissuespecific gene expression. J. Biol. Chem. 275, 38949–38952. Pikkarainen, S., Tokola, H., Kerkela, R., Ruskoaho, H., 2004. GATA transcription factors in the developing and adult heart. Cardiovasc. Res. 63, 196–207. Shiojima, I., Komuro, I., Mizuno, T., Aikawa, R., Akazawa, H., Oka, T., Yamazaki, T., Yazaki, Y., 1996. Molecular cloning and characterization of human cardiac homeobox gene CSX1. Circ. Res. 79, 920–929. Silva, G.V., Litovsky, S., Assad, J.A., Sousa, A.L., Martin, B.J., Vela, D., Coulter, S.C., Lin, J., Ober, J., Vaughn, W.K., Branco, R.V., Oliveira, E.M., He, R., Geng, Y.J., Willerson, J.T., Perin, E.C., 2005. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 111, 150–156. Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., Gabbiani, G., 1986. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell. Biol. 103, 2787–2796. Sobue, K., Sellers, J.R., 1991. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J. Biol. Chem. 266, 12115–12118. Valentine, B.A., Cooper, B.J., de Lahunta, A., O’Quinn, R., Blue, J.T., 1988. Canine X-linked muscular dystrophy. An animal model of Duchenne muscular dystrophy: clinical studies. J. Neurol. Sci. 88, 69–81. Vulliet, P.R., Greeley, M., Halloran, S.M., MacDonald, K.A., Kittleson, M.D., 2004. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet 363, 783–784.
S. Gofflot et al. / Veterinary Immunology and Immunopathology 125 (2008) 225–233 Waltregny, D., de Leval, L., Glenisson, W., Ly Tran, S., North, B.J., Bellahcene, A., Weidle, U., Verdin, E., Castronovo, V., 2004. Expression of histone deacetylase 8, a class I histone deacetylase, is restricted to cells showing smooth muscle differentiation in normal human tissues. Am. J. Pathol. 165, 553–564.
233
Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., Lassar, A., 1991. The myoD gene family: nodal point during specification of the muscle cell lineage. Science 251, 761–766.