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Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate... Background: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. Methods: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). Results: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific pro- teins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immor- talized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. Conclusions: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency. Keywords: Caspase-3 activation, Congenital muscular dystrophy, Immortalization of myogenic cells, Laminin-α2-deficiency, Myotube, Telomerase Background muscles, laminin-α2 assembles with laminin-β1and -γ1 Congenital muscular dystrophy Type1A (MDC1A) is an to form laminin-211. Heterotrimeric laminins that include autosomal recessive disease caused by mutations in the laminin-α2 have been termed merosins, and MDC1A has LAMA2 gene that encodes the extracellular protein thus also been known as merosin-deficient congenital mus- laminin-α2 [1]. Mutations that result in complete loss of cular dystrophy. Laminin-α2 has multiple binding partners laminin-α2 function result in severe neuromuscular in both the extracellular matrix and on the plasma mem- dysfunction, whereas mutations that result in partial loss of brane [3] so that loss of laminin-α2 is accompanied by both function are associated with less severe disease [2]. In skeletal structural deficits and aberrant cell signaling. Primary cultures of myogenic cells from human MDC1A patients have proven useful for analyzing molecular mecha- * Correspondence: millerjb@bu.edu nisms of MDC1A pathogenesis in skeletal muscle. For Departments of Neurology and Physiology & Biophysics, Neuromuscular Biology & Disease Group, Boston University School of Medicine, Boston, MA, example, myotubes formed in primary cultures of human USA MDC1A myoblasts in the absence of exogenous laminin Full list of author information is available at the end of the article © 2013 Yoon et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoon et al. Skeletal Muscle 2013, 3:28 Page 2 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 show both a several-fold increase in caspase-3 activity stable virus-producing cell lines after selection with and increased cell death compared to myotubes formed 0.5 mg/mL G418 or hygromycin (EMD Biosciences, from healthy control myoblasts [4]. The increased caspase- San Diego, CA, USA). Infections were done with 2 μg/mL 3 activity in MDC1A myotubes in vitro appears to re- polybrene (Sigma-Aldrich). Clonal colonies were grown capitulate the similarly increased caspase-3 activity seen from the immortalized population by limiting dilution in the skeletal muscles of laminin-α2-deficient mice and culture, and clonally-related cells were analyzed for CD56 human MDC1A patients in vivo [5-9]. Thus, aberrant expression by flow cytometry and for fusion potential in activation of caspase enzymatic activity is a cell autono- differentiation medium. Several independent clonal lines mous property of laminin-α2-deficient myotubes. The were isolated from each immortalized population and aberrant caspase activation and cell death in muscle expanded for further assays. Telomere length and tel- cells of MDC1A model systems is mediated by a BAX/ omerase activity were assayed as before [13,16]. KU70-dependent signaling pathway [4]. Importantly, in- hibition of aberrant cell death in the skeletal muscles of Human myogenic cells laminin-α2-deficient mice leads to a significant amelior- Table 1 summarizes the human myogenic cells used in ation of pathology, including a several-fold increase in this study. All human cells were obtained from German lifespan and improved motor behavior [4,10,11], thereby or USA biobanks (Table 1 and described below). All demonstrating that aberrantly increased cell death is cells were anonymized prior to receipt and no personal both a significant contributor to the overall pathology identifications were available to us. The cells had been and a potential therapeutic target in human MDC1A. produced prior to our study from muscle biopsies The use of primary cultures of human MDC1A myo- collected under protocols approved by the appropriate genic cells to analyze pathogenetic mechanisms has been institution that included informed donor consent and constrained both by the small number of donors and by approval to publish results in accordance with stan- the limited replication capacity (typically approximately dards of the Helsinki Declaration [17,18]. Because our 50 to 60 population doublings) of human myogenic cells studies were of human cells that were obtained from in primary culture. However, the replication limits of cell banks and for which personal identification data human myogenic cells can be overcome through forced were not obtainable by us, the studies were classified expression of CDK4 and hTERT [12-14]. Using this tech- as exempt from Human Studies review by the Boston nique, we now report the preparation and analysis of University Institutional Review Board in accordance immortalized, clonal lines of human MDC1A myogenic with USA Department of Health and Human Services policy cells. We found that the immortalized cells not only (http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46. retained the capacity to differentiate into myotubes but html#46.101, accessed November, 4, 2013). also showed the aberrant activation of caspase activity Primary MDC1A myoblasts from two different patients, as seen in primary cultures. This is the first report of designated as strains 38/03 and 96/04, were provided by immortalized human myogenic cells that recapitulate such the Muscle Tissue Culture Collection (MTCC) at the a marked pathological change. Thus, these immortalized University of Munich (http://www.baur-institut.de/ MDC1A myogenic cells can provide an essentially unlim- forschung/muskelbank/, accessed November 4, 2013) ited number of cells for study of MDC1A pathogenetic and were obtained from 4-month-old and 8-month-old mechanisms, as well as for the identification and in vitro male donors, respectively. Each donor had a clinical validation of therapeutic targets and strategies, including diagnosis of MDC1A with absence of laminin-α2[4].As by high-throughput screening. controls, we analyzed primary myoblasts of a healthy 36-year-old man (unpublished strain 2/08, provided by Methods the MTCC), as well as myoblasts derived from a biceps Immortalization and cell cloning biopsy of a healthy 60-year-old woman, termed 15Vbic Immortalization of myoblasts and isolation of myogenic [17,18]. As a disease control, we analyzed myoblasts clones was performed as previously described [12-14]. In derived from a biceps biopsy of a 67-year-old man with brief, mouse CDK4 and hTERT cDNAs were inserted facioscapulohumeral dystrophy (FSHD), termed 15Abic into pBabe vectors containing neomycin- and hygromycin- [16-18]. Primary 15Abic and 15Vbic cells were prepared by resistance genes, respectively. LoxP sites were included and obtained from the Sen. Paul D. Wellstone Cooperative in the hTERT vector to allow optional excision of the Research Center for FSHD (http://www.umassmed.edu/ hTERT expression cassettebyCre recombinase. To wellstone/materials.aspx, accessed November 4, 2013) and produce retroviral vectors, these plasmids were trans- immortalization of these 15Abic and 15Vbic cells was fected into the Phoenix ecotropic packaging cell and reported previously [16]. Due to the young age of the the virus-containing supernatant was used to infect the MDC1A donor, it was not possible to obtain control amphotropic packaging cell line PA317 [15] to obtain myoblasts from age-matched donors. After immortalization, Yoon et al. Skeletal Muscle 2013, 3:28 Page 3 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Table 1 Primary and CDK4 + hTERT immortalized myogenic cells used in this study Disease status Donor, age Source Type Name MDC1A Male, 4 months MTCC Munich Primary 38/03 This work Immortalized 38/03-ct4 MDC1A Male, 8 months MTCC Munich Primary 96/04 This work Immortalized 96/04-ct8 FSHD Male, 67 years Wellstone Primary 15Abic Ref 16 Immortalized 15Abic-ct24 Healthy control Female, 60 years Wellstone Primary 15Vbic Ref 16 Immortalized 15Vbic-ct16 Healthy control Male, 36 years MTCC Munich Primary 2/08 This work Immortalized 2/08-ct7 each clonal line was given a new identifier consisting of Antibodies and immunocytochemistry the original name followed by ‘-ct’ (for CDK4 + hTERT) Myosin heavy chain isoforms were detected with one of and a clone number. Thus, 38/03-ct4 was the fourth three antibodies: (1) mouse mAb F59 [20] used at 1:10 clonal line derived from CDK4/hTERT immortalized dilution of hybridoma supernatant; (2) mouse mAb F20 38/03 cells. Requests for immortalized 38/03-ct4, 96/04-ct8, [21] (used at 1:10; Developmental Studies Hybridoma Bank, and 2/08-ct7 myoblasts (Table 1) should be addressed Iowa City, IA, USA), or (3) rabbit anti-MYH3 (Sigma- to Dr. Peter Schneiderat (Peter.Schneiderat@med.uni- Aldrich, St. Louis, MO, USA). Desmin was detected with muenchen.de); and requests for immortalized 15Abic mouse mAb D1033 (Sigma-Aldrich) used at 1:100 for 1 h. and 15Vbic myoblasts should be addressed to the Activated caspase-3 antibody was from Cell Signaling Director of the Wellstone FSHD Center (charles. Technologies (Beverly, MA, USA; cat. #9661, used at emersonjr@umassmed.edu). 1:400); and KU70 antibody was from Novus Biologicals (Littleton, CO, USA; cat #NB100-1915, used at 1:300). Cell culture Dr. Lydia Sorokin (University of Münster) generously Cells were cultured on Lab-Tek Permanox chamber slides provided the rat anti-laminin-α2 mAb 4H8-2 which (Nalge Nunc, Rochester, NY, USA) coated with 40 μg/mL reacts with an epitope within the L4b globular domain poly-D-Lysine or 1% gelatin. In some cases as noted, slides [22]. Cultures were fixed with 4% formaldehyde or 100% were coated at 2 μg/cm with human placental laminin methanol. Primary antibody binding was visualized with (Sigma-Aldrich cat. #L6274). Proliferating myoblasts were appropriate species-specific secondary antibodies conju- cultured at subconfluence in a high serum growth medium gated to either Alexa Fluor 488 or Alexa Fluor 594 (Life and differentiation was induced as cells neared confluence Technologies, Grand Island, NY, USA). Slides were by switching the cultures to a low serum differentiation imaged using a Nikon E800 microscope (Melville, NY, medium as described [17,18]. Where noted, Laminin-111 USA) with SPOT Software (version 4.1) and SPOT Insight (Sigma-Aldrich cat. #L2020 or BD Bioscience cat. #354239) camera (Diagnostic Instruments,Sterling Heights,MI, USA). was added to the culture medium at 5 μM. Cells were cultured under 5% CO at ambient oxygen concentra- Results and discussion tion (normoxia), except, in some cases as noted, when Using forced expression of CDK4 and hTERT followed cells were cultured under a low oxygen atmosphere of by cell cloning, we first produced immortalized myo- 2% O ,5%CO ,93% N (hypoxia) in gas-tight chambers genic cell lines from primary human myoblasts obtained 2 2 2 as described [19]. from MDC1A (38/03, 96/04), normal control (2/08, 15Vbic), and FSHD (15Abic) donors (Table 1 and Caspase enzyme assays Figure 1). The FSHD cells served as a disease control to Caspase enzymatic activity was measured in cell homoge- determine if pathological changes were disease-specific or nates using either the CaspACE Colorimetric Caspase-3 shared. Though primary myoblast cultures reached a repli- Activity Assay (50 to 100 μg protein per assay; Promega, cative limit at approximately 50 to 60 cumulative popula- Madison, WI, USA) or the more sensitive luminescence- tion doublings, the immortalized cells proliferated based Caspase-Glo 3/7 Assay System (5 μg protein per indefinitely (not shown, compare to [13]). Cells that were assay; Promega) according to the manufacturer’sinstruc- CDK4 plus hTERT immortalized had higher telomerase tions and with signal detection on a Safire2 or Infinite enzymatic activity and maintained longer telomeres at M1000 microplate reader (Tecan, Durham, NC, USA). higher population doublings than either primary cells or Yoon et al. Skeletal Muscle 2013, 3:28 Page 4 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Figure 1 Characterization of immortalized compared to primary myogenic cells. (A) CDK4 + hTERT immortalized cells had higher telomerase enzymatic activity (left) and maintained longer telomeres (right) than primary cells or cells with CDK4 only. Results from 38/03 MDC1A cells (telomerase activity by telomeric repeat amplification protocol assay) and healthy control 2/08 cells (telomere length by hybridization assay) are shown as examples. The cervical carcinoma cell line HeLa served as a positive control. Lanes re-arranged for presentation. PD = population doublings. (B) 100% of CDK4 + hTERT immortalized cells expressed desmin (green), with MDC1A 96/04-ct8 cells shown as an example. (C) CDK4 + hTERT immortalized MDC1A myogenic cells showed normal differentiation by fusing into multinucleate cells that expressed myosin heavy chain (MyHC, red) isoforms. (D) Similar percentages of nuclei were incorporated into multinucleate (≥2 nuclei) myotubes formed from immortalized healthy control, MDC1A, and FSHD myoblasts. Error bars = SD, n =5. cells with CDK4 alone (Figure 1A). Culture under low that the laminin-α2-deficient phenotype was maintained oxygen conditions (2% O ,5%CO ,93% N ) did not in the immortalized MDC1A cultures. 2 2 2 significantly alter proliferation or differentiation of the We next examined whether immortalized MDC1A immortalized normal, MDC1A, or FSHD lines compared myogenic cells also showed the pathological changes that to culture under normoxic conditions (not shown). we had previously found in primary MDC1A cultures [4]. The clonal, immortalized myogenic cells were 100% We first compared KU70 immunostaining patterns in positive for expression of the muscle-specific intermedi- cultures of immortalized control and MDC1A myogenic ate filament protein desmin (Figure 1B), whereas primary cells. We found that KU70 immunostaining was restricted cultures were 70% to 95% desmin-positive (not shown), as to the nuclei of immortalized MDC1A myogenic cells, was consistent with a small proportion of non-myogenic whereas both the cytoplasm and nuclei of immortalized cells in the non-clonal primary cultures. Immortalized normal control cells showed KU70 staining (Figure 2A, B). MDC1A, FSHD, and normal control myogenic cells all Because primary cultures of MDC1A myogenic cells formed multinucleate myotubes when switched to low also show decreased KU70 expression in the cytoplasm serum differentiation medium, and, as in primary cell [4], it is clear that immortalization did not affect this cultures, the percentage of nuclei that were within mul- aberrant property of MDC1A myogenic cells. KU70 is a tinucleate cells was similar for disease and control cul- multifunctional protein with roles in the nucleus, cyto- tures (Figure 1C, D, and not shown). We also confirmed plasm, and perhaps at the cell surface [23]. In the cyto- that myotubes formed from immortalized MDC1A plasm, KU70 normally binds to BAX, thereby inhibiting myoblasts failed to express laminin-α2, whereas myo- BAX activation and subsequent cell death [4,24-26]. tubes formed from immortalized control myoblasts did Loss of KU70 from the cytoplasm would promote BAX express laminin-α2 (Figure 2A, B), thus demonstrating activation and cell death, which is consistent with the Yoon et al. Skeletal Muscle 2013, 3:28 Page 5 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Figure 2 Immortalized MDC1A myogenic cells did not express laminin-α2 and showed an altered distribution of KU70. (A) Laminin-α2 (red) appeared in a punctate pattern in multinucleate myotubes (arrows) formed from immortalized healthy control myoblasts. (B) As expected, no laminin-α2 was found in myotubes (arrows) formed from immortalized MDC1A myoblasts. (C) Immortalized myogenic cells from healthy control donors showed KU70 (green) both in the cytoplasm (arrows) and in nuclei. (D) KU70 (green) was restricted to nuclei of immortalized MDC1A myogenic cells. increased cell death phenotype in laminin-α2-deficient at any one time suggests that onset of cell death was mouse muscles and MDC1A human muscles [5-9]. asynchronous in the differentiating MDC1A cultures. Our next step was to examine caspase-3 activation in Caspase-3-positive cells typically have a short half-life cultures of immortalized MDC1A vs. immortalized normal (<5 h); eventually detach from the culture dish [4]; and and FSHD myogenic cells. Caspase-3 activation is associ- could possibly be replaced by remaining undifferentiated ated with activation of the BAX-mediated pathway of cell myoblasts in the cultures. The mechanism(s) that underlie death in MDC1A cell cultures [4]. Using immunohisto- the progression of cells from a state in which there are chemistry with an antibody specific for the cleaved, enzy- limited signs of pathology (for example, KU70 reduced matically activeformof caspase-3,wefound positive in the cytoplasm) to a state with high level activation of immunostaining in approximately 1% to 3% of the dif- caspase-3 followed by cell death remain to be deter- ferentiated, myosin heavy chain-positive (MyHC) cells mined in future work. in MDC1A cultures (Figure 3A to D). For example, in Finally, to confirm the immunocytochemistry results, one survey of an MDC1A 38/03-ct4 culture after we measured caspase-3 enzymatic activity in differenti- 4 days of differentiation on gelatin, we found caspase-3 ated cultures of MDC1A vs. normal and FSHD myogenic immunostaining in 15 out of 1,084 (1.4%) of the MyHC- cells. After 4 days of differentiation, cultures of MDC1A positive cells in the culture. The caspase-3 immunostain- cells had significantly more caspase-3 enzymatic activity ing in MDC1A cells appeared to often fill the cytoplasm than did cultures of normal control or FSHD cells and was sometimes also in nuclei as expected for on- (Figure 3F). This approximate four- to six-fold increase going cell death (Figure 3A to D). In some cases, the in caspase-3 activity in immortalized MDC1A lines was caspase-3-positive cells appeared to be undergoing de- similar to the increase we saw previously in primary generation as evidenced by fragmented and/or aggregated MDC1A vs. primary normal cultures [4]. We found simi- MyHC staining (Figure 3C, D). Furthermore, nuclei in lar results with two different immortalized MDC1A lines caspase-3-positive cells were often irregularly shaped, (38/03-ct4 and 96/04-ct8) and with two different caspase-3 condensed, or fragmented (Figure 3E), which are signs enzymatic activity assays. Culture under low oxygen did of incipient cell death. We did not see such caspase-3- not alter the extent of caspase-3 activation (not shown). positive cells with aberrant nuclei in differentiated cul- The increased caspase-3 activation in the MDC1A cultures tures of immortalized normal or FSHD myogenic cells was at least partially laminin-dependent, as growth on human placental laminin (which includes laminin-211) (not shown). The finding that only a small fraction of the differenti- or in the presence of mouse laminin-111 was sufficient ated, myosin-expressing cells were positive for caspase-3 to prevent approximately 30% to 50% of the aberrant Yoon et al. Skeletal Muscle 2013, 3:28 Page 6 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 immortalized MDC1A myogenic cells should provide an essentially unlimited source of laminin-α2-deficient cells for future studies. In particular, these cells will be valuable for studies of myogenic cell-autonomous mecha- nisms in MDC1A pathology, including, for example, aber- rant induction of cell death and increased autophagy [4,27]. Combining results from studies of the human MDC1A myogenic cells with results from studies of laminin-α2-deficient mice should be particularly useful for further analyses of disease mechanisms. Pathological changes that arise due to interactions of human MDC1A myogenic cells with motor nerve, vascular, inflammatory, or connective tissue cells could possibly be studied in co- cultures. Xenotransplant models might also be useful if the immortalized MDC1A myogenic cells can form a significant number of innervated myofibers after trans- plant into immunodeficient mice [14]. Finally, the im- mortalized MDC1A cells and the pathological changes in these cells that we have identified should be useful for developing cell-based screening assays, including high-throughput screening, as part of pre-clinical studies to identify therapeutic interventions that reverse MDC1A pathology. Conclusions Immortalized myogenic cells from laminin-α2-deficient MDC1A patients recapitulate aspects of MDC1A path- ology including aberrant induction of caspase-3 and KU70 Figure 3 Caspase-3 activity was aberrantly activated in myotubes relocalization. The immortalized MDC1A cells provide formed from immortalized MDC1A myoblasts. (A, B, C, D) a new resource for studies of pathogenetic mechanisms Activated caspase-3 (green) was found in approximately 1% to 3% of and for screening possible therapeutic approaches in the MyHC-expressing (red) differentiated cells in cultures of immortalized laminin-α2-deficiency. MDC1A cells. Two examples of 38/03-ct4 cultures are shown after 4 days in differentiation medium. (E) Arrows indicate nuclei with aberrant Competing interests morphology that were in caspase-3-positive cells (caspase staining not The authors declare that they have no competing interests. shown). Nearby nuclei in caspase-3-negative cells showed normal morphology. Four fields are shown. (F) Quantification of caspase-3 Authors’ contributions enzymatic activity by two different assays showed that, after 4 days GS and WEW developed the immortalization method, immortalized and in differentiation medium, cultures of immortalized MDC1A cells cloned the normal, MDC1A, and FSHD lines, and assayed telomerase activity (96/04-ct8 and 38/03-ct4) had significantly elevated levels of and telomere length (Figure 3A). SY, MLB, EVS, and JAW carried out the caspase-3 compared to cultures of immortalized healthy control remaining experimental studies (Figures 1B-D, 2, and 3, and data in text) which were planned with JBM. PS provided primary MDC1A and control cells (2/08-ct and 15Vbic-ct16) or immortalized FSHD (15Abic-ct24) myogenic cells and biobanking services. JBM wrote the manuscript. All authors cells. **P <001 (t-test, ‘n’ as shown). participated in editing the manuscript. All authors read and approved the final manuscript. increase in caspase-3 (not shown), a result similar to that Acknowledgements we found previously in primary MDC1A cultures [4]. We thank biopsy donors for their generosity; Dr. Sachiko Homma (Boston University School of Medicine) for much helpful advice; Ms. Katherine Bankert In summary, we have immortalized laminin-α2-deficient for technical assistance with initial experiments; and Dr. Lydia Sorokin MDC1A myogenic cells and shown that the immortalized (University of Münster) for the laminin-α2 mAb. We also thank Dr. Kathryn cells not only retain the capacity for differentiation, but Wagner and Dr. Genila Bibat (Kennedy-Krieger Institute and Johns Hopkins School of Medicine) who obtained 15Abic and 15Vbic biopsies as described also recapitulate cell autonomous pathological changes [17]; and Dr. Jennifer CJ Chen and Ms. Kendal Hanger (University of that have been reported in primary MDC1A myogenic Massachusetts Medical School) who prepared the primary myogenic cell cultures cell cultures, in laminin-α2-deficient mouse muscles, from these biopsies as described [17]. and in human MDC1A muscles [4-9]. Among these Funding changes were a reduction in the amount of KU70 in the Supported by grants from the NIH (R01AR060328 to JBM; R01AR062587 to cytoplasm and aberrant activation of caspase-3 with Peter L. Jones, subcontract to JBM); the Muscular Dystrophy Association associated abnormalities of nuclear morphology. The (#216422 to JBM); and the Boston University Undergraduate Research Yoon et al. Skeletal Muscle 2013, 3:28 Page 7 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Opportunity Program to EVS. Work with FSHD cells was performed within pathological human myoblasts: towards a universal tool for the study of the framework of the Senator Paul D. Wellstone Muscular Dystrophy neuromuscular disorders. Skelet Muscle 2011, 1:34. Cooperative Research Center for FSHD, supported by NIH grant 15. Miller AD, Buttimore C: Redesign of retrovirus packaging cell lines to 5U54HD060848. The Muscle Tissue Culture Collection (MTCC) at the avoid recombination leading to helper virus production. Mol Cell Biol University of Munich is part of the German network on muscular dystrophies 1986, 6:2895–2902. (MD-NET, service structure S1, 01GM0601) funded by the German Ministry of 16. Stadler G, Rahimov F, King OD, Chen JC, Robin JD, Wagner KR, Shay JW, Education and Research (BMBF, Bonn, Germany). MTCC is a partner of Emerson CP, Wright WE: Telomere position effect regulates DUX4 in Eurobiobank (www.eurobiobank.org) and TREAT-NMD (www.treat-nmd.eu). human facioscapulohumeral muscular dystrophy. Nat Struct Mol Biol 2013, Funders had no role in design or implementation of the study. 20:671–678. 17. 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Zhu CH, Mouly V, Cooper RN, Mamchaoui K, Bigot A, Shay JW, Di Santo JP, • Convenient online submission Butler-Browne GS, Wright WE: Cellular senescence in human myoblasts is • Thorough peer review overcome by human telomerase reverse transcriptase and cyclin- dependent kinase 4: consequences in aging muscle and therapeutic • No space constraints or color figure charges strategies for muscular dystrophies. Aging Cell 2007, 6:515–523. • Immediate publication on acceptance 13. Stadler G, Chen JC, Wagner K, Robin JD, Shay JW, Emerson CP, Wright WE: Establishment of clonal myogenic cell lines from severely affected dystrophic • Inclusion in PubMed, CAS, Scopus and Google Scholar muscles - CDK4 maintains the myogenic population. Skelet Muscle 2011, 1:12. • Research which is freely available for redistribution 14. Mamchaoui K, Trollet C, Bigot A, Negroni E, Chaouch S, Wolff A, Kandalla PK, Marie S, Di Santo J, St Guily JL, Muntoni F, Kim J, Philippi S, Spuler S, Levy N, Submit your manuscript at Blumen SC,Voit T,Wright WE,Aamiri A, Butler-Browne G, Mouly V: Immortalized www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

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Copyright © 2013 by Yoon et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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Abstract

Background: Congenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency. Methods: To overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). Results: The immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific pro- teins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immor- talized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice. Conclusions: Immortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency. Keywords: Caspase-3 activation, Congenital muscular dystrophy, Immortalization of myogenic cells, Laminin-α2-deficiency, Myotube, Telomerase Background muscles, laminin-α2 assembles with laminin-β1and -γ1 Congenital muscular dystrophy Type1A (MDC1A) is an to form laminin-211. Heterotrimeric laminins that include autosomal recessive disease caused by mutations in the laminin-α2 have been termed merosins, and MDC1A has LAMA2 gene that encodes the extracellular protein thus also been known as merosin-deficient congenital mus- laminin-α2 [1]. Mutations that result in complete loss of cular dystrophy. Laminin-α2 has multiple binding partners laminin-α2 function result in severe neuromuscular in both the extracellular matrix and on the plasma mem- dysfunction, whereas mutations that result in partial loss of brane [3] so that loss of laminin-α2 is accompanied by both function are associated with less severe disease [2]. In skeletal structural deficits and aberrant cell signaling. Primary cultures of myogenic cells from human MDC1A patients have proven useful for analyzing molecular mecha- * Correspondence: millerjb@bu.edu nisms of MDC1A pathogenesis in skeletal muscle. For Departments of Neurology and Physiology & Biophysics, Neuromuscular Biology & Disease Group, Boston University School of Medicine, Boston, MA, example, myotubes formed in primary cultures of human USA MDC1A myoblasts in the absence of exogenous laminin Full list of author information is available at the end of the article © 2013 Yoon et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoon et al. Skeletal Muscle 2013, 3:28 Page 2 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 show both a several-fold increase in caspase-3 activity stable virus-producing cell lines after selection with and increased cell death compared to myotubes formed 0.5 mg/mL G418 or hygromycin (EMD Biosciences, from healthy control myoblasts [4]. The increased caspase- San Diego, CA, USA). Infections were done with 2 μg/mL 3 activity in MDC1A myotubes in vitro appears to re- polybrene (Sigma-Aldrich). Clonal colonies were grown capitulate the similarly increased caspase-3 activity seen from the immortalized population by limiting dilution in the skeletal muscles of laminin-α2-deficient mice and culture, and clonally-related cells were analyzed for CD56 human MDC1A patients in vivo [5-9]. Thus, aberrant expression by flow cytometry and for fusion potential in activation of caspase enzymatic activity is a cell autono- differentiation medium. Several independent clonal lines mous property of laminin-α2-deficient myotubes. The were isolated from each immortalized population and aberrant caspase activation and cell death in muscle expanded for further assays. Telomere length and tel- cells of MDC1A model systems is mediated by a BAX/ omerase activity were assayed as before [13,16]. KU70-dependent signaling pathway [4]. Importantly, in- hibition of aberrant cell death in the skeletal muscles of Human myogenic cells laminin-α2-deficient mice leads to a significant amelior- Table 1 summarizes the human myogenic cells used in ation of pathology, including a several-fold increase in this study. All human cells were obtained from German lifespan and improved motor behavior [4,10,11], thereby or USA biobanks (Table 1 and described below). All demonstrating that aberrantly increased cell death is cells were anonymized prior to receipt and no personal both a significant contributor to the overall pathology identifications were available to us. The cells had been and a potential therapeutic target in human MDC1A. produced prior to our study from muscle biopsies The use of primary cultures of human MDC1A myo- collected under protocols approved by the appropriate genic cells to analyze pathogenetic mechanisms has been institution that included informed donor consent and constrained both by the small number of donors and by approval to publish results in accordance with stan- the limited replication capacity (typically approximately dards of the Helsinki Declaration [17,18]. Because our 50 to 60 population doublings) of human myogenic cells studies were of human cells that were obtained from in primary culture. However, the replication limits of cell banks and for which personal identification data human myogenic cells can be overcome through forced were not obtainable by us, the studies were classified expression of CDK4 and hTERT [12-14]. Using this tech- as exempt from Human Studies review by the Boston nique, we now report the preparation and analysis of University Institutional Review Board in accordance immortalized, clonal lines of human MDC1A myogenic with USA Department of Health and Human Services policy cells. We found that the immortalized cells not only (http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46. retained the capacity to differentiate into myotubes but html#46.101, accessed November, 4, 2013). also showed the aberrant activation of caspase activity Primary MDC1A myoblasts from two different patients, as seen in primary cultures. This is the first report of designated as strains 38/03 and 96/04, were provided by immortalized human myogenic cells that recapitulate such the Muscle Tissue Culture Collection (MTCC) at the a marked pathological change. Thus, these immortalized University of Munich (http://www.baur-institut.de/ MDC1A myogenic cells can provide an essentially unlim- forschung/muskelbank/, accessed November 4, 2013) ited number of cells for study of MDC1A pathogenetic and were obtained from 4-month-old and 8-month-old mechanisms, as well as for the identification and in vitro male donors, respectively. Each donor had a clinical validation of therapeutic targets and strategies, including diagnosis of MDC1A with absence of laminin-α2[4].As by high-throughput screening. controls, we analyzed primary myoblasts of a healthy 36-year-old man (unpublished strain 2/08, provided by Methods the MTCC), as well as myoblasts derived from a biceps Immortalization and cell cloning biopsy of a healthy 60-year-old woman, termed 15Vbic Immortalization of myoblasts and isolation of myogenic [17,18]. As a disease control, we analyzed myoblasts clones was performed as previously described [12-14]. In derived from a biceps biopsy of a 67-year-old man with brief, mouse CDK4 and hTERT cDNAs were inserted facioscapulohumeral dystrophy (FSHD), termed 15Abic into pBabe vectors containing neomycin- and hygromycin- [16-18]. Primary 15Abic and 15Vbic cells were prepared by resistance genes, respectively. LoxP sites were included and obtained from the Sen. Paul D. Wellstone Cooperative in the hTERT vector to allow optional excision of the Research Center for FSHD (http://www.umassmed.edu/ hTERT expression cassettebyCre recombinase. To wellstone/materials.aspx, accessed November 4, 2013) and produce retroviral vectors, these plasmids were trans- immortalization of these 15Abic and 15Vbic cells was fected into the Phoenix ecotropic packaging cell and reported previously [16]. Due to the young age of the the virus-containing supernatant was used to infect the MDC1A donor, it was not possible to obtain control amphotropic packaging cell line PA317 [15] to obtain myoblasts from age-matched donors. After immortalization, Yoon et al. Skeletal Muscle 2013, 3:28 Page 3 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Table 1 Primary and CDK4 + hTERT immortalized myogenic cells used in this study Disease status Donor, age Source Type Name MDC1A Male, 4 months MTCC Munich Primary 38/03 This work Immortalized 38/03-ct4 MDC1A Male, 8 months MTCC Munich Primary 96/04 This work Immortalized 96/04-ct8 FSHD Male, 67 years Wellstone Primary 15Abic Ref 16 Immortalized 15Abic-ct24 Healthy control Female, 60 years Wellstone Primary 15Vbic Ref 16 Immortalized 15Vbic-ct16 Healthy control Male, 36 years MTCC Munich Primary 2/08 This work Immortalized 2/08-ct7 each clonal line was given a new identifier consisting of Antibodies and immunocytochemistry the original name followed by ‘-ct’ (for CDK4 + hTERT) Myosin heavy chain isoforms were detected with one of and a clone number. Thus, 38/03-ct4 was the fourth three antibodies: (1) mouse mAb F59 [20] used at 1:10 clonal line derived from CDK4/hTERT immortalized dilution of hybridoma supernatant; (2) mouse mAb F20 38/03 cells. Requests for immortalized 38/03-ct4, 96/04-ct8, [21] (used at 1:10; Developmental Studies Hybridoma Bank, and 2/08-ct7 myoblasts (Table 1) should be addressed Iowa City, IA, USA), or (3) rabbit anti-MYH3 (Sigma- to Dr. Peter Schneiderat (Peter.Schneiderat@med.uni- Aldrich, St. Louis, MO, USA). Desmin was detected with muenchen.de); and requests for immortalized 15Abic mouse mAb D1033 (Sigma-Aldrich) used at 1:100 for 1 h. and 15Vbic myoblasts should be addressed to the Activated caspase-3 antibody was from Cell Signaling Director of the Wellstone FSHD Center (charles. Technologies (Beverly, MA, USA; cat. #9661, used at emersonjr@umassmed.edu). 1:400); and KU70 antibody was from Novus Biologicals (Littleton, CO, USA; cat #NB100-1915, used at 1:300). Cell culture Dr. Lydia Sorokin (University of Münster) generously Cells were cultured on Lab-Tek Permanox chamber slides provided the rat anti-laminin-α2 mAb 4H8-2 which (Nalge Nunc, Rochester, NY, USA) coated with 40 μg/mL reacts with an epitope within the L4b globular domain poly-D-Lysine or 1% gelatin. In some cases as noted, slides [22]. Cultures were fixed with 4% formaldehyde or 100% were coated at 2 μg/cm with human placental laminin methanol. Primary antibody binding was visualized with (Sigma-Aldrich cat. #L6274). Proliferating myoblasts were appropriate species-specific secondary antibodies conju- cultured at subconfluence in a high serum growth medium gated to either Alexa Fluor 488 or Alexa Fluor 594 (Life and differentiation was induced as cells neared confluence Technologies, Grand Island, NY, USA). Slides were by switching the cultures to a low serum differentiation imaged using a Nikon E800 microscope (Melville, NY, medium as described [17,18]. Where noted, Laminin-111 USA) with SPOT Software (version 4.1) and SPOT Insight (Sigma-Aldrich cat. #L2020 or BD Bioscience cat. #354239) camera (Diagnostic Instruments,Sterling Heights,MI, USA). was added to the culture medium at 5 μM. Cells were cultured under 5% CO at ambient oxygen concentra- Results and discussion tion (normoxia), except, in some cases as noted, when Using forced expression of CDK4 and hTERT followed cells were cultured under a low oxygen atmosphere of by cell cloning, we first produced immortalized myo- 2% O ,5%CO ,93% N (hypoxia) in gas-tight chambers genic cell lines from primary human myoblasts obtained 2 2 2 as described [19]. from MDC1A (38/03, 96/04), normal control (2/08, 15Vbic), and FSHD (15Abic) donors (Table 1 and Caspase enzyme assays Figure 1). The FSHD cells served as a disease control to Caspase enzymatic activity was measured in cell homoge- determine if pathological changes were disease-specific or nates using either the CaspACE Colorimetric Caspase-3 shared. Though primary myoblast cultures reached a repli- Activity Assay (50 to 100 μg protein per assay; Promega, cative limit at approximately 50 to 60 cumulative popula- Madison, WI, USA) or the more sensitive luminescence- tion doublings, the immortalized cells proliferated based Caspase-Glo 3/7 Assay System (5 μg protein per indefinitely (not shown, compare to [13]). Cells that were assay; Promega) according to the manufacturer’sinstruc- CDK4 plus hTERT immortalized had higher telomerase tions and with signal detection on a Safire2 or Infinite enzymatic activity and maintained longer telomeres at M1000 microplate reader (Tecan, Durham, NC, USA). higher population doublings than either primary cells or Yoon et al. Skeletal Muscle 2013, 3:28 Page 4 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Figure 1 Characterization of immortalized compared to primary myogenic cells. (A) CDK4 + hTERT immortalized cells had higher telomerase enzymatic activity (left) and maintained longer telomeres (right) than primary cells or cells with CDK4 only. Results from 38/03 MDC1A cells (telomerase activity by telomeric repeat amplification protocol assay) and healthy control 2/08 cells (telomere length by hybridization assay) are shown as examples. The cervical carcinoma cell line HeLa served as a positive control. Lanes re-arranged for presentation. PD = population doublings. (B) 100% of CDK4 + hTERT immortalized cells expressed desmin (green), with MDC1A 96/04-ct8 cells shown as an example. (C) CDK4 + hTERT immortalized MDC1A myogenic cells showed normal differentiation by fusing into multinucleate cells that expressed myosin heavy chain (MyHC, red) isoforms. (D) Similar percentages of nuclei were incorporated into multinucleate (≥2 nuclei) myotubes formed from immortalized healthy control, MDC1A, and FSHD myoblasts. Error bars = SD, n =5. cells with CDK4 alone (Figure 1A). Culture under low that the laminin-α2-deficient phenotype was maintained oxygen conditions (2% O ,5%CO ,93% N ) did not in the immortalized MDC1A cultures. 2 2 2 significantly alter proliferation or differentiation of the We next examined whether immortalized MDC1A immortalized normal, MDC1A, or FSHD lines compared myogenic cells also showed the pathological changes that to culture under normoxic conditions (not shown). we had previously found in primary MDC1A cultures [4]. The clonal, immortalized myogenic cells were 100% We first compared KU70 immunostaining patterns in positive for expression of the muscle-specific intermedi- cultures of immortalized control and MDC1A myogenic ate filament protein desmin (Figure 1B), whereas primary cells. We found that KU70 immunostaining was restricted cultures were 70% to 95% desmin-positive (not shown), as to the nuclei of immortalized MDC1A myogenic cells, was consistent with a small proportion of non-myogenic whereas both the cytoplasm and nuclei of immortalized cells in the non-clonal primary cultures. Immortalized normal control cells showed KU70 staining (Figure 2A, B). MDC1A, FSHD, and normal control myogenic cells all Because primary cultures of MDC1A myogenic cells formed multinucleate myotubes when switched to low also show decreased KU70 expression in the cytoplasm serum differentiation medium, and, as in primary cell [4], it is clear that immortalization did not affect this cultures, the percentage of nuclei that were within mul- aberrant property of MDC1A myogenic cells. KU70 is a tinucleate cells was similar for disease and control cul- multifunctional protein with roles in the nucleus, cyto- tures (Figure 1C, D, and not shown). We also confirmed plasm, and perhaps at the cell surface [23]. In the cyto- that myotubes formed from immortalized MDC1A plasm, KU70 normally binds to BAX, thereby inhibiting myoblasts failed to express laminin-α2, whereas myo- BAX activation and subsequent cell death [4,24-26]. tubes formed from immortalized control myoblasts did Loss of KU70 from the cytoplasm would promote BAX express laminin-α2 (Figure 2A, B), thus demonstrating activation and cell death, which is consistent with the Yoon et al. Skeletal Muscle 2013, 3:28 Page 5 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Figure 2 Immortalized MDC1A myogenic cells did not express laminin-α2 and showed an altered distribution of KU70. (A) Laminin-α2 (red) appeared in a punctate pattern in multinucleate myotubes (arrows) formed from immortalized healthy control myoblasts. (B) As expected, no laminin-α2 was found in myotubes (arrows) formed from immortalized MDC1A myoblasts. (C) Immortalized myogenic cells from healthy control donors showed KU70 (green) both in the cytoplasm (arrows) and in nuclei. (D) KU70 (green) was restricted to nuclei of immortalized MDC1A myogenic cells. increased cell death phenotype in laminin-α2-deficient at any one time suggests that onset of cell death was mouse muscles and MDC1A human muscles [5-9]. asynchronous in the differentiating MDC1A cultures. Our next step was to examine caspase-3 activation in Caspase-3-positive cells typically have a short half-life cultures of immortalized MDC1A vs. immortalized normal (<5 h); eventually detach from the culture dish [4]; and and FSHD myogenic cells. Caspase-3 activation is associ- could possibly be replaced by remaining undifferentiated ated with activation of the BAX-mediated pathway of cell myoblasts in the cultures. The mechanism(s) that underlie death in MDC1A cell cultures [4]. Using immunohisto- the progression of cells from a state in which there are chemistry with an antibody specific for the cleaved, enzy- limited signs of pathology (for example, KU70 reduced matically activeformof caspase-3,wefound positive in the cytoplasm) to a state with high level activation of immunostaining in approximately 1% to 3% of the dif- caspase-3 followed by cell death remain to be deter- ferentiated, myosin heavy chain-positive (MyHC) cells mined in future work. in MDC1A cultures (Figure 3A to D). For example, in Finally, to confirm the immunocytochemistry results, one survey of an MDC1A 38/03-ct4 culture after we measured caspase-3 enzymatic activity in differenti- 4 days of differentiation on gelatin, we found caspase-3 ated cultures of MDC1A vs. normal and FSHD myogenic immunostaining in 15 out of 1,084 (1.4%) of the MyHC- cells. After 4 days of differentiation, cultures of MDC1A positive cells in the culture. The caspase-3 immunostain- cells had significantly more caspase-3 enzymatic activity ing in MDC1A cells appeared to often fill the cytoplasm than did cultures of normal control or FSHD cells and was sometimes also in nuclei as expected for on- (Figure 3F). This approximate four- to six-fold increase going cell death (Figure 3A to D). In some cases, the in caspase-3 activity in immortalized MDC1A lines was caspase-3-positive cells appeared to be undergoing de- similar to the increase we saw previously in primary generation as evidenced by fragmented and/or aggregated MDC1A vs. primary normal cultures [4]. We found simi- MyHC staining (Figure 3C, D). Furthermore, nuclei in lar results with two different immortalized MDC1A lines caspase-3-positive cells were often irregularly shaped, (38/03-ct4 and 96/04-ct8) and with two different caspase-3 condensed, or fragmented (Figure 3E), which are signs enzymatic activity assays. Culture under low oxygen did of incipient cell death. We did not see such caspase-3- not alter the extent of caspase-3 activation (not shown). positive cells with aberrant nuclei in differentiated cul- The increased caspase-3 activation in the MDC1A cultures tures of immortalized normal or FSHD myogenic cells was at least partially laminin-dependent, as growth on human placental laminin (which includes laminin-211) (not shown). The finding that only a small fraction of the differenti- or in the presence of mouse laminin-111 was sufficient ated, myosin-expressing cells were positive for caspase-3 to prevent approximately 30% to 50% of the aberrant Yoon et al. Skeletal Muscle 2013, 3:28 Page 6 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 immortalized MDC1A myogenic cells should provide an essentially unlimited source of laminin-α2-deficient cells for future studies. In particular, these cells will be valuable for studies of myogenic cell-autonomous mecha- nisms in MDC1A pathology, including, for example, aber- rant induction of cell death and increased autophagy [4,27]. Combining results from studies of the human MDC1A myogenic cells with results from studies of laminin-α2-deficient mice should be particularly useful for further analyses of disease mechanisms. Pathological changes that arise due to interactions of human MDC1A myogenic cells with motor nerve, vascular, inflammatory, or connective tissue cells could possibly be studied in co- cultures. Xenotransplant models might also be useful if the immortalized MDC1A myogenic cells can form a significant number of innervated myofibers after trans- plant into immunodeficient mice [14]. Finally, the im- mortalized MDC1A cells and the pathological changes in these cells that we have identified should be useful for developing cell-based screening assays, including high-throughput screening, as part of pre-clinical studies to identify therapeutic interventions that reverse MDC1A pathology. Conclusions Immortalized myogenic cells from laminin-α2-deficient MDC1A patients recapitulate aspects of MDC1A path- ology including aberrant induction of caspase-3 and KU70 Figure 3 Caspase-3 activity was aberrantly activated in myotubes relocalization. The immortalized MDC1A cells provide formed from immortalized MDC1A myoblasts. (A, B, C, D) a new resource for studies of pathogenetic mechanisms Activated caspase-3 (green) was found in approximately 1% to 3% of and for screening possible therapeutic approaches in the MyHC-expressing (red) differentiated cells in cultures of immortalized laminin-α2-deficiency. MDC1A cells. Two examples of 38/03-ct4 cultures are shown after 4 days in differentiation medium. (E) Arrows indicate nuclei with aberrant Competing interests morphology that were in caspase-3-positive cells (caspase staining not The authors declare that they have no competing interests. shown). Nearby nuclei in caspase-3-negative cells showed normal morphology. Four fields are shown. (F) Quantification of caspase-3 Authors’ contributions enzymatic activity by two different assays showed that, after 4 days GS and WEW developed the immortalization method, immortalized and in differentiation medium, cultures of immortalized MDC1A cells cloned the normal, MDC1A, and FSHD lines, and assayed telomerase activity (96/04-ct8 and 38/03-ct4) had significantly elevated levels of and telomere length (Figure 3A). SY, MLB, EVS, and JAW carried out the caspase-3 compared to cultures of immortalized healthy control remaining experimental studies (Figures 1B-D, 2, and 3, and data in text) which were planned with JBM. PS provided primary MDC1A and control cells (2/08-ct and 15Vbic-ct16) or immortalized FSHD (15Abic-ct24) myogenic cells and biobanking services. JBM wrote the manuscript. All authors cells. **P <001 (t-test, ‘n’ as shown). participated in editing the manuscript. All authors read and approved the final manuscript. increase in caspase-3 (not shown), a result similar to that Acknowledgements we found previously in primary MDC1A cultures [4]. We thank biopsy donors for their generosity; Dr. Sachiko Homma (Boston University School of Medicine) for much helpful advice; Ms. Katherine Bankert In summary, we have immortalized laminin-α2-deficient for technical assistance with initial experiments; and Dr. Lydia Sorokin MDC1A myogenic cells and shown that the immortalized (University of Münster) for the laminin-α2 mAb. We also thank Dr. Kathryn cells not only retain the capacity for differentiation, but Wagner and Dr. Genila Bibat (Kennedy-Krieger Institute and Johns Hopkins School of Medicine) who obtained 15Abic and 15Vbic biopsies as described also recapitulate cell autonomous pathological changes [17]; and Dr. Jennifer CJ Chen and Ms. Kendal Hanger (University of that have been reported in primary MDC1A myogenic Massachusetts Medical School) who prepared the primary myogenic cell cultures cell cultures, in laminin-α2-deficient mouse muscles, from these biopsies as described [17]. and in human MDC1A muscles [4-9]. Among these Funding changes were a reduction in the amount of KU70 in the Supported by grants from the NIH (R01AR060328 to JBM; R01AR062587 to cytoplasm and aberrant activation of caspase-3 with Peter L. Jones, subcontract to JBM); the Muscular Dystrophy Association associated abnormalities of nuclear morphology. The (#216422 to JBM); and the Boston University Undergraduate Research Yoon et al. Skeletal Muscle 2013, 3:28 Page 7 of 7 http://www.skeletalmusclejournal.com/content/3/1/28 Opportunity Program to EVS. Work with FSHD cells was performed within pathological human myoblasts: towards a universal tool for the study of the framework of the Senator Paul D. Wellstone Muscular Dystrophy neuromuscular disorders. Skelet Muscle 2011, 1:34. Cooperative Research Center for FSHD, supported by NIH grant 15. Miller AD, Buttimore C: Redesign of retrovirus packaging cell lines to 5U54HD060848. The Muscle Tissue Culture Collection (MTCC) at the avoid recombination leading to helper virus production. Mol Cell Biol University of Munich is part of the German network on muscular dystrophies 1986, 6:2895–2902. (MD-NET, service structure S1, 01GM0601) funded by the German Ministry of 16. Stadler G, Rahimov F, King OD, Chen JC, Robin JD, Wagner KR, Shay JW, Education and Research (BMBF, Bonn, Germany). MTCC is a partner of Emerson CP, Wright WE: Telomere position effect regulates DUX4 in Eurobiobank (www.eurobiobank.org) and TREAT-NMD (www.treat-nmd.eu). human facioscapulohumeral muscular dystrophy. Nat Struct Mol Biol 2013, Funders had no role in design or implementation of the study. 20:671–678. 17. Homma S, Chen JCJ, Rahimov F, Beermann ML, Hanger K, Bibat GM, Author details Wagner KR, Kunkel LM, Emerson CP, Miller JB: A unique library of Departments of Neurology and Physiology & Biophysics, Neuromuscular myogenic cells from facioscapulohumeral muscular dystrophy subjects Biology & Disease Group, Boston University School of Medicine, Boston, MA, and unaffected relatives: Family, disease, & cell function. Eur J Hum Genet USA. Department of Cell Biology, University of Texas Southwestern Medical 2011, 20:404–410. Center at Dallas, Dallas, TX, USA. Wellstone Program and Department of Cell 18. Jones TI, Chen JC, Rahimov F, Homma S, Arashiro P, Beermann ML, King OD, & Developmental Biology, University of Massachusetts School of Medicine, Miller JB, Kunkel LM, Emerson CP, Wagner KR, Jones PL: Facioscapulohumeral Worcester, MA USA. 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Zhu CH, Mouly V, Cooper RN, Mamchaoui K, Bigot A, Shay JW, Di Santo JP, • Convenient online submission Butler-Browne GS, Wright WE: Cellular senescence in human myoblasts is • Thorough peer review overcome by human telomerase reverse transcriptase and cyclin- dependent kinase 4: consequences in aging muscle and therapeutic • No space constraints or color figure charges strategies for muscular dystrophies. Aging Cell 2007, 6:515–523. • Immediate publication on acceptance 13. Stadler G, Chen JC, Wagner K, Robin JD, Shay JW, Emerson CP, Wright WE: Establishment of clonal myogenic cell lines from severely affected dystrophic • Inclusion in PubMed, CAS, Scopus and Google Scholar muscles - CDK4 maintains the myogenic population. Skelet Muscle 2011, 1:12. • Research which is freely available for redistribution 14. Mamchaoui K, Trollet C, Bigot A, Negroni E, Chaouch S, Wolff A, Kandalla PK, Marie S, Di Santo J, St Guily JL, Muntoni F, Kim J, Philippi S, Spuler S, Levy N, Submit your manuscript at Blumen SC,Voit T,Wright WE,Aamiri A, Butler-Browne G, Mouly V: Immortalized www.biomedcentral.com/submit

Journal

Skeletal MuscleSpringer Journals

Published: Dec 6, 2013

References