Thank you all for braving the cold weather today and coming today to my thesis talk on “enhancing myoblast fusion for the therapy of muscular dystrophies.”10 s
40s: 53 s. The Muscular dystrophies are a group of diseases characterized by progressive muscle wasting. Descriptions of muscular dystrophies first entered Western clinical literature in the 1830s, and as depicted here you can see an early documentation of the disease in a child whose legs are so weak that he uses his hands to move his legs. Although we have been studying these dystrophies for nearly 200 years, we still have not developed cures for them. My thesis concerns studies on enhancing fusion for the treatment of these disease. Before I present my work, I will give you an overview of skeletal muscle structure and the muscular dystrophies. I will then present you two projects within my thesis, and then give you conclusions.
Skeletal muscle is made up of bundles of long myofibers. Each myofiber is a single cell that formed from the fusion of hundreds of cells. A cross-section of muscle stained with H&E shows this tight packing of the myofibers. Each myofiber is surrounded by a specialized membrane called the sarcolemma. Outside of the sarcolemma, stained in light pink, is the basal lamina and extracellular matrix. In blue are the nuclei. Some of these are the myofiber nuclei that originated from the cells that fused together to make this myofiber. Other nuclei in this picture may represent the satellite stem cells, which are located next to the myofibers but separated from them by a thin layer of basal lamina. These cells are able to regenerate the muscle.
When the muscle is injured, such as when you exercise or suffer acute damage, the myofibers can break and necrose. The Pax7+ satellite cells are then activated to proliferate. The master transcription factors Myf5 and MyoD regulate and promote this proliferation. Then, the satellite cells differentiate and fuse together to form new myofibers. This process is directed by the action of the master transcription factors MyoD and myogenin. After fusion, the myonuclei are located in the center of the myofiber, and are slowly pushed out to the periphery over time. In the case of the muscular dystrophies, the myofibers are inherently weak and susceptible to frequent breakage and attempted cycles of regeneration.
One of the most common muscular dystrophies is Duchenne muscular dystrophy. This disease affects 1:3000 boys. The symptoms seen in this disease are shared among the different forms of the muscular dystrophies. Patients with this disease suffer from progressive muscle weakness due to instability in the sarcolemma. This allows proteins that are normally inside the myofiber to leak into the blood stream and high serum creatine kinase levels are one indication of muscular dystrophy. The breakage of the sarcolemma leads the myofiber to degenerate (point). Satellite cells in are activated to repair the muscle, and you can see an example of a regenerating fiber here, but their capacity to repair is soon exhausted. The necrotic myofibers are then replaced with inflammatory, fibrotic, and adipogenic cells. Eventually, patients experience mortality in their 20s or 30s due to cardiac or respiratory failure. In the 1980s, members of the lab of Louis Kunkel identified that DMD is due to genetic mutations in the gene, dystrophin.
Dystrophin is part of a protein complex that connects the F-actin in the muscle fiber to the extracellular matrix and stabilizes the myofiber during contraction. Without dystrophin, the complex is not completely formed, and contraction leads to breakdown of the sarcolemma, resulting in the diseases DMD and Becker’s MD. Not surprisingly, mutations in other members of this complex have been identified as the primary cause of many other forms of muscular dystrophy. These include mutations in sarcoglycans that result in different limb girdle muscular dystrophies, genes that affect glycosylation of alpha dystroglycan that result in the dystroglycanopathies with muscle and brain phenotypes, and mutations in the extracellular matrix proteins that lead to congenital muscular dystrophies.
1:06Muscle cell fusion can be enhanced for improving the fusion of both endogenous cells and transplanted cells. Enhancing the fusion of endogenous satellite cells can promote the regeneration and growth of the myofibers in cases of dystrophies, and could be used in the different type of diseases. Agents that are found to enhance fusion in this way could also be administered in conjunction with transplanted cells. In the case of transplanted cells, donor cells that contain a functional dystrophin can be transplanted and induced to fuse with the myofibers, thus replacing the missing protein. In theory, this therapy would be a cure, but in practice, the field has found that transplanted myoblast often die or engraft and do not all fuse fibers. Understanding the differentiation and fusion of myoblasts can aid in understanding how to promote cell engraftment and fusion of the various progenitor cell types.Injected cells did not fuse, only a small proportion fused, some die, of those that remain (myoblasts) nat med. 1997
40 sWe hypothesized that enhancing fusion can activate endogenous cells to repair damaged muscle or to improve the efficacy of myoblast transplantation. The goals of my thesis were two-part. The first was to test whether a drug, C-EPO, could enhance endogenous fusion and improve the condition of mdx mice, a mouse model for DMD. The second goal was to understand the process of myoblast differentiation and fusion to aid cell-based therapy. I did this my exploring the role of GPR56 in myoblast differentiation and fusion. I will first tell you about our study of C-EPO.
We saw that C-EPO did not improve the fusion of endogenous satellite cells. In the second part of my thesis, I explored the mechanism of fusion itself, looking specifically at the role of a G-protein coupled receptor, GPR56.
Additionally, mutations in GPR56 were found to cause the neurological disease, bilateral frontoparietal polymicrogyria. Patients with this disease have a cobblestone brain phenotype, and show delays in their motor development. Interestingly, some of the dystroglycanopathies are also characterized by having this same cobblestone brain phenotype. Given that mutations in GPR56 result in the same brain phenotype as loss of adys, it is possible that like adys, GPR56 has an important role in the muscle as well. Taken together with previous data showing expression in fetal muscle cells, as well as putative associations with proteins that could be involved in muscle cells, we went forward with studying GPR56’s role in muscle.
I first began by confirmed GPR56’s expression profile in differentiating mouse myoblasts. I isolated primary mouse myoblasts, and differentiated them. (Explain what cells seen at each stage). I isolating mRNA and protein from these myoblasts at different stages of differentiation. As you can see, GPR56 mRNA expression is tightly regulated, with a peak in expression at D1 of differentiation. This corresponds to the stage where the myoblasts have become myocytes, and also has some early myotubes. GPR56’s protein expression is similar, peak at D1 and decreasing expression while the myotubes are forming into larger tubes. Looking at the expression of the master regulators of differentiation, GPR56 is expressed after the onset of MyoD expression in proliferating cells, but during the time that MyoD expression is maintained during early differentiation and fusion. It is also concomitant with the upregulation of myogenin, which is expressed later during differentiation. As you can see from the phase picture of differentiating cells, myocytes, early myotubes, and possibly undifferentiated myoblasts are all in the culture at D1. To determine which cells specifically express GPR56 during this time, I performed immunohistochemitry on these cultures.
These are some examples showing GPR56 localization in differentiation mouse myoblast cultures. In green is GPR56, blue showing the nuclei. You can see that GPR56 is expressed in mononuclear cells in close juxtaposition to a myotube, looking like it is approaching fusion. A small degree of GPR56 is also detected on the myotube itself, which may be from a recently fused nuclei. To establish whether GPR56 was expressed in myoblasts as well as myocytes, I co-stained the cultures with caveolin-1, which is expressed in myoblasts prior to commitment to differentiation. I found that GPR56 was not expressed in caveolin-1 cells, suggesting that it is only expressed in after myoblasts have commitment to differentiation.
Phase pics. of the diff. cultures, KO cells fused less than WT. Quantified using a FI, counts fused/total. At both D2 and D5, the KO (red) less. The overall size of the myotubes can be quantified by counting the percentage of myotubes with >5 nuclei. Indicates late fusion vs early fusion. As you can see, dec. in the KOs at D2, whereas by D5 catch up. This suggests that the defect is prior to early fusion which corresponds with the expression data for GPR56. Because a defect in fusion = defect in any of the steps preceding and including fusion, we looked at the exp. myogenic txn factors in diff.KOvs WT cells. Maintenance of MyoD is dec. in the KO, whereas myog. seems to be unaffected. FHL1 is highly upregulated in the knockouts. These findings suggest that GPR56 is commitment to differentiation and switch of MyoD to the differentiation program. To confirm that these changes due to loss of GPR56, and not due to the KO myoblasts having dev. in a GPR56-deficient environment, we corroborated these findings in a second model in vitro model of myoblast differentiation.
These pictures show the degree of fusion in uninfected, scrambled, and GPR56 shRNA2 and 3 silenced C212 cells at D5.Stainingfor myosin heavy chain, a contractile protein which can be used to easily outline the myotubes, shows that fusion was definitely decreased in GPR56-silenced cells, especially with oligo 3. Quantification shows a significant decrease in fusion with shRNA3 and a trend towards a decreased in shRNA2. In addition, the myotube size is greatly decreased in both cultures. The next step is to look at the changes in differentiation by gene expression.Double check fusion
Unlike with the primary myoblasts, in the case of silencing we found that MyoD was not affected. However, myogenin expression was decreased in expression by both silencing constructs, as seen by protein and mRNA expression, especially with the third oligo. The decrease in MyoD expression in the myoblasts, coupled with the decrease in myogenin expression here, suggests that the commitment to differentiation and early differentiation may be less efficient in the KO cultures. If this is the case, that would be that GPR56 KO or silenced cells are less likely to exit the cell cycle and remain proliferative.
We found that interestingly, maintenance of Pax7 expression, a transcription factor present in satellite stem cells, was increased in the GPR56 silenced cells during differentiation. A similar phenotype was seen in the KO myoblasts, which exhibited increased proliferation in culture over the course of 10 days, as well as increased PCNA expression.
These studies together highlight two possible roles for GPR56. The first is promotion of the maintenance of myoD for commitment and differentiation. Second is promotion of differentiation through myogenin leading to fusion. As a result, loss of GPR56 results in less cells committing to differentiation and fusing. We then wanted to investigate whether the phenotype seen in vitro was replicated in vivo.
We first looked at the general structure of KO mouse leg muscles compared to WT muscle in 1 month old mice. As you can see, the KO muscle histologically looks like WT muscle. We had seen in vitro that there was a decrease in the overall myotube size. In vivo, this can translate to a difference in myofiber diameter, as less cells fusing in would result in less growth. We quantified the myofiber diameter in WT and KO muscle, but found that there were no difference in size. As the links between GPR56, BFPP, and the dystroglycanopathies suggested a possible presence of dystrophy, we also examined the serum CK levels in KO mice compared to WT mice. We found a very slight, but stat. sig. increase in the sCK levels. This degree of increase is very slight, which is in line with studies of BFPP patients that do not show a clinically significant increase in CK levels. Although we did not see any changes in the myofiber size, the in vitro data suggests that a decrease in myofiber size may be transient and that over time, the myofiber growth “catches up.” In order to investigate the effect of GPR56 loss on myofibers as they are growing, we used a regeneration model of in vivo myofiber growth, which in our case was induced by the administration of the snake venom, cardiotoxin.
Duringregeneration, GPR56 mRNA is expressed transiently in a pattern similar to its expression during differentiation in vitro. Looking at the histology of regeneration at days 4, 6, and 18 after injury, you can see that there is not much difference between the extent of regeneration in the wildtype and the knockout. To confirm whether there was a decrease in myofiber size during fusion, I quantified the myofiber size of the regenerating myofibers, and found that at early regeneration, there is no difference, at D6 there is a trend towards a decrease, whereas at D18 there is again no difference. This phenotype fits in with the data that we see in vitro, where the overall effect is slight and other factors in vivo may compensate for loss of GPR56. To see whether there are any molecular changes in expression due to the loss of GPR56 during regeneration (compensation?), we looked at the expression of different factors in injured WT and KO muscle.
We first looked at the expression of the master transcription factors, Myf5, MyoD, and myogenin. As you can see, MyoD is delayed in expression in the knockouts, whereas myogenin has only a slight delay. Myf5 expression is also delayed and highly upregulated. Myf5 has the capability to compensate for MyoD function, and this data suggests that Myf5 is compensating for a decrease in MyoD expression here. Looking at transcription factors that promote the further fusion of myoblasts, NFATc2 and FHL1, we see that there a delay in these factors is less apparent. At D6, there is an increase in FHL1 expression, similar to the increase in FHL1 expression that we had seen in vitro. Emb MHC? This together with the increase of Myf5 expression suggests the activation of compensatory mechanisms that result in the overall lack of a phenotype in vivo. We then sought to understand the signaling pathways that GPR56 is acting through, to better understand these phenotypes.
As GPR56 has been shown to signal through G-proteins, the first clue toward what GPR56 may be signaling to is can be found by looking at possible downstream signaling pathways of the G-proteins. Activation of GPR56, presumably through ligand binding, will activate the Galpha protein, which leads to disassociation of the Gbeta and Ggamma complex. These can signal together to release calcium activate NFAT to translocate into the nucleus and turn on gene transcription. GPR56 has been shown to activate Galpha 12/13, which can signal through Rho to activate the SRF transcription factor. There is prior evidence that GPR56 signaling may activate these transcription factors. To test whether GPR56 signaling could activate transcription from these promoters, we transfected in GPR56, the luciferase gene with NFAT-RE or SRE in the promoter, which SRF binds to, and a b-gal gene for normalization. We could then assay the amount of luciferase produced to determine whether transcription was activated. One drawback of this assay is that it relies on the presence of ligand to activate GPR56, or leaky expression from the receptor. To eliminate this caveat, I also expressed a truncated GPR56 that lacked the N-terminal inhibitory domain. Truncated forms of GPR56 have been shown to have constitutive activity, so this would eliminate that drawback.
I performed the luciferase assays, and a lot of credit goes to Jamie Doyle, Isabelle Draper, and Alan Kopin at Tufts medical who helped me optimize this assay and use their equipment and reagents. These are the results. On the y-axis is normalized luciferase activity, a measure of the amount of signaling induced by GPR56 for that particular promoter element. On the x-axis is increasing amounts of transfected full-length GPR56 in blue, truncated GPR56 in green, and control empty vector in black. As you can see, the truncated GPR56 strongly activated signaling from the SRE promoter, and more weakly activated signaling from NFAT. Increasing amounts of full-length GPR56 were able to reach maximal levels of SRE signaling, whereas it did not reach maximal levels for NFAT-RE. This suggests that there may be separate ligands that activate these separate pathways, thus increasing the regulation of signaling by GPR56. To confirm whether signaling from these pathways were affected in the GPR56 knockout mice, we identified some transcriptional targets of these pathways during differentiation, and looked at the expression of their targets
During differentiation, RhoA activation by G-proteins results in activation of SRF to translocate into the nucleus, where it turns on transcription of genes involved in proliferation, including the transcription of MyoD, FHl1, and myogenin. As I demonstrated earlier, activation of the NFAT transcription factor family is through induction of calcium release, and the NFAT genes themselves are targets of their own activation. So we looked to see if the expression of these target genes were disturbed in knockout mice compared to wildtype.Emphasize SRE/NFAT role in myogenesis, GPCR connectionRhoA signals to SRF, which induces FHL, MyoD expression.
We examined by quantitative RT-PCR the mRNA expression of these target genes in knockout and wildtype muscle. As you can see, the factors MyoD, FHL1, NFATc2, and NFATc3 are decreased in knockout muscle, whereas NFATc1 is not. This fits in with the hypothesis that GPR56 is signaling through the SRF and NFATc2 and NFATc3 during the early commitment to differentiation.Move legend to outside of the graphs.Trend for NFAT, not stat. sig.
As we saw, NFATc3 and NFATc2 expression was decreased. In addition to their role during myofiber differentiation, the NFATs also have promote myofiber growth and fiber type switching in mature muscle. If you recall the earlier data, there was not evidence of a defect in myofiber size in uninjured muscle. However, there is the possibility that fiber type switching could also be affected in the GPR56 knockout mice. The three main fiber types can be characterized by their myosin heavy chains: Type I, IIA, and IIB.
To quantify whether there were any differences in the different fiber types, we first isolated protein from wildtype and knockout muscle of different ages, and used antibodies against MHC I, IIA, and IIB to detect their total amount after being run on a Western blot. I quantified the bands, and plotted here is the protein expression of the Western bands after normalization to total protein as detected by SYPRO staining. As you can see, although the amounts of MHCs changed as the mice aged, no differences in the total amount of any of the MHCs was seen. This approach is little insensitive, so we confirmed using a more sensitive approach. In this approach, we sectioned the muscle, and stained with laminin to outline the myofibers, and the various myosin heavy chains isoforms. We imaged sequential sections of these staining, stitched them together, and counted the positive myofibers both manually and in an automated fashion. These are sample sequential sections from a knockout mouse. To determine whether there were any differences, I took the ratio of % positive myofibers in KO to the % in WT. If there was an increase in the proportion, then the result would be significantly greater than 1. If there was a decrease, then it would be less than 1. However, we found no differences.Say Quantification Western make sure say total content. Make Western disappear.
In conclusion, our studies on enhancing fusion for the muscular dystrophies have giving some insight into the different strategies for developing therapy. Our use of C-EPO in mdx mice illuminated the importance of several issues that must be considered when developing a therapy. The duration of our treatment was an important issue. Previous studies had used C-EPO only for up to 1 month, whereas we used it for 3 months. We found that bringing the study out to this longer time point was important for seeing the effects of C-EPO on its non-target tissues, such as the blood system. If we were able to deliver the drug directly to the muscle instead of systemically, we may have found that the long-term use of the drug would have been beneficial. In future studies, this drug may still hold promise in applications that maximize its use in the short term. One example of this would be to use it directly with transplanted cells to encourage their proliferation, differentiation, and fusion, which can all occur within one week. Different research groups are currently pursuing this avenue of research. . . . We accomplished the first steps necessary for finding novel factors that could improve engraftment. The next step.Validated the screen, suggesting that other genes identified in the screen may yield useful candidates for the improvement of cell engraftment, and the understanding of the process of mammaliam muscle cell fusion.
Dissertation Defense: Enhancing myoblast fusion for therapy of muscular dystrophies
Enhancing myoblast fusion fortherapy of muscular dystrophies Melissa Wu Thesis Defense January 28, 2013
Overview• Skeletal Muscle Structure• Muscular Dystrophies• Thesis 1. Enhancing Fusion in DMD 2. Investigating Process of Fusion Selections from the clinical works of Duchenne, ed. GV Poore, 1883.• Conclusions
Skeletal Muscle Structure Muscle myofiber Myofiber nuclei Satellite stem cell sarcolemma Skeletal muscle made Hematoxylin and Eosin up of bundles of staining of muscle cross- myofibers sectionSkeletal muscle drawing from MDA website
Satellite Cells Regenerate MuscleSatellite cell activation Differentiation and Fusion Pax7 Myf5 MyoD and myogenin MyoD
Skeletal Muscle Dystrophies: Duchenne Muscular Dystrophy• Affects 1:3300 boys• Progressive muscle weakness – Instability of sarcolemma • High serum creatine kinase – Muscle degeneration – Satellite cells repair, but are exhausted – Inflammation, fibrosis, adipogenesis• Average lifespan ~30 years http://neuromuscular.wustl.edu/pathol/dmdpath.htm
Treatment of Duchenne Muscular Dystrophy Therapeutics that address symptoms Membrane glucocorticoids Muscle growth Vascularization strength Dystrophin replacement therapies Gene therapy Read-through or exon-skipping therapies Cell transplantationDiagnosis and management of Duchenne muscular dystrophy, DMD Care Considerations Working Group, 2009
Enhancing fusion for therapy of DMD 1. Enhance fusion of endogenous satellite cells to improve muscle 2. Transplant cells to introduce dystrophin Enhance endogenous Transplant cells fusion• Can be used in different dystrophies • Works in theory, but often injected cells• Can also be used in conjunction with die upon injection or do not fuse intransplanted cells • Need to understand better natural process of fusion
HypothesisEnhancing fusion can activate endogenous cells to repair damaged muscle or improve the efficacy of myoblast transplantation 1. Test carbamylated erythropoietin (C-EPO) as a therapeutic in mdx mice 2. Explore role of GPR56 in myoblast differentiation and fusion
Carbamylated Erythropoeitin Protects Tissues Without Overstimulating Blood Cells EPO C-EPO X EPO-R β-CR Hematopoiesis Tissue Protection • Stimulates RBC production • Prevents apoptosis • Overstimulation leads to anemia • Limits fibrosis • Stimulates satellite cell proliferation • Stimulates muscle fiber growthModified from slide created by Journal of Clinical Oncology
Does administration of C-EPO improve dystrophic signs in mice with muscular dystrophy? Inject C-EPO 3x weekly intraperitoneally: 0, 50, 100 μg/kg mdx Week 4 12 Assay for: Muscle growth (weight) Changes in histology Sarcolemmal integrity
C-EPO treatment does not affect weight of mdx mice 35 30 Weight (g) 25 0 μg/kg ug/kg 20 50 μg/kg ug/kg 100 μg/kg ug/kg 15 -1 1 3 5 7 9 11 13 Time (Weeks)
C-EPO effects on mdx diaphragm 0 μg/kg 50 μg/kg 100 μg/kg4 weeks12 weeks 50 m
C-EPO Induces a short-term increase in regeneration 70 60 * 0 μg/kg% fibers with centrally located 50 μg/kg 50 * 100 μg/kg 40 nuclei 30 20 10 0 4 weeks 12 weeks
Conclusions• C-EPO increased regeneration in the short-term (4 weeks) – Did not increase average muscle fiber size• No effect on overall health of tissue – Did not reduce fibrosis – Did not improve muscle integrity• Why no improvement? – Anemia noted in C-EPO treated mice, especially at 12 weeks – May have interfered with tissue-protective effects• Overall, the effects of C-EPO treatment are modest
Studying Fusion ProcessEnhancing fusion can activate endogenous cells to repair damaged muscle or improve the efficacy of myoblast transplantation 1. Test C-EPO as a therapeutic in mdx mice 2. Explore role of GPR56 in myoblast differentiation and fusion
Screen to find effectors of fusionProliferating Early myotubes Late myotubes myoblasts (2-5 nuclei) (>15 nuclei) Cerletti et al JCS 2006
G-protein coupled receptor 56: Adhesion receptorNH3 GPS COO- CD9 CD81 G Putatively partners with or q G activates proteins that G have a role in myogenesis RhoA fusion SRE E2F NFAT
GPR56 causes “cobblestone brain” neuropathy Some dystroglycanopathies are characterized by a cobblestone brain phenotype BFPP β α γ δ β normal ?Bilateral frontoparietal polymicrogyria Does GPR56 have a role in the muscle? (BFPP)Piao et al, Science 2004.
GPR56 expression is upregulated during muscle cell fusion in vitroD0 D1 D2 D3 D6mRNA expression GPR56 Relative MyoD myogenin α/β tub D0 D1 D2 D3 D6 D0 D1 D2 D3 D6
GPR56 is expressed in committed myoblasts GPR56 GPR56 caveolin-1 Nuclei
GPR56 is expressed in early differentiation Proliferating Committed Early myotubes Mature myotubes myoblasts myocytes XTranscription Factor Expression Pax7 GPR56 Myf5 NFATc3 SRF MyoD myogenin NFATc1 NFATc2 FHL1
Knockout GPR56 myoblasts fuse less and have dysregulated effectors of differentiation Fusion Index Myotubes with >5 nuclei 100 30 WT KO p < 0.05Fusion Index (%) 80 >5 nuclei (%) 25 WT KO p < 0.05 * 60 20 * 15 p < 0.05 40 10 * 20 5 0 0 D2 D5 D2 D5
GPR56-silenced C2C12 cells fuse less and make smaller myotubes MHC DAPI uninfected scrambled shRNA2 shRNA3 Fusion index Myotubes >5 nuclei 50 30Fusion index (%) % tubes >5 nuclei 40 25 30 20 15 p < 0.01 20 p < 0.01 10 10 0 * 5 0 * p < 0.01 *
Unlike in primary KO myoblasts, MyoD is not affected and myogenin is decreased 1200 Relative mRNA expression 1000 myogenin uninfected 800 scrambled 600 shRNA2 400 shRNA3 200 0 0 2 4 6 Days in differentiation
Increased signs of proliferation seen in GPR56-silenced/KO myoblasts uninfected scrambled shRNA 2 shRNA 3 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 Pax7/ tub Cell proliferation in WT and KO myoblasts 10 WT Number of cells (x106) 8 KO * 6 *** 4 *** 2 *** 0 0 2 4 6 8 10 12 Days in Proliferation
In vitro studies show GPR56 promotes commitment and fusion• GPR56 KO and GPR56 shRNA highlight two roles for GPR56 – Promotion of MyoD maintenance for commitment to differentiation and early fusion – Promotion of differentiation leading to fusion• Loss of GPR56 results in less cells committing to differentiation and initiating early fusion
GPR56 knockout in vivo phenotype WT KO Average Myofiber Diameter Serum Creatine Kinase Serum Creatine Kinase 50 100 *Myofiber Diameter 40 (U/L) 80 30 60 (µm) 20 40 10 20 p = 0.01 0 0 WT KO 0 1 2 3 WT KO
Loss of GPR56 does not alter muscle regeneration WT KO GPR56 mRNA expression Relative mRNA expression 6 5 4 3 2 1 0 0 5 10 Days after Ctx injury Myofiber diameter 50 Myofiber Diameter (µm) WT 40 KO 30 20 10 0 4 6 18 Days after Ctx injury
Defining the GPR56 signaling pathway using luciferase assay • G /G signals to NFAT G G G • G 12/13 to Rho to SRF GEFs PLC-b • Transfect HEK293 with: – GPR56 or truncated RHO GPR56 Ca2 + Ca2 + – Luciferase reporter gene NFAT SRF under various promoters – β-gal gene for normalization SRF NFAT SRE NFAT-RE
GPR56 signals strongly to SRE, less strongly to NFAT-RE * p < 0.05, *** p < 0.001
SRF and NFAT target transcripts in muscle cells ? Gα RhoA SRF FHL MyoD SRF SRE NFAT NFAT Differentiation
MyoD, FHL1, NFATc2, and NFATc3 are decreased in knockout muscle 1.5 WT KORelative mRNA expression 1 * * * * 0.5 0 MyoD FHL1 NFATc1 NFATc2 NFATc3 NFATc4
NFAT targets include genes involved in myofiber specification MProliferation Commitment/ Fusion into Multi-nucleated differentiation early tubes I Myotube/fiber IIA IIB NFATc3 SRF NFATc1 NFATc2
Fiber type proportion is unchanged in KO mice compared to WT mice WT KO WT KO 4 mo 9 mo MHC I MHC IIA MHC IIB Laminin
Summary • Promotes commitment to differentiation and early fusion • Identified a cell surface molecule that can promote signaling to SRF and NFATc2 during differentiation • Loss is compensated by other factors • Does not have a role in mature muscleTranscription Factor Expression Pax7 GPR56 Myf5 NFATc3 SRF MyoD myogenin NFATc1 NFATc2 FHL1
Future implications for DMD therapy• Fusion of endogenous muscle stem cells – Issues to consider: • Duration of treatment • Target tissues • Delivery Method – Perhaps better to use directly with transplanted cells• Studying fusion for therapy – Identified a cell-surface molecule involved in promoting the commitment to differentiation and early fusion of cells – Next step: overexpression further increase engraftment and fusion of transplanted cells – Validation of the initial microarray to identify new players, can study other candidates including other GPCRs
Thanks for your brains and laborC-EPO GPR56 Shire Therapeutics Arthur Tzianabos Gregory Robinson Emanuela Gussoni Ariane Beauvais Chelsea Cherenfant Emanuela Gussoni Michael Lawlor Matt Mitchell Hui Meng Alexandra Lerch Gaggl Isabelle Draper Alan Kopin Jamie Doyle