Serum proteomic profiling reveals fragments of MYOM3 as potential biomarkers for monitoring the outcome of therapeutic interventions in muscular dystrophies

Jérémy Rouillon, Jérôme Poupiot, Aleksandar Zocevic, Fatima Amor, Thibaut Léger, Camille Garcia, Jean-Michel Camadro, Brenda Wong, Robin Pinilla, Jérémie Cosette, Anna M L Coenen-Stass, Graham Mcclorey, Thomas C Roberts, Matthew J A Wood, Laurent Servais, Bjarne Udd, Thomas Voit, Isabelle Richard, Fedor Svinartchouk, Jérémy Rouillon, Jérôme Poupiot, Aleksandar Zocevic, Fatima Amor, Thibaut Léger, Camille Garcia, Jean-Michel Camadro, Brenda Wong, Robin Pinilla, Jérémie Cosette, Anna M L Coenen-Stass, Graham Mcclorey, Thomas C Roberts, Matthew J A Wood, Laurent Servais, Bjarne Udd, Thomas Voit, Isabelle Richard, Fedor Svinartchouk

Abstract

Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DMD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of α-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.

© The Author 2015. Published by Oxford University Press.

Figures

Figure 1.
Figure 1.
Western blot analysis of MYOM3 in pools of sera from subgroups of young (G1) and older (G2) DMD patients as well as young (G3) and older (G4) healthy subjects. (A) normal exposure; (B) boosted exposure. For explanation of groups and subgroups see Table 1. Fifty micrograms of serum proteins were loaded in each well.
Figure 2.
Figure 2.
Expression levels of serum MYOM3 fragments (A) and CK (B) in sera from the entire US cohort including 39 young and 17 older DMD patients as well as 29 young and 18 older healthy controls. To measure levels of the MYOM3 fragments, 50 µg of serum proteins were analysed by Western blot, then band intensities were quantified and expressed in arbitrary units (a.u). The CK enzyme activity in serum is expressed in international units per litre (IU/L). (C) Linear regression analysis between serum levels of the MYOM3 fragments and CK for young patients. (D) Linear regression analysis between serum levels of the MYOM3 fragments and CK for older DMD patients. The linearity of the response by Western blot for MYOM3 is demonstrated in Supplementary material, Figure S2.
Figure 3.
Figure 3.
MYOM3 fragments are specifically present in sera from animal models of DMD. (A) Western Blot analysis of serum from GRMD and healthy dogs. GRMD # 1–4: two months old; # 5–6: 18 months old. Healthy # 1–4: two months old; # 5–6: 18 months old dogs. DMD: control serum from DMD patient. (B) Western Blot analysis of serum from 6 months old mdx and WT mice. WT: C57/BL10 strain.
Figure 4.
Figure 4.
Upper panel: Western blot analysis of the MYOM3 fragments in serum from 3 LGMD2D patients (#1 is 35, #2 is 23 and #3 is 24 years old). Serum from two DMD patients (group G1) and three healthy individuals (group G4) were used as controls. Lower panel: Western blot analysis of the MYOM3 fragments in serum from mouse models of different muscular dystrophies at 1 and 6 months of age. WT: C57BL/6J mouse; mdx (model for DMD); KO-Sgcg: model for LGMD2C; KO-Sgca: model for LGMD2D; KO-Dysf: model for LGMD2B; KO-Capn3: model for LGMD2A.
Figure 5.
Figure 5.
Levels of the MYOM3 fragments (A, B) and CK-M (C, D) in serum from healthy (B, D) and mdx (A, C) mice at different ages as estimated by Western blot analysis. Intensity of the bands (in arbitrary units, a.u.) on different gels was normalized by the respective bands of the positive control (50 µg of serum proteins from the same mdx mouse present on each gel). Fifty micrograms of serum proteins were used for the analysis. Age 0 corresponds to newborn mice. Estimation of the CK-M level by Western blot analysis correlated well with the CK activity (Supplementary material, Fig. S3).
Figure 6.
Figure 6.
Levels of the MYOM3 fragments (A, B) and CK-M (C, D) in serum from healthy (B, D) and mdx (A, C) mice at different time after physical exercise estimated by Western blot analysis. Band intensity on different gels was normalized by the respective bands of the positive control (50 µg of serum proteins from the same mdx mouse present on each gel). Fifty micrograms of mouse serum were used for the analysis.
Figure 7.
Figure 7.
Effect of antisense oligonucleotide-mediated exon-skipping therapy in mdx mice on the serum levels of the MYOM3 fragments and CK-M. Twelve 12-week-old male mdx mice were treated with a single 12.5 mg/kg intravenous dose of Pip6a-PMO. Two, 4 and 8 weeks after injections the animals were sacrificed by groups of four mice and quadriceps and blood samples were taken for analysis. Dystrophin restoration in quadriceps muscles was monitored by Western blot analysis (A) and the efficiency of exon skipping by qPCR analysis (B) (See Materials and Methods section for the details). Levels of the MYOM3 fragments (C) and CK-M (D) were measured by Western blot analysis, and then band intensities were quantified and expressed in arbitrary units (a.u). Additional data confirming the robustness of dystrophin quantification are presented in Supplementary material, Figure S4. (E): Raw P-values (Student's t-test) for the comparison of MYOM3 and CK levels between different groups of mice at different time points. The values below the threshold 0.01 are in yellow, and below 0.05 are in pink.
Figure 8.
Figure 8.
Comparison of different assays for the follow-up of the gene therapy treatment in KO-Sgca mice. (A) Histological analyses (upper panel: HPS stain; lower panel: immunodetection of α-sarcoglycan) of gastrocnemius muscles after treatment with increasing doses of rAAV coding for huSgca (1e11, 5e11 and 1e12 vg). (B) Quantitative analysis of α-sarcoglycan positive fibres after the treatments. (C) Restoration of muscular strength (escape test) 83 days after the treatment. (D) Serum CK and (E) MYOM3 fragments levels at different time after the treatment (mean ± SEM). (F) Raw P-values (Student's t-test) for the comparison of MYOM3 and CK levels between different groups of mice at different time points. The values below the threshold 0.01 are in pink, and below 0.05 are in yellow. PBS, 1e11, 5e11 and 1e12: KO-Sgca mice injected with PBS or the respective dose of the vector. WT: C57BL/6J control mice injected with PBS. Levels of the MYOM3 fragments and CK-M were estimated biweekly by Western blot analysis.

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