Myostatin: a Circulating Biomarker Correlating with Disease in Myotubular Myopathy Mice and Patients

Figure 1. Circulating Myostatin Is Reduced in Mtm1−/y Mice and Improved in Response to Antisense Oligonucleotides Mediated Reduction of Dnm2 (A) Plasma myostatin protein levels (ng/mL) from WT and Mtm1−/y mice. (B) Representative immunoblot of DNM2 protein expression, and GAPDH loading control in TA skeletal muscles, from WT and Mtm1−/y mice, treated with DYN101-m targeting Dnm2 mRNA or ASO control (ASO-Ctrl), at 7 weeks of age. (C) Densitometry analysis from immunoblot for protein expression of DNM2 and GAPDH.. (D) Hanging test performed at 7 weeks of age, maximum time 60 s. Each point represents one mouse. (E) Circulating myostatin protein levels in plasma (ng/mL). (F) Linear regression analysis was performed between plasma myostatin levels from (E) and hanging time (D) in Mtm1−/y mice following ASO-Ctrl (black dots) or DYN101-m administration (red dots). Line of best fit shown, slope = 0.40 ± 0.14, 95% confidence interval (CI) 0.10–0.70, p value displayed. Pearson correlation analysis was also performed (r = 0.6296, ∗p = 0.0119). Each point represents one mouse, n ≥ 5 per group. Results represented as violin plots (A and C–E). Mann-Whitney test performed for DSS analysis. ∗p < 0.0125,…

Figure 2. Circulating Myostatin Levels Respond to Dnm2 Reduction in a Time-Dependent Manner, Correlating with Reduced Disease Severity in Mtm1−/y Mice (A) Combined hanging time (seconds) from all cohorts of Mtm1−/y mice following single injection of antisense oligonucleotide targeting Dnm2 (DYN101-m, red line) or ASO-Ctrl (black line). Individual cohorts were then sacrificed 1, 2, 4, 8, 12, or 16 weeks post injection, n = 18–30 mice. (B) Dnm2 mRNA expression quantified by qRT-PCR analysis, relative to Rpl27 expression, from tibialis anterior (TA) muscles, represented as mean + SD. (C) Circulating myostatin protein levels in plasma (ng/mL), represented as mean + SD. The asterisk (∗) indicates significant difference between Mtm1−/y mice with ASO-Ctrl versus DYN101-m administration for (B) and (C). (D) Hematoxylin and eosin (H&E, upper panel) and succinate dehydrogenase (SDH, lower panel) staining of TA muscles, 4 weeks post single injection. (E) Analysis of fiber size, represented as mean + SEM, n = 2–4 mice per group. (F) Analysis of fibers with altered nuclei positioning, represented as a violin plot, individual mouse data shown. ∗p < 0.0125, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. All…

Figure 3. Underlying Mechanisms Leading to Myostatin Alteration in Mtm1−/y Mice (A) Immunoblot for protein expression of Myostatin prodomain and GAPDH (protein loading control) in TA muscles. (B) Myostatin prodomain expression quantified relative to GAPDH loading control from (A). (C) Immunoblot for protein expression of Myostatin prodomain and ponceau (protein loading control) in plasma. (D) Densitometry analysis of myostatin prodomain expression from (C). (E) mRNA analysis of Pre-miR-27a relative to Rpl27 expression from TA skeletal muscles of WT and Mtm1−/y mice, following weekly injections of DYN101-m targeting Dnm2 or ASO-Ctrl. (F) Mature miR-27 analysis relative to U6+Snord61. Results presented as violin plots, individual mouse data shown. ∗p < 0.0125, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. All results from study 1.

Figure 4. General Alterations of the Myostatin Pathway in Mtm1−/y Mice (A–C) mRNA analysis relative to Rpl27 expression from TA skeletal muscles of wild-type (WT) and Mtm1−/y mice, treated with repeated injections of DYN101-m targeting Dnm2 or ASO-Ctrl, analyzed at 7 weeks of age, for Gdf11 (A), AcvRIIb (B), or Fstn (C). (D) Pre-miR-206 expression relative to Rpl27 expression, in WT, Mtm1−/y mice, and Mtm1−/yDnm2+/− mice at 7 weeks of age. (E) mRNA analysis of pre-miR-206 relative to Rpl27 expression from TA skeletal muscles of WT and Mtm1−/y mice, following weekly injections of DYN101-m targeting Dnm2 or ASO-Ctrl. (F) Mature miR-206 analysis relative to U6+Snord61, following repeated weekly injections of DYN101-m targeting Dnm2 or ASO-Ctrl. Each point represents one mouse, n ≥ 5 per group. Results presented as violin plots; individual mouse data shown. ∗p < 0.0125, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (A–C and F) Study 1 (weekly ASO injections), (E) study 2 (second independent cohort), and (D) study 4 (genetic cross).

Figure 5. Dysregulation of the Myostatin Pathway in Mtm1−/y Mice The members of the myostatin pathway analyzed in this manuscript at the mRNA or protein levels are indicated in this figure. Genes regulation in Mtm1−/y mice is represented by color-coding as follows; upregulated genes (green), downregulated genes (red), or unchanged compared to WT (blue).

Figure 6. Plasma Myostatin Levels Are Reduced in Patients with X-linked and Autosomal Dominant Forms of Centronuclear Myopathy Plasma myostatin protein levels (pg/mL) from adult X-linked CNM (XL-CNM, MTM1 mutations) patients and autosomal dominant CNM (AD-CNM, DNM2 mutations; ages 19–61), and adult human control samples (aged 17–72). Each point represents one patient or control, n ≥ 4 per group. Results presented as violin plots. ∗p < 0.025, ∗∗∗∗p < 0.0001.