Myosin inhibition partially rescues the myofiber proteome in X-linked myotubular myopathy

Figures

• 

  Figure 1. Myosin biochemical and energetic states in patients with XLMTM. (A) Typical fluorescent decays for one XLMTM patient and one control. (B) The proportion of myosin molecules in the DRX state (P1). (C) The number of myosin heads in the SRX state (P2) for all patients and fibers analyzed. (D and E) Their respective ATP turnover lifetimes (T1 in D and T2 in E). (F) The theoretical myosin ATP consumption based on P1, P2, T1 and T2 values. For B–F, in the left panels, circles are individual patient’s average data; while in the right panels, circles are individual muscle fibers. Data are shown as mean ± SD. Unpaired t tests with Welch correction were used to compare groups (level of significance P < 0.05).

• 

  Figure 2. Myofilament and myosin order in patients with XLMTM. (A) A typical x-ray diffraction pattern from one control. Equatorial (1,0 and 1,1) as well as meridional (M3) reflections are presented. (B) Interfilament lattice spacing derived from the 1.0 equatorial reflections (d1,0). (C) The heterogeneity of inter-filament lattice spacing (σD). (D) The equatorial intensity ratio (I1,1 to I1,0 ratio); (E) is M3 spacing (SM3). (F) M3 intensity (IM3). Circles are individual muscle bundles. Means represented by a line also appear on all the panels. Unpaired t tests with Welch correction were used to compare groups (level of significance P < 0.05).

• 

  Figure 3. Myosin biochemical and energetic states in the canine model of XLMTM. Our study included WT control dogs as well as XLMTM-affected dogs expressing a MTM1 gene mutation. These XLMTM dogs were injected systemically with a saline solution (XLMTM) or with 2 different doses of gene therapy (AAVlo or AAVhi). (A) Typical fluorescent decays for one XLMTM dog and one WT. (B) The amount of myosin molecules in the DRX state (P1). (C) The proportion of myosin heads in the SRX state (P2). (D and E) Their respective ATP turnover lifetimes are also displayed (T1 in D and T2 in E). (F) The theoretical myosin ATP consumption. For B–F, in the left panels, circles are individual animals’ average data; in the right panels, circles are individual muscle fibers. Data are shown as mean ± SD. One-way ANOVAs with Tukey post hoc were used to compare groups (level of significance P < 0.05).

• 

  Figure 4. Effect of phosphorylation level on myosin biochemical states in XLMTM dogs. (A and B) The relative change in the number of myosin molecules in the DRX (P1) and SRX state (P2), respectively, in response to vehicle, phosphatase treatment or protein kinase A (PKA) incubation. (C and D) The relative change of their respective ATP turnover time (T1 and T2, respectively). For A–D, The left panels show WT; the right panels show XLMTM dogs. Circles are individual muscle fibers. Means represented by a line also appear on all the panels. One-way ANOVAs with Tukey post hoc were used to compare groups (level of significance P < 0.05).

• 

  Figure 5. Comparative proteomic analysis in XLMTM dogs. (A) Venn diagram depicting detected proteins that were significantly upregulated and downregulated in XLMTM as well as those that remained statistically unchanged between experimental groups. (B) Volcano plot displaying Log2 fold change (Log2FC) against Log10 P value. The top 10 most significant proteins have been annotated. (C) A heatmap was created to illustrate the proteins with the greatest fold change, all proteins included possessed a Log2FC > 1.5. For readability some gene names are used rather than protein names; the protein and gene names can be found in Supplemental Table 2. (D) Proteomaps were generated using Log2 transformed protein abundance values of proteins, which were significantly different between groups. Unpaired 2-tailed t test was used.

• 

  Figure 6. Muscle histological analysis in mice lacking MTM1. Our study involved WT mice as well as mice lacking MTM1 (Mtm1y/–). These animals were either treated with vehicle or mavacamten for 4 weeks (+ mava). (A) The theoretical myosin ATP consumption. (B) Body weight. (C) Soleus muscle mass normalized to body weight. (D) Fiber type proportions. (E and F) Myofiber cross-sectional area separated according to their types (I vs IIA). For A, E, and F, circles are individual myofibers’ data; an unpaired t test with Welch correction was used to compare groups (level of significance P < 0.05). For B and C, circles are individual animals’ data; data are shown as mean ± SD; 2-way ANOVA with Tukey post hoc were used to compare groups (level of significance P < 0.05).

• 

  Figure 7. Comparative proteomic analysis in mice lacking MTM1. We used WT mice and mice lacking MTM1 (Mtm1y/–). These animals were either treated with vehicle or mavacamten for 4 weeks (+ mava). (A–F) Part of the proteomics analyses with the principal component analyses and volcano plots displaying Log2 fold change against Log10 P value.