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. 2017 Oct 27;7(1):14275.
doi: 10.1038/s41598-017-14290-3.

Myostatin Inhibition Prevents Skeletal Muscle Pathophysiology in Huntington's Disease Mice

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Free PMC article

Myostatin Inhibition Prevents Skeletal Muscle Pathophysiology in Huntington's Disease Mice

Marie K Bondulich et al. Sci Rep. .
Free PMC article

Abstract

Huntington's disease (HD) is an inherited neurodegenerative disorder of which skeletal muscle atrophy is a common feature, and multiple lines of evidence support a muscle-based pathophysiology in HD mouse models. Inhibition of myostatin signaling increases muscle mass, and therapeutic approaches based on this are in clinical development. We have used a soluble ActRIIB decoy receptor (ACVR2B/Fc) to test the effects of myostatin/activin A inhibition in the R6/2 mouse model of HD. Weekly administration from 5 to 11 weeks of age prevented body weight loss, skeletal muscle atrophy, muscle weakness, contractile abnormalities, the loss of functional motor units in EDL muscles and delayed end-stage disease. Inhibition of myostatin/activin A signaling activated transcriptional profiles to increase muscle mass in wild type and R6/2 mice but did little to modulate the extensive Huntington's disease-associated transcriptional dysregulation, consistent with treatment having little impact on HTT aggregation levels. Modalities that inhibit myostatin signaling are currently in clinical trials for a variety of indications, the outcomes of which will present the opportunity to assess the potential benefits of targeting this pathway in HD patients.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Treatment with ACVR2B/Fc restores deficits in body weight, grip strength and muscle mass in R6/2 mice. (A) ACVR2B/Fc treatment resulted in a progressive weight gain in both WT and R6/2 mice and prevented body weight loss in R6/2 mice. (B) ACVR2B/Fc treatment resulted in a progressive increase in fore-limb grip strength in both WT and R6/2 mice and prevented grip strength deficits in R6/2 mice. (C) Treatment with ACVR2B/Fc increased muscle mass in both WT and R6/2 mice with the consequence that the R6/2 muscle mass at 12 weeks of age (genders combined) is equivalent to that of wild type mice for quadriceps, gastrocnemius and tibialis anterior hind limb skeletal muscles. Statistical analysis was two-way ANOVA with post-hoc Bonferroni correction (see Table S8 for main effects and Table S9 for multiple comparisons). The statistical significance between values for ACVR2B/Fc treated and vehicle treated R6/2 mice is depicted: *p < 0.05; **p < 0.01; ***p < 0.001. n = 5 mice per gender per genotype. All data presented as means ± SEM.
Figure 2
Figure 2
ACVR2B/Fc treatment prevents muscle fibre atrophy. (A) the lesser fibre diameter of myofibres in the TA and quadriceps of WT and R6/2 mice treated with ACVR2B/Fc or vehicle. Fibre counts were obtained from between 3 and 8 sections per mouse, n = 3 or 4 mice/treatment group. The WT muscle fibre diameters were greater than those for R6/2: quadriceps (F(13,10) = 28.82, p ≤ 0.001), TA (F(15,12) = 10.29, p = 0.008). ACVR2B/Fc treatment increased the fibre diameter in both cases: quadriceps (F(13,10) = 92.8, p ≤ 0.001) and TA (F(15,12) = 12.73, p = 0.004). ACVR2B/Fc treatment increased fibre diameter in the quadriceps (p < 0.001) and the TA (p = 0.061) of R6/2 mice. Representative traces from individual mice are depicted. Statistical analysis was two-way ANOVA with post-hoc Bonferroni correction.
Figure 3
Figure 3
Treatment with ACVR2B/Fc improves muscle function in R6/2 mice. (A–D) Contractile dysfunction of the R6/2 EDL muscles was assessed by measuring twitch tension: the time to peak and half relaxation time and this was corrected by ACVR2B/Fc treatment in both EDL (A,C) and TA (B,D) muscles. (E,F) The maximum tetanic force in mice treated with ACVR2B/Fc and vehicle. The maximum EDL (E) and TA (F) forces were decreased in R6/2 mice and completely rescued in mice treated with ACVR2B/Fc. (G) Examples of motor unit traces and the quantification of functional motor units. The number of functional motor units was reduced in R6/2 EDL muscles and this was restored in treated mice. Statistical analysis was two-way ANOVA with post-hoc Bonferroni correction (see Table S8 for main effects and Table S9 for multiple comparisons). Statistically significant differences between vehicle treated WT and vehicle treated R6/2: # p < 0.05, ### p < 0.001; statistically significant differences between ACVR2B/Fc treated R6/2 and vehicle treated R6/2: *p < 0.05; **p < 0.01; ***p < 0.001. n = 11 WT vehicle (EDL), n = 12 WT vehicle (TA), n = 8 WT ACVR2B/Fc (EDL and TA), n = 14 R6/2 vehicle (EDL and TA), n = 12 R6/2 ACVR2B/Fc (EDL and TA). All data presented as means ± SEM. The baseline WT and R6/2 vehicle treated phenotype data were previously published.
Figure 4
Figure 4
Treatment with ACVR2B/Fc delays end-stage disease, but has no effect on rotarod performance or activity measures. (A) ACVR2B/Fc treatment completely prevented the progressive loss in body weight loss that occurs in R6/2 mice. The effect was such that R6/2 mice were significantly heavier than WT mice at some ages (Table S9). (B) Kaplan-Meier curve showing that ACVR2B/Fc treatment delays end-stage disease in R6/2 mice (Chi = 8.764, p < 0.01 Mantel-Cox log-rank test). (C) ACVR2B/Fc treatment completely prevented the progressive loss in fore-limb as well as the combined fore- and hind-limb grip strength that occurs in R6/2 mice. The effect was such that R6/2 mice were significantly stronger than WT mice at some ages (Table S9) (D) ACVR2B/Fc had no effect on the impairment in R6/2 rotarod performance or hypoactivity. Statistical analysis was two-way ANOVA with post-hoc Bonferroni correction (see Table S8 for main effects and Table S9 for multiple comparisons). The statistical significance between values for ACVR2B/Fc treated and vehicle treated R6/2 mice is depicted: *p < 0.05; **p < 0.01; ***p < 0.001. WT vehicle, n = 13; R6/2 vehicle, n = 17; R6/2 ACVR2B/Fc, n = 14. All data presented as means ± SEM.
Figure 5
Figure 5
ACVR2B/Fc treatment and HTT aggregation. (A) ACVR2B/Fc treatment results in a decrease in the aggregate load in R6/2 TA and quadriceps muscles as assessed by the Seprion ELISA. WT vehicle, n = 5; WT ACVR2B/Fc, n = 10; R6/2 vehicle n = 10; R6/2 ACVR2B/Fc, n = 9. WT background signal derives from the substrate. (B) Number of DAPI stained nuclei per ROI and average nucleus size in DAPI pixels. (C) Number of S830 inclusions per ROI and average inclusion size in pixels. (D) Percentage of S830 signal co-localized with DAPI and percentage of inclusions localized to the nucleus. (E) Number of nuclear S830 inclusions per ROI, the percentage of nuclei with inclusions and average size of nuclear inclusions in pixels (F) Level of expression of Pax7 as fold change from WT. Taqman qPCR values were normalized to the geometric mean of Atp5b, Actb and Sdha. WT vehicle, n = 8; WT ACVR2B/Fc, n = 7; R6/2 vehicle n = 6; R6/2 ACVR2B/Fc, n = 8. Statistical analysis for (B) and (D) was two-way ANOVA with post-hoc Bonferroni correction (see Table S8 for main effects) and for (A), (C), (D) and (E) was Student’s t-test (n = 4/treatment group). Statistical significance for R6/2 vehicle vs R6/2 ACVR2B/Fc is depicted by *p < 0.05; **p < 0.01; ***p < 0.001 and for WT vehicle vs R6/2 vehicle by ###p < 0.001. All data presented ± SEM. WT = wild type, ROI = regions of interest.
Figure 6
Figure 6
The effect of ACVR2B/Fc treatment on genes that are dysregulated in R6/2 muscle. The first column in each panel indicates the change in gene expression in R6/2 muscle as compared to WT. Red squares represent an increase and blue squares, a decrease in expression levels. The second column represents the change in gene expression levels in R6/2 muscle in response to ACVR2B/Fc treatment and the third column indicates when a gene in WT muscle was also significantly changed in response to treatment. All of the depicted genes were changed to statistically significant levels (adjusted p < 0.05) with a fold change greater than 1.5. Genes in bold were changed in both quadriceps and tibialis anterior. The panels represent genes that are (A) increased or (B) decreased in the quadriceps and (C) increased or (D) decreased in the tibialis anterior of R6/2 mice as compared to WT. RNAseq was performed on RNA extracted from n = 10 mice/treatment group.

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