NeuroHeal Reduces Muscle Atrophy and Modulates Associated Autophagy

Abstract

Muscle wasting is an unmet medical need which leads to a reduction of myofiber diameter and a negative impact on the functional performance of daily activities. We previously found that a new neuroprotective drug called NeuroHeal reduced muscle atrophy produced by transient denervation. Aiming to decipher whether NeuroHeal has a direct role in muscle biology, we used herein different models of muscle atrophy: one caused by chronic denervation, another caused by hindlimb immobilization, and lastly, an in vitro model of myotube atrophy with Tumor Necrosis Factor-α (TNFα). In all these models, we observed that NeuroHeal reduced muscle atrophy and that SIRT1 activation seems to be required for that. The treatment downregulated some critical markers of protein degradation: Muscle Ring Finger 1 (MuRF1), K48 poly-Ub chains, and p62/SQSTM1. Moreover, it seems to restore the autophagy flux associated with denervation. Hence, we envisage a prospective use of NeuroHeal at clinics for different myopathies.

Keywords: NeuroHeal; autophagy; proteasome; sirtuin 1; skeletal muscle atrophy.

Figure 1

NeuroHeal treatment reduces muscle atrophy by denervation. (A) Schematic workflow and experimental groups and (B) representative photographs of the gastrocnemius (GA) muscles removed from different experimental groups: control (CTL), injured untreated (Unt.), injured treated with NeuroHeal (NH), and injured treated with NH plus nicotinamide (NAM). Bar graph of the relative average weight of the ipsilateral muscle with respect to the contralateral one from different groups including those animals treated with acamprosate (ACA) or ribavirin (RIB) alone at 28 days post-injury (dpi) (n = 4; one-way ANOVA, * p < 0.05) is shown. (C) Left, representative microphotographs of cross GA muscle sections with Hematoxylin and Eosin (H&E) staining at 28 dpi (scale bar = 100 µm) and, right, representation of cross-sectional area (µm²) mean of all fibers in the GA muscle (n = 4; one-way ANOVA * p < 0.0001). (D) Histogram of the cross-sectional area (µm²) distribution of fibers in the GA muscle of different groups (n = 4; Kruskal–Wallis, Benjamin, Krieger, and Yekutieti post hoc * p < 0.05).

Figure 2

NeuroHeal treatment reduces muscle atrophy by disuse. (A) Schematic workflow and experimental groups and (B) a bar graph of the gastrocnemius (GA) muscle weight ratio between ipsilateral and contralateral sites at day 7 (n = 3–4; t-test, * p < 0.05). (C) Left, representative microphotographs of cross GA muscle sections with H&E staining at day 7 (scale bar = 100 µm) and, right, representation of cross-sectional area (µm²) mean of all fibers in the GA muscle (n = 5; one-way ANOVA * p < 0.05). (D) Histogram of the distribution of the cross-sectional area (µm²) of GA fibers (n = 3–4; Benjamin, Krieger, and Yekutieti post hoc * p < 0.05). (E) Bar graph of the grip strength of the immobilized hindlimb from NeuroHeal treated (NH) and untreated groups (Unt.) obtained at day 0, day 7, and day 14 (n = 5–10; one-way ANOVA, * p < 0.05).

Figure 3

NeuroHeal reduces fiber atrophy produced by Tumor Necrosis Factor-α (TNFα) in vitro. (A) Representative microphotographs of myotubes derived from a C2C12 myoblast cell line culture at day 7 and treated during the last 24 h with different components (control (CTL), NeuroHeal (NH), and Ex-527 as a specific SIRT1 inhibitor (scale bar = 50 µm)). Histogram of the distribution of myotubes diameter (n = 3–6; Kruskal–Wallis, Benjamin, Krieger, and Yekutieti post hoc, * p < 0.05) and (B) a Western blot and the corresponding bar graph showing the analyses of Muscle Ring Finger 1 (MuRF1) protein levels in the different experimental groups at day 7 (n = 3; one-way ANOVA, * p < 0.05).

Figure 4

Ubiquitin-proteasomal degradation is modulated by NeuroHeal in the denervated muscle model. (A) Western blots and associated bar graphs showing the analyses of phosphorylated AKT, AKT, and pP70s6k protein levels in different experimental groups at 7 dpi (control (CTL), injured untreated (Unt.), injured treated with NeuroHeal (NH), and injured treated with NH plus nicotinamide (NAM)) (n = 3–4; one-way ANOVA, * p < 0.05) and (B) Western blots and associated bar graphs showing the analyses of Atrogin-1, MuRF1, and K-48 polyubiquitin chain protein levels in different experimental groups at 7 dpi (n = 3–4; t-test, * p < 0.05).

Figure 5

Autophagy flux is modulated by NeuroHeal in the denervated muscle model. Western blots and the associated bar graphs showing the analyses of phosphorylated ULK1, LC3-II, the ATG5-ATG12 complex, p62, and protein levels in different experimental groups at 7 dpi (control (CTL), injured untreated (Unt.), injured treated with NeuroHeal (NH), and injured treated with NH plus nicotinamide (NAM)) (n = 3–4; one-way ANOVA, * p < 0.05).