Mustn1 ablation in skeletal muscle results in functional alterations

Figures

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  FIGURE 1. Differential mRNA expression between 2‐month‐old WT and KO male mice. Heatmap of selected myogenic differentiation and fusion genes.

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  FIGURE 2. Evaluation of differentiation and fusion gene expression. (A) Expression levels of differentiation factors, including myoblast determination protein 1 (MyoD), myogenin (MyoG), myosin heavy chain 4 (Myh4), and desmin (Des) at 2 and 4 months. (B) Expression of fusion factors, calpain 1 (Capn1), caveolin 3 (Cav3), and cadherin 15 (Cadh15) at the same time points (n = 4/group, ns = non‐significant; unpaired t‐test). Error bars indicate SD derived from four independent PCR runs.

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  FIGURE 3. Determination of forelimb grip strength. Each trial was conducted six times at 1‐min intervals. The experimenter was blinded to the age and genotype of the mice being tested. (A) Raw grip strength data of 2‐ and 4‐month‐old male KO mice and WT counterparts. (B) Normalized grip strength by body weight (n = 10/group, **p < 0.01; Welch's t‐test).

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  FIGURE 4. Single limb force analysis. (A) Experimental setup wherein a mouse traverses a runway fitted with two platforms “P” that are integrated with a force plate “FP”. (B) Corresponding force plate outputs, characterizing two forelimb “F” and two hindlimb “H” forces. “Fx” is fore‐aft plane and “Fz” is vertical plane. (C) and (D) delineate the vertical forces exerted by the forelimb and hindlimb, respectively, at 2 and 4 months of age. Boxplots illustrate the distribution of vertical forces. Each data point in linear graphs signifies an individual trial (n = 5/group, *p < 0.05; linear mixed model).

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  FIGURE 5. Evaluation of ex vivo isometric contractile force. (A) Absolute isometric force production of the soleus muscle across various frequencies for the 2‐ and 4‐month WT and KO mice. (B) Physiological cross‐sectional area (PCSA) comparison between WT and KO (n = 8/group, *p < 0.05; Mann–Whitney test). (C) Specific isometric force production of the soleus muscle, normalized by PCSA, at the corresponding frequencies. Upon normalization of the contractile force using PCSA, the 2‐month‐old KO mice showed a significant difference in specific force at 20 Hz, whereas the 4‐month‐old KO mice revealed a more marked decrease at 20 and 60 Hz (n = 8/group, ***p < 0.001; **p < 0.01; *p < 0.05; two‐way ANOVA; Mann–Whitney test). Data represented by mean ± SEM.

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  FIGURE 6. Isometric fatigue test. (A) Fatigue curve represents the performance of the soleus muscle during 300 contractions executed over a period of 5 min for the 2‐ and 4‐ month WT and KO mice. (B) The percent initial force produced is displayed for specified contraction numbers. The protocol revealed significant variations between the KO and WT mice in both age groups (n = 8/group, ****p < 0.0001; ***p < 0.001; *p < 0.05; two‐way ANOVA). Data represented by mean ± SEM.

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  FIGURE 7. Skeletal muscle fiber typing. Immunofluorescence images of the soleus muscle taken at 2 and 4 months of age (A–L), indicating the various fiber (Type I, Type IIa, and Type IIb) type compositions. (Fi = Fibular). (M) Quantitated analysis of stained sections for each muscle fiber type at 2‐months and (N) 4 months (n = 3/group, *p < 0.05; Welch's t‐test). Data represented by mean ± SEM.