Inhibition of Mitochondrial Fission Protein Drp1 Ameliorates Myopathy in the D2-mdx Model of Duchenne Muscular Dystrophy

Figure 1:. Study design schematic. (Illustrated using BioRender)

Figure 2:. Mdivi-1 treatment improves muscular strength and attenuates muscle damage in D2-mdx mice. A) Body weights over a 5-week period. B) Tissue weights were reported at the time of tissue collection expressed as absolute weight (grams). Gastrocnemius (Gastroc), tibialis anterior (TA), quadriceps (Quad), and heart; C) Grip strength expressed in average peak force/body weight; D) Hang wire impulse testing expressed in time (sec) x body weight (grams). E) Creatine kinase activity assay from serum collected via cardiac stick. Data are presented as Mean ± SEM. N=4–8 mice per group. * P < 0.05; ** P < 0.01 significant difference between groups. ## P < 0.01; ### P < 0.001 and #### P < 0.0001 significant main effect of DMD. & P < 0.05 significant main effect of Mdivi-1.

Figure 3:. Mdivi-1 inhibits Drp1-Mediated mitochondrial fission and improves mitochondrial dynamics in skeletal muscle from D2-mdx mice. A) Drp1 (Ser616) phosphorylation. B) Total Drp1 protein expression. C) Ratio of phospho Drp1 (Ser616) over total Drp1. D) Protein expression of mitochondrial fission markers. E) Protein expression of mitochondrial fusion markers. F) Representative immunoblots for D and E. Data are presented as Mean ± SEM. N=4–8 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 significant difference between groups. # P < 0.05; ## P < 0.01 significant main effect of DMD. & P < 0.05; & P < 0.01 significant main effect of Mdivi-1.

Figure 4:. Mdivi-1 treatment lowers autophagy markers, but does not alter mitophagy, mitochondrial biogenesis or content markers in skeletal muscle from D2-mdx mice. A) Protein expression of autophagy and mitophagy markers. B) PGC1α protein expression. C) Protein expression of oxidative phosphorylation complexes. D) VDAC protein expression. Data are presented as Mean ± SEM. N= 7–8 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 significant difference between groups.

Figure 5:. Mdivi-1 treatment improves subsarcolemmal, but not intermyofibrillar mitochondrial morphology in skeletal muscle from D2-mdx mice. A) representative TEM images. B) Ratio of Subsarcolemmal (SS) damaged mitochondria over total mitochondria. C) SS mitochondrial circumference. D) SS mitochondrial roundness. E) SS mitochondrial aspect ratio. F) Intermyofibrillar (IMF) damage mitochondria over total mitochondria. G) IMF mitochondrial circumference. H) IMF mitochondrial roundness. I) IMF mitochondrial aspect ratio. Data are presented as Mean values ± SEM. N= 3–4 mice with 5 TEM images quantified per animal (13,000x magnification). * P < 0.05; ** P < 0.01 significant difference between groups.

Figure 6.. Mdivi-1 treatment has minimal effect on mitochondrial respiration in skeletal muscle from D2-mdx mice A) Representative graph of oxygen consumption rates (OCR). B) Mitochondrial oxygen consumption rate (OCR). C) spare capacity. D) Respiratory control ratio. Data are presented as Mean ± SEM. N = 5–8 mice per group. * P < 0.05; ** P < 0.01 significant difference between groups.

Figure 7:. Mdivi-1 treatment did not alter mitochondrial hydrogen peroxide production in skeletal muscle from D2-mdx mice. A) Pyruvate/malate (Complex I-supported mH2O2 production). B) Succinate/rotenone (Complex II-supported mH2O2 production). C) pyruvate/malate/antimycin (Complex III-supported mH2O2 production). and D) pyruvate/rotenone (Pyruvate Dehydrogenase Complex-supported mH2O2 production). Data are presented as Mean values ± SEM. N= 5–8 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 significant difference between groups.

Figure 8:. Mdivi-1 treatment reduces lipid peroxidation in skeletal muscle D2-mdx mice. A) 4-HNE protein expression. B) Correlation between 4-HNE protein expression and grip strength. C) Correlation between 4-HNE protein expression and hangwire time. Data are presented as Mean values ± SEM. D) GPx4 mRNA expression. N= 7–8 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 significant difference between groups.

Figure 9:. Mdivi-1 treatment improved protein/gene expression of fibrosis and inflammation markers in skeletal muscle from D2-mdx mice. A) Collagen 1 (Col1a1) and Fibronectin 1 (FN1) protein expressions. B) Correlation between 4-HNE protein expression and Col1a1 protein expression. C) IL-6 mRNA expression. D) IL-1β mRNA expression. Data are presented as Mean values ± SEM. N= 6–8 mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001 significant difference between groups.