Date of Award

5-2024

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Kai Zou

Second Advisor

Alexey Veraksa

Third Advisor

Changmeng Cai

Abstract

Duchenne Muscular Dystrophy (DMD) is the most common and severe muscular dystrophy manifested by progressive muscle wasting and weakness. Mitochondrial dysfunction and reactive oxygen species (ROS) accumulation are early pathological features of DMD and precede myopathy. A fine balance of mitochondrial dynamics (fission and fusion) is crucial to maintain mitochondrial function and skeletal muscle health. We recently reported excessive activation of Dynamin-Related Protein 1 (Drp1)-mediated mitochondrial fission in two animal models of DMD. Mitochondrial fission inhibitor‐1 (Mdivi‐1) is an effective pharmacological inhibitor of Drp1-mediated mitochondrial fission and has been shown benefits in numerous diseases. The objective of the study was to examine the effects of Mdivi-1 on skeletal muscle mitochondrial quality control, morphology, function and muscle strength in D2-mdx mouse model of DMD. Methods: 9-10-week-old male D2-mdx mice were treated with either Mdivi-1 (I.P., 40 mg/kg BW, D2-mdx/Mdivi-1) or vehicle (2% DMSO in PBS, D2-mdx/VEH) for 5 weeks. D2-wildtype mice treated with vehicle (WT/VEH) were used as the control (n=8/group). In addition, in order to evaluate whether Mdivi-1 had any adverse effects on phenotypes in normal healthy mouse, we added another group with Mdivi-1 injections in wildtype mice (WT/Mdivi-1, n=4) and measured all behavioral tests, body weight and tissue weights. Muscle strength was measured using a grip strength meter and hang wire test. Mitochondria were isolated from fresh quadricep muscles collected 24 hours after the last injection to measure mitochondrial respiration and H2O2 emission. Transmission Electron Microscopy was used to image mitochondrial structure in soleus muscle. Gastrocnemius muscles were collected for immunoblot analysis. Finally, serum was collected for blood creatine kinase concentration. Results: Grip strength was significantly lower in muscles from D2-mdx mice compared to WT controls (P = 0.01) but was improved in D2-mdx/Mdivi-1 mice (P = 0.05). Serum creatine kinase level was higher in D2-mdx mice compared to WT (P = 0.006) and reduced with Mdivi-1 treatment (P = 0.079). Mitochondrial fission protein markers, Drp1(Ser616) phosphorylation and Fis1, were markedly higher in skeletal muscles from D2-mdx compared to WT (P= 0.009 and <0.0001) but were attenuated with Mdivi-1 treatment (P = 0.041 and 0.029), indicating a rebalance of mitochondrial dynamics. This rebalance subsequently led to improvement in skeletal muscle mitochondrial structure in D2-mdx/Mdivi-1 mice, with evidence of lower numbers of damaged mitochondria and reduced circularity (P = 0.040 and 0.045). Although not statistically significant, ADP and FCCP-stimulated mitochondrial respiration were enhanced by 93.8% and 92.4% in D2-mdx/Mdivi-1 mice compared to D2-mdx/VEH. In addition, lipid peroxidation marker, 4-HNE was significantly higher in D2-mdx mice in comparison to WT (P = <0.0001), but was attenuated with Mdivi-1 treatment (P = 0.08). Conclusion: These data demonstrate that inhibition of Drp1-mediated mitochondrial fission is effective in improving muscular strength in D2-mdx mice, which is at least partly due to enhanced mitochondrial dynamics, structure and attenuated lipid peroxidation. Mdivi-1 may be a viable treatment for DMD.

Comments

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