IMPACT OF DRP1 ACTIVATION, UNFOLDED PROTEIN RESPONSE INDUCTION AND FGF21 LEVELS IN DMD PROGRESSION

Duchenne muscular dystrophy (DMD) is a severe genetic progressive muscle-wasting disease. The transmission of this pathology is X-linked recessive with an incidence of at least 1:5000 newborn boys. Patients typically present the first symptoms related to motor impairments very early in childhood and if not adequately treated, they die prematurely because of cardiac or respiratory complications. Mutations in the DMD gene that lead to the complete loss of dystrophin protein are responsible for this very severe phenotype. Dystrophin is a subsarcolemmal protein that not only provides structural integrity and functional stability to the muscle fiber during contraction but also plays crucial roles in cell signaling pathways. In muscle, dystrophin deficiency triggers a cascade of consequences, extending from myofiber necrosis and chronic inflammation to reduced and aberrant muscle regeneration which culminates in excessive deposition of fibrotic and adipose tissue. In addition, mitochondrial dysfunctions are recognized as a hallmark of DMD since dystrophic muscles have been reported to exhibit several metabolic impairments, reduced mitophagy, defective mitochondrial biogenesis, disrupted Ca2+ homeostasis, and altered mitochondrial dynamics. To date, there is no definitive cure for DMD, despite remarkable signs of progress have been made in developing new therapeutic strategies. However, therapeutic interventions aimed at targeting secondary pathological mechanisms to preserve muscle function are still urgently needed. A precise balance between mitochondrial fission and fusion processes is necessary to maintain a functional mitochondrial network, especially in muscle which is a highly metabolically active tissue that requires a continuous energy supply for muscle contraction. However, only a few studies have addressed mitochondrial dynamics in DMD, highlighting a dramatic fragmentation of the mitochondrial network. In this context, we hypothesize that mitochondrial dynamics may represent a potential therapeutic target for DMD. For this PhD project, we used an mdx-PhAM mouse model, characterized by intrinsically fluorescent and photoconvertible mitochondria, to precisely analyze mitochondrial network morphology. We demonstrated that mdx muscle fibers display a disorganized and less interconnected mitochondrial network. Drp1, the master regulator of mitochondrial fission, and its receptors located on the mitochondrial membrane, such as Fis1 and Mff, are responsible for mitochondrial network fragmentation. We found that all these pro-fission proteins are upregulated in dystrophic muscles from 12 weeks of age. Further analysis revealed that Drp1 activity was enhanced in mdx muscles compared to WT controls, as evidenced by its increased levels in the mitochondrial fraction of mdx muscles, as well as by its increased interaction with Fis1. This unbalanced fission could promote mitochondrial stress and therefore enhance a specific transcriptional program known as UPR, which could impact dystrophic muscle degeneration. Consistently, we revealed a significant upregulation of proteins associated with the mitochondrial-specific UPR (UPRmt) and a muscle-specific induction of the stress mitokine FGF21. FGF21 detrimental effects have been reported concerning bone loss in mdx mice however it could also impact fibro-adipogenic progenitors differentiation considering that these cells express FGF receptor. We therefore decided to inhibit Drp1 with daily intraperitoneal injections of Mdivi-1 and we observed beneficial effects at functional and histological levels. Inflammation and fibrosis were also remarkably reduced. In parallel, we observed a reduction in muscle levels of FGF21 after the treatment, suggesting a role of this myokine also in establishing dystrophic damage. Mdivi-1 also improved mitochondrial activity and preserved the myogenic potential of dystrophic muscle satellite cells. We also focused on another strategy to modulate Drp1-mediated excessive fission in mdx mice injecting an AAV-vector encoding for a validated Drp1 shRNA. We observed that the viral genome was still present after 11 weeks from the injection, suggesting a therapeutic action in preserving muscles. Indeed, AAV-shDrp1 had a positive effect on in situ muscle fragility and hypertrophy, possibly due to the observed reduction of fibrotic markers. In conclusion, the results of my PhD project reveled that Drp1 is an emerging target in DMD and its modulation is a promising approach to counteract muscular dystrophy progression.