The Mitochondria Fission Protein DRP1 is Required for Muscle Mass Maintenance
Year: 2017
Type: dissertation
Abstract: The skeletal muscle accounts for almost 40% of total body mass and represents a major site of metabolic activity. Skeletal muscle is a dynamic tissue: exercise and hormonal stimulation lead to an increase in protein synthesis and fiber size, a process called hypertrophy. Conversely, the loss of muscle mass, named atrophy, is the result of an increase in protein degradation. Muscle atrophy can occur in several pathological conditions like cancer, diabetes, chronic heart failure, AIDS, and during aging. Mitochondria play a key role in muscle homeostasis, because they provide its high energy demand. Indeed, atrophying conditions are characterized by alterations of the mitochondrial network (Romanello et al., 2010). So, the maintenance of a functional mitochondrial population is important for tissues that are highly structured and metabolically active such as neurons, cardiac and skeletal muscle. In these tissues, mitochondria quality control depends only on mitochondria shaping machinery which include fusion and fission processes. Mitochondrial fusion leads to the formation of an interconnected network, facilitating the redistribution of metabolites, proteins and mtDNA. On the contrary, fission generates isolated and fragmented mitochondria allowing the specific removal of dysfunctional and damaged organelles via mitophagy. The importance of mitochondria fusion and fission in skeletal muscles was underlined by transcriptomic and proteomic analysis showing the progressive loss during ageing of mitochondrial proteins involved in these mechanisms (Ibedunjo et al., 2013). The key player of mitochondria fission is Dynamin Related protein-1, Drp1, a cytosolic guanosine triphosphatase (GTPase) that is recruited to the outer mitochondria membrane (OMM) of damaged mitochondria, where it assembles into multimeric ring complexes inducing mitochondria fragmentation (Smirnova et al., 2001). Recent findings indicate that muscle-specific Drp1 overexpression triggers muscle mass loss and decreases exercise performance (Touvier et al., 2015), confirming previous data demonstrating that overexpression of the fission machinery in skeletal muscle is sufficient to induce mitochondrial dysfunction and muscle atrophy (Romanello et al., 2010). Since overexpression studies are often confounded by off target effects, we decided to use a loss of function approach to study the role of Drp1 in skeletal muscle mass maintenance. Therefore, we generated a muscle-specific Drp1 knock-out mouse model (MLC1f-Drp1 KO). The conditional deletion of Drp1 resulted in a lethal phenotype and an important muscle atrophy. Muscle mass loss is mediated by a decrease in protein synthesis mainly due to the activation of Unfolded Protein Response (UPR) pathway that blocks general translation through eif2α phosphorylation. UPR led also to the up-regulation in skeletal muscle of the cytokine FGF21. The observed FGF21 induction resulted in metabolic changes such as hypoglycemia, growth hormone (GH) resistance and decreased insulin-like growth factor IGF-1 expression in the liver, and a consequent reduced animal size. In addition, Drp1 ablation triggered protein degradation through the induction of FoxO3, a major regulator of protein breakdown. In particular, FoxO3 is the responsible of the increased expression of Atrogin1 and MuRF1, two muscle-specific E3 ubiquitin-ligases, and MUSA1, a novel ubiquitin-ligase, meaning that the Ubiquitin-Proteasome system is activated in knock-out animals. Moreover, real-time PCR analyses revealed the induction of several genes implicated in different step of the autophagic flux, suggesting that also Autophagy-Lysosome system contributes to protein degradation in MLC1f-Drp1 knock-out. Considering the severity of the phenotype, we decided to investigate the role of Drp1 in adult muscles through the generation of muscle-specific inducible Drp1 knock-out mice (HSA-Drp1 KO). We surprisingly found that Drp1 deletion in adulthood resulted in body weight loss secondary to muscle atrophy. Moreover, knock-out mice appeared weaker compared to controls. Indeed, both absolute and normalized force were affected in Drp1-deleted animals, suggesting that muscle weakness was not dependent on muscle atrophy. In addition, the absence of mitochondria division impaired mitochondria size and morphology leading to the presence of enlarged mitochondria with a reduced functionality. In fact, mitochondria membrane potential and respiration efficiency were affected in HSA-Drp1 KO muscles. In order to explain the mechanism involved in muscle atrophy, we found that the activation of UPR pathway, and of degradative systems involved in protein breakdown are conserved between conditional and inducible models. However, in vivo protein synthesis rate is not affected during adulthood. Muscle weakness, UPR activation and mitochondria dysfunction can be triggered by an impairment in calcium physiology. We analyzed calcium homeostasis in flexor digitorum brevis (FDB)-isolated fibers and found that the deletion of Drp1 impaired sarcoplasmic reticulum (SR)-calcium release, which is prevented by the inhibition of mitochondrial calcium uptake through mitochondrial calcium uniporter (MCU) silencing. Importantly, these alterations precede muscle mass loss, suggesting that dysregulation of calcium homeostasis can be the cause of the severe phenotype. These findings indicate that mitochondrial dynamics and mophology are important for the maintenance of the physiology of calcium signaling at least in skeletal muscle. Finally, knock-out mice analyzed after 7 months from Drp1 deletion showed signs of precocious aging and a severe muscle mass loss. Moreover, the chronic Drp1 ablation led to myofibers degeneration and regeneration, as shown by the accumulation of centrally nucleated fibers. All together, our results, strongly support the involvement of mitochondrial morphology and/or function in the activation of signaling pathways that control muscle mass. Thus, therapeutic interventions that maintain a fine equilibrium of mitochondrial fusion/fission processes in needed to preserve muscle mass and prevent muscle wasting.
Author: Giulia Favaro
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2783280509