Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs' regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.

Metabolic rescue of muscle and muscle stem cells in muscle disease

Peiling Luan

Duchenne muscular dystrophy (DMD), caused by the mutation of dystrophin gene, is an X-linked disorder that affects 1 in 3500 males, leading to progressive muscle degeneration and eventually resulting in premature death. Increasing evidence indicates that DMD and age-related sarcopenia share clinical hallmarks, such as decreased metabolic activity of both myofibers and muscle stem cells (MuSCs), which results in defective MuSC function and impaired muscle regeneration. In my Ph.D. thesis, I focused on manipulating mitochondrial function, such as mitophagy, and lipid metabolism, particularly sphingolipid metabolic pathways, to restore muscle and MuSC functions in DMD and age-associated sarcopenia. My thesis consists of three projects: Rescue of mitophagy improves muscle function in DMD. In this project, we identified defective mitophagy as a hallmark of muscular dystrophy. Rescue of dysfunctional mitophagy by dietary supplementation of Urolithin A (UA), a metabolite present in fruit extracts and a potent activator of mitophagy, improved muscle function in mdx mice. The beneficial effects of mitophagy restoration observed in dystrophic animals were attributed to the improvement in mitochondrial function, structural integrity of muscles, restoration of MuSC activity, and reduction in general inflammation and fibrosis. Sphingolipid depletion improves muscle regeneration during ageing. We first established the link between sphingolipid metabolism and ageing by demonstrating that sphingolipids accumulate in skeletal muscle upon aging. We reduced skeletal muscle sphingolipid accumulation by treating aged mice with myriocin, a selective inhibitor of serine pamitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid de novo synthesis pathway. Sphingolipid depletion increased MuSC regenerative ability through enhancing the proliferation and differentiation capacities of MuSCs, ultimately leading to the prevention of age-related decline in muscle mass and function. Inhibition of sphingolipid synthesis reverts muscular dystrophy. By studying skeletal muscle transcript profiles of human muscular dystrophies, we identified upregulation of sphingolipid biosynthetic pathway as a universal feature of human patients affected with muscular dystrophy. To rescue the aberrant sphingolipid metabolism, we treated mdx mice with myriocin and showed that inhibition of sphingolipid generation enhanced muscle function, leading to amelioration of DMD. Furthermore, we uncovered a novel role of sphingolipid depletion in macrophage polarization and in reconstruction of the mdx MuSC niche, resulting in improved regenerative capacity of MuSCs. In summary, our work establishes the essential roles of mitophagy and sphingolipid metabolism in maintaining muscle and MuSC function. Restoration of mitophagy and blockade of sphingolipid generation could therefore be attractive treatment strategies to delay the progression of DMD and age-related sarcopenia.

EPFL2020

Metabolic control of muscle and muscle stem cell function

Hongbo Zhang

Skeletal muscle composed of myofibers and a small amount of muscle stem cells (MuSCs). It plays important roles in energy metabolism. Some of the key metabolites, such as NAD+ and acetyl-CoA were recently found to regulate muscle and MuSCs function. Most of these functions are related to the cellular mitochondria. In my PhD thesis projects, we aimed at exploring the link between NAD+ levels, mitochondrial function, muscle structural homeostasis and MuSCs senescence. We also investigated the impact of acetyl-CoA on MuSCs activation and proliferation, through the control of protein acetylation. These projects mainly focus on three aspects: Rejuvenation of adult stem cells by NAD+ repletion. Aging is accompanied by SCs senescence. However, the initial signaling events mediating SCs senescence are still not well defined. We demonstrate the importance of mitochondrial activity as a pivotal modulator of MuSCs senescence. MuSCs from aged mice have reduced NAD+ levels, mitochondrial content and capacity for oxidative respiration. Importantly, the induction of the mitochondrial unfolded protein response (UPRmt), subsequent to increasing cellular NAD+ with the precursor nicotinamide riboside (NR), prevents MuSCs senescence and improves muscle function and regeneration in aged mice. NR also prevents MuSCs dysfunction in the mdx mouse, a known mouse model of accelerated MuSCs senescence. Extending these observations to other SC pools and on the organism as a whole, we demonstrate that NR delays neural and melanocyte SCs senescence, while also increasing mouse lifespan. Control of MuSCs function by acetyltransferase KAT2A. MuSCs are essential for skeletal muscle homeostasis and repair. The complete mechanism controlling MuSCs maintenance and differentiation, however, remains elusive. We found that the acetyltransferase KAT2A expression is essential for myogenic differentiation of MuSCs. KAT2A loss-of-function in cells blocks MuSCs differentiation, and in mice impairs muscle regeneration after damage. The regulation of KAT2A in MuSCs function might rely on its ability to enzymatically acetylate other proteins, and in particular the transcription factor PAX7. NAD+ repletion improves muscle function in muscular dystrophy. Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations that lead to progressive muscle degeneration. Our bioinformatics analysis in mice and DMD patients suggests a role for NAD+ in protecting the muscle from metabolic and structural degeneration. Validating these findings, we reveal that the mdx mouse is characterized by reductions in muscle NAD+ levels, concurrent to increased PARP activity and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the key enzymes for NAD+ consumption and biosynthesis. Replenishing the NAD+ pool by dietary NR supplementation benefits muscle function in mdx and mdx/Utr-/- mice, an effect replicated in C. elegans models of DMD. The beneficial effects of NAD+ repletion in muscular dystrophy are pleiotropic and rely on the improvement in mitochondrial function, structural protein expression and on reductions in general PARylation, inflammation and fibrosis. In summary, our work demonstrates the critical role of NAD+ and Acetyl-coA in maintaining muscle and MuSCs function. Repletion of NAD+ levels, by NR administration, and/or boosting the function of acetyltransferase KAT2A might be attractive strategies to maintain muscle homeostasis and MuSCs function in aging and muscular dystrophy.

EPFL2016

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans

Xu Wang

Using an unbiased high-throughput microRNA (miRNA)-silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA-204-5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA-204-5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA-204-5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC-1 alpha (3.71-fold; p < .01), predicted as an miR-204-5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA-204-5p silencing in C2C12 myotubes. Silencing of miRNA-204-5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA-204-5p silencing. Finally, miRNA-204-5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA-204-5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA-204-5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA-204-5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

2020