Progress Update: Novel Therapeutics for FSHD: Mitochondria-targeted Antioxidants

Posted by Friends of FSH Research on Feb 17, 2024

Update by Dr. Heher
see grant Novel Therapeutics for FSHD: Mitochondria-targeted Antioxidants

Metabolic stress can be understood as a situation where the energy-producing factories within our cells, called mitochondria, are not working as they should. Mitochondria generate energy to keep our muscles and other tissues functioning properly. When they malfunction, it can lead to an imbalance in the production of certain molecules called reactive oxygen species (ROS). These ROS are highly reactive chemical compounds that, when produced excessively cause oxidative stress, which can damage our cells and cause a gradual energy deficit. So, metabolic stress due to mitochondrial dysfunction essentially means that there is trouble in the energy-producing process within our cells, which is a particular problem in muscle, where energy demand is variable and can be very high.

Muscles affected by Facioscapulohumeral Muscular Dystrophy (FSHD) exhibit a variety of metabolic disruptions. While oxidative stress through dysfunctional mitochondria has been recognized as contributor to muscle issues triggered by the FSHD-causative DUX4 protein, the underlying mechanisms leading to prolonged metabolic strain in FSHD remain unclear. Oxidative stress can be alleviated by antioxidants, compounds that can counteract ROS. Antioxidants have shown some success in improving muscle function in clinical trials in FSHD patients.

In this project, we speculated that the efficacy of these therapies could be enhanced by gaining a deeper understanding of the mechanisms driving oxidative stress generation in FSHD. Therefore, this work aimed to pinpoint the specific biochemical sources of the surplus reactive oxygen species (ROS) observed in FSHD muscle cells. The primary objective was to investigate whether the generation of mitochondrial radical oxygen species (mitoROS) due to mitochondrial dysfunction is a central mechanism underlying FSHD pathology/symptoms. Additionally, we sought to explore the involvement of DUX4 in inducing oxidative stress within mitochondria and assess the therapeutic potential of compounds targeting mitochondria for treating FSHD to develop and evaluate more targeted antioxidant-based treatments.

In the first year of the project, we identified mitochondria as the main source of ROS in muscle cells from FSHD patients. We found that alterations in mitochondrial function occur early in the disease process, with increased production of mitoROS driven by DUX4-induced changes in a part of the mitochondrial energy-producing system that is called the respiratory chain. In the respiratory chain, oxygen is used to then allow energy production in a process called oxidative phosphorylation (OXPHOS). This process makes mitochondria the major energy producer, but also the main oxygen consuming system in a cell. Interestingly, oxidative stress was further exacerbated when muscle cells making DUX4 were grown under oxygen levels reflecting the muscle oxygenation in muscle in people (physiological), which is much lower than atmospheric oxygen levels. DUX4 caused even more oxidative stress and muscle cell death in a condition were oxygen levels are very low, as might transiently occur in muscle after heavy exercise (hypoxia). This is relevant as working muscles are subject to constant changes in oxygen availability, for example when we exercise. When oxygen levels decrease, muscles typically adjust their metabolism by utilizing alternative compounds to produce energy instead of relying on oxygen, which is normally efficiently used in the mitochondria. This adaptation process is known as metabolic switching and is controlled by a cellular sensing mechanism known as the molecular response to hypoxia. Hypoxia-inducible factor 1α (HIF1α) plays a crucial role in coordinating this response, and we found that its regulation is disrupted by DUX4. Since ROS produced from mitochondria (mitoROS) can also impair HIF1α, we tested if antioxidants that specifically localize to the mitochondria are more potent in reducing DUX4 toxicity compared to conventional non-targeted general antioxidants. Indeed, supplementation with mitochondria-targeted antioxidants proved more effective in mitigating cell death caused by DUX4 under physiological oxygen conditions, reducing oxidative stress and counteracting HIF1α signaling perturbation in muscle cells derived from FSHD patients.

In year 2 of the project, our focus shifted towards identifying the precise mechanism by which FSHD mitochondria produce excess ROS – and how FSHD cell metabolism differs from that of muscle cells from FSHD-unaffected people. We discovered that mitoROS are released from specific sites within the mitochondrial respiratory chain, inhibition of which strongly reduces build-up of oxidative stress and cell death caused by DUX4. The central role of this mechanism in FSHD pathogenesis is further underscored by our observation that DUX4 toxicity is attenuated in cells lacking a functional mitochondrial respiratory chain, thus relying on non-oxygen consuming pathways to generate energy. Having established the precise location of ROS release from the respiratory chain in the mitochondria allowed us to further refine our antioxidant-based approach by using compounds that localize not just within mitochondria, but act on distinct sites within the mitochondrial respiratory chain. For example, targeting defective FSHD mitochondria with mitochondria-targeted coenzyme Q10 (mitoQ10), a known modifier of the respiratory chain, proved particularly effective in reducing oxidative stress and cell death in response to DUX4.

Our findings were further supported by analyses of gene expression in muscle biopsies from FSHD patients, which revealed dysregulation of genes related to mitochondrial function (particularly oxidative metabolism through OXPHOS), oxidative stress and glycolytic, non-oxidative (i.e. not using OXPHOS) metabolism.

Finally, by directly measuring the types of energy generation occurring in muscle cells (respirometry), we found that FSHD cells produce less energy than unaffected controls, and display a metabolic mismatch between the energy production rates via oxidative (OXPHOS) versus non-oxidative means. Specifically, mitochondrial energy production through OXPHOS is impaired, which is the most effective way to produce energy in cells. Consequently, FSHD cells were also found to have altered levels of compounds associated with energy production and metabolism (the metabolome), specifically under physiologically relevant oxygen levels.

As a result of this comprehensive investigation, we have identified new potential pathomechanisms of FSHD that are related to metabolic switching in response to varying oxygen availability. Further, targeted antioxidant-based approaches such as the use of respiratory chain modifiers like mitoQ10 are being explored as potential therapeutic candidates for FSHD, with the aim of alleviating disease symptoms and improving patient outcomes.