Exploring the interaction between mitochondrial dynamics and autophagy: can we counteract accumulation of dysfunctional mitochondria in FSHD muscle?

Report by Dr. Heher
See also Exploring the interaction between mitochondrial dynamics and autophagy: can we counteract accumulation of dysfunctional mitochondria in FSHD muscle?

Skeletal muscle is the largest tissue in the human body, responsible for movement, posture, and metabolic regulation. Given its regular use, skeletal muscle cells are particularly reliant on efficient cellular quality control mechanisms. Autophagy is a natural cellular process that involves the degradation and recycling of old or dysfunctional cellular components through controlled breakdown of organelles and proteins for subsequent reuse of their building blocks. This process plays a crucial role in maintaining skeletal muscle health and function. A specialized form of autophagy, termed mitophagy, specifically targets and degrades damaged or dysfunctional mitochondria, the energy-producing organelles within cells. Accumulation of damaged mitochondria can be detrimental to the cell, leading to impaired energy production, increased oxidative stress, and activation of cell death pathways. We, and others, have shown that metabolic stress and mitochondrial dysfunction are features of FSHD, and there is subtle evidence that autophagy is also impaired. In this project, we aim to investigate:

1. How autophagy (cell cleaning) and mitophagy (clearance of damaged mitochondria) are affected in FSHD.

2. Whether modulation of autophagy and mitophagy is a potential therapeutic.

Initially, we analyzed whether genes involved in energy production through mitochondria, autophagy, and mitophagy showed different activity in muscles from FSHD patients. We found that many genes differentially regulated between FSHD patients and unaffected controls were related to mitochondrial metabolism. Particularly the tricarboxylic acid (TCA) cycle (energy production), fatty acid production, and mitochondrial electron transport (energy source within mitochondria), alongside impairment of the primary metabolic pathway called glycolysis (sugar breakdown) are affected. Interestingly, comparison of gene activity between inflamed and non-inflamed FSHD muscles from the same patients revealed that muscles without inflammation also had different activity of genes related to autophagy and mitophagy. On the other hand, inflamed FSHD muscles showed more activity in genes related to mitochondria-induced cell death, mitochondrial dysfunction, and responding to damage from oxidative stress (a by-product of mitochondrial energy production). This suggests that early changes in muscle metabolism and cellular recycling processes such as auto- and mitophagy might be important factors in FSHD disease progression (now published in https://doi.org/10.1093/hmg/ddad175).

We have also performed assays to study mitochondrial function in several cell models derived from FSHD patients, using respirometry which measures oxygen consumption (with the vast majority being consumed by mitochondria to make energy). FSHD muscle cells have defects in both glycolytic (i.e. converting sugar into energy) and mitochondrial energy production. Importantly, these processes are also impaired when we made unaffected muscle cells produce the FSHD-causative protein DUX4. We next induced autophagy in FSHD patient-derived cells by ‘starvation’ (culture without core nutrients and metabolites such as sugars, amino acids or growth factors). FSHD cells undergo cell death at higher levels after starvation than unaffected controls, and retain higher mitochondrial metabolic activity, thus producing more reactive oxygen species. Importantly, this shows that FSHD mitochondria act differently than mitochondria from unaffected cells under conditions that promote autophagy, which could potentially further impair FSHD muscle cell function through oxidative stress.

We also tested how lowering oxygen levels (hypoxia - which also promotes autophagy) affects FSHD patient-derived cells. We found that at, low oxygen levels, FSHD cells underwent cell death at higher levels than unaffected controls and experienced higher oxidative stress. Since autophagy is central to cellular adaptation to low-oxygen conditions, we also looked at the activity of genes involved in autophagy at different oxygen levels. Various key autophagy genes were more active at low oxygen levels in healthy cells. However, this response was weaker or even reversed in FSHD cells, demonstrating that FSHD cells inefficiently adjust to changes in their environment because of abnormal autophagy.

We are currently generating FSHD muscle cell lines (so-called ‘reporter’ cells) where we can track autophagy flux and mitochondrial turnover in real time. These cells will be used to study how inhibition or activation of auto- and mitophagy affects FSHD muscle cells.

Understanding how FSHD cells differ in autophagy and mitophagy will allow us to identify the key mechanisms involved in this metabolic dysregulation of the disease and identify potential therapeutics.