Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is genetic myopathy which is inherited in an autosomal dominant manner. FSHD is the third most common form of muscular dystrophy, affecting approximately 1:7500 individuals and is characterised by progressive skeletal muscle weakness and wasting of the facial, scapular and humeral muscles. FSHD stems from a genetic mutation in a D4Z4 repeat on the qter of chromosome 4 and the shortening of the repeat, coupled with hypomethylation of the 5'–C–phosphate–G–3'’s results in the aberrant expression of protein called double homeobox 4 (DUX4). DUX4 is expressed at very low levels within healthy individuals, however within FSHD patients, DUX4 is expressed in 8% of atrophic myonuclei and 10% of disorganised myonuclei. This expression of DUX4 stimulates a pathological cascade driving oxidative stress, apoptosis, inflammation and impaired myogenesis. To date, there is no cure and effective therapies are limited. Previous research has focused on the inflammation within the disease, however, recent research is showing that oxidative stress may actually be the main pathogenic driver.
There is limited research into antioxidant therapy as a therapy for FSHD and those that have investigated this have had no real success. Antioxidant research regularly focuses on the development of novel antioxidant compounds, often overlooking natural antioxidants within the human body. High-density lipoproteins (HDLs), primarily known for their role in reverse cholesterol transport, also demonstrate potent antioxidant, anti-inflammatory and anti-apoptotic properties. These protective properties have been best demonstrated in atherosclerosis, however also extend to other diseases associated with oxidative stress. Consequently, the aim of this thesis was to investigate the use of HDL-based therapy in FSHD. Based on the now well-characterised cellular protective effects of HDLs, we hypothesise that HDL treatment will reduce DUX4-mediated cellular damage both in vitro and in vivo.
To investigate the effects of HDL treatment, we first had to characterise our in vitro FSHD model. C2C12 (murine skeletal muscle) cells were transfected with DUX4 via a liposomal gene transfer system and assessed for levels of oxidative stress and cell death in both low-, and high, oxygen concentrations to mimic the varying levels of oxidative stress that is characteristic of FSHD skeletal muscle. In both low- and high- oxygen culture environments, DUX4 expression increased oxidative stress and cell death. DUX4 also impaired myotube formation and disturbed myoblast organisation, confirming the model was phenotypically active. To confirm that DUX4 was increasing susceptibility to oxidative stress, transfected cells were exposed to 30 μM hydrogen peroxide (H2O2) to stimulate an oxidative stress response. DUX4 demonstrably rendered cells more susceptible to oxidative stress as determined by levels of reactive oxygen species, cell death and myotube formation. Excitingly, treatment with a physiological concentration of HDL (21 µM) for 16 hours alleviated DUX4-mediated oxidative stress and cell death in both low-, and high, oxygen conditions. Moreover, HDL treatment was able to improve myotube organisation in the more damaging high oxygen conditions.
As HDLs had proven protective in a model of early stage FSHD, we next wanted to investigate their ability to protect against DUX4-mediated cellular damage in a progressive model of FSHD. For this study, a lentiviral gene transfer system was utilised as this leads to chronic DUX4 expression, where DUX4 can diffuse to nearby nuclei and further induce DUX4 expression. HDL treatment was able to significantly abrogate DUX4-mediated oxidative stress, cell death and myotube formation within this progressive FSHD model. Interestingly, low-density lipoproteins (LDLs), which were used as a lipoprotein control, significantly increased DUX4-mediated cellular damage, indicating that increased circulating LDL cholesterol may exacerbate the disease process in FSHD patients.
As HDLs were shown to be protective in vitro, we next wanted to investigate their ability to protect against DUX4-mediated cellular damage in vivo. C57Bl/6 mice received an intramuscular injection of DUX4 lentiviral particles into the hind limb and either received 21 µM HDL treatment or vehicle control (1xPBS). Interestingly, DUX4 induced sex-specific effects on cellular damage and the protective effects of HDLs were also sex-specific. HDL treatment decreased cytosolic DNA/RNA oxidative damage in males, however had no effect in females. Conversely, HDL treatment increased levels of lipid peroxidation in males. HDL treatment had no obvious effect on oxidative stress markers in females, however it did preserve DUX4 positive atrophic fibre diameter, whereas this had no effect in males. Moreover, HDL treatment was able to reduce levels of apoptosis in both males and females, indicating that HDLs effect are target specific within each sex.
The results obtained from this study have identified that HDL-based therapies offer promising results in vitro, however their use in vivo is more complex and requires more attention. Our results indicate that in vivo, HDL particles are potentially being remodelled and there are sex-specific effects. There are hints that HDLs could be protective, especially against changes in skeletal muscle architecture and apoptosis (cleaved caspase 3), however, more essential research is required. Fundamentally, we have shown that in vitro, HDLs are protective, but as other research has found when it comes to in vivo, HDL-therapy needs component by component, and sex-dependent experiments to fully understand the mechanisms in vivo.