Programme Contents

ACTN3 genotype influences androgen receptor signalling and skeletal muscle mass regulation in health and disease

Muscle wasting due to ageing, disease or chronic illness is a debilitating condition associated with a reduction in quality of life and life expectancy. α-Actinin-3 (ACTN3) is a cytoskeletal protein integral to muscles contractile properties that interacts with a wide array of structural, metabolic and signalling proteins. Homozygosity for the null allele (577XX) results in ACTN3 deficiency in 1 in 5 humans worldwide and is associated with reduced muscle mass and sprint/power performance in elite athletes and the general population.  ACTN3 deficiency is also a known risk factor for falling in the elderly and a genetic modifier of muscle disorders.

Using an Actn3 knockout (KO) mouse that models ACTN3 deficiency in humans we have demonstrated the traits of reduced muscle mass and strength. We are exploring the mechanisms resulting in the reduced mass. Muscle mass regulation involves a complex network of pathways including PI3K/Akt/mTOR and Androgen Receptor (AR) via androgen signalling. Androgens such as testosterone signal skeletal muscle hypertrophy through activation of the AR signalling and PI3K/Akt/mTOR pathways. Studies now show ACTN3 genotype influences muscle mass through regulation of the PI3K/Akt/mTOR pathway. A study of elite Russian athletes (209) showed significantly higher testosterone in male and female athletes carrying the ACTN3 R-allele with ACTN3 genotype explaining >12.5% of variation in testosterone levels (Ahmetov et al., 2014). The α-actinins are known primary co-activators and enhancers for AR activity (Huang et al., 2004) and are known to interact with key players in the PI3K/Akt/mTOR signalling pathway; PI3K, PIP2 and mTOR (Lek and North 2010; Norman et al., 2014). This link between ACTN3 genotype and testosterone levels in elite athletes has focussed our studies on these pathways but how ACTN3 deficiency influences these pathways has not been explored.

Microarray analyses have shown a significant reduction in AR levels at a transcript (∼25%) in Actn3 KO muscles, including transcript expression of androgen responsive genes Odc1, Amd2, Smox and Itgb1bp3. AR protein levels in both skeletal muscles and testes were also greatly reduced in the Actn3 KO. Localisation of AR shown by IHC is also altered, while circulating testosterone levels were unchanged. Effects of androgen deprivation were also investigated by a castration model (N=6 per genotype/treatment) to determine how α-actinin-3 deficiency would influence muscle wasting. Mice were given pre-emptive analgesia (buprenorphine 0.1mg/kg), anaesthetized with isoflurane before receiving either sham or castration surgery. Mice were euthanised 12 weeks post-surgery. Our pilot castration studies show that androgen deprivation may be detrimental to α-actinin-3 deficient individuals with greater response to muscle atrophy.

We have also explored protein synthesis by surface sensing of translation (SUnSET) pathways including PI3K/Akt/mTOR by Western blotting analyses. A sub group of mice were given an intraperitoneal injection of either puromycin (0.04 μmol/g) [WT n=8, KO n=7] or vehicle only (PBS) [WT n=6, KO n=6] were sacrificed, 30 minutes post procedure. Intriguingly, male Actn3 deficient mice also demonstrate increased levels of protein synthesis (P <0.01)specifically in the PI3K/AKT/mTOR pathways. Preliminary findings suggest an up-regulation of TGFβ pathway members including SMAD2, 3 and 4.

These findings suggest ACTN3 genotype influences muscle mass regulation through reduced AR availability and altered regulation of these pathways. Understanding how ACTN3, PI3K/Akt/mTOR and AR signalling interact, we will provide insights into muscle wasting conditions and their treatments.