Systemic delivery of a mitochondria targeted antioxidant partially preserves limb muscle mass and grip strength in response to androgen deprivation
Introduction
Maintaining a critical amount of muscle mass is important for physical function and overall health (Powers et al., 2016; Srikanthan and Karlamangla, 2014; Srikanthan et al., 2016). In males, a decrease in testicular androgen production (hypogonadism) contributes to the loss of muscle mass that occurs during aging and various pathological conditions (Bhasin et al., 1997; Bhasin et al., 2005). Indeed, a longitudinal study showed that older males with higher levels of testosterone maintained lean mass over a 4.5 year period (LeBlanc et al., 2011). While hypogonadism impacts various muscle groups, the loss of limb muscle is particularly important as these muscles comprise a majority of total muscle mass (Kim et al., 2002), and they are the primary muscle involved with functional tasks such as walking and climbing stairs. Androgen replacement is the only effective pharmacological therapy to blunt the loss of limb muscle in hypogonadal individuals (Ferrando et al., 2002; Ferrando et al., 2003), but it is not a universal option due to side effects (e.g. enhanced malignant tumor growth and adverse cardiac events) (Metzger and Burnett, 2016; Fowler and Whitmore, 1982; Amos-Landgraf et al., 2014). Therefore, it is imperative to define new treatments that can preserve limb muscle mass in response to androgen deprivation.
It is thought that androgens regulate muscle mass by signaling through the androgen receptor (Ophoff et al., 2009; Serra et al., 2013). While this appears to be true for certain muscles (e.g. levator ani) (Serra et al., 2013), the androgen receptor is dispensable for regulating mass of the limb muscles (Altuwaijri et al., 2004; Ueberschlag-Pitiot et al., 2017). For example, the presence of androgens themselves, not a functional androgen receptor, regulated mass of the tibialis anterior (TA) muscle in rodents (Ueberschlag-Pitiot et al., 2017). More recently, myofiber-specific deletion of the androgen receptor did not prevent androgen-mediated growth of limb muscles in female mice (Sakakibara et al., 2021). While androgens do not appear to regulate limb muscle mass via the androgen receptor, the pathways by which androgens mediate limb muscle mass, particularly when androgen production is compromised, remain almost completely unknown, limiting therapeutic options.
Because the TA muscle mass is sensitive to androgens in an androgen receptor independent manner (Ueberschlag-Pitiot et al., 2017; Sakakibara et al., 2021), our laboratory has characterized many of the intramuscular signaling events that change in this muscle in response to androgen deprivation (Steiner et al., 2017; Rossetti et al., 2018; Rossetti et al., 2019; Rossetti et al., 2020). Specifically, we showed that markers of impaired mitochondrial quality were elevated in the TA in response to androgen deprivation including higher reactive oxygen species (ROS) content (e.g. H2O2) and ROS reactive byproducts (e.g. 4-hydroxynonenol; 4HNE). These markers coincided with markers of mitochondrial degradation pathway activation comprising BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and the PTEN-induced kinase 1 (PINK1)/PARKIN pathways, which clear damaged or dysfunctional mitochondria via the autophagy/lysosomal system (Serra et al., 2013; Rossetti et al., 2018, 2019; Rossetti et al., 2018a, Rossetti et al., 2018b). Accordingly, we and others have shown markers of autophagy/lysosomal pathway were also elevated in the limb muscle in response to androgen deprivation, including an increase in the ratio of the lipidated (II) to non-lipidated (I) forms of microtubule-associated protein light chain 3 (LC3) and a decrease in p62 protein content (Steiner et al., 2017; Rossetti et al., 2019; Rossetti and Gordon, 2017). Although these markers of impaired mitochondrial quality and increased ROS were not present at all time points throughout the day, these markers were inversely related to TA mass when present (Rossetti et al., 2018a, Rossetti et al., 2018b), suggesting they could be contributing to limb muscle atrophy. The markers of increased ROS were of particular interest given that high levels of ROS, especially ROS generated by mitochondria, contribute to muscle atrophy in other conditions (e.g. limb muscle immobilization and mechanical ventilation) (Min et al., 2011; Powers et al., 2011). Therefore, the purpose of this study was to determine whether systemic administration of an antioxidant preserved limb muscle mass in response to androgen deprivation. We provide evidence that systemic administration of an antioxidant that targets the mitochondria partially preserved limb muscle mass and grip strength in response to androgen deprivation.
Section snippets
Study #1: antioxidant gene expression
The TA muscle samples analyzed for diurnal antioxidant gene expression were generated from a study previously conducted by our laboratory (Rossetti et al., 2019). In brief, male C57Bl/6NHsd mice (14 weeks of age) were purchased from Envigo (Indianapolis, IN) and subjected to either a sham or castration surgery. Mice recovered for 8 weeks prior to sacrifice. At sacrifice, tissues were harvested from a subset of mice from each treatment group every 4 h beginning at the onset of the dark cycle
Antioxidant defense gene expression is lower in the TA muscle following androgen deprivation
The mRNA content of superoxide dismutase 1 and 2 (Sod1 & Sod2), which scavenge free superoxide radicals in the cytosol and mitochondria (Fukai and Ushio-Fukai, 2011), respectively, was overall lower in the TA muscle throughout the diurnal cycle following androgen deprivation (Fig. 1A and B; p ≤ 0.001). The mRNA content of catalase and glutathione peroxidase 1 (Gpx1), which detoxify hydrogen peroxide predominantly in the cytosol, was also overall lower in the TA muscle throughout the diurnal
Discussion
The decrease in androgen production that occurs in response to various pathological conditions contributes to the long-term atrophy of limb skeletal muscles (Ferrando et al., 2003; Steiner et al., 2017; White et al., 2013a; White et al., 2013b). Similar to previous work in other atrophic conditions (Min et al., 2011; Powers et al., 2011), we show that systemic administration of a mitochondria targeted antioxidant can partially preserve limb muscle mass and grip strength following androgen
Funding
The National Institute of Health (grants R01AG064951, R56AG067754, R21AR077387) supported BFM. The funding source had no role in the design or execution of this study.
Acknowledgements
The authors would like to thank Frederick Peelor for analysis of myofibrillar and mitochondrial fractional synthetic rates.
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