Regulation of Limb Muscle Mass during Hypogonadism

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy-mediated protein breakdown pathway that persist throughout the diurnal cycle. The regulatory signals that contribute to the increase in autophagy markers remain ill defined. The purpose of Aim 1 was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. We hypothesized that androgen deprivation will increase signals that are known to induce the initiation and progression of the autophagy process. To do this, male mice were subjected to a castration surgery or a sham surgery to serve as a control. Seven-weeks post-surgery, a subset of mice from each group were sacrificed every 4 hrs over a 24 hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. The phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice in a manner consistent with an overall increase in autophagy. Regulatory signals that induce activation and the progression of autophagy were also increased including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). We also observed an increase in the protein expression pattern of p53 and the mRNA of p53 target genes Cyclin Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These findings characterize novel changes in autophagy regulatory signals in the limb skeletal muscle following androgen deprivation. Although the factors that underlie the increase in autophagy markers in the muscle in response to androgen deprivation remain unknown, we have shown markers of reactive oxygen species (ROS) were elevated in the TA of mice following androgen depletion. These markers coincided with markers of mitochondrial degradation pathways, suggesting impaired mitochondria may be generating excess ROS. As mitochondrial derived ROS can induce muscle atrophy at least in part through autophagy activation, the purpose of the study in Aim 2 was to determine whether treatment with an antioxidant blunts preserves limb muscle mass and reduces markers of autophagy and mitophagy in response to androgen depletion. We hypothesize that an antioxidant will preserve limb muscle mass and reduce markers of autophagy and mitophagy in response to androgen deprivation. To determine this, male mice were subjected to a castration or sham surgery to serve as a control. Three days post-surgery, a subset of castrated mice remained untreated while the other castrated mice began treatment with a more general antioxidant resveratrol (RSV) or the mitochondrial specific antioxidant, MitoQ. Mice were sacrificed 8-weeks post-surgery. Treatment with MitoQ, but not RSV, partially blunted the loss of lean mass and forelimb grip strength in response to androgen deprivation. MitoQ, but not RSV, also partially preserved mass of the limb muscles. Despite this partial preservation, markers of autophagy and mitophagy were largely unaffected by antioxidant treatment. These data show that treatment with a mitochondrial targeted antioxidant can partially preserve limb muscle mass in response to androgen deprivation without altering markers of autophagy or mitophagy.