The reduced mechanical loading of space flight is associated with decreased skeletal muscle fiber cross sectional area (CSA) and force-generating capacity. Muscle atrophy and weakness may then impede mission objectives, increase the risk of injury, and increase susceptibility to damage upon reloading. Muscular alterations with spaceflight are therefore a critical limitation to performance during space flight missions and a serious health concern for astronauts. Recent evidence suggests that oxidative stress is increased in microgravity and contributes to skeletal muscle atrophy. Oxidative stress is classically defined as an imbalance between reactive oxygen species (ROS) production and removal by endogenous antioxidants. ROS are produced primarily from the mitochondria as well as nonphagocytic NAPDH-oxidases (Nox) in skeletal muscle and can have various harmful effects without adequate antioxidant sequestration. ROS have been shown to activate different proteolytic pathways during skeletal muscle disuse and may induce translocation of the muscle specific neuronal nitric oxide synthase-µ (nNOSµ) splice variant from the sarcolemma to the cytosol leading to muscle atrophy. The purpose of this investigation was to develop novel, targeted countermeasures to abolish the effects of oxidative stress and prevent muscle atrophy during microgravity.
The experimental design consisted of a series of studies utilizing a seven day hindlimb-unloading (HU) rat model of microgravity to evaluate the therapeutic effectiveness of different antioxidants/ROS-inhibitors. The rat models were used to test two specific aims: 1) Determine the efficacy of countermeasures that raise HSP70 against unloading-induced muscle atrophy; and 2) Identify the contribution of renin-angiotensin II signaling to oxidative stress and muscle atrophy with mechanical unloading by utilizing the angiotensin-II receptor blocker, losartan.
EUK-134 abolished skeletal muscle atrophy as measured by muscle mass and Type I fiber CSA. Inhibition of muscle atrophy was related to increased expression of nNOS at the membrane in conjunction with associated proteins, dysferlin and caveolin-3. In addition, EUK-134 increased anabolic signaling via the serine/threonine-specific protein kinase, Akt. Specifically, Akt serine 473 and mTOR serine 2448 phosphorylation were increased in HU-EUK-134 animals and p70-S6k threonine 389 phosphorylation was preserved.
More targeted peptidyl inhibitors of ROS also prevent muscle atrophy with mechanical unloading and suggest the source of ROS production. The peptidyl NADPH-oxidase-2 (Nox2) inhibitor, gp91 ds-tat, and a peptidyl mitochondrial ROS inhibitor, SS-31, are being utilized during 7D hindlimb unloading protocols, and data indicates that both inhibitors improve soleus muscle mass and fiber CSA. This indicates that the source of ROS may be both Nox2 and mitochondria during simulated microgravity.
The angiotensin II receptor blocker, Losartan, prevents the reduction in soleus muscle fiber CSA following 7D of hindlimb unloading. Angiotensin II has been identified as a potent stimulus for ROS production in skeletal muscle via the G-protein coupled angiotensin type 1 receptor (AT1R). The AT1 receptor blocker, Losartan is therefore also being utilized for molecular targeting of ROS. Data collected suggests that Losartan is effective in preventing the reduction in soleus muscle fiber CSA.