br Materials and methods br Results br Discussion
Materials and methods
Discussion It is intriguing that NAC improved muscle lactate levels and clinical performance probably related to its antioxidant and anti-inflammatory effect, but it was not able to improve mitochondrial function at normoxia. In general, mitochondria dysfunction, oxidative stress and inflammation are related events . Ischemia/reperfusion (I/R) results in insufficient oxygen supply, mitochondriopathy leading to reduced AT13387 supply, oxidative stress, and inflammation, which lead to the clinical signs of peripheral artery disease . However, most of our knowledge in this sense came from acute I/R . We here demonstrated that these could be unrelated events in a long-term model. The choice of antioxidant in our study could partially explain this apparent paradox. The prevention of mitochondrial ROS formation by NAC was not associated with a decrease in the phosphorylation of eNOS in a model of endothelial I/R . In the same way, the beneficial effects of NAC in ischemic heart failure are partially mediated by the reduction of PKA activity . Thus, some of the effects induced by ROS and the protective effects of NAC are independent of mitochondrial dysfunction and this corroborates our findings. Additionally, NAC is not a mitochondrial-targeted antioxidant, and the effects of such a kind of antioxidant could be highly different from NAC. For example, MITO-Tempol was able to scavenge mitochondrial superoxide and preserved mitochondrial respiration in the model of ligation of the femoral artery . Finally, this could be a temporal related event, and since muscle was evaluated at a single time point it is possible that the improvement in oxidative damage and inflammation is an early event and posteriorly an improvement in mitochondrial respiration could be observed. The lack of effect of NAC in improving oxygen consumption could be linked to its inability to modulate pathways associated with mitochondrial regeneration. In our model, at least late after the induction of ischemia, it did not appear that modulation of autophagy or mitophagy occurs. Previous studies elucidate the ability of NAC to attenuate ischemia injury through inhibiting autophagy , , however, recently it was demonstrated that an increase in autophagy and mitophagy are important players during muscle regeneration after ischemia . Our results did not support a major role for the modulation of autophagy or mitophagy on the protective effects of NAC. In addition, NAC could not up-regulate mitochondrial biogenesis that could be another possible mechanism to explain NAC effects in this model. For example, it was recently demonstrated that NAC could attenuate oxidative damage caused by exercise, but not necessarily was beneficial for mitochondrial biogenesis , and this is in accordance to our findings. Despite of this, NAC was able to decrease oxygen consumption under hypoxia. It was observed an increase in ex vivo oxygen consumption under hypoxic conditions, and a decrease ex vivo oxygen consumption under normoxia. This reflects mitochondria adaptation to hypoxia. Continuous hypoxia increased several different aspects of mitochondrial function in the myocardium as well as skeletal muscle that includes COX activity and oxygen consumption , . Besides been an adaptive response to hypoxia, it could also increase the production of ROS, thus the effect of NAC on oxygen consumption could be related to a decreased ROS production by mitochondria. In this context, a decrease of oxygen consumption, mainly under hypoxic conditions, is observed after treatment with hydrogen sulfide . This gas is able to decrease citochrome C activity, decreasing oxygen consumption, thus protecting hypoxic tissues in different animal models of diseases , , . It is demonstrated that NAC could interact with H2S , thus we supposed that NAC could directly decrease oxygen consumption under hypoxia, but we could not demonstrate such kind of NAC effect in an in vitro assay.