A wealth of data suggests AET as a key intervention for prevention and treatment in cardiology. Recent reports demonstrate protection provided by AET against HF-induced skeletal myopathy 
. However, the molecular mechanisms by which AET delay or reverse skeletal muscle myopathy in HF remain elusive. Several key findings emerge from this study, in which we analyzed the contribution of AET in preventing plantaris atrophy in sympathetic hyperactivity induced-HF mice: (a) progressive skeletal muscle loss in α2A
ARKO mice was associated with increasingly oxidative stress and proteasomal activity; (b) UPS overactivation and oxidative damage were detected when plantaris muscle atrophy was established in 7 month-old α2A
ARKO mice; (c) AET efficiently reestablished plantaris phenotype, UPS and oxidative stress to WT levels. In human HF: (d) increased skeletal muscle proteasome activity suggests overactivation of UPS and (e) AET restored proteasome activity to healthy control levels.
Overwhelming evidence demonstrates a striking association between disease-induced skeletal muscle atrophy and UPS activation 
, which also occurs in HF 
. In fact, ubiquitination of skeletal muscle contractile proteins has been suggested in HF 
. In line with these findings, we demonstrate that plantaris atrophy in HF mice is associated with UPS overactivation and that these phenomena display interesting relationship with severity of the disease, since skeletal myopathy was associated with worsening cardiac function and clinical signs of HF.
At 3 months of age, when α2A
ARKO mice display preserved cardiac function and exercise tolerance, plantaris hypertrophy is observed in comparison with WT, which might be explained by β2
-adrenoceptor overactivation due to sympathetic hyperactivity, as previously demonstrated 
. Moreover, decreased proteasomal activity in 3 month-old α2A
ARKO mice indicates reduced skeletal muscle proteolysis, favoring muscle hypertrophy. This finding might also be related to β2
-adrenoceptor overactivation due to sympathetic hyperactivity, which activates hypertrophic signaling pathways besides inhibiting UPS activation in skeletal muscle in atrophic states 
. At 5 months of age, skeletal muscle hypertrophy was no longer observed and proteasomal activity was similar between α2A
ARKO and WT. However, when severe HF was established in 7 month-old α2A
ARKO mice, plantaris muscle atrophy and substantial proteasomal overactivation were detected. Among the possible mechanisms contributing to these observations, oxidative stress should be highlighted, since increasing lipid hydroperoxidation and protein carbonylation from 3 to 7 months of age were observed in α2A
ARKO mice and redox unbalance is known to modulate UPS activation and leads to skeletal muscle atrophy.
Increased oxidative stress arises from imbalance between pro- and antioxidant activity 
and is depicted in skeletal muscle under catabolic or dysfunctional states 
. Importantly, while increased oxidative stress through superoxide dismutase (SOD) deletion accelerates aging-induced skeletal muscle atrophy 
, antioxidant treatment effectively attenuates skeletal muscle loss in a cancer model 
. Therefore, strong evidence of redox imbalance-induced skeletal muscle atrophy supports our hypothesis that oxidative damage is a major determinant of skeletal muscle loss in HF.
UPS modulation by redox balance depends upon oxidative damage extension. Protein damage by mild or moderate redox imbalance increases UPS substrate availability, causing elevation of proteasome activity 
. Conversely, severe oxidative damage impairs substrate tagging by E3 ligases and causes proteasomal dysfunction due to accumulation of non-degradable aggregates 
, resulting in overall UPS inactivation. Therefore, we suggest that skeletal muscle oxidative stress reaches moderate levels in 7 month-old α2A
ARKO mice, since accumulation of lipid hydroperoxides and carbonylated proteins were observed concomitantly with UPS overactivation.
Studies suggest proteasome inhibition as a treatment against skeletal muscle loss 
. However, potentially dangerous effects of such intervention must be considered, since the UPS is a major effector of the protein quality control mechanism in all cells 
. In fact, cardiac dysfunction occurs when the proteasome is inhibited in vivo
. In contrast, AET undoubtedly promotes beneficial effects in several tissues, including cardiac and skeletal muscles in HF 
. Thus, we have recently shown that AET prevents skeletal muscle atrophy in our experimental model 
, and here we extend our findings to the preventive effect of AET on oxidative stress and UPS overactivation.
Restoration of redox balance by AET is probably driven by antioxidant defense, such as augmented activity of free radical scavengers and reduced levels of inflammatory cytokines 
. Accordingly, we demonstrate here that AET reduced skeletal muscle lipid hydroperoxidation and protein carbonylation, accounting for reduced intracellular stress and relief of UPS overload. Therefore, reduced UPS activation by AET possibly occurred due to improvements in redox balance. These results suggest that AET ultimately counteracts increased protein degradation by the UPS.
Besides UPS overactivation by oxidative stress, it may also be purposed that redox unbalance is involved in HF-induced skeletal myopathy by reducing skeletal muscle regenerative capacity through disturbance of satellite cell pool or differentiation rate 
. Indeed, direct negative effects of oxidative stress on skeletal muscle satellite cells have been reported 
. Furthermore, it has been shown that HF patients present depressed IGF-1 signaling in skeletal muscle 
and that anabolic effects of IGF-1 are partially attributed to satellite cells activation 
. Following this rationale and considering that satellite cells activation is the leading process mediating muscle regeneration, it is also reasonable to speculate that anti-atrophic effects of AET could be blunted in our model, even though we observed several beneficial outcomes. In this sense, investigation of satellite cells participation in cardiac cachexia is a promising topic for future studies.
In human HF, AET also improved aerobic capacity (VO2
peak) and work economy, which were mainly due to skeletal muscle improvements, since cardiac function did not differ between sedentary and trained HF patients (28±5 vs. 35±2% are EF values in sedentary and trained HF patients, respectively; p>0.05). Importantly, skeletal muscle UPS overactivation is suggested by increased proteasome activity in sedentary HF patients, corroborating findings of a recent study that presented increased abundance of MuRF1 in skeletal muscle in a larger population of HF patients, regardless of age 
. This same study also showed that MuRF1 expression is reduced by AET, which goes in line with our finding that AET reverted 26 S Proteasome overactivation in HF patients.
Increased proteasomal activity was not accompanied by increased protein carbonylation, indicating that our HF patients displayed only mild skeletal muscle oxidative stress, but sufficient to induce myofibrillar protein damage and proteasomal activation. It is important to highlight that all HF patients were under β-blockade, ACE inhibition and statins, which independently provide antioxidant effects 
and may have relieved skeletal muscle oxidative stress. However, we reinforce the role of AET in HF treatment by showing that even optimal drug treatment does not improve aerobic capacity and could not maintain skeletal muscle proteasomal activity, which is clearly achieved by AET.
Our study shows that AET reduced oxidative stress, UPS overactivation and prevented skeletal muscle atrophy in HF mice, however, it does not provide direct evidence of cause-effect among these findings. However, our hypothesis that relieved oxidative stress counteracts UPS overactivation is partly supported by the literature 
. One might argue that our sympathetic hyperactivity-induced HF model is not the most similar to human HF, however, we provided strong evidence that the progression of HF in our model recapitulates many aspects of human HF 
. Since enrolled HF patients were under optimal pharmacological therapy, we could not isolate the effects of AET. Additionally, small biopsy fragments didn’t allow further exploration of UPS modulation and evaluation of mild oxidative stress indicators, such as lipid hydroperoxidation. Left ventricular FS values in WT mice were lower than found in our previous work 
, which probably occurred due to the anesthetic agent used in the present study (halothane presently used vs. isoflurane previously used 
). Even though halothane depresses cardiac contractility in a higher degree than isoflurane 
, we were able to reproduce the same pattern of cardiac dysfunction in our HF model and the effects of exercise training 
In conclusion, we provide evidence that AET prevents skeletal muscle oxidative stress and UPS activation in HF mice, which probably contributes to prevention of skeletal myopathy. The clinical relevance of the present investigation is demonstrated by attenuation in skeletal muscle proteasome activity in exercise-trained HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen AET as an efficient non-pharmacological tool for HF therapy.