We have previously demonstrated that Ang II-induced skeletal muscle wasting is related to anorexigenic and food intake-independent catabolic effects of Ang II that lead to increased protein degradation and apoptosis of muscle [4
]. Our present study showed that the catabolic effect of Ang II is mediated, at least in part, via increased superoxides produced by NADPH oxidase.
A potential link between ROS and physiological effects of Ang II was first suggested by the demonstration that Ang II increased NAD(P)H activity and superoxide production in cultured vascular smooth muscle cells [18
], and that the effect of Ang II on blood pressure was reduced by the administration of superoxide dismutase [19
]. Subsequent studies provided evidence for an important role for ROS and particularly superoxide in Ang II-induced signaling, contributing to cardiac myocyte and vascular smooth muscle cell hypertrophy, endothelial dysfunction, hypertension, and insulin resistance [20
]. However, there are very few studies on the effect of Ang II on skeletal muscle. Russell et al.
demonstrated that murine C2C12 myotubes produced ROS in response to Ang II. This effect was inhibited by diphenyleneiodonium, implying the participation of NADPH oxidase, and was accompanied by increased protein degradation which was blunted by the use of antioxidants [22
]. In addition, the expression of Nox2
] subunits of the NADPH oxidase has been detected in skeletal muscle, suggesting that these NADPH oxidase subunits have a functional role in regulating skeletal muscle oxidative stress. In contrast to the results of Russell et al.
our laboratory has not detected significant expression of Ang II receptors on C2C12 cells (Delafontaine, unpublished results), which is consistent with other reports [25
] and with our prior studies that have indicated that the wasting effect of Ang II is mediated at least in part via intermediate cytokines such as IL-6 and serum amyloid A [26
]. Furthermore, we have previously shown that Ang II markedly increases urinary corticosterone levels in the mouse and that the glucocorticoid inhibitor RU486 blunted Ang II-induced wasting [27
]. Our current study demonstrates that irrespective of the downstream mediators of Ang II-induced wasting an increase in oxidative stress appears to be critically required for this effect of Ang II. Ang II-induced oxidative stress was accompanied by an increase in muscle 20S proteasome activity (), and these effects were blocked in p47phox−/−
mice. Interestingly, we did not find that the hypertensive response to Ang II was inhibited in p47phox−/−
mice, in contrast to a previous report from Landmesser et al.
] However, it is pertinent to note that Landmesser et al.
used a lower dose of Ang II (486 ng/kg/min vs. 1500 ng/k/min in our study) and that the hypertensive response to Ang II in their study was only partially blocked in p47phox−/−
mice. Our results indicate that the inhibition of Ang II-induced muscle atrophy in the p47phox−/−
mice was independent of Ang II effects on systolic blood pressure. This is consistent with our prior data [4
], which showed that the reduction in body weight induced by Ang II was not altered by normalization of blood pressure with hydralazine, but was markedly blunted by the Ang II AT1 receptor antagonist, losartan.
It is of note that our data does not exclude the possibility of Ang II-induced superoxide formation from other sources such as mitochondria. NADPH oxidase-dependent superoxide production in response to Ang II may be very important in diseases such as hypertension. Thus, vascular activation of superoxide production was attenuated in the aorta of p47phox−/−
mice infused with Ang II [28
]. However, this enzyme complex is not the only cellular source of superoxide that can be affected by this peptide. Ang II activates mitochondrial ROS formation in endothelial [29
], vascular smooth muscle cells and in rat aorta in vivo
]. Furthermore, there is positive feedback among enzymes producing ROS. Thus, ROS are able to directly activate NADPH oxidase to produce more ROS in vascular smooth muscle cells [31
]. It is been speculated that NADPH oxidase-induced ROS could directly stimulate the mitochondria [30
]. Indeed, myocardial mitochondrial ATP-sensitive potassium channels are activated by cytosolic superoxides derived from NADPH oxidase [32
], and opening of these channels leads to mitochondrial ROS release [33
]. Further studies are required to determine potential cross-talk mechanisms involving NADPH oxidase and mitochondria and their contribution to Ang II-dependent atrophy signaling.
Skeletal muscle wasting is a major contributor to negative outcomes in conditions such as cancer, chronic kidney disease and advanced congestive heart failure. Our findings indicate that Ang II-induced catabolic effects that lead to skeletal muscle wasting are redox-dependent and involve NADPH oxidase. The implication that superoxide formation is part of the signaling pathways contributing to skeletal muscle atrophy could be important in developing new drugs targeting muscle wasting in different conditions.