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Introduction: Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II type 2 receptor (AT2R) potentiates skeletal muscle stem (satellite) cell (SC) differentiation and muscle regenerative capacity. However, mechanisms whereby AT2R regulates SC differentiation, and its potential involvement in cachexia development remain unknown. Methods: AT2R siRNA was transfected to primary cultured SCs and the changes in AT2R downstream signaling pathways were analyzed by phosphoprotein array, on which 1318 unique antibodies against various phosphoproteins are spotted. The identified signaling pathway was pharmacologically and genetically manipulated both in vitro and in vivo, and the skeletal muscle regenerative capacity was analyzed by qRT-PCR, western blotting and immunohistochemistry. Results: We found that 20 phosphoproteins were upregulated (>1.4 fold change) and 28 were downregulated (<0.7 fold change) after AT2R knockdown. Among these proteins, we found an increase in p-Akt (Ser473) and p-GSK-3β (Ser9) during SC differentiation both in vitro (SC culture) and in vivo (injury-induced skeletal muscle regeneration), suggesting an activation of β-catenin pathway. Consistently, β-catenin TCF/LEF reporter (TOPFlash) assay showed an increase in β-catenin activity both in vitro and in vivo. siRNA-mediated AT2R knockdown in cultured SCs significantly decreased p-Akt (Ser473) and p-GSK-3β(Ser9) phosphorylation and TOPFlash activity, and importantly, inhibited SC differentiation. Treatment of AT2R knocked-down SCs with GSK-3βinhibitor BIO or Wnt3A restored β-catenin activity, resulting in the restoration of SC differentiation. These data indicate that AT2R positively regulates SC differentiation via Akt/GSK-3β/β-catenin pathway. Importantly, we found that skeletal muscle regeneration was reduced (decreased regenerating myofiber size and myogenin/desmin expression) in a mouse myocardial infarction model of CHF, concomitant with marked reduction in AT2R expression and decreased β-catenin activity. In these mice, hindlimb injection of BIO restored β-catenin activity and muscle regenerative capacity. Conclusions: These data indicate that AT2R/Akt/GSK-3β/β-catenin pathway plays a critical role in regulating SC differentiation and muscle regeneration, and could be a novel therapeutic target in wasting disorders such as CHF and CKD. [ABSTRACT FROM AUTHOR]

Introduction: Patients with advanced congestive heart failure (CHF) or chronic kidney disease (CKD) often have increased angiotensin II (Ang II) levels and cachexia. We previously demonstrated that Ang II infusion in rodents causes skeletal muscle wasting, likely contributing to cachexia in CHF and CKD. We also demonstrated that Ang II type 1 receptor (AT1R) activation in skeletal muscle stem (satellite) cells (SCs) inhibits SC proliferation and muscle regenerative capacity. We hypothesize that Ang II-mediated SC inhibition contributes to the development of muscle wasting in high Ang II condition. Methods: We generated tamoxifen-inducible, SC-specific AT1R knockout mice (SC-AT1R-/-; generated by crossing SC-specific Pax7CreERand AT1R floxed mice). By breeding SC-AT1R-/-and control mice with Cre-reporter mouse line, SCs and their progenies were labeled with EYFP. These mice were infused with Ang II (1.5 μg/kg/min) and muscle regeneration and wasting were analyzed by qRT-PCR, western blotting and immunohistochemistry. Results: Ang II reduced skeletal muscle regeneration in vivo, indicated by the reduced number of regenerating myofibers in hindlimb muscles after cardiotoxin injury (71.9% decrease vs. control, p<0.001), and by the reduction of SC proliferation/differentiation markers MyoD and myogenin (56.5% and 62.5% decrease, respectively, by qRT-PCR, p<0.001). In contrast, hindlimb muscles of SC-AT1R-/-mice were protected against Ang II-mediated reduction in skeletal muscle regenerative capacity; the number of regenerating myofibers and expression of MyoD and myogenin were restored. Ang II inhibited primary cultured SC proliferation via AT1R signaling in vitro, and SC proliferative capacity was restored in AT1R null SCs. Importantly, we found that there was a significant recovery from Ang II-induced muscle wasting in SC-AT1R-/-mice. SC lineage tracing (EYFP labeling) and gene expression analyses revealed that AT1R-deficient SCs had significantly higher regenerative capacity in high Ang II condition, leading to increased number of newly formed/repaired myofibers (12.4±2.7% of total myofibers in SC-AT1R-/-and ND in control, p<0.001), resulting in the restoration of muscle mass and cross sectional area in SC-AT1R-/-mice. Conclusions: These data indicate that inhibition of AT1R signaling in SCs could have a therapeutic potential to treat muscle wasting in chronic diseases with high Ang II, such as CHF and CKD. [ABSTRACT FROM AUTHOR]

Introduction: Skeletal muscle atrophy occurs via two main systems, namely, autophagy and ubiquitin-proteasome system (UPS). Although we have previously shown that angiotensin II (Ang II) induces skeletal muscle atrophy via UPS activation, little is known about Ang II-mediated autophagy in skeletal muscle. Therefore, we aimed to investigate whether Ang II modulates autophagy in skeletal muscle in mice. Methods: FVB mice (10 -12 weeks-old) were infused with Ang II (1.0 μg/kg/day) for 7 days; pair-fed (PF) group was infused with saline and served as control. Tibialis anterior (TA) muscle was used for western blot, RT-qPCR, mitochondrial respiration assay, as well as enzymatic activity, and extensor digitorum longus muscle was used for Transmission Electronic Microscopy (TEM). Results: Ang II infusion reduced mouse body weight (12%, p=0.0001) and caused muscle loss (p&lt0.05). Ang II inhibited phosphorylation of UKL1 at Ser317 (p-ULK1), leading to suppressed p-beclin1 (Ser14) and p-ATG14 (Ser29), critical proteins to initiate autophagosome formation. Ang II also blunted the LC3B-II conversion and increased p62/SQTSM1 protein expression (hallmark of autophagosome cargo accumulation), whereas lysosomal enzymatic activities (cathepsins B and L) were not altered. These data indicate that Ang II impairs the initial step of autophagosome formation. TEM analysis revealed that Ang II caused an accumulation of mitochondria with abnormal cristae and swollen morphology, likely due to reduced autophagosomes. These changes were associated with increased PINK1 protein expression, decreased PGC-1α gene and protein expressions. Importantly, Ang II markedly reduced the oxygen consumption rate in isolated mitochondria. These data strongly indicate that Ang II impairs mitochondrial function and its clearance, which may potentially induce muscle wasting. Conclusions: Here we show that Ang II inhibits critical molecules involved in the initiation of autophagy, which leads to suppression autophagosome formation (e.g. reduced LC3B-II conversion). This likely promotes an accumulation of damaged mitochondria and ubiquitinated proteins. Our data strongly suggest that autophagy is critical to induce skeletal muscle deregulation in Ang II-infused mice and raises the possibility of a new therapeutic target in wasting disorders such as heart failure. [ABSTRACT FROM AUTHOR]