Title: Role of Autophagy in the control of muscle mass
Abstract: Protein degradation in skeletal muscle cells is essentially mediated by the activity of two highly conserved pathways, the ubiquitin-proteasome and the autophagy-lysosome pathway.
In the ubiquitin-proteasome pathway, target proteins are conjugated to multiple ubiquitin moieties and ubiquitin-tagged proteins are degraded within the proteasome complex (Lecker et al., 2006; Mammucari et al., 2007). The ubiquitin-proteasome system is constitutively active in normal skeletal muscle and is responsible for the turnover of most soluble and myofibrillar muscle proteins.
In the autophagy-lysosome system, portions of cytoplasm and cell organelles are sequestered into vacuoles, called autophagosomes, that are delivered to the lysosomes for the degradation of their content by acidic hydrolases (Lum et al., 2005). Also the autophagy system is constitutively active in skeletal muscle.
The ubiquitin-proteasome system is constitutively active in muscle but its activity increases significantly during muscle atrophy due to activation of two ubiquitin-ligases: Atrogin-1/Mafbx and Murf1 (Gomes et al., 2001). The activation of these two genes is regulated by the transcription factor FoxO3. This factor is normally phosphorylated and inactivated by AKT / PKB. Conversely when this pathway is suppressed (eg during muscle atrophy), FoxO3 translocates into the nucleus where it can transactivate its target genes (Sandri et al., 2004; Stitt et al., 2004).
Alteration of autophagy has been observed in various myopathies caused by genetic defects of lysosomal components, e.g. Pompe's and Danon's disease, or by drugs that inhibit lysosomal function, such as chloroquine (Shintani and Klionsky, 2004).
During muscle atrophy induced by various debilitating conditions (such as fasting and diabetes), there is activation of several genes, named Atrophy-Related-Genes or “Atrogenes”. Among the atrogenes, two most-induced are two ubiquitin-ligases, Atrogin-1 and Murf1. Several autophagy genes belong to the “Atrogenes”. These genes are: LC3, GABARAP and BNIP3.
During the first part of my PhD we focused on the transcriptional regulation of the autophagy genes. Our hypothesis was that FoxO3 can coordinate the ubiquitin-proteasome and the autophagy-lysosome system.
To characterize the mechanisms that control the autophagic/lysosomal pathway during muscle atrophy in vivo, we first determined whether the Akt/mTOR pathway is involved in the regulation of some of autophagy-related genes.
During starvation and denervation, two different models of muscle wasting, the Autophagy-Related-Genes are induced. Moreover these autophagy-related genes are suppressed by Akt, and acute activation of Akt in transgenic mice inhibits autophagy in atrophying muscle. Importantly mTOR pathway did not appear to play a significant role in the activation of the autophagic/lysosomal pathway during muscle atrophy. Indeed the regulation of autophagy-related genes and the formation of autophagic vesicles are not induce either by rapamycin, an inhibitor of mTOR, or by knocking down of mTOR. These findings are in agreement with previous studies (Kochl et al., 2006; Mordier et al., 2000; Sarkar et al., 2007; Yamamoto et al., 2006).
We used gain- and loss-of-function experiments to determine the role of FoxO3 in the autophagic/lysosomal pathway. These experiments found two novel FoxO3 targets that regulate autophagy. LC3 and Bnip3 promoters contain several potential FoxO binding sites and ChIP (Chromatin-ImmunoPrecipitation) experiments on atrophying muscles showed that FoxO3 binds chromatin of their promoters in specific sites. The regions of FoxO3 interaction were cloned upstream luciferase gene and functional studies confirmed that FoxO3 transactivates LC3 and BNIP3 genes. Moreover, loss-function experiments showed that BNIP3 upregulation is necessary for autophagy induction in adult muscle.
Finally, we asked whether the induction of autophagy is secondary to the activation of the ubiquitin-proteasome system. Inhibition of ubiquitin-proteasome system by pharmacological or genetic approach, did not affect autophagy, suggesting that the two degradation pathways are independently controlled by FoxO3 (Mammucari et al., 2007). Thus, FoxO3 coordinates the two major proteolytic systems of the cell.
In the second part of my PhD I focused my studies on the role of basal autophagy in skeletal muscle homeostasis.
It is known that excessive activation of autophagy aggravates muscle wasting by removing portion of cytoplasm, proteins, and organelles (Dobrowolny et al., 2008; Mammucari et al., 2007; Wang et al., 2005; Zhao et al., 2007). Conversely, inhibition of lysosome-dependent degradation causes myopathies like Pompe and Danon diseases, and autophagy inhibition is thought to play a role in many myopathies with inclusions or with abnormal mitochondria (Levine and Kroemer, 2008; Temiz et al., 2009).
To understand the exact role of autophagy in physiology of skeletal muscle we have generated conditional knockout for Atg7 gene to block autophagy specifically in skeletal muscle.
The Atg7 protein is crucial for the formation of the autophagy vesicles by the activations of different Atg proteins and for the formation of the autophagosome.
To understand the role of the autophagy in adult skeletal muscle, Atg7 floxed mice were crossed with mice that express the Cre-recombinase under the muscle-specific promoter Myosin light chain 1f.
Muscle-specific deletion of Atg7, resulted in profound muscle atrophy, accumulation of protein aggregates that are positive for p62/SQSTM1 and age-dependent decrease in force. Moreover Atg7 null muscles showed accumulation of abnormal mitochondria, distension of sarcoplasmic reticulum, sarcomere disorganization, and formation of aberrant concentric membranous structures. Moreover, muscle loss is more exacerbated in autophagy knockout mice during denervation and fasting. These results suggest that the autophagy flux is important to preserve muscle mass and to maintain myofiber integrity. Moreover Atg7 null muscles showed activation of endoplasmic reticulum chaperones, such as BiP, as well as the phosphorylation of eIF2α, suggesting an ongoing unfolded protein response. The failure of protein-folding quality control in Atg7 null mice induces endoplasmic reticulum stress which can generate ROS, and suppression of protein synthesis which can contribute to muscle atrophy (Masiero et al., 2009).
To further confirm our findings in adulthood, we generated a tamoxifen-inducible muscle-specific Atg7 knockout mice. In this case, the floxed Atg7 mice were crossed with mice expressing the Cre-recombinase fused with a modified estrogen receptor, under the control of a muscle-specific promoter (Human Skletal Muscle). When animals are treated with tamoxifen (an estrogen analogue that has a high affinity for the modified estrogen receptor), the Cre-recombinase is stabilized and can recombinate the loxP site.
Identical results were obtained in inducible Atg7 null muscles. These mice showed p62/SQSTM1 accumulation, muscle atrophy and decrease in force generation. Morphological analyses revealed accumulation of abnormal mitochondria in small atrophic fibers and the number of centrally nucleated fibers were more abundant after acute Atg7 deletion than in non-inducible autophagy-deficient muscles (Masiero et al., 2009).
Our results suggest that inhibition/alteration of autophagy can contribute to myofiber degeneration and weakness in muscle disorders characterized by accumulation of abnormal mitochondria and inclusions.
Publication Year: 2010
Publication Date: 2010-01-27
Language: en
Type: article
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