Mechanism of myostatin action during satellite cell activation and muscle wasting.

Myostatin, a Transforming Growth Factor-beta (TGF-β) superfamily member, has been well characterised as a negative regulator of muscle growth and development. In support, inactivation or mutation of the myostatin gene results in a dramatic increase in skeletal muscle mass, however excess myostatin inhibits myogenesis. Recently, myostatin has also been shown to have a role in post-natal muscle growth. Myostatin regulates activation, proliferation and self-renewal of the muscle satellite cell pool. Moreover, loss of myostatin results in enhanced skeletal muscle regeneration in response to injury, whereas increased post-natal myostatin expression is associated with many skeletal muscle wasting conditions. Furthermore, myostatin has been shown to directly induce cachexia following subcutaneous injection of Myostatin over-expressing cells into mice. Despite studies implicating myostatin in the regulation of post-natal skeletal muscle growth, little is known about the processes through which myostatin activity is regulated or the mechanisms through which myostatin functions. Thus this thesis examines regulation of myostatin activity through proteolytic processing, and signaling mechanisms through which myostatin acts to regulate the satellite cell pool and to promote skeletal muscle proteolysis. In this thesis it is demonstrated that processing and secretion of Myostatin is relatively reduced in differentiated myotubes as compared to proliferating myoblasts. Furthermore, processing of Myostatin is developmentally regulated, with decreased Myostatin processing occurring during foetal muscle development when compared to post-natal adult muscle. It is also demonstrated that mature Myostatin negatively regulates furin promoter activity. Furin protease is critical for the processing of several members of the TGF-β superfamily, thus a mechanism is proposed whereby myostatin negatively auto-regulates its proteolytic processing during development to facilitate the process of myoblast differentiation. It was further demonstrated that over-expression of Pax7 in C3H10T1/2 multipotent cells enhances myogenic conversion in these cells. However, over-expression of Pax7 in C2C12 myoblasts delays the onset of differentiation, concomitant with an increase in the population of quiescent, satellite cell-like reserve cells. Furthermore, treatment with Myostatin down-regulates Pax7 expression, while Pax7 expression was higher in myostatin-null myoblasts as compared to wild-type myoblast cultures. Furthermore, absence of myostatin alters cell heterogeneity, whereby an increase in Pax7+/MyoD- reserve cell populations is observed. Pax7 expression persists longer through differentiation in cultured primary myoblasts from myostatin-null animals when compared to wild-type counterparts. Reserve cell populations were also measured, and consistent with increased expression of Pax7, there is an increased pool of quiescent self-renewed reserve cells in differentiated cultures from myostatin-null mice as compared with wild-type cultures. Taken together, these results suggest that increased expression of Pax7 regulates the self-renewal process of satellite cells, and furthermore, growth factors such as myostatin signal through Pax7 to regulate the self-renewed pool of satellite cells. In this thesis it is shown that myostatin induces cachexia through a mechanism independent of NF-κB. Myostatin treatment results in a cachexia phenotype with a reduction in myotube number and size in vitro, as well as a loss of body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 are reduced, while NF-κB localisation and expression remains unchanged. Expression of the ubiquitin-associated genes Atrogin-1, MuRF-1 and E214k are shown to be up-regulated following Myostatin treatment. The mechanism behind myostatin-mediated cachexia was further investigated. It is shown that myostatin antagonises the IGF-1/PI3-K/AKT hypertrophy pathway by inhibiting AKT phosphorylation, thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. In addition, microarray analysis resulted in the identification of a potentially interesting downstream target gene of myostatin during the induction of cachexia. Initial characterisation of CXXC5, or MM1 as it was renamed, was also performed. Over-expression of MM1 in vitro results in the up-regulation of components of the ubiquitin-proteasome pathway including Atrogin-1, E214k, E220k and RC2. It was further demonstrated that increased expression of MM1 enhances the level of protein ubiquitin-conjugation. Furthermore, over-expression of MM1 results in myotube collapse and the formation of multinucleated myosacs. It was also demonstrated that the MM1-induced myotube collapse results in disruption of the typical myotube microtubule structure. Therefore, these data suggest that MM1 is a muscle wasting-inducing gene which functions in the regulation of myostatin-mediated cachexia. Therefore data presented in this thesis highlights a mechanism through which myostatin is regulated, and further delineates the role of myostatin in controlling several key processes during post-natal myogenesis, namely satellite cell replenishment and skeletal muscle wasting

Myostatin signals through Pax7 to regulate satellite cell self-renewal

Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells

Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3−/− mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3−/− mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3−/− muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3−/− skeletal muscle. The exaggerated ROS in the Smad3−/− muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy

Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3−/− mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3−/− mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3−/− muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3−/− skeletal muscle. The exaggerated ROS in the Smad3−/− muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy

Myostatin Augments Muscle-Specific Ring Finger Protein-1 Expression Through an NF-kB Independent Mechanism in SMAD3 Null Muscle

Smad (Sma and Mad-related protein) 2/3 are downstream signaling molecules for TGF-β and myostatin (Mstn). Recently, Mstn was shown to induce reactive oxygen species (ROS) in skeletal muscle via canonical Smad3, nuclear factor-κB, and TNF-α pathway. However, mice lacking Smad3 display skeletal muscle atrophy due to increased Mstn levels. Hence, our aims were first to investigate whether Mstn induced muscle atrophy in Smad3(-/-) mice by increasing ROS and second to delineate Smad3-independent signaling mechanism for Mstn-induced ROS. Herein we show that Smad3(-/-) mice have increased ROS levels in skeletal muscle, and inactivation of Mstn in these mice partially ablates the oxidative stress. Furthermore, ROS induction by Mstn in Smad3(-/-) muscle was not via nuclear factor-κB (p65) signaling but due to activated p38, ERK MAPK signaling and enhanced IL-6 levels. Consequently, TNF-α, nicotinamide adenine dinucleotide phosphate oxidase, and xanthine oxidase levels were up-regulated, which led to an increase in ROS production in Smad3(-/-) skeletal muscle. The exaggerated ROS in the Smad3(-/-) muscle potentiated binding of C/EBP homology protein transcription factor to MuRF1 promoter, resulting in enhanced MuRF1 levels leading to muscle atrophy