Smad3 is a key intracellular signaling mediator for both TGF-β and Myostatin, two major regulators of skeletal muscle growth. Previous published work has revealed pronounced muscle atrophy together with impaired Satellite Cell (SC) functionality in Smad3-null muscles. In the present study, we have further validated a role for Smad3 signaling in skeletal muscle regeneration. Here, we show that Smad3-null mice had incomplete recovery of muscle weight and myofiber size after muscle injury. Histological/Immunohistochemical analysis suggested impaired inflammatory response and reduced number of activated myoblasts during the early stages of muscle regeneration in the M. tibialis anterior muscle of Smad3-null mice. Nascent myofibers formed after muscle injury were also reduced in number. Moreover, Smad3-null regenerated muscle had decreased oxidative enzyme activity and impaired mitochondrial biogenesis, evident by the down-regulation of the gene encoding TFAM, a master regulator of mitochondrial biogenesis. Consistent with known Smad3 function, reduced fibrotic tissue formation was also seen in regenerated Smad3-null muscle. In conclusion, Smad3 deficiency leads to impaired muscle regeneration, which underscores an essential role of Smad3 in post-natal myogenesis. Given the negative role of Myostatin during muscle regeneration, the increased expression of Myostatin observed in Smad3-null muscle may contribute to the regeneration defects.

Background and purpose:  There are very few studies of functional and rehabilitation outcomes in patients with spinal cord injury (SCI) owing to infarction. Methods:  Retrospective chart review of consecutive admissions to a tertiary medical unit specializing in SCI rehabilitation, Melbourne, Australia. All admissions between 1 January 1995 and 31 December 2008 with a recent onset of SCI owing to ischaemia were included. Outcome measures included the following: demographic characteristics, American Spinal Injury Association (ASIA) Impairment Scale (AIS), length of stay (LOS), medical complications, accommodation, support services, continence, mobility and Functional Independence Measure (FIM) motor scores. Outcome measures recorded at admission, discharge and at 12 months post discharge. Results:  Forty-four patients were admitted for rehabilitation (men = 26, 59%), with a median age of 72 years (interquartile range [IQR], 62-79). On admission, 41 (93%) patients had paraplegia. The majority of patients (n = 33, 75%) had an incomplete SCI. Aetiology was vascular in 19 (43%) patients, idiopathic in 11 (25%) and other in 14 (33%). The median LOS in rehabilitation was 85 days (IQR, 24-129). The most common complications were pain (n = 34, 77%), urinary tract infection (n = 25, 57%), spasticity (n = 12, 27%), cardiac failure (n = 11, 25%) and pneumonia (n = 9, 20%). At rehabilitation discharge most patients (n = 35, 80%) had no change in their AIS grade. Despite this, the FIM motor subscale on admission (median = 28; IQR, 21-34) had significantly improved (P < 0.0000) by discharge (median = 66; IQR 42-78). Conclusion:  Despite their comorbidities and limited change in AIS, these patients had significant improvement in functional abilities during impatient rehabilitation.

Classically, PPARβ/δ function was thought to be restricted to enhancing adipocyte differentiation and development of adipose-like cells from other lineages. However, recent studies have revealed a critical role for PPARβ/δ during skeletal muscle growth and regeneration. Although PPARβ/δ has been implicated in regulating myogenesis, little is presently known about the role, and for that matter the mechanism(s) of action of PPARβ/δ in regulating postnatal myogenesis. Here we report for the first time, using a PPARβ/δ-specific ligand (L165041) and the PPARβ/δ-null mouse model, that PPARβ/δ enhances postnatal myogenesis through increasing both myoblast proliferation and differentiation. In addition, we have identified Gasp-1 (Growth and differentiation factor associated serum protein-1) as a novel downstream target of PPARβ/δ in skeletal muscle. In agreement, reduced Gasp-1 expression was detected in PPARβ/δ-null mice muscle tissue. We further report that a highly conserved PPAR responsive element (PPRE) within the 1.5kb proximal Gasp-1 promoter region is critical for PPARβ/δ regulation of Gasp-1. Gasp-1 has been reported to bind to and inhibit the activity of Myostatin; consistent with this, we find enhanced secretion of Gasp-1, increased Gasp-1 Myostatin interaction and significantly reduced Myostatin activity upon L165041-mediated activation of PPARβ/δ. Moreover, we have analyzed the ability of hGASP-1 to regulate myogenesis, independent of PPARβ/δ activation. Results reveal that hGASP-1 protein treatment enhances myoblast proliferation and differentiation, whereas silencing of hGASP-1 results in defective myogenesis. Taken together these data reveal that PPARβ/δ is a positive regulator of skeletal muscle myogenesis, which functions through negatively modulating Myostatin activity via a mechanism involving Gasp-1.

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish that is of conservation concern in North America and Asia. Data on Pacific lamprey population structure are scarce and conflicting, impeding conservation efforts. We optimized 12 polymorphic microsatellite loci for the Pacific lamprey. Three to 13 alleles per locus were observed in a sample of 51 fish collected from the West Fork Illinois River, Oregon. Observed heterozygosity ranged from 0.235 to 0.902 and expected heterozygosity ranged from 0.214 to 0.750. Cross-species amplification produced 8 to 12 polymorphic loci in four other Entosphenus species and in the western brook lamprey (Lampetra richardsoni). Two loci appear to be diagnostic for distinguishing Entosphenus from Lampetra. These markers will be valuable for evaluating population structure and making conservation decisions for E. tridentatus and other lamprey species.

Ubiquitination-mediated proteolysis is a hallmark of skeletal muscle wasting manifested in response to negative growth factors, including myostatin. Thus, the characterization of signaling mechanisms that induce the ubiquitination of intracellular and sarcomeric proteins during skeletal muscle wasting is of great importance. We have recently characterized myostatin as a potent negative regulator of myogenesis and further demonstrated that elevated levels of myostatin in circulation results in the up-regulation of the muscle-specific E3 ligases, Atrogin-1 and muscle ring finger protein 1 (MuRF1). However, the exact signaling mechanisms by which myostatin regulates the expression of Atrogin-1 and MuRF1, as well as the proteins targeted for degradation in response to excess myostatin, remain to be elucidated. In this report, we have demonstrated that myostatin signals through Smad3 (mothers against decapentaplegic homolog 3) to activate forkhead box O1 and Atrogin-1 expression, which further promotes the ubiquitination and subsequent proteasome-mediated degradation of critical sarcomeric proteins. Smad3 signaling was dispensable for myostatin-dependent overexpression of MuRF1. Although down-regulation of Atrogin-1 expression rescued approximately 80% of sarcomeric protein loss induced by myostatin, only about 20% rescue was seen when MuRF1 was silenced, implicating that Atrogin-1 is the predominant E3 ligase through which myostatin manifests skeletal muscle wasting. Furthermore, we have highlighted that Atrogin-1 not only associates with myosin heavy and light chain, but it also ubiquitinates these sarcomeric proteins. Based on presented data we propose a model whereby myostatin induces skeletal muscle wasting through targeting sarcomeric proteins via Smad3-mediated up-regulation of Atrogin-1 and forkhead box O1.

AIMS/HYPOTHESIS: Although myostatin-null (Mstn (-/-)) mice fail to accumulate fat in adipose tissue when fed a high-fat diet (HFD), little is known about the molecular mechanism(s) behind this phenomenon. We therefore sought to identify the signalling pathways through which myostatin regulates accumulation and/or utilisation of fat. METHODS: Wild-type, Mstn (-/-) and wild-type mice treated with soluble activin type IIB receptor (sActRIIB) were fed a control chow diet or an HFD for 12 weeks. Changes in gene expression were measured by microarray and quantitative PCR. Histological changes in white adipose tissue were assessed together with peripheral tissue fatty acid oxidation and changes in circulating hormones following HFD feeding. RESULTS: Our results demonstrate that inactivation of myostatin results in reduced fat accumulation in mice on an HFD. Molecular analysis revealed that metabolic benefits, due to lack of myostatin, are mediated through at least two independent mechanisms. First, lack of myostatin increased fatty acid oxidation in peripheral tissues through induction of enzymes involved in lipolysis and in fatty acid oxidation in mitochondria. Second, inactivation of myostatin also enhanced brown adipose formation in white adipose tissue of Mstn (-/-) mice. Consistent with the above, treatment of HFD-fed wild-type mice with the myostatin antagonist, sActRIIB, reduced the obesity phenotype. CONCLUSIONS/INTERPRETATION: We conclude that absence of myostatin results in enhanced peripheral tissue fatty acid oxidation and increased thermogenesis, culminating in increased fat utilisation and reduced adipose tissue mass. Taken together, our data suggest that anti-myostatin therapeutics could be beneficial in alleviating obesity.

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of "cachectic-like" muscle wasting in mice. The mechanism of action of Myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of Myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which Myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind Myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with Myostatin promoted dramatic myotubular atrophy. Mechanistically, Myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated FOXO1 and FOXO3. We further show that addition of Myostatin results in enhanced activation of Atrogin-1 and MURF1 and reduced expression of both Myosin Light Chain (MYL) and Myosin Heavy Chain (MYH). In addition, we found that Myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and Atrogin-1. Therefore, these data suggest that the mechanism through which Myostatin promotes muscle wasting is very well conserved between species, and that Myostatin-induced human myotube atrophy is mediated through inhibition of IGF/PI3-K/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-β (TGF-β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-)) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2)) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.

Epac1 and Epac2 bind cAMP and mediate cAMP-dependent activation of Rap1. cAMP is produced in neutrophils in response to many chemoattractants. This second messenger plays a key role in the regulation of the functions of neutrophils. However, it is still not known whether Epacs are expressed in human neutrophils. We found that stimulation of PLB-985 cells differentiated into neutrophil-like cells, human neutrophils with 8CPT-2Me-cAMP (a selective activator of Epacs), or FK (a diterpene that augments the intracellular level of cAMP) led to GTP-loading of Rap1. Epac1 mRNA was expressed in UND and DF PLB-985 cells, but Epac1 protein was only detected in DF PLB-985 cells. In human neutrophils, the Epac1 transcript was present, and Epac1 protein could be detected by Western blot analysis if the cells had been treated with the serine protease inhibitor PMSF. FK induced adhesion of PLB-985 cells and human neutrophils on fibrinogen, a ligand for β2 integrins. Interestingly, in DF PLB-985 cells, but not in human neutrophils, 8CPT-2Me-cAMP induced β2 integrin-dependent adhesion. The failure of 8CPT-2Me-cAMP to induce β2 integrin-dependent human neutrophil adhesion could be explained by the fact that this compound did not induce a switch of the β2 integrins from a low-affinity to a high-affinity ligand-binding conformation. We concluded that Epac1 is expressed in human neutrophils and is involved in cAMP-dependent regulation of Rap1. However, the loading of GTP on Rap1 per se is not sufficient to promote activation of β2 integrins.