Myostatin Induces DNA Damage in Skeletal Muscle of Streptozotocin-induced Type 1 Diabetic Mice*

Background: Uncontrolled type 1 diabetes leads to DNA damage and skeletal muscle atrophy. Results: STZ-induced Foxa2 up-regulates Mstn leading to DNA damage via p63/REDD1 pathway in skeletal muscle. Conclusion: Mstn is a target of Foxa2. Blocking Mstn can attenuate DNA damage in the diabetic muscle. Significance: The findings reveal a mechanism of induction of Mstn and DNA damage during diabetes. One of the features of uncontrolled type 1 diabetes is oxidative stress that induces DNA damage and cell death. Skeletal muscle atrophy is also considerable in type 1 diabetes, however, the signaling mechanisms that induce oxidative stress culminating in muscle atrophy are not fully known. Here, we show that in Streptozotocin-induced diabetic wild type mice, hypo-phosphorylation of Akt, resulted in activation of Foxa2 transcription factor in the muscle. Foxa2 transcriptionally up-regulated Myostatin, contributing to exaggerated oxidative stress leading to DNA damage via p63/REDD1 pathway in skeletal muscle of Streptozotocin-treated wild type mice. In Myostatin−/− mice however, Streptozotocin treatment did not reduce Akt phosphorylation despite reduced IRS-1 signaling. Moreover, Foxa2 levels remained unaltered in Myostatin−/− mice, while levels of p63/REDD1 were higher compared with wild type mice. Consistent with these results, relatively less DNA damage and muscle atrophy was observed in Myostatin−/− muscle in response to Streptozotocin treatment. Taken together, our results for the first time show the role of Foxa2 in Myostatin regulation in skeletal muscle in diabetic mice. Altogether, these results demonstrate the mechanism by which Myostatin contributes to DNA damage in skeletal muscle of the diabetic mice that would lead to myofiber degeneration.

One of the features of uncontrolled type 1 diabetes is oxidative stress that induces DNA damage and cell death. Skeletal muscle atrophy is also considerable in type 1 diabetes, however, the signaling mechanisms that induce oxidative stress culminating in muscle atrophy are not fully known. Here, we show that in Streptozotocin-induced diabetic wild type mice, hypo-phosphorylation of Akt, resulted in activation of Foxa2 transcription factor in the muscle. Foxa2 transcriptionally up-regulated Myostatin, contributing to exaggerated oxidative stress leading to DNA damage via p63/REDD1 pathway in skeletal muscle of Streptozotocintreated wild type mice. In Myostatin ؊/؊ mice however, Streptozotocin treatment did not reduce Akt phosphorylation despite reduced IRS-1 signaling. Moreover, Foxa2 levels remained unaltered in Myostatin ؊/؊ mice, while levels of p63/REDD1 were higher compared with wild type mice. Consistent with these results, relatively less DNA damage and muscle atrophy was observed in Myostatin ؊/؊ muscle in response to Streptozotocin treatment. Taken together, our results for the first time show the role of Foxa2 in Myostatin regulation in skeletal muscle in diabetic mice. Altogether, these results demonstrate the mechanism by which Myostatin contributes to DNA damage in skeletal muscle of the diabetic mice that would lead to myofiber degeneration.
Type 1 diabetes is caused by lack of insulin secretion in the body due to pancreatic ␤ cell damage. The increased levels of glucose in circulation dysregulate the prooxidant-antioxidant balance, thus enhancing oxidative stress and reducing antioxidant levels. The exaggerated level of reactive oxygen species (ROS) 2 increases protein degradation and reduces protein syn-thesis eventually leading to skeletal muscle wasting. In addition to proteins, DNA is also susceptible to damage by ROS during type 1 diabetes. Increased ROS, especially hydroxyl (-OH) and superoxide (O 2 . ) radicals can damage DNA by addition to double bonds of DNA bases resulting in increased 8-oxo-dG levels, a mutagenic base by-product used as a marker for oxidative stress. A recent study conducted on type 1 diabetic patients has shown that 8-OHdG levels and level of carbonylated proteins were increased during type 1 diabetes (1). Additionally, DNA damage consists of single and double strand breaks and AP (apurinic/apyrimidinic) sites. The cellular DNA repair machinery comprising base excision repair and nucleotide excision repair is able to repair the above mentioned lesions (2). However, DNA repair mechanisms are also affected by ROS resulting in DNA fragmentation and eventually necrosis of cells. Indeed alterations in the DNA repair capacity and index have been observed in diabetic patients (3). In recent years, regulated in development and DNA damage responses (REDD1), a component of stress response and a developmentally regulated transcriptional target of p63 and p53 (4), has been identified to be involved in oxidative stress-induced DNA damage in skeletal muscle during chronic hypoxia (5). Myostatin (Mstn), a growth and differentiation factor, has been associated closely with skeletal muscle wasting. Our laboratory has shown recently that Mstn can induce ROS and in turn Anti-oxidant enzymes in skeletal muscle through TNF-␣ and NADPH oxidase (Nox) in a feed-forward manner (6). Previously it was reported that Mstn expression and level was up-regulated in skeletal muscle of rodents with type 1 diabetes induced by Streptozotocin (STZ) (7,8,9). Furthermore, Mstn expression was attenuated by insulin administration in STZ-induced type 1 diabetic mice (7). However, the signaling mechanisms by which high glucose levels induce Mstn expression are still unknown.
Since Mstn levels are increased in skeletal muscle during STZ-induced type 1 diabetes and Mstn also acts as a pro-oxidant, the aim of this study was to investigate how Mstn levels are up-regulated during STZ-induced diabetes and whether Mstn can cause DNA damage under hyperglycemic conditions.
Here, we present evidence that STZ-induced hyperglycemia up-regulated Mstn via Foxa2 transcription factor, which in turn induced ROS via TNF-␣ and Nox. The excessive ROS levels caused by high glucose and elevated Mstn levels led to p63/ REDD1 regulated DNA damage. Using in vitro techniques, we demonstrate that Mstn induced single-strand and doublestrand breaks in DNA, eventually leading to DNA fragmentation in muscle cells via p63/REDD1 pathway. Furthermore, inactivation or inhibition of Mstn in skeletal muscle or cells attenuated DNA damage by regulating the DNA damage/repair mechanisms.

EXPERIMENTAL PROCEDURES
Animals-7-week-old C57Bl/6J male mice (WT) were obtained from National University of Singapore-Centre for Animal Resources. Mstn Ϫ/Ϫ male mice (7-week-old) were obtained as previously described (6) and maintained at Nanyang Technological University Animal house. All animals had free access to chow diet and water. All experimental procedures were approved by the Institutional Animal Care and Use Committee, Singapore.
Reagents and Proteins-STZ was purchased from Sigma-Aldrich. Mstn protein containing conditioned medium was obtained from the Mstn expressing CHO cell line (10); control conditioned medium is denoted as CCM and conditioned medium containing Mstn is denoted as CMM in this report. To antagonize Mstn, we used Ant1, a C-terminal truncation of Mstn protein which is a dominant negative (Mstn mimetic) protein.
Ant1 was produced and purified as previously published (11).
Induction of Type 1 Diabetes (Hyperglycemia) by STZ Injection-To generate an acute type 1 diabetes mouse model, WT and Mstn Ϫ/Ϫ mice were intraperitoneally injected with a single high-dose (concentration 22.5 mg/ml) of STZ (150 mg/kg body weight). Mice injected with an equal volume of sodium citrate buffer were maintained as control groups. Nonfasting body weight and random blood glucose levels were determined on the day of injection and subsequently on Day 1,2,4,5,7,8,9,11,12,13,14,15,16,and 18. Mice were fasted for 9 -10 h and body weight and fasting blood glucose was measured on Day 3, 6, 10, and 17. WT-Control and -STZ injected groups are denoted as WT-C and WT-STZ, respectively, while Mstn Ϫ/Ϫ groups are denoted as Mstn Ϫ/Ϫ -C and Mstn Ϫ/Ϫ -STZ in this report. Mice were sacrificed on Day 4, 7, 11, and 18 and hind limb muscles were collected.
Hematoxylin and Eosin Staining-Tibialis anterior muscle sections (10 m) were stained with hematoxylin and eosin as described previously (12). Images were taken at 10ϫ magnification using a Leica upright microscope, equipped with Image Pro Plus software.
Treatment of C2C12 and Primary Myoblasts-STZ at two different concentrations (STZ1 ϭ 0.25 mg/ml; STZ2 ϭ 1 mg/ml) was used to treat proliferating C2C12 myoblasts and primary myoblasts isolated from WT and Mstn Ϫ/Ϫ mice for 48 h. The conditioned medium from CHO cells (CMM) was found to have Mstn at a concentration of 3.5 ng/ml (as determined by Enzyme Immuno Assay (EIA) (Immundiagnostik AG)). Two different concentrations of CMM (CMM1-7 ng; CMM2-17.5 ng) were used to treat C2C12 and primary myoblasts in proliferation medium for 48 h. Ant1 at a concentration of 1 ng/ml was used to pretreat cells 1 h before STZ or CMM treatment.
RT-qPCR (Reverse Transcriptase Quantitative Polymerase Chain Reaction)-RNA was isolated from Gastrocnemius muscle and liver tissue, and RT-qPCR was performed exactly as described in Sriram et al. (6). The forward and reverse primers used are available upon request.
Protein Isolation-Protein lysates from Gastrocnemius muscles and myoblasts were made as previously described (6). Cytoplasmic and nuclear fractions from Biceps femoris muscles were isolated as previously described (16). The protein concentrations were measured by Bradford's assay (17).
Western Blot Analysis-Western blotting was performed as described previously (6). List of primary and secondary antibodies are available upon request.
Electrophoretic Mobility Shift Assay-The Foxa2 binding site was identified using the TFSEARCH tool. The oligonucleotides containing the Foxa2 binding site on mouse Mstn promoter (5Ј-TTTTTTCCCTCAAATATTTGTTTTAGTAACAA-3Ј) were labeled at the 3Ј-end with Biotin Tetra-ethylene glycol (Sigma-Aldrich). The nuclear extracts from WT-C and WT-STZ Biceps femoris muscle were used for the assay. The electrophoretic mobility shift assays were performed using the Lightshift Chemiluminescent EMSA kit (Thermo Scientific) as previously described (6).
Chromatin Immunoprecipitation (ChIP) Assay-HepG2 cells were transfected with 1.7 kb mouse Mstn promoter construct (1.7P) (18) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). ChIP assay was performed according to the published protocol (19). The following set of primers was used for PCR: Foxa2 forward primer 5Ј-GTCAGCTCTTCCTAGTTTTTACTTCTC-3Ј and Foxa2 reverse primer 5Ј-TCCTTTAAGACTTGGAGTGCTGT-3Ј. The resulting PCR products were electrophoresed on 1.5% agarose gel and stained with ethidium bromide.
Luciferase Assay-C2C12 myoblasts were transfected with either pGL3-basic and pFLAG-CMV2 empty vectors or 1.7 kb mouse Mstn promoter construct (1.7P) and pFLAG-CMV2 or 1.7P and pFLAG-Foxa2, together with the control Renilla luciferase vector pRL-TK using Lipofectamine 2000 (Invitrogen), per the manufacturer's guidelines. Sixteen hours after transfection, the medium was replaced with fresh proliferation medium and the myoblasts were incubated for a further 24 h. Luciferase assays were performed using the Dual Luciferase Assay System, as per the manufacturer's protocol (Promega). Relative luciferase activity was measured in triplicate using the Fluoroskan Ascent Microplate Fluorometer and Luminometer (Thermo Fisher Scientific Inc.).
Preparation of Muscle Homogenates for Enzyme Assays-Quadriceps muscle homogenates were made according to a previously published protocol (6), and total protein concentration was measured by Bradford's assay (17).
Estimation of Lipid Peroxidation Product-The lipid peroxidation product (malonaldehyde) was determined as described previously (20).
Estimation of Superoxide Dismutase and Glutathione Peroxidase-The activity of Superoxide dismutase was estimated as described by Sriram et al. (6). Glutathione peroxidase enzyme assay was performed by the modified method of Rotruck et al. (21) as described previously (6).
Estimation of Reduced Glutathione-Reduced glutathione levels were determined by the method of Moron et al. (22).
Analysis of Intracellular ROS Production-ROS production was assayed in C2C12 myoblasts treated with STZ during proliferation as previously described (6) using the fluorescent dye, CM-H2DCFDA (Molecular Probes).
Myoblast Proliferation Assay-Myoblast proliferation assay was performed as previously described (6) using the methylene blue photometric end point assay (23).
Immunohistochemistry for REDD1 and OGG1-OCT-embedded Tibialis anterior muscles were sectioned and fixed with 4% paraformaldehyde in PBS. The immunohistochemistry protocol for REDD1 or OGG1 primary antibodies was followed as per manufacturer's instructions (Proteintech). Images of the stained sections were taken at 5ϫ magnification using a Leica upright microscope.
Immunocytochemistry (ICC) for 8-oxo-dG-C2C12 and primary myoblasts were seeded at a density of 15,000 cells/cm 2 . Next day, proliferation medium with or without STZ1, STZ2, CCM, CMM1, CMM2, and with or without pretreatment with Ant1 was added to the myoblasts and incubated for 48 h. The immunostaining for 8-oxo-dG was performed according to manufacturer's instructions (Trevigen Inc.). Images (DAPIblue; 8-oxo-dG-green) were taken at 10ϫ magnification using a Leica upright microscope.
Comet Assay-C2C12 myoblasts were grown and treated as described in the previous section. A single cell suspension was made and comet assay was performed as previously described (24). The alkaline lysis method was used to detect the combination of single strand breaks, double strand breaks, and alkali-labile sites in the DNA, and neutral lysis method was performed to detect only DNA double strand breaks. Using propidium iodide (PI), the comets were visualized and at least 50 comet images/slide and 3 slides/ treatment were examined. Comet image analysis software was used to quantify various parameters.
Statistical Analysis-The p value was calculated using ANOVA, and p Ͻ 0.05 was considered as significant. Five or seven mice for each treatment were used for various experiments. The results are presented as mean Ϯ S.E. of three independent experiments.

STZ Treatment Induced Muscle Atrophy in
Mice-STZ treatment has been shown to induce hyperglycemia in rodents (25), hence we injected mice with STZ to establish a type 1 diabetes model. The results showed maximum induction of ROS, indicated by expression of TNF-␣ (Fig. 1A) and Nox1 (Fig. 1B) and anti-oxidant enzymes (data not shown) on Day 7 in STZtreated muscles; hence, further experiments were performed using Day 7 STZ-treated muscles. Furthermore by Day 7, the percentage loss of Gastrocnemius and Quadriceps muscle weights normalized to body weight were significantly reduced in WT-STZ and Mstn Ϫ/Ϫ -STZ groups when compared with their respective control groups (data not shown).
Histology of Tibialis anterior muscle revealed that muscle fiber number was reduced in the WT-STZ and Mstn Ϫ/Ϫ -STZ muscle as compared with respective controls (Data not shown). The muscle fiber cross-sectional area (CSA) was significantly decreased in WT-STZ muscle when compared with WT-C muscle (Fig. 1C). Specifically, in WT-C muscle, ϳ40% of muscle fibers only had an area Յ2500 m 2 ; however, in the WT-STZ muscle, ϳ64% were found to have an area Յ2500 m 2 . In Mstn Ϫ/Ϫ -C muscle, ϳ60% of the muscle fibers had an area Ն2500 m 2 , while in the Mstn Ϫ/Ϫ -STZ muscle, ϳ50% fibers had an area Յ2500 m 2 (Fig. 1C). These results confirmed that STZ treatment led to extensive skeletal muscle atrophy in WT mice and relatively less muscle atrophy in Mstn Ϫ/Ϫ mice.
Foxa2 Mediated Up-regulation of Mstn in Response to STZ-The expression and levels of Mstn and its downstream targets, p-Smad2/3 (( Fig. 1D (i), (ii), and (iii)) were significantly up-regulated in WT-STZ muscle (lane 2) when compared with WT-C muscle (lane 1). To investigate the mechanism involved in STZinduced Mstn transcription, an in silico analysis was performed on the 1.7 kb upstream sequence of the mouse Mstn gene to identify various transcription factor binding sites. The sequence analysis identified a putative Foxa2 binding site (5Ј-CAAATATTTGTT-3Ј) within the 1.7 kb sequence of the mouse Mstn promoter. Foxa2 has been shown to regulate glucose homeostasis and glucose-induced insulin release (26). RT-qPCR and Western blot analysis of Foxa2 indicated that Foxa2 mRNA expression and protein level ( Fig. 2A (i) and (ii)) was significantly up-regulated in WT-STZ muscles (lane 2) and barely detectable in Mstn Ϫ/Ϫ muscles even upon STZ treatment (lanes 3 and 4). Next, to determine whether high glucose levels induced by STZ can enhance Foxa2 binding to Mstn promoter, electrophoretic mobility shift assay was performed. As shown in Fig. 2B (i), STZ treatment in WT mice led to increased Foxa2 binding as indicated by the shifted band (lane 3). Furthermore, when nuclear extracts from WT-STZ Biceps femoris muscles were incubated with increasing concentrations of competitor oligos, the disappearance of the shifted band was observed ( Fig. 2B (ii)lanes 3 and 4). The specificity of Foxa2 binding was confirmed using Foxa2 specific antibody; the results showed a supershift of the Foxa2 specific band Myoblasts transfected with 1.7 kb mouse Mstn promoter construct showed an ϳ8.0-fold increase in Luciferase activity, when compared with myoblasts transfected with the empty vectors (Fig. 2D). A further significant increase in Luciferase activity was observed in myoblasts transfected with both Foxa2 expression vector and 1.7 kb mouse Mstn promoter construct when compared with myoblasts transfected with control vectors (Fig. 2D). These data confirm that Foxa2 regulates transcription of Mstn gene.
Western blot analysis for p63, REDD1, and OGG1 was also performed on protein lysates obtained from STZ-treated C2C12 cells with gain or loss of Foxa2 expression. As shown in Fig. 4C (i) and (ii), overexpression of Foxa2 reduced the levels of REDD1 in the untreated (lane 2) and STZ-treated (lanes 4 and 6) cells; while OGG1 levels were increased upon overexpression of Foxa2 and STZ2 treatment (lane 6). In agreement with these results, p63 and REDD1 levels were higher in cells transfected with Foxa2-siRNA and treated with STZ1 as compared with control (lanes 3, 4- Fig.  4D (i) and (ii)). However, higher concentration of STZ (STZ2) reduced the levels of p63 and REDD1 even in cells transfected with Foxa2-siRNA (lane 6) when compared with Foxa2-siRNA transfected and STZ1-treated cells. No significant differences were observed in OGG1 levels in Foxa2-siRNA transfected and STZtreated cells in comparison to control (Fig. 4D).
The effect of STZ or Mstn on DNA damage in C2C12 myoblasts was also evaluated by immunocytochemistry for 8-oxo-dG, a sensitive marker of ROS-induced DNA damage. The intensity of 8-oxo-dG staining was increased upon STZ1 (Fig. 5A) and CMM1 (7 ng) (Fig. 5B) treatment, while Ant1 pre-treatment reduced the staining intensity. In primary myoblasts isolated from WT and Mstn Ϫ/Ϫ mice, STZ (Fig. 5C) and CMM (Fig. 5D) treatment increased DNA damage and Ant1 pre-treatment was able to protect WT myoblasts from DNA damage, even upon treatment with higher concentrations of STZ (STZ2) or CMM (CMM2-17.5 ng). In Mstn Ϫ/Ϫ myoblasts, there was no change in the intensity of 8-oxo-dG staining upon treatment with two different concentrations of STZ (Fig. 5C) or CMM (Fig. 5D). These results indicated that STZ and Mstn induced DNA damage in myoblasts that was attenuated by inhibition of Mstn.

STZ and CMM Cause Single Strand Breaks and Double Strand Breaks in DNA in Proliferating Myoblasts-Comet
assay was performed to detect single strand breaks and double strand breaks in DNA. The arrows in Fig. 6A (i) (alkaline lysis) and (ii) (neutral lysis) indicate DNA fragmentation and damage, as detected by the increased comet tail length and height, in C2C12 myoblasts caused by STZ. Pretreatment with Ant1 reduced the comet formation to the level similar to untreated myoblasts (quantitation data not shown).
brought the levels of these parameters back to levels similar to CCM-treated myoblasts (Fig. 6, C and D).
STZ Treatment Altered Genes Involved in DNA Repair in Vivo-In muscles, Western blot analyses for XRCC1, a protein involved in repair of single strand breaks in DNA, and Histone H2A.X, involved in DNA double strand break repair were performed. The results indicated that XRCC1 protein levels were significantly reduced in WT-STZ muscles (lane 2), while there was no change observed in Mstn Ϫ/Ϫ muscles (lanes 3 and 4), when compared with WT-C muscles (lane 1) (Fig. 7A (i) and (ii)). The level of phosphorylated Histone H2A.X was increased and total Histone H2A.X was correspondingly decreased in WT-STZ muscles (lane 2) when compared with WT-C muscles (lane 1) (Fig. 7A (i) and (ii)). Mstn Ϫ/Ϫ -C and Mstn Ϫ/Ϫ -STZ muscles (lanes 3 and 4, respectively) showed higher levels of phosphorylated and total Histone H2A.X when compared with WT-C muscles (lane 1) (Fig. 7A (i) and (ii)).

STZ and CMM Alter p63/REDD1 Signaling and DNA Repair Genes in Proliferating Primary
Myoblasts-The effect of STZ and Mstn on p63 and REDD1 levels and DNA repair genes was also analyzed in STZ-(data not shown) or Mstn-treated (Fig. 7B (i) and (ii)) WT and Mstn Ϫ/Ϫ primary myoblasts. The results show that p63 levels were decreased in CMM1-and CMM2treated WT myoblasts in a dose-dependent manner (lanes 2 and 3), while only CMM2 treatment reduced p63 level in Mstn Ϫ/Ϫ myoblasts (lane 9) (Fig. 7B (i) and (ii)). CMM1 and CMM2 treatment increased OGG1 protein levels (lanes 2 and 3), while Ant1 pretreatment rescued this increase (lanes 4 -6), when compared with CCM-treated WT primary myoblasts (lane 1). In Mstn Ϫ/Ϫ myoblasts, the increase in OGG1 levels on CMM treatment (lanes 8 and 9) was lesser than CMM-treated WT myoblasts. CMM2 treatment on WT myoblasts reduced REDD1 and p-Histone H2A.X levels (lane 3), while Ant1 pretreatment rescued the levels (lanes 4 -6). CMM2 treatment on FIGURE 5. STZ-induced DNA damage in proliferating myoblasts is attenuated by antagonizing Mstn. A, immunocytochemistry was performed for 8-oxo-dG on proliferating C2C12 myoblasts treated for 48 h with or without STZ1 and with or without pre-treatment of Ant1. The fluorescence for 8-oxo-dG (green) and DAPI (blue) was viewed under a Leica upright microscope and images were taken at 10ϫ magnification. Increased fluorescence is directly proportional to increased DNA damage; scale bar represents 100 m (n ϭ 3). B, 8-oxo-dG immunostaining in proliferating C2C12 myoblasts treated for 48 h with CCM, CMM1, Ant1ϩCCM or Ant1ϩCMM1. Images were taken as mentioned above in A; scale bar represents 100 m (n ϭ 3). Immunostaining for 8-oxo-dG was performed on WT and Mstn Ϫ/Ϫ primary myoblasts treated for 48 h during proliferation with STZ1, STZ2, Ant1 alone, Ant1ϩSTZ1, or Ant1ϩSTZ2 (C) or with CCM, CMM1, CMM2, Ant1ϩCCM, Ant1ϩCMM1, or Ant1ϩCMM2 (D). Representative images showing an increase or decrease in 8-oxo-dG staining. Images were taken as mentioned above in A; scale bar represents 100 m (n ϭ 3).

DISCUSSION
Type 1 diabetes is characterized by lack of insulin, hyperglycemia and oxidative stress. In this report, we elucidate the mechanisms by which STZ treatment leads to an increase in Mstn expression through activation of Foxa2 transcription factor. Enhanced Mstn contributes to ROS that causes DNA damage mediated by p63/REDD1 in the muscle of diabetic mice.
Over the recent years, various studies have shown that Mstn expression is increased in STZ treated mice (7-9), however the mechanism behind the increase in Mstn levels was not identified. Since, our results presented here showed an increase in Mstn RNA and protein levels, we surmised that Mstn could be regulated at the transcriptional level during type 1 diabetes. Indeed, in silico analysis of Mstn 5Ј-upregulatory sequences revealed the presence of a binding site for Foxa2, a transcription factor regulated by insulin (30). Previous studies demonstrated that in the absence of insulin, Foxa2 activity and level was enhanced in the liver. Foxa2 has been reported to regulate genes implicated in glucose (26) and lipid metabolism in the liver (28).
Our results indicate that in the muscle from STZ-treated WT mice, the Foxa2 expression and protein was up-regulated ( Fig Previously it has been reported that insulin signaling inhibits Foxa2 activity by phosphorylation mediated by Akt (28,30). Accordingly, Akt phosphorylation was down regulated upon STZ treatment in WT mice (Fig. 3A (i) and (ii), lanes 1 and 2) leading to higher Foxa2 and Mstn levels. These findings not only indicate that one of the transcriptional targets of Foxa2 is Mstn but also extend the role of Foxa2 in skeletal muscle of diabetic mice. However, despite developing overt diabetes, the levels of Foxa2 were significantly lower in Mstn Ϫ/Ϫ muscle even after treatment with STZ, which we propose could be due to enhanced Akt signaling in these mice (Fig. 3A (i) and (ii), lanes  3 and 4). Furthermore, Foxa2 has been reported to auto regulate its expression through a positive feedback loop (31). Such an autoregulatory mechanism would help maintain increased levels of Foxa2 in STZ-treated WT mice and lower levels in Mstn Ϫ/Ϫ mice.
In line with earlier findings (32,33,34), increased ROS was observed (and thus Anti-oxidant enzymes) in skeletal muscles of WT diabetic mice via TNF-␣, Nox1 (Fig. 1, A and B) and lipid peroxidation (data not shown). However, our results revealed no change in these parameters in Mstn Ϫ/Ϫ -STZ mice (Fig. 1, A  and B) indicating that in the absence of Mstn these mice resisted the induction of ROS by STZ. ROS are potent molecules that are toxic to the cells; if uncontrolled, lead to protein degradation as well as DNA damage. It is evident that STZinduced ROS can lead to DNA damage (35,36). In this communication, we show for the first time that significantly higher levels of Mstn-induced ROS led to DNA damage and inhibited DNA repair systems in the skeletal muscle. Earlier, it has been suggested that STZ also has a direct effect on myoblasts manifested by an up-regulation in ROS and inhibition of myoblast proliferation (29). Regardless of direct or indirect effect, we noticed not only an increase in ROS but also Foxa2 in STZtreated myoblasts thus recapitulating in vivo effects of STZ on muscle.
Earlier, Andican and Bursac reported oxidative DNA damage during STZ-induced type 1 diabetes with a concomitant increase in oxidized DNA bases like 8-oxo-dG in the liver of STZ treated diabetic rats (37). Furthermore, 8-oxo-dG content was found to be higher in the skeletal muscle of diabetic patients (38). Similarly, occurrence of higher levels of OGG1 has been reported in pancreas of diabetic patients that was correlated with DNA damage (39). Consistently, our results demonstrate that both STZ and Mstn can cause an increase in 8-oxo-dG and OGG1 indicative of DNA damage and repair respectively and inhibition of Mstn partially reversed these changes. FIGURE 6. STZ or CMM treatment leads to single strand breaks and double strand breaks in DNA in proliferating myoblasts. A, comet assay was performed by alkaline lysis method (i) and neutral lysis method (ii) on C2C12 myoblasts treated with STZ1, Ant1, or Ant1ϩSTZ1 for 48 h during proliferation. Representative images showing the head and tail of comets detected by PI stain during various treatments. DNA fragmentation is observed by the formation of tail in the comet as indicated by arrows. Comet images were taken using a Leica upright microscope (n ϭ 3). B, comet assay was performed by alkaline lysis method (i) and neutral lysis method (ii) on C2C12 myoblasts treated with CCM, CMM1, CMM2, Ant1ϩCCM, Ant1ϩCMM1, or Ant1ϩCMM2 for 48 h during proliferation. Representative images showing the head and tail of comets detected by PI stain during various treatments. DNA fragmentation is observed by the formation of tail in the comet as indicated by arrows. Comet images were taken using a Leica upright microscope. Quantitative analysis of various parameters of comet assay following alkaline lysis (C) and neutral lysis (D); comet length and comet height (C i) and (D i), comet mean intensity (C ii), head mean intensity (C ii) and (D ii), % DNA in head and % DNA in tail (C iii) and (D iii), tail length (C iv) and (D ii), tail area (C iv), tail moment and olive moment (C v) and (D iv). *, p Ͻ 0.05, **, p Ͻ 0.01, ***, p Ͻ 0.001, ****, p Ͻ 0.0001 when compared with CCM-treated myoblasts;ˆ, p Ͻ 0.05,ˆˆ, p Ͻ 0.01,ˆˆˆ, p Ͻ 0.001,ˆˆˆˆ, p Ͻ 0.0001 when compared with CMM1-or CMM2-treated myoblasts (n ϭ 3).
REDD1 is known to regulate cellular ROS through p63 and hence plays an important role in stress response and modulation of growth factors (4). Here, our results have established Mstn-mediated down-regulation of REDD1 and p63 levels via ROS production. However, recently, Hulmi et al. reported an increase in p63 and REDD1 levels in STZ-treated mice at week 1 of STZ treatment plausibly due to higher dose of STZ (180 mg/kg) and/or the type of muscles used in the study (8). Our results indicated that the basal levels of REDD1 and p63 were higher in Mstn Ϫ/Ϫ mice probably in part due to higher levels of AMP-activated protein kinase (AMPK) in these mice (40) because AMPK was shown to stimulate REDD1 in vitro (41). Another reason could be due to the elevated IGF-1 signaling in Mstn Ϫ/Ϫ mice, since IGF-1 treatment was reported to up-regulate the protein levels of REDD1 in muscle (42). Nevertheless, deficiency of p63 is associated with inefficient DNA repair (43) as well as with reduced transcription of REDD1 (4). Consistently, in vitro experiments confirmed Foxa2-mediated up-regulation of Mstn upon STZ treatment leading to DNA damage via p63/REDD1 signaling (Fig. 4, C and D).
At the cellular level, both 8-oxo-dG staining and comet assay showed that both concentrations of CMM were able to induce DNA single strand breaks, while only a higher concentration of CMM (CMM2) was able to induce double strand breaks in DNA. Furthermore, XRCC1, involved in single strand break repair, was decreased in WT-STZ muscle (Fig. 7A (i) and (ii)) indicating that in the diabetic condition due to enhanced ROS, an increase in DNA single strand break would occur and the repair would be impaired due to lower levels of the repair enzyme. XRCC1 is a critical enzyme involved in various stages of DNA repair via its interactions with other repair proteins. An increase in ROS is known to down-regulate XRCC1 that results in inefficient single strand break repair and build-up of repair intermediate products (44). In the Mstn Ϫ/Ϫ mice, the levels of XRCC1 remain unchanged even after STZ treatment suggesting that single strand break repair mechanism was not compromised in these muscles.
Histone H2A.X, involved in double strand break repair, was activated upon STZ treatment in WT mice thus indicating the response of the cell survival system against the DNA damage due to increased oxidative stress in these mice. The basal levels of both H2A.X and p-H2A.X in Mstn Ϫ/Ϫ muscles were higher than WT muscles, which remained unaltered even after STZ treatment. It is difficult to predict the significance of this result however a recent report by Turinetto et al. suggests a role of p-H2A.X in self-renewal of mouse embryonic and induced pluripotent stem cells that is independent of DNA damage response function (45). Overall, our findings suggest that deletion/inhibition of Mstn abrogates DNA damage in the diabetic muscle/cells.
Our results showed that STZ administration in Mstn Ϫ/Ϫ mice resulted in high glucose levels and greater decrease in body weights initially when compared with the WT mice (data not shown). This anomaly could be due to higher gluconeogenesis in these mice. In fact, the expression of genes involved in gluconeogenesis like PEPCK and G6P was elevated in the Mstn Ϫ/Ϫ mice relative to the WT mice (data not shown), which is in agreement with Wang et al. (46). As demonstrated previously, Mstn Ϫ/Ϫ mice have reduced amount of adipose tissue (47) indicating that muscle would be the predominant source to provide precursors for the enhanced gluconeogenesis following  STZ treatment, thus accounting for the increased muscle loss observed in Mstn Ϫ/Ϫ mice. However, even though Mstn Ϫ/Ϫ mice exhibit higher glucose levels upon STZ treatment, appreciable DNA damage was not observed in these mice. The inactivation/inhibition of Mstn was not able to improve the primary defect of type 1 diabetes but was able to rescue skeletal muscle from oxidative stress-induced DNA damage to a certain extent.
In summary, our results illustrate the mechanism of Mstn regulation in type 1 diabetes and Mstn-mediated DNA damage in skeletal muscle. Our findings also indicate that inhibition of Mstn could be an effective preventive measure to mitigate DNA damage in diabetic muscle.