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Journal of Cell Science logoLink to Journal of Cell Science
. 2015 May 1;128(9):1707–1717. doi: 10.1242/jcs.158964

Bit-1 is an essential regulator of myogenic differentiation

Genevieve S Griffiths 1, Jinger Doe 2, Mayumi Jijiwa 3, Pam Van Ry 2, Vivian Cruz 2, Michelle de la Vega 3, Joe W Ramos 3, Dean J Burkin 2, Michelle L Matter 1,3,*
PMCID: PMC4446732  PMID: 25770104

Abstract

Muscle differentiation requires a complex signaling cascade that leads to the production of multinucleated myofibers. Genes regulating the intrinsic mitochondrial apoptotic pathway also function in controlling cell differentiation. How such signaling pathways are regulated during differentiation is not fully understood. Bit-1 (also known as PTRH2) mutations in humans cause infantile-onset multisystem disease with muscle weakness. We demonstrate here that Bit-1 controls skeletal myogenesis through a caspase-mediated signaling pathway. Bit-1-null mice exhibit a myopathy with hypotrophic myofibers. Bit-1-null myoblasts prematurely express muscle-specific proteins. Similarly, knockdown of Bit-1 expression in C2C12 myoblasts promotes early differentiation, whereas overexpression delays differentiation. In wild-type mice, Bit-1 levels increase during differentiation. Bit-1-null myoblasts exhibited increased levels of caspase 9 and caspase 3 without increased apoptosis. Bit-1 re-expression partially rescued differentiation. In Bit-1-null muscle, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 9 and caspase 3 is decreased. Bcl-2 re-expression rescued Bit-1-mediated early differentiation in Bit-1-null myoblasts and C2C12 cells with knockdown of Bit-1 expression. These results support an unanticipated yet essential role for Bit-1 in controlling myogenesis through regulation of Bcl-2.

KEY WORDS: Bit-1, Caspase 3, Differentiation, Myogenesis

INTRODUCTION

Bit-1 (also known as peptidyl-tRNA hydrolase 2; PTRH2) is part of an integrin-specific signaling pathway responsible for the survival effects of cell–extracellular matrix interactions (Griffiths et al., 2011; Jan et al., 2004). It mediates attachment-dependent cell survival through an interaction with FAK (also known as PTK2) and subsequent activation of the PI3K–AKT–NFκB pathway. Upon integrin-mediated attachment, Bit-1 expression induces Bcl-2 transcription (Griffiths et al., 2011) and blocks the intrinsic mitochondrial apoptotic pathway. When integrins are not ligated, Bit-1 complexes with Groucho/TLE family proteins to promote apoptosis in a variety of cell types including myoblasts (Bouchentouf et al., 2007; Jan et al., 2004). Bit-1 is phosphorylated by PKD1, and Bit-1 expression impairs ERK phosphorylation (Biliran et al., 2008; Kairouz-Wahbe et al., 2008). Thus, Bit-1 is a phosphoprotein that can modulate NFκB and ERK signaling and thereby regulate integrin-mediated apoptotic signals.

Initial characterization of Bit-1-null mice revealed that they are smaller than wild-type littermates, develop a runting syndrome and die within the first 2 weeks of life. Indeed, normal development is impaired in the Bit-1 nulls, as kidney subcapsular glomeruli and epaxial muscle fiber diameters are smaller than those of controls (Kairouz-Wahbe et al., 2008). Recently, we reported that a homozygous mutation in the Bit-1 gene causes infantile-onset multisystem neurologic, endocrine and pancreatic disease (IMNEPD) with corresponding muscle weakness (Hu et al., 2014). The more severely affected patient became wheelchair dependent owing to muscle weakness and ataxia by age 15. However, the physiological function of Bit-1 has not been fully investigated in any tissue, including skeletal muscle.

Skeletal muscle differentiation is a multi-step process that requires myoblast progenitor cells to withdraw from the cell cycle, commit to a myogenic phenotype and differentiate into multinucleated myofibers that comprise mature muscle cells (Anderson, 1998; Grounds, 1991; Lluís et al., 2006). Signal transduction pathways that regulate apoptosis are also crucial in controlling cell differentiation. For example, caspase 3 activation is necessary for the maturation of skeletal muscle (Fernando and Megeney, 2007; Larsen et al., 2010; Li and Yuan, 2008). Caspase 3 might also regulate differentiation indirectly by targeting and inactivating proteins that are involved in stem cell renewal (Fujita et al., 2008; Janzen et al., 2008). Other apoptotic regulators also play a role in muscle differentiation, including Bcl-xL (encoded by BCL2L1) and the PI3K–AKT pathway (Briata et al., 2011; Majmundar et al., 2011; Murray et al., 2008; Wilson and Rotwein, 2007). The mechanism that activates these biochemical pathways in a non-death setting is not yet completely elucidated.

Here, we investigated the physiological function of Bit-1 using knockout (KO) mice. We find that Bit-1 levels increase during differentiation in wild-type (WT) mice, suggesting that Bit-1 plays a key role in myogenesis. Indeed, Bit-1-null mice exhibited a myopathy with hypotrophic myofibers without affecting apoptosis. This myopathy is due to early differentiation that occurs in the absence of Bit-1 expression. Both Bit-1 and its downstream effector Bcl-2 rescued Bit-1-mediated early differentiation. These findings reveal that Bit-1, which regulates integrin-mediated apoptosis, can be engaged in the completely different function of muscle differentiation.

RESULTS

Loss of Bit-1 promotes early myogenesis

We investigated in detail the phenotype of Bit-1-null mice with particular focus on skeletal muscle. Bit-1 KO mice display reduced muscle mass and smaller gastrocnemius muscle fibers. As such, we reasoned that myogenesis might be altered in Bit-1 KOs. Hematoxylin and eosin (H&E)-stained gastrocnemius muscle tissue from Bit-1 KO mice at postnatal day 7 (P7) demonstrated smaller myofibers and an increased number of nuclei (Fig. 1A). Because Bit-1 mediates cell survival and apoptosis in tumor cells (Griffiths et al., 2011; Jan et al., 2004), we examined apoptosis in KO and WT tissue sections by TUNEL staining. Apoptosis was similar in KO and WT gastrocnemius muscle tissue at P7 (Fig. 1A, lower graph). Mean myofiber cross-sectional area and longitudinal fiber width were significantly decreased in Bit-1 KO gastrocnemius muscle tissue compared to age-matched WT littermates at P7 (Fig. 1B). Muscle diameter, as measured by Feret's diameter, was significantly decreased in Bit-1 KO tibialis anterior muscle at P0 and P7 and in tricep muscle at P0 (Fig. 1C, upper graph). The mean tricep and tibialis anterior muscle weights at P7 were significantly decreased in the KO mice compared to those of age-matched WT littermates (Fig. 1C, lower graph). The smaller myofibers and increased number of nuclei observed in Bit-1 KO tissue might be due to abnormal differentiation. Therefore, to examine the effect of Bit-1 ablation on myogenesis, primary myoblast cultures were generated from 7-day-old Bit-1 KO and WT mice. Upon isolation of KO myoblasts and prior to induction of differentiation, the expression of the differentiation-specific proteins troponin T (encoded by TNNT2) and myosin II (also known as MYH2) was substantially increased in Bit-1 KO myoblasts relative to WT myoblasts (Fig. 1D). The number of troponin-positive cells was significantly increased in Bit-1 KO myoblasts compared to WT myoblasts at day 0 pre-differentiation (Fig. 1E,F). These findings suggest that loss of Bit-1 was permissive to inducing myoblast differentiation irrespective of growth-stimulating conditions (i.e. high serum).

Fig. 1.

Fig. 1.

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Bit-1-null mice exhibit a myopathy with hypotrophic muscle fibers. (A) H&E-stained gastrocnemius muscle tissue sections (top, cross-sections; bottom, longitudinal). Scale bars: 10 µm (upper panels), 50 µm (lower panels). Lower graph, Apoptag-stained gastrocnemius muscle tissue sections for detection of apoptotic myofibers. A comparable proportion of apoptotic myoblasts was observed for both WT and Bit-1 KO. n = 3000 (for both Bit-1 KO and WT). (B) Top graphs show quantification of myofibril number (y-axis) versus myofibril cross-sectional area (x-axis, left) and mean cross-sectional area (right) of Bit-1 KO and age-matched WT littermate controls at P7. Bottom graphs show quantification of mean longitudinal fiber width (left; n = 3000 for both Bit-1 KO and WT) and mean muscle weight (right; n = 5 for both Bit-1 KO and WT) of Bit-1 KO and age-matched WT littermate controls. **P<0.001; ***P<0.0001. (C) Quantification of triceps and tibialis anterior (TA) muscle diameter using minimal Feret's diameter analysis at P0 and P7 of Bit-1 KO and age-matched WT littermate controls (top graph; n = 3000 for both Bit-1 KO and WT). Mean muscle weight (bottom graph; n = 5 for both Bit-1 KO and WT) of Bit-1 KO and age-matched WT littermate controls at P0 and P7. *P<0.05. (D) Primary myoblast cultures were derived from WT and Bit-1 KO mice (n = 3 for each genotype). Western blot analysis for the differentiation-specific proteins troponin T and myosin II at pre-differentiation day 0 (cultured in high-serum medium) is shown. Tubulin was used as a loading control. The data are representative of three independent experiments. (E) Bit-1-null myoblasts express the differentiation-specific marker troponin T earlier than WT age-matched littermate controls. Immunostaining for troponin T (red) and staining of DNA (DAPI, blue) was performed at pre-differentiation day 0 (40×). The data are representative of three independent experiments. (F) Quantification of isolated primary Bit-1 KO myoblasts and WT age-matched littermate controls at pre-differentiation day 0. n = 3 for each genotype. **P<0.01 (Student's t-test). (G) Western blot of pro-caspase 3 and cleaved caspase 3 in primary myoblasts isolated from Bit-1 KO and age-matched WT littermates at day 0 pre-differentiation. Actin was used as a loading control. The blot is representative of three independent experiments. (H) Apoptag-stained primary myoblasts isolated from Bit-1 KO and WT age-matched controls at day 0 pre-differentiation (40×). (I) Quantification of apoptotic cells. Apoptosis was comparable for KO and WT controls at day 0 pre-differentiation. n = 3 for each genotype. Data presented in bar graphs show the mean±s.d.

Caspase 3 activity is increased in Bit-1-null myoblasts without increased apoptosis

We next assessed caspase 3 activity in isolated Bit-1 KO and WT skeletal myoblasts prior to inducing differentiation. Indeed, caspase 3 activity was increased in Bit-1 KO myoblasts compared to WT myoblasts prior to inducing differentiation (Fig. 1G). Caspase 3 activity is required and sufficient for myoblast differentiation (Fernando et al., 2002). Our data suggest that the early differentiation in Bit-1 KO myoblasts might be due to increased caspase 3 activity. To rule out the possibility that caspase 3 activity enhanced apoptosis in these cells, we assayed for apoptosis by TUNEL staining. A comparable proportion of apoptotic cells were observed for WT and Bit-1 KO myoblasts prior to inducing differentiation (Fig. 1H,I). These observations suggest that elevated caspase 3 activity in the Bit-1 KO promotes premature differentiation in the early stages of skeletal myogenesis. The increase in caspase 3 activity does not promote apoptosis.

To further delineate the effect of Bit-1 on myogenesis, we isolated myoblasts from Bit-1 KO and WT age-matched littermates and transiently transfected them with WT Bit-1 (Fig. 2A, upper blot). WT, WT-Bit-1-transfected or Bit-1 KO myoblasts were placed in low serum to induce differentiation over 7 days. Caspase 3 activity was assessed by immunoblotting at differentiation day 3. As expected, Bit-1 KO myoblasts showed enhanced active caspase 3 compared to WT controls (Fig. 2A, lower blot). A partial reduction in increased caspase 3 activity was obtained with expression of WT Bit-1 (Fig. 2A, lower blot). Fluorometric analyses of caspase 3 activity revealed a 1.7-fold increase in caspase 3 activity at pre-differentiation day 0 in the isolated Bit-1 KO myoblasts compared to WT controls (Fig. 2B, upper graph). A partial rescue of Bit-1 KO myoblasts with WT Bit-1 expression reduced caspase 3 activity to midway between KO and WT activity levels (Fig. 2B, upper graph).

Fig. 2.

Fig. 2.

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Expression of Bit-1- in Bit-1- null primary myoblasts inhibits early differentiation and reduces caspase 3 activity. (A) Western blot analyses. The top blot shows Bit-1 expression levels in WT and Bit-1-null myoblasts and the expression of WT Bit-1 in isolated KO myoblasts at pre-differentiation day 0. Actin was used as a loading control. The bottom blot shows pro-caspase 3 and cleaved caspase 3 as assessed at differentiation day 3 (differentiation medium) in myoblasts from Bit-1 KO and age-matched WT littermate controls or Bit-1-null myoblasts transfected with WT Bit-1. The 19- and 17-kDa fragments are indicative of caspase 3 cleavage, which is more abundant in Bit-1 KO myoblasts. The data are representative of three independent experiments. (B) Top graph, a sharp increase in caspase 3 activity, as determined by using a fluorometric protease assay that directly measures caspase 3 activity, was found at pre-differentiation day 0 and differentiation days 3, 5 and 7 in Bit-1 KO myoblasts (open squares) compared to age-matched WT littermates (circles). Expression of WT Bit-1 (triangles) reduced caspase 3 activity to mid-way between KO and control levels. Bottom graph, assessment of BrdU incorporation in Bit-1 KO (open squares) and WT (circles) myoblasts indicates that there is a sharp decrease in BrdU incorporation at pre-differentiation day 0 and differentiation days 3, 5 and 7 in the Bit-1 KO myoblasts. Expression of Bit-1 (triangles) reduced BrdU incorporation to mid-way between the KO and WT control levels. (C) Quantification of myoblast apoptosis at day 3 of differentiation. Apoptosis was comparable for Bit-1 KO and WT controls (n = 5 for each myoblast type). (D) Quantification of the percentage of cells expressing troponin and myogenin (%Troponin and %Myogenin, respectively) and the fusion index (%). Bit-1 KO myoblasts demonstrated earlier and increased troponin T and myogenin expression in addition to increased myoblast fusion at pre-differentiation day 0 and over three days of differentiation. n = 5 for each genotype. *P<0.01 for Bit-1 KO versus WT or WT Bit-1 (Student's t-test). Quantitative data show the mean±s.d.

To confirm that Bit-1 KO myoblasts withdraw from the cell cycle prior to inducing differentiation, colormetric analysis of proliferating myoblasts as assessed by BrdU incorporation was determined. Bit-1 KO myoblasts incorporated approximately half as much BrdU as WT myoblasts at day 0 pre-differentiation (Fig. 2B, lower graph). In contrast, expression of WT Bit-1 induced a partial rescue by increasing BrdU incorporation to midway between Bit-1 KO and WT levels (Fig. 2B, lower graph). A comparable proportion of apoptotic cells were observed for both WT and Bit-1-null myoblasts during differentiation (Fig. 2C). Moreover, re-expressing Bit-1 in Bit-1 KO myoblasts returned differentiation to control levels (Fig. 2D). Therefore, Bit-1 KO myoblasts are withdrawing from the cell cycle and beginning to differentiate even in high serum. Expression of Bit-1 blocks these cells from cell cycle withdrawal.

Myogenin accumulation is delayed in caspase 3-null primary myoblasts (Fernando et al., 2002), whereas Bit-1 KO primary myoblasts induce early expression of myogenin at day 0 pre-differentiation and increased myogenin accumulation during differentiation (Fig. 2D). Bit-1 KO primary myoblasts express higher troponin levels and have an increased fusion index compared to control WT myoblasts. In addition, re-expression of Bit-1 returns troponin levels and the fusion index back to control levels (Fig. 2D). Taken together, these findings point to Bit-1 as a negative regulator of differentiation that acts by modulating caspase 3 activity.

siRNA-mediated knockdown of Bit-1 promotes early differentiation in a C2C12 differentiation model

To test whether it was specifically the loss of Bit-1 in KO skeletal muscle that led to premature differentiation (rather than other developmental effects), we explored whether knockdown of Bit-1 in a myoblast cell line had similar results. C2C12 cells were engineered to express reduced levels of Bit-1, normal levels of Bit-1 or increased levels of Bit-1 (Fig. 3B) and induced to undergo differentiation in low serum (Fernando et al., 2002). To assess muscle differentiation, we examined the expression of the differentiation markers troponin T, myosin II and myogenin. C2C12 cells transfected with Bit-1-targeting small interfering RNA (siRNA) showed premature differentiation as demonstrated by enhanced troponin T expression at pre-differentiation day 0 and differentiation day 1 (Fig. 3A) compared to normal differentiation in C2C12 cells transfected with scrambled siRNA (Fig. 3A). Myogenin expression was comparable in C2C12 cells transfected with Bit-1-targeting siRNA or scrambled siRNA (Fig. 3A). In contrast, C2C12 cells overexpressing Bit-1 showed increased levels of troponin T on differentiation day 3, indicating delayed differentiation (Fig. 3A). Similarly, immunostaining for and quantification of the differentiation-specific marker troponin T revealed that loss of Bit-1 promoted troponin expression at pre-differentiation day 0 and enhanced expression at differentiation days 2 and 3 compared to that of scrambled siRNA controls (Fig. 3C,D). Increased myogenin expression was also observed at differentiation day 1 in C2C12 cells transfected with Bit-1 siRNA compared to those transfected with scrambled siRNA (Fig. 3C,E). In contrast, immunostaining of cells overexpressing Bit-1 showed delayed troponin expression, which was visible at differentiation day 3 (Fig. 3C,D). Finally, upon induction of differentiation, a significant increase in myotube formation was evident at earlier time-points in C2C12 cells transfected with Bit-1 siRNA compared to those transfected with scrambled siRNA (Fig. 3F; compare a fusion index of 90% versus 70%, knockdown to control, respectively). Upon differentiation of C2C12 myoblasts overexpressing Bit-1, a delay in myotube formation was evident (Fig. 3F). Notably, during normal C2C12 differentiation, Bit-1 expression increases as differentiation progresses (Fig. 3G), indicating a differentiation-specific role for Bit-1. These findings confirm that loss of Bit-1 promotes early differentiation whereas Bit-1 overexpression delays differentiation as measured by the expression of the differentiation-specific markers troponin, myosin II and myogenin and by the fusion index.

Fig. 3.

Fig. 3.

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Loss of Bit-1 expression induces differentiation in growing C2C12 cells. (A) C2C12 cells were transfected with either a scrambled siRNA, Bit-1 siRNA or wild-type Bit-1 (Bit-1 OverExp.) and analyzed by western blotting for the differentiation-specific proteins troponin T and myogenin at pre-differentiation day 0 (cultured in high-serum medium) and differentiation days 1 and 3 (cultured in low-serum differentiation medium). An increase in troponin T expression was found in C2C12 cells with low Bit-1 expression (Bit-1 siRNA) at pre-differentiation day 0. Myogenin expression levels were comparable between scrambled siRNA, Bit-1 siRNA and Bit-1 overexpressing cells. Tubulin was used as a loading control. The data are representative of three independent experiments. (B) Western blot analysis of Bit-1 expression levels in scrambled siRNA, Bit-1 siRNA and Bit-1 overexpressing cells at pre-differentiation day 0. Tubulin was used as a loading control. The data are representative of three independent experiments. (C) Immunostaining for troponin T (red) and myogenin (green), and staining of DNA (DAPI, blue) was performed at pre-differentiation day 0 and differentiation days 1 and 3 (40×). The data are representative of three independent experiments. Scale bars: 100 µm. (D–F) Quantification of the percentage of cells expressing troponin (%Troponin, D) and myogenin (%Myogenin, E) and the percentage fusion index (F). The percentage fusion index was assessed as a percentage of cells containing two or more nuclei within a differentiated myotube. Data show the mean±s.d. n = 5 per genotype. *P<0.05; **P<0.01; ***P<0.001 for Bit-1 KO versus Scr siRNA (Student's t-test). (G) Bit-1 and troponin expression levels were assessed during normal C2C12 myogenic differentiation over 3 days under low-serum conditions. Tubulin was used as a loading control. The blot is representative of three independent experiments.

siRNA-mediated knockdown of Bit-1 promotes increased caspase 3 activity in a C2C12 differentiation model

We next examined how loss of Bit-1 affected caspase 3 activity in C2C12 cells during differentiation. Proliferating C2C12 cells transfected with Bit-1 siRNA (Fig. 3B) or scrambled siRNA (Fig. 3B) were placed in low-serum medium and allowed to differentiate for 3 days. At the relevant time-points, cells were washed extensively to ensure lysates were free of non-adherent apoptotic cells. Immunoblotting demonstrated enhanced caspase 3 activity at day 0 pre-differentiation in C2C12 cells transfected with Bit-1 siRNA compared to those transfected with scrambled siRNA (Fig. 4A). To confirm the immunoblotting analysis, a fluorometric assay of caspase 3 activity was performed. Fluorometric analyses revealed a twofold increase in caspase 3 activity at pre-differentiation day 0 and a threefold increase within 24 h, which declined as differentiation progressed in the C2C12 cells transfected with Bit-1 siRNA (Fig. 4B, left graph). Thus, loss of Bit-1 increased caspase 3 activity prior to inducing differentiation.

Fig. 4.

Fig. 4.

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Caspase 3 activity is increased in growing C2C12 cells transfected with Bit-1-targeting siRNA. (A) C2C12 cells were transfected with either a scrambled siRNA or Bit-1 siRNA and, 48 h later, were analyzed by western blotting for pro-caspase 3 and cleaved caspase 3 at pre-differentiation day 0 (high serum) and differentiation (Diff) days 1 and 3 (differentiation medium). The 19- and 17-kDa fragments are indicative of caspase 3 activation, which is more abundant at pre-differentiation day 0 in C2C12 cells with Bit-1 knockdown. The data are representative of three independent experiments. (B) Left graph, a sharp increase in caspase 3 activity, as determined by a fluorometric protease assay that directly measures caspase 3 activity, was found at pre-differentiation day 0 and differentiation days 1 and 3 in C2C12 cells expressing low levels of Bit-1 (Bit-1 siRNA; open squares) compared to cells treated with scrambled siRNA (Scr siRNA; black circles). Re-expression of WT Bit-1 (black triangles) reduced caspase 3 activity back to control levels. Right graph, assessment of BrdU incorporation in C2C12 cells transfected with Bit-1 siRNA and scrambled siRNA controls demonstrate that there is a sharp decrease in BrdU incorporation at pre-differentiation day 0 and differentiation days 1 and 3 in C2C12 cells expressing low levels of Bit-1 (open squares). Re-expression of Bit-1 (black triangles) reduced BrdU incorporation back to control levels. (C) Quantification of C2C12 apoptosis at day 3 of differentiation (cultured in differentiation medium). Apoptosis was comparable for Bit-1 siRNA and scrambled siRNA controls (n = 3 for each). (D) Left graph, caspase 8 activation, as determined by a fluorometric protease assay that directly measures caspase 8 activity, displayed a minimal increase upon induction of differentiation, which was similar in the Bit-1 siRNA (open squares) and scrambled siRNA (black circles) C2C12 cells. Cells were incubated in low-serum medium with a caspase 8 selective inhibitor (z-LETD.fmk) or DMSO-carrier control and assessed over 3 days of differentiation. Right graph, treatment of C2C12 cells with a caspase 8 selective inhibitor did not change BrdU incorporation in differentiating C2C12s. (E) Quantification of the percentage of cells expressing troponin and myogenin (%Troponin and %Myogenin, respectively) and the percentage fusion index. C2C12 cells expressing knockdown levels of Bit-1 demonstrated earlier and increased troponin T and myogenin expression in addition to increased myoblast fusion (%Fusion) over 3 days of differentiation. Re-expression of WT Bit-1 returned the percentage of cells expressing troponin or myogenin and the percentage fusion index to control levels. Incubation with a caspase 8 selective inhibitor did not significantly alter C2C12 differentiation. n = 5 for each condition: Bit-1 siRNA, Scrambled siRNA, Bit-1 siRNA+WTBit-1. *P<0.01 for Bit-1 KO versus Scr siRNA (Student's t-test). Quantitative data show the mean±s.d.

Re-expression of Bit-1 rescues caspase 3 activity to wild-type levels

Rescue of C2C12 cells in which Bit-1 levels had been knocked down with WT Bit-1 re-expression, returned caspase 3 activity back to control levels at pre-differentiation day 0 and during differentiation (Fig. 4B, left graph). A comparable proportion of apoptotic cells was observed for C2C12 myoblasts transfected with Bit-1 siRNA or scrambled siRNA (Fig. 4C). Caspase 3 activity is necessary for myoblast differentiation, whereas caspase 8 activity has a minimal effect (Fernando et al., 2002). Fluorometric analysis of caspase 8 activity showed an increase in activity during differentiation in agreement with previous reports (Fernando et al., 2002; Fig. 4D, left graph); however, cells transfected with Bit-1 siRNA or scrambled siRNA had a similar trend (Fig. 4D, left graph). Treatment with a pharmacological inhibitor selective for caspase 8 (z-LETD.fmk) completely inhibited caspase 8 activity in all cells (Fig. 4D, left graph). These findings suggest that, upon Bit-1 knockdown, increased caspase 3 activity but not caspase 8 promotes myogenic differentiation.

Upon differentiation, myoblasts withdraw from the cell cycle and differentiate into multinucleate myofibers. Colorimetric analysis of proliferating cells as assessed by BrdU incorporation was determined during differentiation. C2C12 cells transfected with Bit-1 siRNA demonstrated significantly reduced proliferation at differentiation day 1 compared to scrambled siRNA control (Fig. 4B, right graph). Rescue of C2C12 cells in which Bit-1 expression had been knocked down with re-expression of WT Bit-1 returned BrdU levels back to control levels during differentiation (Fig. 4B, right graph). Treatment with the caspase 8 inhibitor z-LETD.fmk did not alter the observed cell cycle withdrawal in the Bit-1-knockdown C2C12s or in the corresponding controls (Fig. 4D, right graph). Moreover, rescue of C2C12 cells in which Bit-1 levels had been knocked down, with WT Bit-1 re-expression returned differentiation back to control levels as shown by decreased percentages of cells expressing troponin or myogenin, and decreased fusion index (Fig. 4E). Taken together, these findings demonstrate that prior to inducing differentiation, C2C12 cells with reduced levels of Bit-1 are withdrawing from the cell cycle and beginning to differentiate. Thus, loss of Bit-1 promotes myoblast differentiation through increased caspase 3 activity and not through caspase 8.

Bcl-2 levels are reduced in Bit-1-null skeletal muscle

The Bcl-2 family regulates caspase activity by blocking release of cytochrome c and inhibiting caspase 9 and 3 activation. The BH4 domain of Bcl-2 and Bcl-xL binds to the C-terminal part of Apaf-1 and inhibits caspase 9 activation and therefore caspase 3 activity (Hu et al., 1998; Huang et al., 1998; Pan et al., 1998). We have reported that Bit-1 functions to upregulate Bcl-2 expression in a HeLa tumor cell line (Griffiths et al., 2011). Because we observed an increase in caspase 3 activity in Bit-1 KO skeletal muscle tissue, in isolated Bit-1 KO primary myoblasts and in C2C12 cells transfected with Bit-1 siRNA, we next examined Bcl-2 levels in gastrocnemius muscle from Bit-1 KO and aged-matched WT littermates (Fig. 5A). In Bit-1 KO muscle tissue, Bcl-2 levels are reduced, suggesting that Bcl-2-mediated inhibition of caspase 3 activity is decreased. Consistent with this result, knockdown of Bit-1 expression in C2C12 cells also reduces Bcl-2 levels (Fig. 5B). Our data suggests that loss of Bit-1 decreases Bcl-2 levels and promotes increased caspase 3 activity. This increase in caspase 3 activity would, thereby, initiate the early differentiation observed in isolated Bit-1 KO myoblasts and in C2C12 cells transfected with Bit-1 siRNA.

Fig. 5.

Fig. 5.

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Bcl-2 expression rescues Bit-1-null-induced early differentiation. (A) Western blot of Bit-1 KO and age-matched WT littermate control gastrocnemius muscle tissue. Bit-1 KO muscle cannot achieve WT levels of Bcl-2 expression. Tubulin was used as a loading control. The blot is representative of three independent experiments. (B) C2C12 cells were transfected with either a scrambled siRNA or Bit-1 siRNA or Bit-1 siRNA and Bcl-2 plasmid and, 48 h later, were analyzed by western blotting for Bit-1 and Bcl-2 expression. Tubulin was used as a loading control. The blot is representative of three independent experiments. (C) A sharp increase in caspase 3 activity, as determined by a fluorometric protease assay that directly measures caspase 3 activity, was found at pre-differentiation day 0 and differentiation days 1 and 3 in C2C12 cells expressing low levels of Bit-1 (Bit-1 siRNA; open squares) compared to scrambled siRNA (Scr siRNA; black squares) or scrambled siRNA and Bcl-2 (Scr siRNA/Bcl-2; black triangles). Re-expression of Bcl-2 (Bit-1 siRNA/Bcl-2; open circles) reduced caspase 3 activity back to control levels. (D) Quantification of the percentage of cells expressing troponin (%Troponin) and percentage fusion index. Re-expression of Bcl-2 in C2C12 cells expressing low levels of Bit-1 reduced troponin T and the percentage fusion back to control levels over 3 days of differentiation. n = 5 for each condition: Bit-1 siRNA, scrambled siRNA, scrambled siRNA+Bcl-2, Bit-1 siRNA+Bcl-2. *P<0.01 for Bit-1 KO versus Bcl-2 (Student's t-test). Quantitative data show the mean±s.d.

Bcl-2 expression rescues early myogenesis of Bit-1-null cells

Rescue of C2C12 cells in which Bit-1 levels had been knocked down, with Bcl-2 re-expression, returned caspase 3 activity to control levels at pre-differentiation day 0 and during differentiation (Fig. 5C). Re-expression of Bcl-2 in these cells also reduced differentiation-specific troponin expression (Fig. 5D, left graph) and fusion index (Fig. 5D, right graph) to control levels during differentiation. Similarly, Bit-1-null myoblasts demonstrated decreased Bcl-2 expression levels (Fig. 6A). Re-expression of Bcl-2 in Bit-1-null myoblasts rescued caspase 3 activity back to control levels (Fig. 6B). Moreover, caspase 9 cleavage was increased in Bit-1-null myoblasts compared to WT controls (Fig. 6C). Caspase 9 activity was higher in Bit-1-null myoblasts, and re-expression of Bcl-2 in Bit-1-null myoblasts decreased caspase 9 activity to WT control levels (Fig. 6D). The pattern of caspase 9 activity was similar to that of caspase 3. These findings suggest that Bit-1 regulates skeletal muscle differentiation by modulating Bcl-2 expression and, therefore, downstream caspase activity.

Fig. 6.

Fig. 6.

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Bcl-2 expression rescues Bit-1-null-mediated early myogenesis. (A) Western blot analysis of Bcl-2 expression levels in isolated WT and Bit-1 KO myoblasts at pre-differentiation day 0. Actin was used as a loading control. (B) An increase in caspase 3 activity, as determined by a fluorometric protease assay that directly measures caspase 3 activity, was found at pre-differentiation day 0 and differentiation days 1 and 3 in Bit-1 KO myoblasts (open circles) compared to age-matched WT littermates (closed diamonds). Expression of Bcl-2 (open squares) reduced caspase 3 activity to control levels. n = 5 for each genotype. *P<0.01 for Bit-1 KO versus Bcl-2 (Student's t-test). (C) Pro-caspase 9 and cleaved caspase 9 were increased in isolated Bit-1 KO myoblasts at pre-differentiation day 0. Tubulin was used as a loading control. (D) An increase in caspase 9 activity, as determined by a fluorometric protease assay that measures caspase 9 activity, was found at pre-differentiation day 0 and differentiation days 1 and 3 in Bit-1 KO myoblasts (open circles) compared to age-matched WT littermates (closed diamonds). Expression of Bcl-2 (open squares) reduced caspase 9 activity towards control levels. n = 5 for each genotype. *P<0.01 for Bit-1 KO versus Bcl-2 (Student's t-test). Quantitative data show the mean±s.d.

DISCUSSION

In this study, we provide evidence that Bit-1, a regulator of Bcl-2, is a mediator of muscle differentiation. Upon examination and further characterization of the skeletal muscle of Bit-1-null mice, we found these mice developed a muscle myopathy involving hypotrophic myofibers. The observed early differentiation in Bit-1-null muscle was due to decreased Bcl-2 expression and subsequent increased caspase 3 activity without increased apoptosis. Re-expression of Bit-1 or of Bcl-2 blocked premature differentiation of Bit-1-null myoblasts. Our data demonstrate for the first time that Bit-1 plays a regulatory role in normal muscle differentiation outside of its function in regulating apoptosis.

Muscle differentiation requires myoblast precursor cells to withdraw from the cell cycle and differentiate into multinucleated myofibers (Anderson, 1998; Grounds, 1991; Lluís et al., 2006). Several of the biochemical pathways that regulate apoptosis also play key roles in terminal cell differentiation. Bcl-2 family members, including Bcl-xL, are required for skeletal muscle differentiation (Murray et al., 2008). Caspase 3 is another such mediator and is also required for skeletal muscle differentiation (Fernando et al., 2005; Fernando et al., 2002; Li and Yuan, 2008). These findings point to a crucial regulatory role of genes that control the intrinsic mitochondrial apoptotic pathway in muscle differentiation. However, how such signaling pathways are regulated is not yet fully understood.

We have previously identified Bit-1 as a regulator of integrin-mediated apoptosis (Griffiths et al., 2011; Jan et al., 2004). Integrins are a family of transmembrane receptors that bind to extracellular matrix (ECM) proteins. These receptors promote cell–ECM adhesion and mediate adhesion-initiated signal transduction pathways. A subset of integrins is essential for myogenesis, including the β3 and α7 integrins (Burkin and Kaufman, 1999; Liu et al., 2011). The β3 integrin acts on myogenic differentiation through activation of p130Cas and MAPK (Kawauchi et al., 2012). α7 integrin mutations result in congenital myopathies in humans and induce muscular dystrophy in mice (Hayashi et al., 1998; Mayer et al., 1997). Exactly how downstream signals are activated by integrin attachment to promote muscle differentiation is not yet fully known. Integrin adhesion activates the tyrosine kinase focal adhesion kinase (FAK), which in turn activates a number of downstream signaling pathways that promote cell proliferation, survival and muscle differentiation. Integrin-dependent myoblast differentiation requires FAK activation (Luo et al., 2009). We have previously reported that Bit-1 interacts in a complex with FAK in a myoblast cell line (Griffiths et al., 2011). We propose that integrins signal through Bit-1 to control myoblast differentiation by linking adhesion and apoptosis pathways. The molecular mechanism of how integrins induce Bit-1 signaling is currently under investigation.

Our data show that the myopathy characterized by hypotrophic myofibers observed in Bit-1 nulls is due, in part, to increased caspase 3 signaling. We have previously reported that loss of Bit-1 expression induces increased caspase 3 activity in normal endothelial cells and cancer cells (Griffiths et al., 2011). Our data support previous findings on the essential role of caspase 3 activity for muscle differentiation (Fernando et al., 2002) and extend them by demonstrating that Bit-1 negatively regulates caspase 3 activity during muscle differentiation. We show here that loss of Bit-1 promotes increased caspase 3 activity in myoblasts even under pre-differentiation high-serum conditions. Although re-expression of Bit-1 in the primary Bit-1-null myoblasts showed minimal effect on returning caspase 3 levels to control levels we did observe a significant rescue of caspase 3 activity upon re-expression of Bit-1 in C2C12 cells that had been treated with Bit-1 siRNA. Moreover, in both cell types, re-expression of Bit-1 induced a significant rescue of the differentiation phenotype. The reduced effect on caspase 3 activity by re-expressing Bit-1 in the primary Bit-1-null myoblasts might be due to the lower transfection efficiency that occurs with primary cells. Our findings are in agreement with the report that caspase 3 overexpression induces myoblast differentiation under pre-differentiation high-serum conditions (Fernando et al., 2002). To determine whether the increase in early differentiation upon loss of Bit-1 was due to other caspases such as caspase 8, we used a pharmacological inhibitor for caspase 8. Pharmacological inhibition of caspase 8 did not block the observed early differentiation in C2C12 myoblasts with siRNA knockdown of Bit-1. Although the pharmacological inhibitor tested is selective and not specific, caspase 8 did not appear to play a role. This finding is in agreement with previous reports that myoblast differentiation requires caspase 3 but not caspase 8 (Fernando et al., 2002). Furthermore, caspase 9, an initiator caspase in the intrinsic mitochondrial apoptotic pathway, plays a non-apoptotic role by activating caspase 3 in C2C12 myoblast differentiation (Murray et al., 2008). Our data demonstrate that caspase 9 processing and activity increased in Bit-1-null myoblasts similar to caspase 3 activity, suggesting that caspase 9 is the intermediate step that activates caspase 3 during differentiation. Our data also confirm that similar to caspase 9 and 3, Bit-1 functions in a non-apoptotic role during myogenesis.

We have shown that Bit-1 regulates caspase 3 activity through its effects on Bcl-2. The Bcl-2 family controls caspase activity by inhibiting cytochrome c release and subsequent caspase 3 activation. The BH4 domain of Bcl-2 and Bcl-xL binds to the C-terminal region of Apaf-1, inhibiting caspase 9 activation and subsequent caspase 3 activity (Hu et al., 1998; Huang et al., 1998; Pan et al., 1998). Bcl-xL overexpression blocks myoblast differentiation by inhibiting downstream caspase activation (Murray et al., 2008). It is therefore possible that other Bcl-2 family members like Bcl-xL could mediate the effects of Bit-1 in muscle differentiation. This remains to be determined. We have reported previously that upon integrin-mediated attachment, Bit-1 upregulates Bcl-2 expression (Griffiths et al., 2011). We report here that Bcl-2 levels were significantly reduced in gastrocnemius muscle from Bit-1-null mice compared to age-matched WT littermate controls. Moreover, re-expression of Bcl-2 in Bit-1-null myoblasts returned caspase 3 activity to WT control levels and rescued early differentiation. Bcl-2 re-expression in these cells also resulted in a decrease in caspase 9 activity to WT control levels. These findings suggest that Bcl-2-mediated inhibition of caspase 3 activity is decreased in Bit-1-null muscle through modulation of the initiator caspase 9 and might be, at least in part, the reason that the Bit-1-null mice present with hypotrophic myofibers. Therefore, Bit-1 regulates myoblast differentiation by controlling Bcl-2 expression to restrain caspase 3 in a non-death setting.

Overexpression of Bcl-2 ameliorates muscular dystrophy disease progression in an integrin ligand laminin-α2-null mouse model of muscular dystrophy (Dominov et al., 2005) and in a mouse model of oculopharngeal muscular dystrophy (Davies and Rubinsztein, 2011). It will be informative to determine whether overexpression of Bit-1 in laminin-α2-null mice might attenuate disease progression in these mice. Functional and physiological studies are needed in these mice to demonstrate the therapeutic potential of Bit-1 overexpression. Recently, we used whole exome sequencing to identify mutations in Bit-1 that cause progressive congenital muscle weakness in humans (Hu et al., 2014), suggesting that Bit-1 plays a key role in muscle function. Our data here point to Bit-1 as an important regulator of muscle differentiation. Bit-1 is a potential new therapeutic target for muscle disease such as muscular dystrophy. We postulate that Bit-1 is the missing signal between integrins and the intrinsic mitochondrial apoptotic pathway in muscle differentiation.

MATERIALS AND METHODS

Animals

Mouse tissue was obtained from C57BL/6 Bit-1−/− and age-matched WT littermates. All animals received care in compliance with the principles of laboratory animal care and use formulated by the Institutional Animal Care and Use Committee.

Genotyping

Genotyping of Bit-1-null mice has been described previously (Kairouz-Wahbe et al., 2008). Briefly, DNA for genotyping was isolated from 7-day-old mouse tails or toes, and genotypes were determined by PCR using the primer combinations G2F/G1R and G2F/30d; G2F (5′-TGGGTCTTTGAATCAACTAG-3′), G1R (5′-ACATGCCACAAGCAACTCCA-3′), 30 d (5′-TTTGAGACCCTATCACTCCACACG-3′). PCR was performed under conditions of initial 2 min denaturation at 95°C, followed by 30 cycles consisting of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min with a final extension of 72°C for 5 min.

H&E staining

Gastrocnemius muscles from Bit-1 KO and WT mice were embedded in optimal cutting temperature (OCT) cutting compound (Sakuara, Torrance, CA) and sectioned to 8 µm using a LeicaCM 1850 cryostat. Sections were placed onto prewashed Surgipath slides (Surgipath, Richmond, IL). Sections were stained with H&E and used to determine myofiber cross-sectional area and longitudinal fiber width.

Myofiber area determination

H&E-stained slides were used to determine myofiber cross-sectional area and longitudinal myofiber width. A minimum of 1000 fibers per animal from three Bit-1 KO and three WT mice were counted. Myofiber cross-sectional area was determined with a Zeiss Axioskop 2 Plus fluorescent microscope, and images were captured with a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Minimal Feret's diameter

Quantitative determination of muscle fiber cross-section diameter was obtained using minimal Feret's diameter analysis for triceps and tibialis anterior muscles of Bit-1 KO and age-matched WT litter-mate controls at P0 and P7. Using a Leica CM1850 cryostat, 10-µm sections of Tissue-TEK OCT-compound-embedded tissues from mice were placed on Surgipath microscope slides (Surgipath Medical Industries). H&E staining was performed on triceps and tibialis anterior muscles from Bit-1 KO and WT age-matched littermates at P0 and P7. Images were taken using an Olympus Fluoview FV1000 Laser Confocal Microscope and blinded evaluation of sections was performed. Pictures of triceps or tibialis anterior sections were taken at 100× using a Zeiss Axioskop 2 Plus fluorescent microscope, Zeiss AxioCam HRc digital camera and Axiovision 4.8 software. Compiled images were used to reconstruct a view of the entire tricep or tibialis anterior muscle as described previously (Van Ry et al., 2014). This compilation was used for calculating minimum Feret's diameter. There was a minimum of n = 3 mice for each treatment group and a minimum of n = 3000 fibers.

Myoblast isolation

Primary myoblasts were isolated from Bit-1 KO and WT mice from gastrocnemius skeletal muscle as described previously (Megeney et al., 1996). Isolated muscle was treated with Pen-strep (Invitrogen) for 10 min at 37°C to prevent bacterial contamination during culture. Muscle tissue was rinsed three times in PBS and minced in Ham's F-12 medium supplemented with 20% FBS and 2% Pen-strep, 1% L-glutamine and 1% non-essential amino acids (Invitrogen).

Fusion index

To determine the fusion index, differentiation cultures were analyzed microscopically as described previously (Quach et al., 2009). A 20× objective and phase contrast (Zeiss Axioskop 2 Plus microscope) were used to analyze random fields of WT control, Bit-1-null primary myoblasts or C2C12 myoblasts. Myotubes with three or more nuclei were counted. The fusion index was then determined as the percentage of nuclei in myotubes compared with the total number of nuclei in the field. Approximately 100 myotubes were counted per dish. Measurements were performed in triplicate in three independent experiments per genotype.

Cell culture

The C2C12 myoblast cell line was maintained in DMEM supplemented with 15% FBS, 1% Pen-strep, 1% MEM non-essential amino acids and 1% L-glutamine. Cells were incubated at 5% CO2, 37°C. For differentiation assays, medium was changed to DMEM supplemented with 1.5% horse serum, 1% Pen-strep, 1% MEM non-essential amino acids and 1% L-glutamine and incubated at 5% CO2, 37°C. Primary myoblasts were isolated from Bit-1-null and age-matched WT littermates as described above and induced to differentiate in low-serum medium.

Caspase 8 inhibition

For caspase 8 inhibition, primary KO and WT myoblasts or C2C12 cells were treated with 20 µM inhibitor selective for caspase 8 (z-LETD.fmk; BD Pharmingen) or negative control peptide (Z-FA-FMK; BD Pharmingen) for 3 h prior to differentiation, when fresh inhibitor or control peptide was added. Fresh inhibitor or control peptide was added every 24 h throughout the experiments.

Bit-1 RNA interference

A Dharmacon siGENOME Smartpool (Thermo Fisher Scientific) consisting of four Bit-1-specific siRNAs and control scrambled siRNAs were used to knock down Bit-1 expression levels. For transient transfection experiments, 2×105 C2C12 cells were transfected with 25 µM of Bit-1 SMARTpool using the Lipofectamine 2000 transfection reagent (Invitrogen). At various time-points, cells were subjected to immunostaining or enzymatic assays as described below. Cell lysates were also collected at corresponding time-points for immunoblotting. For overexpression and re-expression experiments, 3 µg of the WT Bit-1 or WT Bcl-2 constructs were transfected into KO and WT primary myoblasts or C2C12 cells using Lipofectamine 2000.

Immunoblotting

Isolated primary Bit-1 KO myoblasts, aged-matched WT littermate control primary myoblasts and C2C12 cells were lysed with lysis buffer containing 50 mM Tris-HCl, 3 mM EDTA, 0.5% Triton X-100 pH 7.0, 0.5 mM dithiothreitol and protease inhibitors (Complete Protease Inhibitor Tablets, Roche Applied Science). Cell lysates were sonicated and cleared by centrifugation at 18,000 g for 10 min at 4°C. Equal amounts of cell lysates were resolved on 4–12% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to Immobilon-P nylon membrane (Millipore Corp., Bedford, MA) and immunoblotted. Membranes were blocked for 1 h with blocking buffer (3% BSA/PBS-T) and incubated with various antibodies at a dilution of 1∶1000 unless specified otherwise, including rabbit polyclonal anti-Bit-1 antibody (developed at Washington Biotechnology, Columbia, MD using the Bit-1 amino acid sequence GPADLIDKVTAGHLKL), mouse anti-troponin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit anti-myogenin antibody (Santa Cruz Biotechnology Inc.), anti-myosin (Abcam), anti-microtubulin (BioLegend), anti-caspase 3 (Cell Signaling) or anti-caspase 9 (Cell Signaling) for 1 h. Membranes were washed and incubated with Li-Cor secondary antibodies (anti-rabbit-IgG IR 800 CW and anti-mouse-IgG IR 680 CW; Li-Cor) for 1 h. Immunoblots were analyzed using Odyssey software, provided with the Li-Cor system.

Caspase fluorometric assays

Primary Bit-1 KO and WT myoblasts and C2C12 cells were analyzed for caspase 3 activity, caspase 8 activity or caspase 9 activity by using fluorometric assays (BioVision Inc., San Francisco, CA) as per the manufacturers' instructions. Briefly, 1×105 cells were plated per well in a 96-well plate. Cells were exposed to non-differentiation or differentiation medium prior to being lysed and incubated with DEVD-AFC substrate for 1 h at 37°C. Fluorescence was assessed using a plate reader with 400/505 nm excitation/emission. Cells incubated with a caspase 8 inhibitor (BD Pharmingen) at a concentration of 20 µM were used as a negative control for caspase 8 activity.

BrdU proliferation assay

Proliferation rates of Bit-1 KO primary myoblasts, WT primary myoblasts and C2C12 cells were determined using a BrdU incorporation assay (Cell Signaling) according to the manufacturer's instructions. Bit-1 or WT primary myoblasts or C2C12 cells were exposed to non-differentiation or differentiation medium prior to analysis using the BrdU kit reagents. Cells were washed three times, fixed and incubated with an anti-BrdU antibody for 1 h at room temperature. Cells were next washed three times and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at room temperature, followed by addition of TMB substrate. After a 30-min incubation, a STOP solution was added and absorbance was read at 450 nm in a plate reader. Cells incubated in the presence of a caspase 8 selective inhibitor (BD Biosciences) at a concentration of 20 µM were used as a negative control.

Immunostaining

Bit-1 and WT primary myoblasts or C2C12 cells were fixed in 4% paraformaldehyde, permeabilized and incubated with an anti-troponin T or anti-myogenin antibody (Santa Cruz), followed by an Alexa-Fluor-594- or Alexa-Fluor-488-conjugated secondary antibody (Molecular Probes) and counterstained with DAPI. Images were obtained using a Zeiss Axiovert 200 m fluorescent microscope.

APOPtag

Isolated primary myoblasts from Bit-1 KO and aged-matched WT littermates or gastrocnemius muscle tissue were fixed in 1% paraformaldehyde in PBS, permeabilized in 70% ethanol and stained with ApopTag reagent (apoptosis in situ detection kit; Millipore, Temecula, CA) and propidium iodide as per the kit protocol. Images were obtained using a Zeiss Axiovert 200 m fluorescent microscope.

Statistical analyses

Myofiber cross sectional area analysis, fusion index, ELISA and densitometric readings were subjected to statistical analysis. Differences between the mean values and the densitometric readings were analyzed by ANOVA followed by Bonferroni's test for multiple comparisons between pairs or a Student's t-test. Values of P<0.05 indicated statistical significance. At least three independent experiments were performed for each data set and combined for statistical analysis.

Acknowledgements

We thank S. Young-Robbins and Anna Leychenko for expert technical assistance.

Footnotes

Competing interests

The authors declare no competing or financial interests.

Author contributions

G.S.G. performed caspase activity assays, siRNA experiments and western blotting, and quantified differentiation in myoblasts and C2C12 cells. J.D., M.J., P.V.R. and V.C. performed H&E staining and analyzed hypotrophic fibers in mice. M.d.l.V. performed C2C12 differentiation analysis and quantification. J.W.R., D.J.B. and M.L.M. designed experiments, analyzed data and wrote the manuscript.

Funding

This work was supported in part by grants from the National Institutes of Health [grant numbers RO1-GM104984 to M.L.M.; NCRR P20-RR016453 to M.L.M.; R01-GM088266 to J.W.R.; R01AR064338-01 to D.J.B.]; and a grant from the Ingeborg Foundation (M.L.M.). Deposited in PMC for release after 12 months.

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