Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) transcription complexes have been individually implicated in myogenesis, but their genetic interaction has not previously been addressed. Using MEF2A, c-Jun and Fra-1 chromatin immunoprecipitation sequencing (ChIP-seq) data and predicted AP-1 consensus motifs, we identified putative common MEF2 and AP-1 target genes, several of which are implicated in regulating the actin cytoskeleton. Because muscle atrophy results in remodelling or degradation of the actin cytoskeleton, we characterized the expression of putative MEF2 and AP-1 target genes (Dstn,Flnc, Hspb7, Lmod3 and Plekhh2) under atrophic conditions using dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock protein b7 (Hspb7) was induced by Dex treatment and further analyses revealed that loss of MEF2A using siRNA prevented Dex-regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun expression reduced Dex-mediated upregulation of Hspb7 whereas AP-1 depletion enhanced Hspb7 expression. In vivo, expression of Hspb7 and other autophagy-related genes was upregulated in response to atrophic conditions in mice. Manipulation of Hspb7 levels in mice also impacted gross muscle mass. Collectively, these data indicate that MEF2 and AP-1 confer antagonistic regulation of Hspb7 gene expression in skeletal muscle, with implications for autophagy and muscle atrophy.

Muscle atrophy is a phenomenon associated with reduced muscle fibre number and size caused by increased proteolysis and decreased protein synthesis (Romanick et al., 2013). In the elderly, muscle wasting is referred to as sarcopenia (Morley et al., 2001); in patients with cancer, AIDS or other chronic diseases, muscle atrophy is referred to as cachexia (Kotler, 2000). Improving or maintaining muscle mass in these populations has a profound impact on the ‘health span’ of individuals. For example, there is evidence that cachexia in cancer patients directly affects the time to tumour progression and disease recurrence (Kadar et al., 2000; Prado et al., 2009). In the ubiquitin proteasome pathway, the forkhead box protein (FoxO) family of transcription factors activates muscle atrophy through induction of two E3 ubiquitin ligases, MAFbx/atrogin-1 and MuRF1 (also known as TRIM63) (Sandri et al., 2004). Current treatment programs for muscle atrophy include activating the serine/threonine protein kinase (Akt) pathway, which induces muscle hypertrophy by inactivating FoxO proteins (Stitt et al., 2004). However, Akt can be inhibited by myostatin, a member of the transforming growth factor β (TGF-β) superfamily (Trendelenburg et al., 2009), superseding Akt activation as a treatment option. A new antibody recently characterized to bind to both members (A and B) of the myostatin/activin type II receptor (ActRII) induces hypertrophy in a muscle wasting model in vivo (Lach-Trifilieff et al., 2014). Additionally, targeting ActRIIB in cancer cachexia models can prevent atrophy, which results in prolonged survival without tumour manipulation (Zhou et al., 2010).

The autophagy pathway is an alternative mechanism of protein degradation that has also been implicated in muscle wasting. Foxo3, unlike other members of the FoxO family, is able to regulate autophagy in addition to the ubiquitin–proteasome pathway (Mammucari et al., 2007; Zhao et al., 2007). Several possible autophagy pathways have been identified in muscle, two of which are macroautophagy and chaperone-mediated autophagy (CMA). Although both processes ultimately lead to protein degradation in the lysosome, they achieve this through different mechanisms. In CMA, heat shock cognate 70 (Hsc70) targets proteins directly to the lysosome (Chiang et al., 1989). Macroautophagy requires de novo synthesis of autophagosomes in a multistep process that involves autophagy-related protein (Atg) family members. Autophagy is required for muscle homeostasis, as demonstrated by the muscle atrophy in mouse knockout models that lack proteins involved in autophagosome formation, such as Atg5 and Atg7 (Masiero et al., 2009; Raben et al., 2008). Aged muscle shows decreased autophagy and therefore build-up of protein aggregates (Demontis and Perrimon, 2010). LC3B (Map1lc3b) is an Atg protein that provides a useful readout for autophagy because it is post-translationally modified as it becomes part of the autophagosome (Kabeya et al., 2000). First, pro-LC3B is cleaved by Atg4 to form cytosolic LC3B-I. Atg7 then lipidates LC3B-I to form LC3B-II, which can form part of the autophagosome. Using samples from various atrophic mouse models, LC3B was shown to be strongly upregulated (Lecker et al., 2004). Furthermore, Foxo3 can directly regulate several autophagy-related genes, including that encoding LC3B (Mammucari et al., 2007; Zhao et al., 2007). A form of autophagy termed chaperone-assisted selective autophagy (CASA) merges the chaperone-mediated and macroautophagy pathways. In CASA, Hsc70 forms a complex with Bag3, Hspb8 and E3 ubiquitin ligase CHIP to identify protein aggregates and target them to the autophagosome (Arndt et al., 2010).

Myocyte-enhancer factor 2 (MEF2) is a member of the MADS-box family of transcription factors found in many tissues, including skeletal and cardiac muscle (Edmondson et al., 1994; Lin et al., 1997). MEF2 functions in a homo- or heterodimer complex with four different MEF2 isoforms in vertebrates (MEF2A–MEF2D), which bind to the consensus sequence [C/TTA(A/T)4TAG/A]. Previously we have shown that MEF2A can target a shared subset of genes in C2C12 myoblasts, an in vitro model of skeletal myogenesis, and in primary cardiomyocytes (Wales et al., 2014). Gene ontology (GO) analysis contained terms enriched for actin cytoskeleton organization and actin filament-based processes. In addition, many of these cytoskeletal MEF2 target genes were enriched for activator protein 1 (AP-1) cis elements, suggesting the possibility of combinatorial control. AP-1, consisting of Jun homodimer or Jun–Fos family heterodimers, recognizes the consensus sequence TGAG/CTCA (Angel et al., 1987). Global analysis of myoblast determination protein (MyoD) target genes in skeletal myoblasts likewise showed that AP-1 motifs are prominent in neighbouring sequences (Blais et al., 2005; Cao et al., 2010). Neighbouring MEF2 and AP-1 cis elements have recently been shown to be enriched in macrophages and neurons (Ma and Telese, 2015; Nagy et al., 2013). Several AP-1 subunits have been implicated in myogenesis. In particular, c-Jun antagonizes MyoD transcriptional activity in vitro (Bengal et al., 1992; Li et al., 1992). Using high throughput data, Blum et al. (2012) reported that c-Jun and MyoD coordinate muscle enhancers, indicating a more complex role for AP-1 in muscle than previously anticipated (Blum et al., 2012). Additionally, the Fos family member Fra-2 is thought to play a role in maintenance of the skeletal muscle satellite cell population (Alli et al., 2013).

Although MEF2 and AP-1 have individually been shown to function in the myogenic program, their potential interaction has not been documented. Additionally, although loss of MEF2 and AP-1 have been implicated in loss of sarcomere integrity during development and satellite cell-mediated muscle regeneration (Hinits and Hughes, 2007; Potthoff et al., 2007; Windak et al., 2013), the combined role of these factors in muscle atrophy has not been investigated. Here, we document that MEF2 and AP-1 regulate several genes associated with the actin cytoskeleton. Among the gene products, the small heat shock protein, Hspb7 is implicated in muscle atrophy.

Identification of biological processes associated with MEF2A and AP-1 recruitment

MEF2 and AP-1 are transcription complexes involved in myoblast proliferation and differentiation, yet whether they regulate common or non-overlapping target genes during differentiation has not been thoroughly explored. To address this, we utilized a previously reported MEF2A dataset from chromatin immunoprecipitation combined with exonuclease digestion (ChIP-exo) for differentiating C2C12 myogenic cells (48 h in differentiation medium; DM) (Wales et al., 2014) and compared these binding events with data from c-Jun (Blum et al., 2012) and Fra-1 (Wold group, ENCODE) ChIP in combination with sequencing (ChIP-seq), which was likewise carried out in myogenic cells. Fra-1 is primarily associated with bone development (Eferl et al., 2004; Fleischmann, 2000) and c-Jun has been implicated in many tissues, including muscle (Bengal et al., 1992; Blum et al., 2012; Li et al., 1992). Both the c-Jun and Fra-1 ChIP-seq data were completed in C2C12 myoblasts in growth medium (GM), making these data comparable with our previously generated MEF2A dataset. The c-Jun dataset contained 9778 binding events and the Fra-1 dataset contained 6507. To determine the percentage of shared binding sites across these datasets we used a MEF2A-centric analysis (Fig. 1A). From this analysis we observed that the majority of MEF2A binding sites (69%) were independent of c-Jun or Fra-1 recruitment, yet 17% of MEF2A-bound DNA also contained c-Jun recruitment and 12% contained both c-Jun and Fra-1. Fra-1 and MEF2A alone shared few binding sites (2%).

Fig. 1.

Comparison of MEF2A and AP-1 target genes in skeletal muscle. (A) Percentage overlap between MEF2A, c-Jun, and Fra-1. The total number of MEF2A ChIP-seq binding events is 2783. All datasets were converted to the mm9 genome and then overlapping peaks were identified from individual bed files for MEF2A, c-Jun and Fra-1 using the UCSC intersect function. (B) Functional roles for MEF2A alone, MEF2A and c-Jun, and MEF2A with c-Jun and Fra-1. Using the datasets from 1A, GREAT analysis revealed GO terms for biological processes. The total number of genes per GO term are indicated to the right of each GO term.

Fig. 1.

Comparison of MEF2A and AP-1 target genes in skeletal muscle. (A) Percentage overlap between MEF2A, c-Jun, and Fra-1. The total number of MEF2A ChIP-seq binding events is 2783. All datasets were converted to the mm9 genome and then overlapping peaks were identified from individual bed files for MEF2A, c-Jun and Fra-1 using the UCSC intersect function. (B) Functional roles for MEF2A alone, MEF2A and c-Jun, and MEF2A with c-Jun and Fra-1. Using the datasets from 1A, GREAT analysis revealed GO terms for biological processes. The total number of genes per GO term are indicated to the right of each GO term.

Functional roles for shared MEF2A and AP-1 binding sites were identified using the Genomics Regions Enrichment of Analysis Tool (GREAT), which revealed enriched GO terms for biological processes, the top ten of which are depicted in Fig. 1B. The GO terms were ranked by binomial raw P-value; the number of genes within each GO term is indicated. DNA enriched for MEF2A-alone was associated with the better-known functions of MEF2 such as actin-filament-based processes and skeletal muscle tissue development, but also with cell development (blue). There were no GO terms identified for MEF2A and Fra-1. However, MEF2A and c-Jun had GO terms for striated muscle development, vascular development and heart morphogenesis (purple). The complete list of GO terms is given in Supplementary Dataset S1. Fewer GO terms were enriched for genes associated with all three factors and these were related to the actin cytoskeleton and negative regulation of smooth muscle cell proliferation (black).

Because the MEF2A dataset was obtained from differentiating myoblasts, and given that several other AP-1 components apart from c-Jun and Fra-1 could be associated with MEF2 target genes, we determined the location of AP-1 consensus sequences containing the sequence TGAGTCA using cisGenome and allowing zero mismatches. From the mm9 genome, this search identified 264,537 AP-1 consensus sites. We focused on putative MEF2A and AP-1 binding sites within ±10 kb of the transcription start site (TSS) of the single nearest gene and observed that 11 out of these 76 genes are associated with the molecular function ‘cytoskeleton protein binding’ (Table S1). We then assessed the recruitment of MEF2A, c-Jun or Fra-1and the presence of AP-1 consensus sequences near five of these genes, encoding the proteins destrin (Dstn), filamin C (Flnc), heat shock protein family, member 7 (Hspb7), leiomodin 3 (Lmod3) and pleckstrin homology domain containing family H, member 2 (Plekhh2). Dstn is an actin-depolymerizing protein (Carlier et al., 1997) and FlInc, a muscle-specific filamin (Thompson et al., 2000) that promotes the cross-linking of actin. We had also previously identified the genes encoding Hspb7 (a small heat shock protein) and Lmod3 (tropomodulin family member) as MEF2 target genes in cardiac muscle (Wales et al., 2014). Human nemaline myopathy has been associated with mutations in Lmod3 (Cenik et al., 2015; Garg et al., 2014; Yuen et al., 2014). Hspb7 expression is higher in mdx (a genetic model of muscular dystrophy) mice than in normal mice and is also enhanced in ageing muscle (Doran et al., 2006, 2007). Recently, a skeletal muscle-specific conditional Hspb7 knockout model was reported to result in progressive myopathy (Juo et al., 2016). The role of Plekhh2 appears to be to stabilize the cortical actin cytoskeleton (Perisic et al., 2012). The recruitment pattern of MEF2A, c-Jun, Fra-1 and any AP-1 consensus sequences is indicated in images from UCSC (Fig. S1). The overall trends demonstrate several points. First, MEF2A was performed using ChIP-exo, which involves exonuclease digestion prior to sequencing, therefore MEF2A peaks are more defined. This indicates one of the advantages of ChIP-exo over conventional ChIP-seq. Second, the scale of c-Jun and Fra-1 recruitment differ dramatically, which could indicate differential antibody affinities or distinct DNA binding affinities of AP-1 family members. Third, MEF2A and c-Jun or Fra-1 show similar recruitment patterns to a subset of putative target genes. Of these five MEF2A target genes, c-Jun was recruited to an overlapping or neighbouring binding event near Flnc, Hspb7 and Plekhh2 (Fig. S1). Fra-1 and MEF2A recruitment only overlapped at the promoter of Flnc, and Fra-1 was detected within the second intron of Hspb7, where c-Jun also showed recruitment (Fig. S1). This in silico analysis identifies a potential interplay between AP-1 and MEF2 in the transcriptional control of target genes involved in muscle stability.

Actin cytoskeletal genes are regulated by MEF2A and AP-1

Aside from c-Jun and Fra-1, AP-1 has many family members that are expressed in muscle, including Fra-2, JunD, c-Fos and several others. We assessed the expression pattern of MEF2A and AP-1 family members during C2C12 differentiation (Fig. 2A). As myogenin and MEF2A levels increased during myogenesis, only one AP-1 subunit (Fra-2) also increased. c-Jun, JunD and, most dramatically, Fra-1 levels were reduced in DM. To move forward in determining a role for MEF2 and AP-1 in myogenesis, we focused on Fra-2 and c-Jun because they are expressed in myoblasts and have been shown to have a role in myogenesis (Alli et al., 2013; Bengal et al., 1992; Blum et al., 2012; Li et al., 1992). Additionally, the Fra-2–c-Jun heterodimer is one of the main AP-1 binding complexes present in C2C12 differentiation (Andreucci et al., 2002). Fra-2 is present in different isoforms and subject to post-translational modification by ERK (Alli et al., 2013; Andreucci et al., 2002). To date, Fra-2 ChIP-seq data is not available.

Fig. 2.

MEF2A and AP-1 regulation of actin cytoskeletal genes. (A) MEF2A and AP-1 protein expression during myogenesis. C2C12 cells were allowed to differentiate from myoblasts (GM) to 48 h DM. (B) Expression pattern of MyoG, Dstn, Flnc, Hspb7, Lmod3 and Plekhh2 during C2C12 differentiation in GM and 48 h DM. Values were calculated using the ΔΔCt method and normalized to β-actin (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Efficiency of knockdown of MEF2A, Fra-2 and c-Jun in C2C12 myocytes (48 h DM) using siRNA-mediated gene silencing. C2C12 cells were transfected with siRNA and allowed to differentiate for 48 h before western blot analysis. scr indicates a scrambled siRNA control. (D) siRNA-mediated knockdown of MEF2A, Fra-2 and c-Jun at 48 h DM was carried out to assess changes in target gene expression. Data were analysed as in B. (E) C2C12 cells were transfected with GFP–Tam67 using calcium phosphate. RNA was isolated at the indicated time points and analysed via qRT-PCR as in B. (F) Western blot depicting expression of GFP-tagged Tam67.

Fig. 2.

MEF2A and AP-1 regulation of actin cytoskeletal genes. (A) MEF2A and AP-1 protein expression during myogenesis. C2C12 cells were allowed to differentiate from myoblasts (GM) to 48 h DM. (B) Expression pattern of MyoG, Dstn, Flnc, Hspb7, Lmod3 and Plekhh2 during C2C12 differentiation in GM and 48 h DM. Values were calculated using the ΔΔCt method and normalized to β-actin (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Efficiency of knockdown of MEF2A, Fra-2 and c-Jun in C2C12 myocytes (48 h DM) using siRNA-mediated gene silencing. C2C12 cells were transfected with siRNA and allowed to differentiate for 48 h before western blot analysis. scr indicates a scrambled siRNA control. (D) siRNA-mediated knockdown of MEF2A, Fra-2 and c-Jun at 48 h DM was carried out to assess changes in target gene expression. Data were analysed as in B. (E) C2C12 cells were transfected with GFP–Tam67 using calcium phosphate. RNA was isolated at the indicated time points and analysed via qRT-PCR as in B. (F) Western blot depicting expression of GFP-tagged Tam67.

The expression of cytoskeletal genes during myogenesis was determined using quantitative reverse transcription (qRT)-PCR in GM (myoblasts) and at 48 h in DM (myocytes) (Fig. 2B). During C2C12 differentiation, the expression of each gene except Dstn increased. To confirm MEF2A recruitment to Dstn, Flnc, Hspb7, Lmod3 and Plekhh2, ChIP-qPCR was performed in growth conditions (GM) and during differentiation (48 h DM). During differentiation, MEF2A was recruited to each gene, compared with a control region upstream of SMA (Fig. S2). MEF2A recruitment to Lmod3 and Hspb7 was the most significant and reflects the ChIP-seq data (Fig. S1). Fra-2 and c-Jun recruitment were also assessed at the same regions as MEF2A (Figs S1 and S2). c-Jun was moderately recruited to Flnc, Hspb7 and Plekhh2 in both GM and 48 h DM equally but was not detected near Dstn or Lmod3. Fra-2 enrichment was detected for all genes at 48 h DM, with the strongest recruitment observed on Flnc (Fig. S2).

To determine whether actin cytoskeletal genes are sensitive to the loss of AP-1 and MEF2 we utilized siRNA-mediated gene silencing. MEF2A is the predominant MEF2 subunit in differentiating myogenic cells and our previous ChIP-exo data was completed using a MEF2A antibody; therefore, we initially used siRNA targeting MEF2A. Because AP-1 could function in Jun–Fos or Jun–Jun homodimers and because in muscle c-Jun and Fra-2 have been documented to be crucial factors in regulating myoblast proliferation (Alli et al., 2013; Bengal et al., 1992), we used siRNA targeting c-Jun and Fra-2 (Fig. 2C). Depletion of MEF2A resulted in downregulation of Hspb7 and Lmod3 (Fig. 2D). Dstn, Flnc and Plekhh2 showed MEF2A recruitment (Fig. S2) but no differential expression with MEF2A knockdown, possibly reflecting some level of redundancy in the MEF2 family. A reduction in c-Jun did not significantly affect target gene expression, although decreases in Flnc, Plekhh2 and Fra-2 were observed. Similarly, although Fra-2 expression was partially reduced, this did not result in dramatic changes in gene expression, potentially indicating AP-1 functional redundancy.

To account for partial knockdown with siRNA technology or the involvement of other AP-1 family members in expression of these genes, we exogenously expressed GFP–Tam67, a potent dominant negative form of c-Jun that lacks the transactivation domain but contains the DNA binding and dimerization domains. Previously, Tam67 has been used as a dominant negative of the AP-1 complex and, as such, is a useful tool to use when redundancy of the complex AP-1 subunits is suspected (Hennigan and Stambrook, 2001). We assessed expression of the five target genes in growth conditions (0 h DM) and after 24 and 72 h in DM. Tam67 expression had marginal effects on Dstn, Flnc, Hspb7, Lmod3 and Plekhh2 expression at 0 and 24 h in DM (Fig. 2E). However, after 72 h in DM, all five genes were robustly upregulated by Tam67 expression. Expression of GFP–Tam67 is depicted in Fig. 2F. Together these data indicate that a subset of MEF2A target genes are transcriptionally regulated by AP-1.

Atrophy induced by age or dexamethasone modulates expression of MEF2A and AP-1 cytoskeletal target genes

The cytoskeleton is integrally linked to the contractile unit of the myofibril, the sarcomere, which is mainly comprised of α-actin and myosin. During muscle atrophy (a phenomenon observed in sarcopenia, cachexia and various genetic diseases) the cytoskeleton and components of the sarcomere become degraded, resulting in overall muscle loss and weakness. Because these five proteins (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) are involved in the actin cytoskeleton, to varying degrees, we next determined whether expression of the corresponding MEF2 and AP-1 target genes changes under atrophic conditions. To determine whether these genes were differentially expressed in growing postnatal muscle compared with mature adult muscle we isolated RNA from the gastrocnemius and quadriceps of 8- and 63-week-old mice (Fig. 3A). In the gastrocnemius, Hspb7 was upregulated with age. In the quadriceps, Dstn, Flnc and Hspb7 were upregulated.

Fig. 3.

Atrophy induced by aging or dexamethasone causes changes in MEF2A and AP-1 cytoskeletal target genes. (A) RNA from the gastrocnemius and quadriceps of 8- and 63-week-old C57BL/6 mice was isolated for qRT-PCR analysis. Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3 except for Flnc expression in the gastrocnemius sample where n=2, mean±s.e.m., *P<0.05). (B) Dex treatment of myotubes induces atrophy and modulates the expression of MEF2A and AP-1 target genes. C2C12 cells were allowed to differentiate for 72 h and then treated with Dex for 6 h (upper graph) or 24 h (lower graph) at the indicated concentrations. Values were calculated using the ΔΔCt method and normalized to Gapdh.

Fig. 3.

Atrophy induced by aging or dexamethasone causes changes in MEF2A and AP-1 cytoskeletal target genes. (A) RNA from the gastrocnemius and quadriceps of 8- and 63-week-old C57BL/6 mice was isolated for qRT-PCR analysis. Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3 except for Flnc expression in the gastrocnemius sample where n=2, mean±s.e.m., *P<0.05). (B) Dex treatment of myotubes induces atrophy and modulates the expression of MEF2A and AP-1 target genes. C2C12 cells were allowed to differentiate for 72 h and then treated with Dex for 6 h (upper graph) or 24 h (lower graph) at the indicated concentrations. Values were calculated using the ΔΔCt method and normalized to Gapdh.

In cell culture, muscle atrophy can be replicated by treatment with Dex, a synthetic glucocorticoid. To model glucocorticoid-induced atrophy, C2C12 myoblasts were allowed to differentiate for 72 h in DM, and then treated with Dex (Fig. 3B). In this analysis we included two E3 ubiquitin ligases, MAFbx and MuRF1, which are associated with muscle atrophy and serve as positive controls for the process. These E3 ligases promote atrophy and ubiquitinate proteins for degradation. In muscle, MuRF1 directly targets myosin and myosin binding proteins for degradation, contributing to sarcomere loss (Clarke et al., 2007; Cohen et al., 2009). After 6 or 24 h of treatment with Dex, MAFbx and MuRF1 were upregulated. Treatment with Dex for 6 h increased Dstn and Hspb7 expression and decreased Flnc. Expression of Lmod3 and Plekhh2 was unchanged. After 24 h treatment with Dex these trends were similar; however, Hspb7 was upregulated fivefold, equivalent to the degree of MAFbx and MuRF1 induction.

MEF2A and c-Jun and Fra-2 regulate atrophy-induced Hspb7 expression

Hspb7 was the most dynamically regulated gene during ageing in skeletal muscle and was also highly inducible in response to Dex treatment; therefore, we pursued the regulation of this gene by MEF2A, c-Jun and Fra-2 in more detail. A previous study had identified target genes of the glucocorticoid receptor (GR) in Dex-treated C2C12 myotubes (Kuo et al., 2012). Interestingly, this study found GR recruitment to an intron of Hspb7. Using the UCSC browser we plotted the MEF2A, c-Jun, Fra-1 and AP-1 peaks and compared them with GR recruitment reported by Kuo et al. (2012) within the Hspb7 gene (Fig. 4A). GR was recruited following Dex treatment to within the second intron of Hspb7, which contained c-Jun and Fra-1 enrichment peaks. We utilized Dex treatment to further determine how MEF2A, AP-1 and GR might contribute to Hspb7 expression.

Fig. 4.

Changes in Hspb7 expression induced by dexamethasone are modulated by MEF2A and AP-1. (A) UCSC genome browser image depicting recruitment of MEF2A (blue), Fra-1 (brown; C2 FOSL1), c-Jun (green) and glucocorticoid receptor (GR, red) to Hspb7. AP-1 consensus sequences are indicated by vertical black lines. (B) Dex treatment of myotubes strongly upregulated Hspb7 protein expression. C2C12 cells were treated with 10 μM Dex for the indicated times. Actin was used as a loading control. (C,D) Exogenous expression of MEF2A, Fra-2 or c-Jun with Dex treatment in growth conditions (C) or 72 h DM (D). C2C12 cells were transfected with the indicated construct using calcium phosphate and allowed to recover. Cells were then treated with 10 μM Dex for 24 h after 24 h in GM (C) or after 48 h in DM (D). (E) Loss of MEF2A, Fra-2 or c-Jun affects the induction of Hspb7 expression by Dex treatment. Cells were transfected with the indicated siRNA and allowed to differentiate for 48 h, after which they were treated with 10 μM Dex for 24 h. (F) At 48 h, DM C2C12 cells were treated with 10 μM Dex for 24 h and prepared for ChIP-qPCR analysis. Enrichment of GR or MEF2A was detected at the 2nd intron or 3′-end, respectively. Data were calculated using the percentage input method.

Fig. 4.

Changes in Hspb7 expression induced by dexamethasone are modulated by MEF2A and AP-1. (A) UCSC genome browser image depicting recruitment of MEF2A (blue), Fra-1 (brown; C2 FOSL1), c-Jun (green) and glucocorticoid receptor (GR, red) to Hspb7. AP-1 consensus sequences are indicated by vertical black lines. (B) Dex treatment of myotubes strongly upregulated Hspb7 protein expression. C2C12 cells were treated with 10 μM Dex for the indicated times. Actin was used as a loading control. (C,D) Exogenous expression of MEF2A, Fra-2 or c-Jun with Dex treatment in growth conditions (C) or 72 h DM (D). C2C12 cells were transfected with the indicated construct using calcium phosphate and allowed to recover. Cells were then treated with 10 μM Dex for 24 h after 24 h in GM (C) or after 48 h in DM (D). (E) Loss of MEF2A, Fra-2 or c-Jun affects the induction of Hspb7 expression by Dex treatment. Cells were transfected with the indicated siRNA and allowed to differentiate for 48 h, after which they were treated with 10 μM Dex for 24 h. (F) At 48 h, DM C2C12 cells were treated with 10 μM Dex for 24 h and prepared for ChIP-qPCR analysis. Enrichment of GR or MEF2A was detected at the 2nd intron or 3′-end, respectively. Data were calculated using the percentage input method.

At the protein level, Dex treatment upregulated Hspb7 expression in the late stages of differentiation but not under growth conditions (Fig. 4B). Next, the effect of exogenous expression of MEF2A, Fra-2 or c-Jun in combination with Dex treatment was assessed in growth conditions and in 72 h myotubes. Under growth conditions, Hspb7 expression was only enhanced by the combined overexpression of MEF2A and Dex treatment. Dex treatment alone could not induce induction of Hspb7 (Fig. 4C). In myotubes, Dex treatment consistently upregulated Hspb7 expression, except when c-Jun was ectopically expressed. This further indicated the repressive role of AP-1 at this locus (Fig. 4D). By 48 h in DM, endogenous expression of MEF2A was significantly higher (Fig. 2A). However, premature expression of MEF2A under the conditions for exogenous expression contributed to upregulation of Hspb7 at this time point, and this was enhanced by Dex treatment.

Finally, to determine whether MEF2 and AP-1 are necessary for Dex-induced upregulation of Hspb7, C2C12 myoblasts were transfected with siRNA targeting MEF2A, Fra-2 or c-Jun, allowed to differentiate for 48 h and then treated with Dex to determine whether Hspb7 expression was affected (Fig. 4E). Under Dex treatment, loss of MEF2A prevented Dex-dependent induction of Hspb7 expression; however, loss of Fra-2 or c-Jun upregulated Hspb7 and this was enhanced by Dex treatment. Of note, when expressed exogenously, c-Jun was able to block Hspb7 induction whereas Fra-2 could not, yet loss of either of these proteins led to the upregulation of Hspb7 expression (Fig. 4D,E). As we previously showed, c-Jun and Fra2 function as a heterodimer in skeletal muscle (Andreucci et al., 2002), therefore this disparity could be related to the relative transcriptional potency of these transcription factors: c-Jun is a robust transcriptional regulator and can form a homodimer in the absence of Fra-2, whereas Fra-2 is a weaker transcriptional activator that has no intrinsic transactivation domain and relies on recruitment of the Jun heterodimeric partner for bringing transactivation properties to the AP-1 complex (Suzuki et al., 1991; Wisdon and Verma, 1993). Together, these data indicate the possibility of antagonistic regulation of Hspb7 by AP-1 and MEF2. Also, MEF2 could function in combination with GR recruitment for upregulation of Hspb7. To address the possible involvement of GR, we performed ChIP-qPCR and assessed GR recruitment to the second intron of Hspb7 after Dex treatment (48 h DM plus 24 h 10 μM Dex). Indeed, we observed a substantial recruitment of GR to the Hspb7 locus after Dex treatment, in agreement with ChIP-seq data generated by Kuo et al. (2012), but no change in MEF2A binding at the 3′-end (Fig. 4F).

Characterization of the role of Hspb7 in muscle atrophy

Small heat shock proteins have a documented role in protecting the cytoskeleton during stress (Garrido et al., 2012). Hspb7 (also known as cvHsp) is highly expressed in skeletal and cardiac muscle (Krief et al., 1999) and has been linked to cardiac morphogenesis, cardiomyopathies and skeletal muscle integrity in the adult (Chiu et al., 2012; Juo et al., 2016; Rosenfeld et al., 2013). In other cell types, Hspb7 has been shown to prevent protein aggregation (Eenjes et al., 2016; Minoia et al., 2014) and to localize to nuclear speckles (Vos et al., 2009), a sub-nuclear location in which pre-mRNA is spliced.

Using an HA-tagged Hspb7 expression construct we observed that exogenous expression of Hspb7 did not influence myogenic induction in vitro, as shown by the lack of change in MyoG expression (Fig. 5A), in agreement with Juo et al. (2016) who showed that Hspb7 does not contribute directly to myogenesis. Hspb7 has been associated with autophagy but the mechanism is unclear (Vos et al., 2010). Bag3 is a component of CMA that shows enriched expression in striated muscle, and Bag3-null mice develop myopathies (Homma et al., 2006). Interestingly, Hspb7 and Bag3 single nucleotide polymorphisms (SNPs) have been associated with heart failure (Garnier et al., 2015). Hspb7 has also been shown to interact with Hspb8 (Sun et al., 2004), an autophagy-related protein that interacts with Bag3 (Carra et al., 2008). Therefore, we investigated whether Hspb7 could affect autophagy by using the conversion of LC3B-I to LC3B-II as a molecular readout for this process. We observed that LC3B-II levels were not significantly affected by Hspb7 overexpression; however, exogenous Hspb7 protein was rapidly degraded within 24 h whereas GFP protein levels were maintained (Fig. 5A). We hypothesized that the turnover of Hspb7 protein could indicate its involvement in autophagy and subsequent degradation in the autolysosome. To confirm whether the loss of exogenous Hspb7 demonstrated in Fig. 5A could be via autophagy we monitored endogenous Hspb7 proteins levels in response to treatment with rapamycin, an mTOR inhibitor. Endogenous Hspb7 was reduced after 24 h of rapamycin treatment (Fig. 5B), indicating that Hspb7 could be degraded within the autophagosome.

Fig. 5.

Role of Hspb7 in skeletal muscle atrophy. (A) Overexpression of Hspb7 in differentiating C2C12 myoblasts. Cells were transfected with Hspb7–HA and allowed to differentiate for the indicated times. Extracts were prepared for western blot. (B) Rapamycin treatment decreases Hspb7 expression. Myotubes (72 h) were treated with rapamycin (10 μg/ml) for the indicated times. Protein extracts were then prepared for western blot analysis. (C) Myoblasts transfected with EYFP–Hspb7 were treated with rapamycin (10 μg/ml) for 6 h and then assessed for LC3B co-localization (red). Hoechst stain was used to visualize nuclei. (D) C2C12 cells were transfected with Hspb7–HA or control and treated with rapamycin (10 μg/ml) for 6 h in GM, 48 h post-transfection. Protein extracts were then prepared for western blot. (E) C2C12 cells were prepared as in D. Cells were treated with either BafA alone (200 nM) or BafA plus rapamaycin (10 μg/ml) for 6 h. (F) Quantification of LC3B-II expression was relative to actin. Values were quantified in ImageJ.

Fig. 5.

Role of Hspb7 in skeletal muscle atrophy. (A) Overexpression of Hspb7 in differentiating C2C12 myoblasts. Cells were transfected with Hspb7–HA and allowed to differentiate for the indicated times. Extracts were prepared for western blot. (B) Rapamycin treatment decreases Hspb7 expression. Myotubes (72 h) were treated with rapamycin (10 μg/ml) for the indicated times. Protein extracts were then prepared for western blot analysis. (C) Myoblasts transfected with EYFP–Hspb7 were treated with rapamycin (10 μg/ml) for 6 h and then assessed for LC3B co-localization (red). Hoechst stain was used to visualize nuclei. (D) C2C12 cells were transfected with Hspb7–HA or control and treated with rapamycin (10 μg/ml) for 6 h in GM, 48 h post-transfection. Protein extracts were then prepared for western blot. (E) C2C12 cells were prepared as in D. Cells were treated with either BafA alone (200 nM) or BafA plus rapamaycin (10 μg/ml) for 6 h. (F) Quantification of LC3B-II expression was relative to actin. Values were quantified in ImageJ.

To investigate whether Hspb7 associates with autophagosomes, we generated an EYFP–Hspb7 construct and determined the co-localization of Hspb7 with LC3B under rapamycin treatment in myoblasts (Fig. 5C). After 6 h of rapamycin treatment, LC3B was detected in puncta throughout the cell and in large aggregates, where EYFP–Hspb7 was also observed.

Because rapamycin affected endogenous Hspb7 protein levels and Hspb7 and LC3B co-localize, we hypothesized that Hspb7 could be directly involved in autophagic flux. Related family member Hspb8 was able to induce LC3B-II accumulation in previous studies, but Hspb7 had no effect (Vos et al., 2010) even though these studies were carried out without pharmacological activation of autophagy and without lysosome inhibitors, which are probably necessary to interpret autophagic flux. Therefore, we treated cells with rapamycin in the presence or absence of Hspb7–HA (Fig. 5D). It was observed that rapamycin alone induced LC3B-II levels but this was reduced with Hspb7–HA expression. Changes in LC3B-II levels without lysosomal blockade are invoked by either increased or decreased autophagic flux; therefore, we next used BafA in combination with rapamycin and Hspb7 exogenous expression to determine whether the rate of autophagy was altered by Hspb7 expression. Interestingly, we saw reduced LC3B-II levels (Fig. 5E, quantified in Fig. 5F), indicating the possibility that Hspb7 reduces autophagy.

A role for Hspb7 in skeletal muscle autophagy in vivo

Based on the above data, we suspected that Hspb7 is induced under muscle atrophy and has a potential role in autophagy. Although we propose that Hspb7 is involved in mature muscle homeostasis, our in vitro experiments were limited to early growth conditions because exogenous expression of Hspb7 or siRNA was lost in cultured myotubes (>72 h DM). Therefore, to determine whether Hspb7 is associated with autophagy in mature muscle we used a previously characterized in vivo model of autophagy via 24 h fasting of mice (Ju et al., 2010). Twenty-four hours prior to fasting, colchicine (0.4 mg/kg/day) was injected to serve as an autophagic block and this treatment was repeated every 24 h (Fig. 6A). Under colchicine plus fasting conditions, Hspb7 expression was increased in the quadriceps and gastrocnemius muscles but not the heart (Fig. 6B). We also assessed expression of Hspb7 and autophagy-related genes in the tibialis anterior (TA) muscle under these conditions and observed an upregulation of Map1lc3b, p62, MyoG Atg7, Foxo1, Foxo3 and Hspb7 (Fig. 6C).

Fig. 6.

Hspb7 expression is associated with autophagy. (A) Experimental design for in vivo experiments. 6- to 8-week-old C57BL/6 mice were treated with saline, colchicine (0.4 mg/kg/day) or colchicine plus fasting (24 h). (B) Hspb7 expression in the heart, quadriceps (Quad) and gastrocnemius (Gastroc). Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Changes in autophagy genes in response to fasting. RNA was isolated from the TA for qRT-PCR analysis. Data were analysed as in B.

Fig. 6.

Hspb7 expression is associated with autophagy. (A) Experimental design for in vivo experiments. 6- to 8-week-old C57BL/6 mice were treated with saline, colchicine (0.4 mg/kg/day) or colchicine plus fasting (24 h). (B) Hspb7 expression in the heart, quadriceps (Quad) and gastrocnemius (Gastroc). Values were calculated using the ΔΔCt method and normalized to Gapdh (n=3, mean±s.e.m., *P<0.05, **P<0.01). (C) Changes in autophagy genes in response to fasting. RNA was isolated from the TA for qRT-PCR analysis. Data were analysed as in B.

To determine whether Hspb7 has a protective role during muscle atrophy induced by starvation we exogenously expressed an HA-tagged Hspb7 construct into the TA muscle using electroporation. These were ‘paired’ experiments whereby the TA muscle of one leg received the Hspb7–HA expression vector while the contralateral leg TA received the control vector, thus serving as a control in the same animal (Fig. 7A). Interestingly, total mass of the TA was reduced with Hspb7–HA expression in the colchicine plus fasting condition (Fig. 7B). Total RNA was isolated for qRT-PCR analysis of autophagy-related genes (Fig. 7C). Under control conditions (saline), Hspb7–HA did not alter expression of Map1lc3b, p62, Foxo1 or Foxo3. These genes were upregulated in response to colchicine and fasting. However, in Hspb7–HA transfected limbs, induction of Map1lc3b, p62 and Foxo1 was weaker, although expression was not significantly different from the paired leg control (Fig. 7C). At the protein level, we noticed that mice from the colchicine plus fasting condition that did not receive Hspb7–HA showed an upregulation of total LC3B (note LC3B-I in lanes 7–9) compared with non-fasted, saline conditions (Fig. 7D). This change in protein level correlates with increased mRNA expression of Map1lc3b (Fig. 7C). Hspb7–HA expression showed weaker, though not significantly different, expression of total LC3B compared with the paired leg controls from the same mice (Fig. 7D).

Fig. 7.

Hspb7 regulates gross muscle mass and LC3B expression. (A) Experimental design for in vivo experiments. Hspb7–HA was electroporated into the TA muscle while the contralateral TA muscle received the control plasmid (pCAGGSnHC). Mice were then treated with saline or colchicine plus fasting for the indicated times (colchicine, 0.4 mg/kg/day; fasting, 24 h). (B) Total TA muscle mass under the indicated experimental conditions (n=6; mean±s.e.m., **P<0.01). (C) In vivo gene expression during colchicine plus fasting with exogenous expression of Hspb7–HA or control in paired leg experiment. Saline, n=6; colchicine plus fasting, n=5; mean±s.e.m.; *P<0.05, **P<0.01 compared with saline control. Values were calculated using the ΔΔCt method and normalized to the geometric mean of β-actin, Gapdh, Tbp and Rps26. (D) Samples from C were analysed using western blot to determine level of exogenous expression of Hspb7–HA and LC3B. (E) TA muscles were electroporated with tandem LC3 and Hspb7–HA or tandem LC3 and control plasmid and allowed to recover for 3 days before being treated with saline or colchicine plus fasting, as depicted in A. Transverse sections of the muscle were then prepared for analysis of tandem LC3 localization. (F) Experimental design of siRNA experiments. Three independent siRNAs targeting Hspb7 were pooled into a final concentration of 450 pmol (150 pmol of each) and electroporated into the TA muscle. Control siRNA (scr) was electroporated into the contralateral control TA muscle. At 2 days post-electroporation, colchicine was administered daily for a further 2 days. (G) Expression of Hspb7 protein levels in the TA muscle after siRNA knockdown. (H) Total TA muscle mass under the indicated experimental conditions (n=3; mean±s.e.m., *P<0.05).

Fig. 7.

Hspb7 regulates gross muscle mass and LC3B expression. (A) Experimental design for in vivo experiments. Hspb7–HA was electroporated into the TA muscle while the contralateral TA muscle received the control plasmid (pCAGGSnHC). Mice were then treated with saline or colchicine plus fasting for the indicated times (colchicine, 0.4 mg/kg/day; fasting, 24 h). (B) Total TA muscle mass under the indicated experimental conditions (n=6; mean±s.e.m., **P<0.01). (C) In vivo gene expression during colchicine plus fasting with exogenous expression of Hspb7–HA or control in paired leg experiment. Saline, n=6; colchicine plus fasting, n=5; mean±s.e.m.; *P<0.05, **P<0.01 compared with saline control. Values were calculated using the ΔΔCt method and normalized to the geometric mean of β-actin, Gapdh, Tbp and Rps26. (D) Samples from C were analysed using western blot to determine level of exogenous expression of Hspb7–HA and LC3B. (E) TA muscles were electroporated with tandem LC3 and Hspb7–HA or tandem LC3 and control plasmid and allowed to recover for 3 days before being treated with saline or colchicine plus fasting, as depicted in A. Transverse sections of the muscle were then prepared for analysis of tandem LC3 localization. (F) Experimental design of siRNA experiments. Three independent siRNAs targeting Hspb7 were pooled into a final concentration of 450 pmol (150 pmol of each) and electroporated into the TA muscle. Control siRNA (scr) was electroporated into the contralateral control TA muscle. At 2 days post-electroporation, colchicine was administered daily for a further 2 days. (G) Expression of Hspb7 protein levels in the TA muscle after siRNA knockdown. (H) Total TA muscle mass under the indicated experimental conditions (n=3; mean±s.e.m., *P<0.05).

To further define the role of Hspb7, we utilized a tandemly tagged LC3 plasmid containing both GFP and mRFP (Kimura et al., 2007). GFP becomes degraded in the acidic lysosomal compartment and, therefore, the difference in fluorescence corresponds to autophagosomes or autolysosomes, which appear yellow (GFP+ and RFP+) or red (GFP− and RFP+), respectively. We electroporated this plasmid into the TA muscle of mice with Hspb7–HA and initiated a protocol of colchicine plus fasting, as depicted in Fig. 6A. Compared with saline, mice treated with colchicine plus fasting showed upregulation of LC3 puncta with the control plasmid (Fig. 7E). True LC3 puncta corresponding to autophagosomes or autolysosomes can be difficult to determine using this method because exogenous LC3 is prone to aggregation (Kuma et al., 2007) and, indeed, we did see minimal aggregation at the periphery of myofibres with saline and control plasmids. Hspb7 has been shown to prevent protein aggregation in vitro (Eenjes et al., 2016), therefore we predicted that Hspb7 could also reduce LC3 aggregation. Interestingly, we observed an overall trend whereby co-expression of Hspb7–HA with tandem-LC3 resulted in enhanced aggregation of LC3 (Fig. 7E).

We next utilized siRNA to knockdown Hspb7in vivo, in combination with 2 days of colchicine treatment (Fig. 7F). Loss of Hspb7 protein was detected most clearly with colchicine treatment (Fig. 7G). Interestingly, conditions with a significant reduction of Hspb7 protein levels were associated with an increase in muscle mass (Fig. 7H). Collectively, these data suggest a possible involvement of Hspb7 in regulation of overall muscle mass and autophagic flux. Further studies are required to fully characterize the complex function of Hspb7 in muscle in vivo.

From our previous study, in which we identified novel MEF2A target genes in skeletal and cardiac muscle (Wales et al., 2014), we observed that AP-1 consensus cis elements were enriched by MEF2A binding events and, based on GO term analysis, there was indication that MEF2A regulates the actin cytoskeleton (Potthoff et al., 2007; Wales et al., 2014). In the work presented here, we show through bioinformatic and biochemical analyses that MEF2 and AP-1 share a number of putative common target genes related to the establishment and maintenance of the actin cytoskeleton, some of which was validated using ChIP-qPCR and knockdown analysis. In particular, we focused on Hspb7 under conditions of muscle atrophy and observed that AP-1 and MEF2 antagonistically regulate expression of this gene. Moreover, experiments in mice and in cultured muscle cells suggest a role for Hspb7 in autophagy, having possible implications for muscle atrophy.

MEF2 and AP-1 regulation of the actin cytoskeleton and muscle atrophy

MEF2 and AP-1 are ubiquitous transcription factors yet their potential interaction at the transcriptional level has not been studied. Based on our results, MEF2 and AP-1 appear to inversely regulate Hspb7, wherein MEF2 promotes expression and AP-1 represses it. We also identified several other potential common target genes that MEF2 and AP-1 might co-operatively or competitively regulate. Based on GO term analysis, Fra-1 and MEF2A do not share a significant number of target genes compared with MEF2A and c-Jun (Fig. 1). This could indicate that Fra-1 and MEF2 have fundamentally different functions and also that Fra-1 associates with another Jun family member such as JunB or JunD to target differential AP-1 target genes under proliferative conditions. Interestingly, MEF2 and c-Jun were enriched for several muscle-related GO terms. This could reflect the role of c-Jun in priming muscle-specific enhancers with MyoD (Blum et al., 2012) and possibly MEF2. Fra-1 and c-Jun exclusive targets were mainly associated with angiogenesis and the response to bacterium, the latter being associated with inflammation and cytokine production, one of the traditional roles of AP-1 (Hess et al., 2004; Shaulian and Karin, 2002). The bioinformatic analysis demonstrates that analysis of differential transcription factors can reveal distinct roles of cooperative and exclusive biological functions.

A striking feature of Fra-1 and c-Jun recruitment is that the anti-inflammatory GR also binds within the same location of the second intron of Hspb7 (Fig. 4A). GR and AP-1 competitive regulation is not a new phenomenon and was observed several decades ago on the gene encoding collagenase I (Jonat, 1990). Subsequently, many other genes have been shown to be regulated by GR and AP-1. Interestingly, GR cooperates with a Jun homodimer but inhibits Jun–Fos heterodimers (Adcock and Caramori, 2001; Herrlich, 2001). Additionally, AP-1 has been shown to potentiate GR recruitment by promoting accessible chromatin in epithelial cells (Biddie et al., 2011); however, in the case of Hspb7 expression, AP-1 and GR do not synergize. In the case of Hspb7, we observed that loss of c-Jun or Fra-2 induces Hspb7 expression upon Dex treatment, indicating that a Jun–Fos dimer is involved in regulation of this gene. Moreover, our studies implicate a widespread level of cooperativity between AP-1 and MEF2 that warrants further investigation.

In striated muscle, the actin cytoskeleton stabilizes the sarcomere in concert with the costamere, which tethers the Z-line of the sarcomere to the sarcolemma (Ervasti and Campbell, 1993). This facilitates sarcomere stabilization and connects the actin cytoskeleton to the extracellular matrix via the costamere, but it also has roles in other cell processes including migration, adhesion and gene expression (Zheng et al., 2009). Therefore, identifying pathways that mediate actin cytoskeletal gene expression has implications for different types of muscle disease. MEF2 has a well-established role in sarcomere organization, because it regulates key target genes associated with the costamere and sarcomeric proteins (Ewen et al., 2011; Hinits and Hughes, 2007; Potthoff et al., 2007). There are fewer studies that have investigated the role of AP-1 in sarcomere integrity; however, in cardiomyocytes c-Jun has been shown to have an important role in promoting sarcomere gene expression and sarcomere integrity (Windak et al., 2013). Interestingly, destabilization of the actin cytoskeleton triggers c-Jun activity in vitro and represses glucocorticoid receptor activity (Oren et al., 1999). The myofibres in mouse models of cachexia are associated with a defective sarcolemma, similar to that seen in muscular dystrophies (Acharyya et al., 2005); therefore, common structural and cytoskeletal defects could contribute to various pathologies. In cancer cachexia, expression of a dominant negative AP-1 factor Tam67 or AP-1 and NF-κB double inhibitor can prevent loss of muscle mass (Moore-Carrasco et al., 2006, 2007). By contrast, JunB was found to be universally downregulated in models of atrophy (Lecker et al., 2004) and loss of AP-1 factors in denervation-induced muscle atrophy prevents upregulation of MAFbx and MURF1 (Choi et al., 2012). Dysregulation of AP-1 in cancer could therefore not only modulate proliferation and metastasis (Eferl and Wagner, 2003) but also impact muscle health, which is known to be modulated in cancer cachexia (Dodson et al., 2011).

A role for Hspb7 in muscle disease and sarcopenia

To date, it has been thought that the role of small heat shock proteins, including Hspb7, is stabilization of the cytoskeleton under stress conditions. Hspb7 can interact with α-filamin (Krief et al., 1999), filamin C (Juo et al., 2016), the cytoskeleton in tachypaced cardiomyocytes (Ke et al., 2011) and myofibrils in response to cardiac ischemia (Golenhofen et al., 2004). However, based on studies by Vos and colleagues, it appears that Hspb7 could have a role in autophagy and protein degradation, making its role more extensive than previously thought (Vos et al., 2009, 2010). Recently, Hspb7 has been shown to prevent aggregate formation, and not to contribute to aggregate clearance (Eenjes et al., 2016). Our study confirms that Hspb7 is upregulated with age, and also in the context of GR-mediated atrophy and fasting conditions in vivo. These observations agree with the previously stated idea that Hspb7 has a protective role (Doran et al., 2007); however, our data indicate that after rapamycin treatment in vitro the exogenous expression of Hspb7 localizes with LC3B and decreases autophagic flux. The rate of autophagy is considered to be a delicate balance because too much or too little results in different molecular deficiencies; therefore, the ability of Hspb7 to modulate this process in muscle could have pivotal consequences in the context of sarcopenia and other forms of muscle atrophy.

Because impairment of autophagy is believed to be associated with sarcopenia (Rubinsztein et al., 2011), our findings could have important implications in future therapies that seek to target autophagy in muscle wasting. Deficits in satellite cell numbers has also recently been associated with decreased autophagy (García-Prat et al., 2016), which could implicate a role for Hspb7 in satellite cell biology. Although Hspb7 is primarily associated with autophagy (Vos et al., 2010) it could have uncharacterized roles in proteasomal degradation pathways in other tissues. Additionally, the induction of Hspb7 via glucocorticoids could result in a subsequent maladaptive role for Hspb7 if signalling is prolonged. Skeletal muscles deficient in Hspb7 do not show defects in myogenesis but still develop myopathies postnatally, indicating that Hspb7 has a potentially crucial role in skeletal muscle integrity in adults (Juo et al., 2016). It would be interesting to further investigate this in the context of ageing or fasting. Hspb7 is highly expressed in the heart, and a crucial role for Hspb7 in heart development has been shown in zebrafish (Rosenfeld et al., 2013). Mutations in Hspb7 are correlated with cardiomyopathies and, co-incidentally, Hspb7 SNPs within the second intron (where GR and AP-1 recruitment was observed) are associated with cardiomyopathies (Garnier et al., 2015; Matkovich et al., 2010). In our studies, although Hspb7 was induced in skeletal muscles in response to fasting it was not upregulated in the heart (Fig. 7). This could indicate that Hspb7 is induced to protect tissues in response to different forms of stress. The importance of MEF2, AP-1 and GR in Hspb7 regulation in the heart could also be related to cardiac development and it would be interesting to determine whether Hspb7, and the GR–MEF2–AP-1 signaling axis as highlighted here, has a role in cardiac hypertrophy or other cardiomyopathies.

In conclusion, MEF2 and AP-1 have historically been assigned fairly distinct functions in tissue-specific gene expression and growth control, respectively. Here, we implicate them together in the antagonistic or cooperative control of cytoskeletal genes such as Hspb7 in skeletal muscle. These studies also highlight the role of the actin cytoskeleton and its regulation by MEF2 and AP-1 transcriptional regulators, which may be of particular importance for muscle function and pathology.

Cell culture

C2C12 myoblasts were obtained from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with high glucose and L-glutamine (Hyclone) supplemented with 10% foetal bovine serum (HyClone) and 1% penicillin/streptomycin (Invitrogen). C2C12 myoblasts were induced to differentiate in differentiation medium containing DMEM/high glucose/L-glutamine supplemented with 2% horse serum (Hyclone) and 1% penicillin/streptomycin for the indicated times. Cells were maintained in an humidified, 37°C incubator at 5% CO2 and replenished with fresh medium every 24 h. Pharmacological drug treatments were completed for the indicated times.

Transfections

C2C12 myoblasts were transfected using the calcium phosphate precipitation method. Cells were then harvested at 48 h post-transfection or the medium was changed to DM.

Plasmids

Expression plasmids for pMT2–MEF2A, pCMV–c-Jun, pcDNA3.1-Fra-2, pCMV–dsRed2, pcDNA–GFP, GFP–Tam67 have been described previously (Alli et al., 2013; Hennigan and Stambrook, 2001; Perry et al., 2009). pCAGGSnHC–HSPB7-HA was described by Lin et al. (2014). EYFP–Hspb7 was sub-cloned from Hspb7–HA into EYFP–pcDNA3 using XhoI and EcoRI.

Antibodies and reagents

Rabbit polyclonal MEF2A antibody has been previously described (Cox et al., 2003). The following antibodies were purchased from Santa Cruz Biotechnology: actin (sc-1616), dsRed (sc-33354), MEF2A (sc-313X; used in ChIP), donkey anti-goat IgG-HRP (sc-2020), Fra-2 (sc-604), c-Jun (sc-1694), GFP (sc-9996), MCK (sc-365046) and MyoD (sc-304). Anti-LC3B was from Cell Signaling (2775). Myogenin and HA monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank. The remaining antibodies were as follows: Hspb7 (Abcam, ab150390), glucocorticoid receptor (Abcam, ab3579), rabbit IgG (Millipore, 12-370). Dexamethasone (sc-29059) was used at a concentration of 10 μM, unless otherwise indicated. DMSO was used as a volume control. Rapamycin (10 μg/ml) and BafA1 (200 nM) were purchased from Santa Cruz Biotechnology (sc-3504 and sc-201550).

siRNA transfection of C2C12 myoblasts

Knockdown of target genes was carried out using siRNAs obtained from Sigma-Aldrich; targets are listed in Table S2. In C2C12 myoblasts, siRNA was transfected at the following concentrations: MEF2A (30 nM), c-Jun (50 nM) and Fra-2 (50 nm).

Immunoblots

Cells were washed with 1× PBS and lysed in NP-40 lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA, 100 mM NaF and 10 mM Na pyrophosphate) containing protease inhibitor cocktail (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich) and 1 mM sodium orthovanadate (Bioshop). Protein concentrations were determined by Bradford assay (Bio-Rad). Twenty micrograms of total protein were resolved on 10% SDS–PAGE and then transferred onto Immobilon-FL PVDF membrane (Millipore) for 1 h or overnight. Nonspecific binding sites were blocked using 5% milk in PBS or TBST. Membranes were incubated with primary antibodies overnight at 4°C in 5% milk in PBS or 5% BSA in TBST. Horseradish peroxidase-conjugated secondary antibody was added for 1 h at room temperature. Protein was detected with ECL chemiluminescence reagent (Pierce). For cyto-nuclear fractionation, the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit (78833) was used.

Chromatin immunoprecipitation

Methods were carried out as described previously described (Wales et al., 2014) with the addition of a third immunoprecipitation wash buffer (IP wash buffer III; 20 mM Tris pH 8.1, 250 mM LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA).

RNA extraction

Total RNA was extracted from C2C12 myoblasts using the RNeasy Plus kit (Qiagen) and Qiashredder (Qiagen). RNA isolated from tissue was extracted using Trizol (Invitrogen). RNA was converted to cDNA using Superscript III (Invitrogen) according to the manufacturer's instructions.

Quantitative PCR

SybrGreen (BioRad or ABM) was combined with 2.5 μl gDNA or cDNA and 500 nM primers in a final volume of 20 μl. cDNA was diluted 1:10 prior to use. Each sample was prepared in triplicate and analysed using Rotor-Gene Q (Qiagen). Parameters for qRT-PCR using BioRadwere were 30 s 95°C, [5 s 95°C, 30 s 60°C]×40 cycles. Parameters for qRT-PCR using ABM were 10 m 95°C, [3 s 95°C, 30 s 60°C]×35 cycles. Parameters for ChIP-qPCR were 5 min 95°C, [5 s 95°C, 15 s 60°C]×40 cycles. Fold enrichment (ChIP-qPCR) and fold change (qRT-PCR) were quantified using the ΔΔCt method. Primers used in qRT-PCR and ChIP-qPCR are listed in Tables S3 and S4, respectively.

Bioinformatics

AP-1 consensus sequences were mapped using cisGenome. GREAT (default settings) identified GO terms based on DNA sequences obtained from available datasets: GSE61207 for MEF2A; ENCSR000AIK for Fra-1; and GSE37525 for c-Jun. All datasets were converted to mm9 using UCSC LiftOver. Overlapping binding sites were determined using the UCSC Table Browser Intersect function using default settings.

Animal care

For ageing experiments, 63- and 8-week-old C57BL/6 male mice were obtained from Jackson Lab or Charles River, respectively. Mice were sacrificed using cervical dislocation in accordance with the Institutional Animal Care and Use Committee of York University. For autophagy experiments, 6- to 8-week-old C57BL/6 male mice were sacrificed using cervical dislocation in accordance with University of Ottawa Animal Care and Use Committee. Autophagic flux was induced using a combination of daily intraperitoneal colchicine administration (0.4 mg/kg/day) and fasting as described by Ju et al. (2010) with the following changes: (1) mice were placed on a 24 h fast; (2) 3 days of recovery were allowed after ptfLC3, Hspb7–HA or pCAGSSnHC electroporation.

Electroporation of plasmid DNA into mouse tissue

Plasmid DNA was prepared as follows: twenty-five micrograms of Hspb7–HA or empty vector (pCAGGSnHC) in sterile half saline were injected into the right and left TA muscles of 6- to 8-week-old C57BL/6 male mice, respectively. Co-electroporation of tfLC3 and Hspb7–HA or empty vector was carried out using equal amounts of 12.5 μg. After injection, muscles were subject to electrical stimulation using an electroporation system (ECM 830, BTX) with electroporation array needle (model no.533, BTX) programmed at 80 V, 6 pulses, 50 ms/pulse and 500 ms interval between pulses. Tissues were harvested immediately after cervical dislocation and homogenized with 1 ml Trizol in a Lysing Matrix D tube (6913100, MP Biomedical) with MagNA Lyser (03358968001, Roche) programmed at 17,000 rpm. Three homogenizations of 20 s were carried out, between which the samples were chilled on ice for 30 s. Tissue homogenates were then prepared for RNA and protein extraction using the Trizol protocol (Invitrogen).

Electroporation of siRNA into mouse tissue

Prior to injection of siRNA, the right TA muscle was injected with 25 μl of 0.4 unit/μl hyaluronidase (LS005477, Worthington) and digested for 1 h. siRNA targeting Hspb7 was pooled at 150 pmol to give a final amount of 450 pmol. The paired leg received 450 pmol of non-targeting scrambled siRNA (scr). The siRNAs used are listed in Table S2. At 2 days post-electroporation, saline or colchicine (0.4 mg/kg/day) was administered intraperitoneally and then every 24 h for a further 2 days.

Immunofluorescence

C2C12 myoblasts were fixed and permeabilized using 90% methanol at −20°C for 6 min. The protocol used for immunofluorescence has been previously described (Ehyai et al., 2015). Anti-LC3B (Cell Signaling, 2775) was used at a 1:200 dilution. Hoechst 33342 stain was added for 30 min to visualize nuclei (1:10,000; Sigma-Aldrich). Images were captured using a Carl Zeiss Axio Observer.Z1 (Photometrics Evolve 512 EMCCD camera). Cells were imaged under a 63× (Plan-Apochromat; 1.40 NA in oil) objective, using ZEN image acquisition software 2.0. Immunofluorescent sections of TA muscle were prepared as follows: TA muscles were harvested and frozen within Cryomatrix (6769006, Thermo Scientific) in liquid nitrogen-chilled isopentane; 10 μm sections of TA muscles were affixed to positive charged slides and fixed with 4% paraformaldehyde for 10 min at room temperature. Sections were rinsed with PBS and mounted with Prolong Gold anti-fade with DAPI. Images were acquired using a Zeiss Observer Z1 microscope.

Statistics

Data are presented as mean±s.e.m. Statistical analysis was carried out using two-tailed unpaired Student's t-test.

We would like to thank G. Sweeney (York University, Toronto, Canada) for providing the tandem LC3 plasmid (tflc3) and Y. Nakamura (University of Chicago, Chicago, USA) for providing Hspb7–HA.

Author contributions

S.W.T. completed most of the data acquisition, analysis and interpretation. D.Y. and J.G. completed in vivo experiments, which were then analysed by S.W.T. A.B. assisted in bioinformatic analysis. A.F. assisted with in vitro immunofluorescence and imaging, ChIP-qPCR and Tam67 experiments. S.W.T., A.B. and J.C.M. designed experiments and wrote the manuscript.

Funding

This work was supported by a grant from the Canadian Institutes of Health Research to J.C.M.

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Competing interests

The authors declare no competing or financial interests.

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