During muscle regeneration, the transcription factor Pax7 stimulates the differentiation of satellite cells (SCs) toward the muscle lineage but restricts adipogenesis. Here, we identify HDAC4 as a regulator of Pax7‐dependent muscle regeneration. In HDAC4‐deficient SCs, the expression of Pax7 and its target genes is reduced. We identify HDAC4‐regulated Lix1 as a Pax7 target gene required for SC proliferation. HDAC4 inactivation leads to defective SC proliferation, muscle regeneration, and aberrant lipid accumulation. Further, expression of the brown adipose master regulator Prdm16 and its inhibitory microRNA‐133 are also deregulated. Thus, HDAC4 is a novel regulator of Pax7‐dependent SC proliferation and potentially fate determination in regenerating muscle.
HDAC4 is shown to promote damage‐induced satellite cell (SC) proliferation and muscle regeneration in vivo by stimulating Pax7 expression. It also prevents aberrant lipid accumulation and induction of the brown fat transcription factor Prdm16 in regenerating muscle.
Loss of HDAC4 in SCs reduces expression of Pax7 and its target genes, which in turns inhibits SC proliferation.
Knockdown of HDAC4‐regulated Lix1, a Pax7 target, decreases the proliferative rate of SCs.
HDAC4 inactivation in SCs leads to a prominent reduction of microRNA‐133 and concomitant increase in its target, Prdm16, a master regulator of brown adipogenesis.
Skeletal muscle fulfills diverse functional demands and manages mechanical stress by modifying contractile and metabolic properties of myofibers and replacing those damaged via regenerative adult stem cells. The on‐demand and activity‐dependent muscle remodeling is achieved by elaborate reprogramming of muscle gene transcription . In myofibers, histone deacetylase 4 (HDAC4) is a critical factor that connects neural activity to the muscle transcriptional programs. Inactivation of HDAC4 suppresses denervation‐induced muscle atrophy and synaptic gene expression while increases re‐innervation , , , . These findings highlight a central regulatory role of HDAC4 in activity‐dependent muscle remodeling.
Muscle regeneration triggered by myofiber damage is another key adaptive program required for maintaining muscle integrity. Satellite cells (SCs), the principal muscle stem cells, are essential for damage‐induced muscle regeneration , . The paired box transcription factor Pax7 is the master regulator of SCs . Genetic ablation of Pax7 leads to a loss of SC pool and severely impaired muscle regeneration , , . Regeneration failure in Pax7 knockout (KO) mice is also accompanied by accumulation of lipids in muscle . Intriguingly, SCs have been shown to potentially develop into non‐muscle lineages . Recent studies reveal a shared lineage of muscle and brown adipose tissue  and SCs have the potential to differentiate into brown adipocytes . In this program, microRNA‐133 (miR‐133), which targets and inhibits the master regulator of brown adipose tissue Prdm16, restricts SCs toward the muscle lineage . How Pax7 regulates SC activation and fate commitment during muscle regeneration remains incompletely understood. Here, we provide evidence that HDAC4 is a novel regulatory component in Pax7‐dependent SC expansion and fate determination critical for adult muscle regeneration.
Results and Discussion
HDAC4 is required for satellite cell expansion in damaged muscle
We previously observed in muscle biopsy from an ALS patient that HDAC4 is localized not only to myofiber nuclei but also centrally located nuclei, a marker for newly regenerating fibers . Moreover, expression of HDAC4, similar to Pax7, progressively increased in tibialis anterior (TA) muscles damaged by cardiotoxin (CTX) (Fig 1A and B). These observations indicate that HDAC4 might be involved in muscle regeneration. To investigate the possibility, we generated satellite cell (SC)‐specific HDAC4 knockout (KO) mice by crossing HDAC4Lox mice with a tamoxifen (Tmx)‐inducible CreERT2 transgene that was placed at the endogenous Pax7 locus (Pax7‐CreERT2). HDAC4Lox;Pax7‐CreERT2 and control HDAC4Lox mice were treated with Tmx to activate Cre‐mediated excision of HDAC4 in SCs , followed by CTX injection in TA and gastrocnemius (GA) muscles to activate SCs and muscle regeneration. The analyses of SCs purified by fluorescence‐activated cell sorting (FACS) showed the efficiency of HDAC4 KO by this method is approximately 60–80% (Supplementary Fig S1A–C).
During FACS analyses, we noted that the number of activated SCs was significantly reduced in HDAC4 KO mice, whereas non‐SC cells were slightly increased (Fig 1C), suggesting that SC expansion might be impaired. To determine whether HDAC4 is required for injury‐stimulated SC activation, we quantified Pax7‐positive SCs in TA muscles treated with CTX. Immunostaining showed that the frequency of Pax7‐positive cells as well as the staining intensity of Pax7 was much lower in HDAC4 KO compared to CTX‐treated control muscles (Fig 1D and E, and Supplementary Fig S1D). MyoD‐positive activated SCs were similarly reduced in HDAC4 KO muscle (Fig 1F). In contrast, numbers of quiescent SCs in uninjured muscle were not significantly different in control and HDAC4 KO muscle, indicating that HDAC4 regulates SC expansion during regeneration (Supplementary Fig S1E and F). Supporting this conclusion, RNA analyses revealed that expression of genes required for SC activation and muscle differentiation, including Pax7, MyoD, Myf5, and Myogenin (MyoG), were all markedly reduced in damaged HDAC4 KO muscles (Fig 1G). A modest reduction of these genes was detected in saline‐treated HDAC4 KO muscle, likely due to mild muscle injury caused by the injection procedure, which generates local activation of SCs. Further, intraperitoneal injection of Tmx alone did not significantly reduce the expression of Pax7 and myogenic factors in uninjured muscle of HDAC4 KO mice (Supplementary Fig S2A). Together, these results show that HDAC4 inactivation impairs SC expansion induced by injury.
HDAC4 regulates the expression of Pax7 and its target genes in activated SCs
We next investigated whether HDAC4 regulates gene transcription program in SCs. SCs were isolated from CTX‐injured control and HDAC4 SC KO muscles, cultured in growth medium (GM) and subjected to RNA analysis. We found that the expression of the SC‐master transcription factor, Pax7, was significantly reduced in HDAC4 KO SCs (Fig 2A). Immunoblot analysis confirmed its reduction in HDAC4 KO SCs (Fig 2B). Supporting a Pax7 deficiency, the expression of Myf5, a well‐established Pax7 target, was also reduced (Fig 2A). This reduction of Pax7 and Myf5 is specifically caused by the loss of HDAC4, as their expression was not changed in activated SCs or damaged TA muscles isolated from HDAC4lox/lox and HDAC4lox/lox; Pax7‐CreERT2 mice without Tmx treatment (Supplementary Fig S2B and C). Importantly, the expression of MyoD was not affected in HDAC4 KO SCs (Fig 2A), consistent with a previous report that MyoD is not regulated by Pax7 . MyoG, a downstream effector of muscle differentiation, was modestly decreased in HDAC4 KO SCs. To further determine the role of HDAC4 in Pax7‐dependent gene transcription, we assessed a set of Pax7 target genes previously identified , . As shown in Fig 2A, Pax7 target genes including Lix1, Mest, PlagL1, and Cipar1 were all reduced in HDAC4 KO SCs. In contrast, ID2 and ID3 were unchanged in HDAC4 KO SCs. Conversely, Igfbp2, a gene negatively regulated by Pax7 , was increased in HDAC4 KO SCs. We further analyzed Pax7 target gene expression in regenerative TA muscle. In addition to Myf5 (Fig 1G), Lix1 and Mest were significantly reduced in regenerative HDAC4 KO muscle, whereas other Pax7 targets were unaffected (Fig 2C). Together, these results show that HDAC4 positively regulates Pax7 and a subset of Pax7 target genes in activated SCs.
HDAC4 regulates SC proliferation
The reduction of Pax7‐positive cells in injured HDAC4 KO muscle indicates a defect in SC expansion. To assess whether HDAC4 regulates SC proliferation, SCs were purified from CTX‐treated control and HDAC4 SC KO muscle, cultured in GM, and pulse‐labeled with BrdU. HDAC4 KO SCs showed greater than twofold reduction in proliferation (Fig 3A and B). Apoptosis, as indicated by active caspase‐3, was similar between control and HDAC4 KO SCs (Fig 3C). To determine whether HDAC4 regulates differentiation, purified SCs were cultured in differentiation medium (DM) and assessed by the expression of myosin heavy chain. Both control and KO SCs underwent efficient differentiation although quantification revealed a modest but statistically significant reduction in HDAC4 KO SCs (Supplementary Fig S3A and B). Collectively, these results indicate that HDAC4 mainly regulates SC proliferation.
Lix1 is required for Pax7‐dependent SC proliferation
Although ID3 was proposed as a Pax7 target required for SC proliferation , its expression in SCs or regenerative muscle was not affected by HDAC4 inactivation (Fig 2A and C). Our analysis of a limited set of Pax7 target genes has identified Lix1 (limb expression 1) and Mest (mesoderm‐specific transcript) as two genes whose expressions were strongly suppressed in both HDAC4 KO SCs and muscles (Fig 2A and C). To test the possibility that they might be the factors that promote SC proliferation, we knocked down Lix1 or Mest in SCs isolated from CTX‐activated muscle. These siRNAs effectively suppressed Lix1 or Mest expression (Supplementary Fig S3C and D). We found that Lix1 KD significantly reduced SC proliferation without inducing apoptosis (Fig 3D–F), whereas Mest KD had no effect (Supplementary Fig S3E). Expression of Pax7 and myogenic factors were unaffected by Lix1 KD (Supplementary Fig S3C). These results indicate that Lix1 is a Pax7 target gene required for efficient SC proliferation.
HDAC4 is required for efficient damage‐induced muscle regeneration
We next determined whether muscle regeneration is impaired in HDAC4 SC KO mice. At 8 days post‐CTX treatment, control muscles underwent proper regeneration as illustrated by relatively uniform myofibers that are positive for embryonic myosin (eMyHC) and centrally localized nuclei (Fig 4A). In HDAC4 SC KO muscle, the regenerating fibers appeared to be smaller and interspersed with infiltrated cells. Quantification using eMyHC staining confirmed a reduction of regenerative fibers in the HDAC4 KO mice (Fig 4B). At 5 weeks post‐CTX treatment, HDAC4 KO muscles showed reduced muscle size, whereas the overall muscle structure appeared to be normal (Supplementary Fig S4A), indicating delayed muscle regeneration. Supporting these results, regeneration‐associated muscle hypertrophy was significantly inhibited in HDAC4 KO TA muscle (Fig 4C). In Pax7 SC‐specific KO animals, the muscle regeneration defect becomes more apparent when Cre‐mediated recombination (driven by Tmx‐inducible Cre) was sustained by continuous administration of Tmx and muscle was injured by repeated cycles of CTX injection , . Adapting this experimental condition, we found that muscle regeneration was indeed more profoundly impaired in HDAC4 SC KO muscles, as indicated by a significant reduction in muscle size (Fig 4D). In SC‐specific Pax7 KO mice, regeneration failure is accompanied by accumulation of lipids in muscle . Analysis of HDAC4 SC KO muscles also revealed aberrant increase in neutral lipid stained by Oil Red O (Fig 4D and Supplementary Fig S4B). Further analysis showed that Oil Red O‐stained cells were located between regenerating myofibers (Supplementary Fig S4C), indicating an expansion of interstitial adipocytes in HDAC4 SC KO muscle. Collectively, these data show that HDAC4 SC KO impairs muscle regeneration.
HDAC4 inactivation increases brown adipocytes in regenerative muscle and deregulates miR‐133
SC has the potential to develop into brown adipose tissue, a fate that is regulated by miR‐133 . miR‐133 targets and inhibits Prdm16, the master transcription factor for brown fat adipogenesis , , . Inhibition of miR‐133 can reprogram SCs toward the brown adipose lineage . To investigate whether HDAC4 regulates miR‐133 expression, we assessed mature miR‐133a and two other muscle microRNAs, miR‐1a and miR‐206, both of which repress Pax7 expression and SC proliferation . As shown in Fig 4E, expression of miR‐133a, but not miR‐1a or miR‐206, was reduced in HDAC4 KO SCs. Consistent with a reduction in miR‐133a, a significant induction of Prdm16 and a brown fat marker, UCP1, were observed in HDAC4 KO SCs. Supporting these findings, UCP1‐positive cells were increased in HDAC4 KO muscle (Fig 4F and G). Collectively, these results indicate that HDAC4 restricts the expression of the miR‐133–Prdm16 axis important for the brown adipocyte lineage determination.
In this study, we provide evidence that HDAC4 is required for efficient muscle regeneration induced by injury. The characterization of HDAC4 KO has revealed a significant reduction in Pax7 expression in SCs (Fig 2A and B). Indeed, subsets of Pax7 target genes were significantly mis‐regulated in HDAC4 KO SCs (Fig 2A). Surprisingly, siRNA knockdown of HDAC4 in purified and activated SCs in vitro did not cause a reduction in Pax7 expression (Supplementary Fig S5A). These results indicate that regulation of Pax7 by HDAC4 is indirect and requires an intact muscle microenvironment in vivo. Further supporting an indirect mechanism, HDAC4 knockdown in C2C12 myoblasts did not affect acetyl‐histone H4 (K8) levels in the promoter regions of Pax7 or Lix1 (Supplementary Fig S5B). It is of interest to note that HDAC4 can regulate cytokine production , which might influence the inflammatory program important for muscle regeneration . How HDAC4 in SCs communicates with other components of the muscle regeneration program to affect Pax7 expression awaits further studies.
The differential requirement of Pax7 target genes on HDAC4 could reflect the up‐regulation of Pax3 in HDAC4 KO SCs and regenerating TA muscle (Fig 2A and D). This finding indicates that HDAC4 selectively regulates Pax7, but not Pax3, expression in SCs. Pax3 is highly related to Pax7 and can regulate similar sets of genes , . Thus, the elevated Pax3 might partially compensate for Pax7 and maintain the expression of common targets. The differential dependence of Pax7 target genes on HDAC4 led us to focus on Lix1 and Mest, which are down‐regulated in both HDAC4 KO SCs and damaged muscles, as candidates that mediate Pax7‐dependent SC proliferation. We found that knockdown of Lix1, but not Mest, suppressed the proliferation of activated SCs (Fig 3E and Supplementary Fig S3E). This finding suggests that Pax7‐dependent SC proliferation involves Lix1. The biochemical function of Lix1 is largely unknown. Its Drosophila orthologous, Lowfat (Lft), is a component of the FAT‐signaling pathway that regulates planar cell polarity (PCP) . Interestingly, PCP plays an important role in SC expansion . Whether Lix1 and HDAC4 regulate SC proliferation via the PCP pathway requires further investigation.
Supporting its role as a positive regulator of Pax7, HDAC4 KO led to defects in SC proliferation and muscle regeneration. Similar to Pax7 KO mice, regenerative muscles in HDAC4 KO mice also showed aberrant lipid accumulation (Fig 4D). Interestingly, we found that HDAC4 KO in SCs reduces expression of miR‐133a, a negative regulator of the brown fat transcription factor Prdm16. Consistent with this finding, the expression of Prdm16 and a marker for brown adipocytes, UCP1, were both elevated in HDAC4 KO SCs (Fig 4E). An increase in UCP1‐positive cells was also observed in HDAC4 KO regenerating muscle (Fig 4F and G). These findings are consistent with a previous report that miR‐133 suppresses SC differentiation to brown adipocytes . Our study therefore suggests that HDAC4 is required for maintaining miR‐133a in SCs and thereby inhibits SC differentiation toward the brown adipose lineage. Interestingly, miR‐133 over‐expression can increase Pax7 levels in primary preadipocytes , indicating that miR‐133 positively regulates Pax7 expression. Accordingly, HDAC4 could increase Pax7 expression in SCs via miR‐133. Given the instructive role of miR‐133 in brown adipose determination , , , this scenario implies a regulatory role of HDAC4 in SC fate determination. The physiological relevance of SC plasticity is not known. By directing some activated SCs toward an adipose lineage, this arrangement could potentially provide metabolic advantages to regenerative muscle. Since HDAC4 is regulated by multiple physiological and pathological conditions, its activity in SCs might influence the composition of muscle fibers and adipose tissue in regenerative muscle. Further studies are required to determine how HDAC4 regulates miR‐133 expression and its impact on SC fate determination in response to patho‐physiological conditions.
Materials and Methods
HDAC4Lox mice were provided by Dr. E. Olson . Pax7CreERT2 mice were purchased from Jackson Laboratory (B6;129‐Pax7tm2.1(cre/ERT2)Fan/J). Wild‐type C57BL/6 mice were also from Jackson Laboratory. Conditional strains contain HDAC4Lox; Pax7‐CreERT2 (KO) or HDAC4Lox (control). Tamoxifen (Tmx; Sigma; T5648) treatment for Cre recombinase activation was performed by intraperitoneal injection to both control and HDAC4 KO mice (200 μl of 10 mg/ml in corn oil). Mouse gender, age, numbers, and Tmx treatment are indicated in each figure legend. To induce muscle degeneration/regeneration, mice were anesthetized and injected with 50 μl of 10 μM cardiotoxin (Sigma; C9759) in one site of TA muscle or in two sites of GA muscle. All mice were housed at the Duke University mouse facilities in accordance with the IACUC.
MCC designed and performed experiments and wrote the manuscript; SR conducted mouse genotyping, immunostaining, and quantification of data. RH performed ChIP experiments and analyzed data. BW helped with the animal works. MK provided technical assistances and edited the manuscript. CMF provided critical reagents, discussions and edited the manuscript. TPY supervised the project and edited the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Materials and Methods
We thank Dr. Eric Olson for the generous gift of HDAC4‐flox mouse. We also thank Mr. Thomas Fitzpatrick for quantification image data, Dr. Jae‐woo Lee for MF20 antibody, and Ms. Minsi Zhang for discussions. This work was supported by 276662 (MDA) to MCC, AR060042 (NIAMS, NIH) to CMF and AR055613 (NIAMS, NIH) to TPY.
- © 2014 The Authors