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PPARδ regulates satellite cell proliferation and skeletal muscle regeneration

PPARδ regulates satellite cell proliferation and skeletal muscle regeneration Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that play important roles in development and energy metabolism. Whereas PPARδ has been shown to regulate mitochondrial biosynthesis and slow-muscle fiber types, its function in skeletal muscle progenitors (satellite cells) is unknown. Since constitutive mutation of Pparδ leads to embryonic lethality, we sought to address this question by conditional knockout (cKO) flox/flox of Pparδ using Myf5-Cre/Pparδ alleles to ablate PPARδ in myogenic progenitor cells. Although Pparδ-cKO mice were born normally and initially displayed no difference in body weight, muscle size or muscle composition, they later developed metabolic syndrome, which manifested as increased body weight and reduced response to glucose challenge at age nine months. Pparδ-cKO mice had 40% fewer satellite cells than their wild-type littermates, and these satellite cells exhibited reduced growth kinetics and proliferation in vitro. Furthermore, regeneration of Pparδ-cKO muscles was impaired after cardiotoxin-induced injury. Gene expression analysis showed reduced expression of the Forkhead box class O transcription factor 1 (FoxO1) gene in Pparδ-cKO muscles under both quiescent and regenerating conditions, suggesting that PPARδ acts through FoxO1 in regulating muscle progenitor cells. These results support a function of PPARδ in regulating skeletal muscle metabolism and insulin sensitivity, and they establish a novel role of PPARδ in muscle progenitor cells and postnatal muscle regeneration. Keywords: Cre/LoxP, skeletal muscle, stem cell, proliferation, differentiation, self-renewal Background other physiological signaling pathways [2-4]. In addition, Skeletal muscle is the most abundant tissue in mam- skeletal muscle mass is always in a state of hypertrophy mals, making up 45% to 55% of total body mass in or wasting, based on relative use or disuse, respectively humans, and plays important roles in body movement [5,6]. Skeletal muscle has superior capacity to regenerate and metabolic regulation. Muscle is made up of different itself upon injury [7]. fiber types which have different metabolic requirements Skeletal muscle plasticity is mainly maintained by a that affect the whole body energy homeostasis of the subset of cells known as satellite cells [8,9]. These animal [1]. Type 1 fibers are classified as slow fibers and cells, located beneath the basal lamina of the muscle use oxidative metabolism as a fuel source, making them fiber, are normally maintained in a quiescent state. highly fatigue-resistant. Conversely, type 2 fibers are Satellite cells become activated when the muscle classified as fast fibers, use mainly glycolytic metabolism becomes damaged through injury or normal activity. and are less resistant to fatigue. Type 2 fibers are further Once activated the cells will reenter the cell cycle and broken down into three subtypes, known as types 2a, 2x undergo a few rounds of division, then differentiate and fuse with existing muscle fibers to rebuild the and 2b, that express corresponding myosin heavy chain (MyHC) isoforms and have decreasing resistance to fati- damaged area. Satellite cells in the quiescent state gue. Notably, skeletal muscles are plastic, and fiber-type express paired-box transcription factor 7 (Pax7) [10]. switching occurs in response to changes in activity and After activation the cells will express Pax7 and myo- genic differentiation antigen 1 (MyoD) concurrently while the cells undergo a few rounds of division (pro- * Correspondence: skuang@purdue.edu liferation). These proliferating cells eventually with- Department of Animal Sciences, Purdue University, 901 West State Street, draw from the cell cycle and either return to West Lafayette, IN 47907, USA Full list of author information is available at the end of the article © 2011 Angione et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Angione et al. Skeletal Muscle 2011, 1:33 Page 2 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 quiescence (self-renewal) through downregulation of cell types and animal species [28,29]. These receptors MyoD or differentiate through downregulation of Pax7 must form heterodimers with the retinoid X receptor and upregulation of myogenin. Thus expression of (RXR) before they can bind to specific recognition Pax7 and MyoD distinguishes the status of a cell, sequences, called PPAR response elements (PPREs), + - whether it is self-renewing (Pax7 /MyoD ), proliferat- which are located in the promoter and intron regions of + + - + ing (Pax7 /MyoD ) or differentiating (Pax7 /MyoD ), a wide variety of target genes [21,30]. The PPARs acti- vate or repress transcription through the recruitment of respectively [11-13]. Notably, researchers in several coactivators and corepressors. recent studies have demonstrated that the choice PPARδ has been shown to be important for the proper between self-renewal and differentiation of newly divided satellite cells is dynamically regulated [14-16]. function of skeletal muscle in gain- and loss-of-function Whereas most proliferating myoblasts divide symmetri- studies. Synthetic compound-mediated activation or cally, a subpopulation of cells can divide asymmetri- overexpression of the Pparδ gene in mice causes an cally to give rise to both self-renewal and increase in the oxidative capacity of the muscle resulting differentiating progenies [14-16]. from an increase in the number of type 1 oxidative Myogenic factor 5 (Myf5) is also important for skeletal fibers and a decrease in the number of type 2 glycolytic muscle development and satellite cell function. Myf5 fibers [31,32]. These increases in oxidative capacity have transcripts can first be detected at E8 in the developing been shown to contribute to an organism’s overall exer- embryo and are important for specifying the cells of the cise endurance. Along with increases in oxidative capa- muscle lineage [17]. Myf5-Cre lineage tracing labels a city, transgenic mice also show a decrease in overall majority of satellite cells, which will have become com- body fat content and individual adipocyte size. Mice mitted to the myogenic lineage. However, 10% of satel- with constitutively active Pparδ can maintain a normal lite cells remain Myf5-negative and are thought to be a body weight even when challenged with a high-fat diet, population of more primitive, uncommitted stem cells whereas their wild-type littermates become obese when that can give rise to committed cells through asym- fed the same diet. By contrast, skeletal muscle-specific metric cell division [15]. Myf5-null satellite cells are knockout of Pparδ seems to cause a decrease in oxida- defective in transient proliferation prior to differentia- tive capacity and makes mice prone to obesity and tion [18,19]. Intriguingly, investigators in recent studies metabolic disorders [33]. This loss of oxidative capacity have demonstrated that brown adipocytes, but not white could be due in part to reduced expression of PGC1a, whichisknownto playaroleintype1fiberformation adipocytes, are derived from Myf5 lineage progenitors and maintenance. These results suggest that PPARδ [20]. Therefore, Myf5-Cre-mediated conditional knock- out can be used to knock out floxed target genes in plays a role in preventing obesity and the development committed myogenic progenitor cells and their descen- of metabolic disorders. dants (mature skeletal muscles) as well as in brown Until now all the studies on PPARδ in skeletal muscle adipocytes. have focused on mature muscle fibers. It remains to be Peroxisome proliferator-activated receptors (PPARs) determined whether PPARδ plays a role in myogenic are members of the nuclear estrogen receptor superfam- satellite cells and postnatal muscle regeneration. PPARδ ily and have been shown to be important for the proper has been shown to regulate the proliferation and/or metabolism of fatty acids [21]. There are three PPAR maturation of several cell types, including mouse isoforms (a, δ and g), and each plays a specific role in embryonic stem cells, oligodendrocytes, keratinocytes, metabolism [22-24]. The PPARs are expressed in a wide endothelial progenitors and cancer cells [34-38]. How- range of adult tissues, but each has its own tissue-speci- ever, whether PPARδ positively or negatively regulates fic expression patterns. PPARa is highly expressed in proliferation is highly cell type-specific, and the evidence the liver and heart, PPARδ (alsoreferred toasPPARb, presented in the literature has sometimes been contra- PPARb/δ or NR1C2) is highly expressed in the intestine dictory [36]. In the current study, we used a Cre/LoxP- and liver, and PPARg is highly expressed in both brown based conditional mutation approach to remove Pparδ and white adipose tissues [21-23,25,26]. The three PPAR from the satellite cells to examine its function in muscle isoforms are ligand-activated receptors that are activated progenitor cells. We show herein that PPARδ is an by fatty acids, fatty acid derivatives and a variety of syn- important regulator of satellite cell proliferation in vitro thetic compounds [21]. The ligand-binding domains of and of muscle regeneration in vivo. PPARs vary slightly, resulting in specific affinity for fatty acids and synthetic compounds [27]. For example, Materials and methods GW501516 is one synthetic compound that has been Animals shown to specifically activate PPARδ with 60- to 1, 000- All experimental procedures involving the use of mice fold selectivity over the other isoforms, depending on were carried out in accordance with Purdue University’s Angione et al. Skeletal Muscle 2011, 1:33 Page 3 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Animal Care and Use Committee. C57BL/6J mice con- minutes using 25 ml of DMEM containing 2% fetal taining LoxP sites flanking exon 4, which encodes the bovine serum (FBS) and 10 mM 4-(2-hydroxyethyl)-1- N-terminal zinc finger of the DNA-binding domain of piperazineethanesulfonic acid, then passed through a f/f the Pparδ gene (Pparδ ), had been generated previously 100-μm filter. After filtration, the cells and muscle deb- [39]. These mice were crossed with Myf5-Cre to gener- ris were pelleted at 250 × g for five minutes and the f/f ate Myf5-Cre/Pparδ offspring for ablation of Pparδ in DMEM was removed. The cells and muscle debris were Myf5 lineage cells (called “Pparδ-cKO” hereinafter) [40]. then plated on noncoated plates with Ham’scomplete +/f f/f Pparδ or Pparδ littermates that did not inherit the medium containing 20% FBS, 4 ng/ml basic fibroblast Myf5-Cre allele were used as controls (called “wild type” growth factor and 1% penicillin/streptomycin (p/s) (10, hereinafter, as these mice express Pparδ normally). Gen- 000 U penicillin/g/ml, 10 mg streptomycin/ml). The otyping was done by PCR to confirm the presence of cells and muscle debris were maintained in an incubator Crealong withthepresenceof floxedand wild-type at 37°C with 5% CO for three days, and 5 ml of Ham’s Pparδ alleles as described by The Jackson Laboratory medium were added each day. On the third day, all cells under mouse stock numbers 007893 and 005897 (Bar and muscle debris (with cells attached) were collected Harbor, ME, USA). into a 15-ml conical tube and digested with 1 ml of 0.025% trypsin for five minutes at 37°C. Dissociated cells Cardiotoxin injection were resuspended in 10 ml of Ham’s complete medium Tibilais anterior (TA) muscles taken from six-week-old and passed through a 30-μm filter, then plated on a col- C57BL/6J mice were injured by injection of cardiotoxin lagen-coated plate. Myoblasts were maintained in Ham’s (CTX) (C3987; Sigma-Aldrich, St Louis, MO, USA) to medium and passed through the filter several times induce muscle regeneration. The animals were first before they became senescent and were discarded. To anesthetized by intraperitoneal injection of 0.2 ml keta- differentiate myoblasts, Ham’s medium was switched to mine cocktail/20 g body weight. Ketamine cocktail con- DMEM containing 5% horse serum with 1% p/s when tains 0.9 ml of ketamine (100 mg/ml), 0.1 ml of xylazine cultures reach 80% confluence. For cell-growth analysis, (100 mg/ml) and 9.0 ml of saline. The hind limbs were primary myoblasts from both Pparδ-cKO and wild-type then shaved to expose the belly of the TA and wiped mice were counted, and 50, 000 cells were seeded into with 70% ethanol. Next we injected 50 μlof10 μM each well of a six-well plate. Cells were removed from CTX into the belly of the TA muscle. The mice were the plate with trypsin and counted with a hemocyt- allowed to recover on a heating pad for about one hour. ometer on days 3, 6 and 9 after the initial plating. The mice were then harvested at days 5 and 14 after injection, and their TA muscles were removed for RNA Isolation and culture of single myofibers extraction and histological examination after being fixed Single myofiber-carrying satellite cells were isolated as in 4% paraformaldehyde. previously described [41]. Fibers were harvested from the soleus (SOL) and extensor digitorum longus (EDL) mus- Glucose challenge cles of mice that were six weeks of age. Whole muscles Glucose tolerance tests were performed on two- and were removed from the hind limbs by careful handling of nine-month-old mice. All mice were fasted for three the tendons only. The SOL and EDL muscles were first hours prior to the start of testing. Blood was collected placed in 5 ml of DMEM containing 0.2% collagenase I from the tip of the tail and tested before (0 minutes) and then into the water bath at 37°C for 40 minutes and 15, 30, 60 and 90 minutes after intraperitoneal (EDL muscle) or 80 minutes (SOL muscle). The fibers injection of glucose (2 g/kg). Blood glucose levels were were then placed into a 6-cm plate with 5 ml of DMEM measured using an ACCU-CHEK Active blood glucose and separated from the tendons by careful manipulation meter system (Roche Diagnostics, Indianapolis, IN, using heat-polished Pasteur pipettes coated with horse USA). Body weight data were collected prior to glucose serum to prevent sticking. After the fibers were sepa- tolerance testing. rated, they were fixed immediately for staining or trans- ferred onto a new plate containing 5 ml of DMEM with Cell culture 20% FBS and 2% chick embryo extract, then placed in the Primary myoblasts were harvested from the limb mus- incubator at 37°C with 5% CO for three days. At the end cles of mice that were six weeks of age. Limb muscles of the three days, the fibers were collected, fixed with 2% were digested in a solution containing 1% collagenase B paraformaldehyde and prepared for staining. (Roche Diagnostics) and 2.4 U/ml Dispase II (neutral protease, grade II; Roche Applied Science) for 30 min- Immunocytochemistry utes in an incubator at 37°C with 5% CO and triturated Primary myoblasts were growninchamber slides for every 15 minutes. The digestion was stopped after 30 staining. The cells were fixed with 2% paraformaldehyde Angione et al. Skeletal Muscle 2011, 1:33 Page 4 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 for ten minutes, then washed three times with PBS covered with a coverslip and allowed to dry overnight. before the primary antibody was added. The primary For nicotinamide adenine dinucleotide, reduced antibodies used were Pax7 (Developmental Studies (NADH) staining, sections were washed briefly in PBS Hybridoma Bank, Department of Biology, University of to remove excess O.C.T. compound. The slides were Iowa,IowaCity, IA USA),MyoD(sc-20; SantaCruz then incubated in a solution containing 100 mg/ml Biotechnology, Santa Cruz, CA, USA), and mAb clones NADH and 0.1 g/ml nitroblue tetrazolium (NBT) for 30 HB287, HB277 and HB283 for MyHC1, MyHC2a and minutes at 37°C. The slides were then washed three MyHC2b, respectively (American Type Culture Collec- times with deionized water. Unbound NBT was removed tion, Manassas, VA, USA). Cells were incubated with from the sections by washing the slides three times each the primary antibody for one hour at room temperature with 30%, 60% and 90% acetone. The sections were then on a shaker. After incubation with the primary antibody, washed with deionized water and mounted. the cells were washed three times with PBS before the secondary antibody was added. The secondary antibo- Gene expression dies used were goat anti-mouse immunoglobulin G2b All gene expression data were gathered by real-time (IgG2b) conjugated with Alexa Fluor 647 dye, goat anti- PCR. RNA was extracted from cell culture, whole-mus- mouse IgG1 conjugated with Alexa Fluor 568 dye, goat cle and adipose tissue using the RNeasy RNA extraction anti-mouse IgM conjugated with Alexa Fluor 488 dye kit (QIAGEN, Germantown, MD, USA). RNA was con- (Molecular Probes/Life Technologies, Carlsbad, CA, verted into cDNA using Moloney murine leukemia virus USA) and daylight Alexa Fluor 488-conjugated goat reverse transcriptase and random hexamer primers. The anti-rabbit (Jackson ImmunoResearch Laboratories, Inc, real-time PCR oligonucleotides are listed in Additional West Grove, PA, USA). The cells were incubated with file 1, Table S1. Samples were then run in a LightCycler secondary antibody and Hoechst dye (a DNA dye) for 480 System (Roche Applied Science) for 40 cycles. Fold -ΔΔCt 30 minutes. The cells were again washed three times changes were calculated using the 2 method. with PBS before being mounted with Dako Fluorescence Mounting Medium (Glostrup, Denmark) and covered Western blot analysis with a coverslip. Images were recorded on a Leica Expression of PPARδ protein in Pparδ-cKO and wild- DMI6000 B inverted fluorescence microscope (Leica type muscles was examined by Western blot analysis as Microsystems, Mannheim, Germany). previously described [32]. Briefly, equal amounts of nuclear proteins extracted under identical conditions Histology were run on SDS-PAGE gels and transferred onto poly- The muscles harvested from the hind limbs were vinylidene difluoride membrane. PPARδ was detected embedded in optimal cutting temperature (O.C.T.) com- with rabbit anti-PPARδ antibody from Santa Cruz Bio- pound (Sakura Finetek USA Inc, Torrance, CA, USA) technology (SC-7197). and quickly frozen in dry ice-cooled isopentane. Cryo- sections were cut to 10-μm thickness and placed on Statistical analysis glass slides. For immunohistochemistry, slides were The number of mice used for each experiment is listed blocked with blocking buffer (5% horse serum, 2% BSA, in the figure legend. Every experiment was performed in 0.2% Triton X-100 and 0.1% sodium azide in PBS) for triplicate. For the glucose tolerance tests, statistical ana- two hours prior to antibody staining. The slides were lysis was based on area under the curve (AUC) using incubated with primary antibodies for one hour at room the trapezoidal rule. All data are presented as means ± temperature, then washed three times with PBS before SEM. P-values were calculated using a two-tailed Stu- incubation with secondary antibody for 30 minutes. dent’s t-test unless otherwise indicated. All values equal After three more washes with PBS, the slides were to 0.05 or less were considered significant and are mounted with Dako Fluorescence Mounting Medium denoted by asterisks in the figures. and covered with a coverslip. Results H & E and NADH staining Characterization of the Pparδ-cKO model Sections (10-μm thickness) were first washed with PBS To generate tissue-specific knockout of Pparδ,weused to remove excess O.C.T. compound, then incubated in mice bearing a floxed exon 4 in the Pparδ gene [39]. hematoxylin for five minutes, washed and incubated in The presence of Cre will excise exon 4, corresponding 1% eosin for 30 seconds. The sections were then dehy- to the N-terminal zinc finger of the DNA binding drated in increasing concentrations of ethanol and domain of PPARδ, and lead to premature stop of trans- mounted with CytoSeal Mounting Medium (Electron lation and abolish the transcriptional activity of the Microscopy Sciences, Fort Washington, PA, USA), resulting truncated PPARδ (Additional file 2, Figure Angione et al. Skeletal Muscle 2011, 1:33 Page 5 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 f/f S1A). The Pparδ mice were bred to mice expressing To confirm the tissue-specific knockout, we collected Cre recombinase under the control of the endogenous RNA samples from adult skeletal muscle, BAT and Myf5 promoter Myf5-Cre (007893; The Jackson Labora- white fat (WAT) and conducted quantitative RT-PCR tory) to generate the Pparδ-cKO mice. Since Myf5 is (qPCR) analysis. The levels of Pparδ in the Pparδ-cKO expressed in the mesoderm during development, we mice compared to their wild-type littermates were expected that PPARδ would be selectively ablated in reduced by 7-fold in TA muscle and by 11-fold in BAT, several mesodermal tissues that express Myf5, including buttheywerenot changedinWAT (Figure1A).These skeletal muscle and brown fat (BAT) [20]. results confirm the tissue-specific knockout and are Figure 1 Characterization of the PPARδ-cKO model. Blue and green bars represent wild-type (WT) and peroxisome proliferator-activated receptor δ conditional knockout (Pparδ-cKO) (MUT) tissues, respectively. (A) Relative expression levels of Pparδ in tibialis anterior (TA) muscle, brown fat and white fat tissues (N = 8 pairs of wild-type and mutant mice) for each tissue type. (B) Quantitative RT-PCR showing the relative expression levels of Pparδ in whole-muscle tissue, proliferating myoblasts, white fat and brown fat in wild-type mice (N = 8 for each tissue type and N = 2 for myoblasts). (C) and (D) Expression levels of Ppara in TA muscle (C) and Pparg in TA, brown fat and white fat tissues (N = 8 for each tissue type) (D). (E) and (F) Expression levels of carnitine palmitoyltransferase 1b (mCPT1b) and Forkhead box class O transcription factor 1 (FoxO1) genes in TA muscles (N = 3). Angione et al. Skeletal Muscle 2011, 1:33 Page 6 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 consistent with the notion that skeletal muscle and BAT observation, qPCR analysis indicated that satellite cell- derive from the Myf5 lineage and WAT derives from a specific Pax7 gene expression was reduced by about Myf5-independent lineage [20]. We further confirmed 40% in the mutant compared to the wild-type TA mus- by Western blot analysis that PPARδ protein levels are cles (Figure 2D). Interestingly, normal satellite cell num- reduced by 60% to 80% in the gastrocnemius muscles of bers were detected at three weeks of age, and a nearly Pparδ-cKO mice compared to those of wild-type mice 20% reduction was detected at age five weeks in the (Additional file 2, Figure S1B). Interestingly, analysis of Pparδ-cKO compared to the wild-type EDL fibers relative Pparδ expression in various wild-type tissues (Additional file 4, Figure S3). Furthermore, the number of 4’, 6-diamidino-2-phenylindole-positive nuclei per indicated that both proliferating myoblasts (derived from satellite cells) and WAT expressed much higher myofiber was reduced in the Pparδ-cKO mice at five levels of Pparδ compared to whole muscle and BAT weeks old but not at three weeks old (Additional file 4, (Figure 1B). Specifically, Pparδ mRNA levels in primary Figure S3). The gradual reduction in satellite cells and myoblasts were about 15-fold greater than those in the differentiated myonuclei in the Pparδ-cKO muscle dur- TA muscles, suggesting a specific role of PPARδ in ing postnatal growth suggests that PPARδ is important myoblast proliferation. In addition, we found that the for satellite cell proliferation and maintenance. levels of Pparg and Ppara remained unchanged in Next we examined whether the satellite cells asso- Pparδ-cKO compared to wild-type tissues (Figures 1C ciated with the isolated fibers were able to proliferate and 1D), confirming that Pparδ-cKO did not elicit com- and differentiate normally using a single-myofiber cul- pensation by other PPAR isoforms. Therefore, any ture paradigm to mimic satellite cell activation in vivo observed phenotypes are due to specific knockout of [13,15]. We cultured isolated fibers for three days in Pparδ in our mouse model. vitro and fixed and stained clusters of myoblasts that To understand how PPARδ functions in muscle, we had proliferated on single fibers with antibodies to Pax7 compared the expression of a number of known PPARδ and MyoD (Figure 2B). Previous studies have established + - + + - + target genes in wild-type and mutant TA muscles from that Pax7 /MyoD ,Pax7 /MyoD and Pax7 /MyoD adult mice. The expression of carnitine palmitoyltrans- cells represent self-renewing, proliferating and differen- ferase 1b (mCPT1b), whose protein regulates the rate- tiating progenies, respectively [11-13]. The relative per- limiting step for b-oxidation of long-chain fatty acids centage of cells in these three categories was examined. [21,24], and Forkhead box class O transcription factor 1 Compared to the wild type, mutant SOL fibers had half + + (FoxO1), a PPARδ target that regulates skeletal muscle as many proliferating cells (Pax7 /MyoD ) and twice as metabolism and progenitor cell function [42-44], were many differentiating cells (MyoD ), whereas the propor- significantly decreased in Pparδ-cKO muscle (Figures 1E tion of self-renewing cells (Pax7 )remainedthe same and 1F). The expression of several other known target between wild-type and mutant fibers (Figure 2E). genes of PPARδ, including Sirt1, UCP1 and PGC1a, was Together, these results show that PPARδ is important not altered in the mutant muscles (Additional file 3, Fig- for maintaining the proliferation of activated myoblasts ure S2). These results confirm that Pparδ-cKO affects and that loss of PPARδ leads to accelerated myogenic candidate target gene expression and identify mCPT1b differentiation. and FoxO1 as potential target genes regulated by PPARδ To further characterize the proliferative defects of in resting skeletal muscles. Pparδ-null myoblasts, we examined the expression of Ki67, a cell proliferation marker, in cultured primary Satellite cell and myoblast proliferation and myoblasts from mutant mice and wild-type littermates differentiation (Figure 3A-B). Real-time PCR analysis confirmed a 60% Adult Pparδ-cKO mice have a reduced number of satel- reduction in Pparδ expression in newly established cul- lite cells, and Pparδ-null myoblasts exhibited reduced tures of Pparδ-cKO compared to wild-type myoblasts proliferation and increased differentiation kinetics. (Additional file 5, Figure S4A). The percentage of Ki67 Because proliferating myoblasts expressed high levels of cells and the Ki67 immunofluorescence intensity of the Pparδ compared to whole muscles (Figure 1B), we Pparδ-mutant myoblasts were threefold less than those investigated the effect of Pparδ mutation on the satellite of wild-type plates (Figures 3C). Conversely, a PPARδ cells in vivo and in myoblasts in culture. Intact single agonist, GW501516, significantly increased wild-type fibers were isolated from the representative slow (SOL) myoblast proliferation (Additional file 5, Figure S4B). and fast (EDL) muscles and stained with Pax7 antibody We also plotted the growth curve of wild-type and to label satellite cells (Figure 2A). At two to three mutant myoblasts at days 3, 6 and 9 after they were pla- months old, the mutant mice had, on average, a 40% ted in culture. Myoblasts from Pparδ-cKO muscle reduction in satellite cell numbers in both EDL and showed a reduced growth rate compared to myoblasts SOL muscles (Figure 2C). Consistent with this from wild-typemuscle(Figure3D). Theseresults Angione et al. Skeletal Muscle 2011, 1:33 Page 7 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 2 Reduced satellite cell number and proliferation in Pparδ-cKO mice. (A) Freshly isolated extensor digitorum longus (EDL) muscle fibers were stained with paired-box transcription factor 7 (Pax7) antibody to label satellite cells. Nuclei were counterstained with 4’, 6-diamidino- 2-phenylindole (DAPI). Scale bar = 25 μm for all panels in parts (A) and (B). (B) Soleus (SOL) muscle fibers were cultured for 72 hours, and the proliferating satellite cells on the fibers were fixed and stained with Pax7 and myogenic differentiation antigen 1 (MyoD) antibodies. Nuclei were + + + ), proliferating cells (Pax7 /MyoD ) and differentiating cells counterstained with DAPI. Satellite cell status is classified as self-renewed cells (Pax7 + + (MyoD ). (C) Quantification of the number of Pax7 satellite cells per single fiber isolated from the EDL and SOL muscles (N = 360 fibers each from the SOL and EDL muscles). **P < 0.0001. (D) Relative expression of the Pax7 gene in tibialis anterior (TA) muscles by quantitative PCR (N = 3). (E) Quantification of the number of self-renewed, proliferating and differentiating cells on SOL fibers after culture for 72 hours (N = 20 fibers for each group). provide compelling evidence that PPARδ positively regu- 14 days, the mice were killed and the TA muscles were lates myoblast growth and proliferation. collected for gene expression and histological analysis. We examined the regenerating regions under a micro- Muscle regeneration after injury is impaired in Pparδ-cKO scope, and the number of regenerating fibers (small, animals centrally located nucleus) and regenerated fibers (large, To examine whether the reduced rates of proliferation eccentrically located nucleus) were counted. Both wild- in the Pparδ-cKO myoblasts result in defects in muscle type and Pparδ-cKO TA muscles underwent regenera- regeneration in vivo, we injured the TA muscles of tion, but they exhibited marked differences (Figures 4A mutant and wild-type animals at six weeks of age. After and 4B). The mutant muscles had much larger areas of Angione et al. Skeletal Muscle 2011, 1:33 Page 8 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 3 Defective growth and proliferation of primary myoblast cells derived from Pparδ-cKO muscles. (A) and (B) Representative images of Ki67 staining that specifically labeled proliferating primary myoblasts: Ki67 staining (red) and 4’, 6-diamidino-2-phenylindole (DAPI) staining (blue). Scale bar = 50 μm. (C) Relative Ki67 signal intensity in wild-type and mutant myoblast cultures. The intensity of Ki67 and DAPI staining and the number of pixels were measured using Photoshop software (Adobe Systems Inc, San Jose, CA, USA). The ratio of Ki67 intensity values to DAPI intensity values was also quantified using Photoshop (N = 3). (D) Growth curve of wild-type and mutant myoblasts after nine days in culture (N = 3). - + small-caliber regenerating fibers that covered almost the differentiated Pax7 /MyoD myocytes in the Pparδ-cKO whole muscle sections, whereas the wild-type TA mus- muscle, together with reduced total myoblast number cles had reduced regenerating areas that were mostly per unit area and decreased size of newly regenerated confined to the center of the section (Figures 4A and fibers (Figures 4F to 4I). In addition, we confirmed by 4B). The number of small regenerating fibers was 30% qPCR that Myogenin gene expression was upregulated more, and the number of large regenerating fibers was in regenerating Pparδ-cKO muscles at this stage (Figure 20% fewer, in Pparδ-cKO muscles compared to wild- 4J). By contrast, Myf5 was expressed at similar levels in type muscles (Figure 4C). In addition, the regenerating the mutant and wild-type muscles. These results indi- Pparδ-cKO TA muscles expressed reduced levels of cate that the observed regenerative defects of Pparδ- MyHC 2b mRNA, the most abundant MyHC isoform in cKO may be due to reduced proliferation and increased the TA muscle (Figure 4D), which further confirms the differentiation kinetics of activated myoblasts. regenerative defects. The Pparδ-cKO muscles were We also examined the expression of mCPT1b and eventually able to regenerate after 30 days, suggesting FoxO1 genes during muscle regeneration at day 5 after that Pparδ-cKO mainly causes a delay in muscle CTX treatment, when damaged muscles are not yet regeneration. regenerated and satellite cell proliferation peaks [8]. Because Pparδ-cKO myoblasts are defective in prolif- Interestingly, expression of mCPT1b was unchanged but eration in vitro, we sought to determine whether such FoxO1 expression was reduced in the Pparδ-cKO mus- defects exist in vivo during muscle regeneration. We cles compared to wild-type muscles during muscle analyzed Pax7 and MyoD expression (Figures 4F and regeneration (Figure 4E), suggesting that PPARδ may 4G) at days 3 to 5 post-CTX treatment, at which stage activate FoxO1 in proliferating myoblasts and that myoblast proliferation peaks. Consistent with our in mCPT1b is a PPARδ target only in mature muscle vitro results, we observed a reduction of proliferating fibers. Together with our earlier observation that Pparδ + + Pax7 /MyoD myoblasts and an increase in is expressed at much higher levels in proliferating Angione et al. Skeletal Muscle 2011, 1:33 Page 9 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 4 Defective regeneration of skeletal muscles and gene expression patterns in Pparδ-cKO mice after injury with cardiotoxin. (A) and (B) Tibialis anterior (TA) muscles from six-week-old wild-type and peroxisome proliferator-activated receptor δ conditional knockout (Pparδ- cKOmice were injected with cardiotoxin (CTX) to induce injury. The mice were allowed to recover for 14 days before their muscles were harvested and processed for staining. Small and large fibers (indicating regenerating and nonregenerating fibers, respectively) were counted. Scale bars = 50 μm for parts (A) and (B) and for parts (F) and (G). (C) Quantification of regenerating and nonregenerating fibers after injury with CTX (N = 3 pairs of mice, with five sections taken from each mouse). (D) Relative mRNA expression level showing reduced myosin heavy chain isoform 2b (MyHC-2b) in mutant muscle after injury with CTX (N = 4). (E) Relative mRNA expression levels of Pparδ target genes in the TA muscle after injury with CTX (N = 4). (F) and (G) Cryosections of regenerating wild-type (F) and mutant (G) mice at day 5 after injury with CTX. The sections are labeled with paired-box transcription factor 7 (Pax7) (red), myogenic differentiation antigen 1 (MyoD) (green) and 4’,6- + - - + + + diamidino-2-phenylindole (DAPI) (blue). Arrows, arrowheads and asterisks indicate examples of Pax7 /MyoD , Pax7 /MyoD and Pax7 /MyoD myoblasts, respectively. (H) The number of total myoblasts labeled by Pax7 and/or MyoD per unit area counted from 8 to 13 areas. (I) + - + + - + Percentage distribution of Pax7 /MyoD , Pax7 /MyoD and Pax7 /MyoD myoblasts in regenerating wild-type and mutant TA muscles at day 5 after injury with CTX. (J) Relative mRNA expression levels of Myf5 and Myogenin in TA muscles at day 3 after injury with CTX (N = 3). *P < 0.05 by single-tailed Student’s t-test. Angione et al. Skeletal Muscle 2011, 1:33 Page 10 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 myoblasts compared to mature muscles (Figure 1B), mAbs (Figures 5A to 5D). We chose the EDL and SOL these results suggest a critical role of PPARδ in satellite muscles to represent fast and slow muscle types, respec- cell function in vivo. tively. To overcome potential bias associated regiona- lized distribution of fiber types within a muscle [45], we Pparδ-cKO does not alter myosin heavy chain isoforms enumerated all muscle fibers in the entire muscle (Fig- Because Pparδ overexpression increases oxidative mus- ures 5A to 5D). No difference in the percentage of each cle fiber types [32], we sought to determine whether fiber type was found between mutant and wild-type ani- Pparδ-cKO causes any changes in fiber-type distribution. mals in either the EDL or SOL muscle at six weeks old We labeled MyHC protein isoforms with a panel of (Figure 5E). To further determine whether there are Figure 5 Normal fiber-type distributions in extensor digitorum longus and soleus muscles of six-week-old Pparδ-cKO mice. (A) through (D) Immunostained sections showing the distribution of fiber types in the extensor digitorum longus (EDL) and soleus (SOL) ("C-” denotes wild- type littermate control and “M-” denotes Pparδ-cKO): myosin heavy chain isoform 1 (MyHC-1) (red), MyHC-2a (blue), MyHC-2b (green), MyHC-2x (black) and laminin (white). The basal lamina surrounding each fiber is shown. Scale bar = 1 mm. (E) Percentage distribution of each fiber type in the EDL and SOL muscles (N = 4 pairs of littermates. Angione et al. Skeletal Muscle 2011, 1:33 Page 11 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 changes in muscle oxidative metabolism without chan- Pparδ-cKO whole muscle and BAT (10% to 20% of ging MyHC isoform expression, we carried out NADH- wild-type levels) is probably due to non-Myf5 lineage tetrazolium reductase (NADH-TR) staining of the SOL cells’ residing in these tissues, such as nerve cells (neural and EDL muscles. NADH-TR staining marks ATPase branches and Schwann cells), vessel-associated cells activity that is correlated to oxidative capacity (type 1 (blood, lymph, endothelial and smooth muscle cells) and fibers arehighlyoxidative anddarklystained,type 2a interstitial connective tissue-associated cells (fibroblasts fibers are intermediate fibers and lightly stained and and white adipocytes). In addition, because Myf5-Cre is type 2b fibers are glycolytic and unstained) (Additional expressed in about 90% of satellite cells [15], a small file 6, Figures S5A to S5D). In agreement with our fraction of Myf5 satellite cells would still have normal MyHC isoform staining experiments, there were no dif- levels of Pparδ expression. We nevertheless observed ferences in NADH activity between the Pparδ-cKO and proliferative defects in satellite cells in vitro and muscle wild-type mice in either the EDL or the SOL muscles regeneration in vivo. Further studies using other Cre (Additional file 6, Figures S5E and S5F). These results lines with improved efficiency of Pparδ deletion in satel- demonstrate that Pparδ-cKO does not lead to changes lite cells would help to better address the muscle-regen- in muscle fiber type or oxidative capacity in young erative defects. animals. Mice lacking PPARδ in their skeletal muscles are born at normal ratios and are viable. These animals do not Old Pparδ-cKO mice become obese and have impaired appear to have any noticeable defects in body weight or glucose clearance response to glucose early in life. There was no difference PPARδ is an important regulator of muscle energy in total fiber number or percentage of each fiber type in metabolism. Therefore, we tested the body weight and representative slow (SOL) and fast (EDL) muscles. How- insulin sensitivity in young and old mutant mice. Young ever, old mice (eight to nine months old) were signifi- Pparδ-cKO mice (two months of age) did not differ sig- cantly fatter than their wild-type littermates and were nificantly in body weight compared to their sex-matched found to have developed glucose intolerance. This phe- wild-type littermates (Figure 6A). However, older notype in the aged mouse is similar to that reported in mutant mice (nine months of age) were about 25% hea- another transgenic mouse model in which conditional vier on average than their wild-type littermates (Figure mutation of Pparδ in mature skeletal muscles is 6B). Next we measured the rate of insulin-mediated glu- mediated by human a-skeletal actin promoter-driven cose clearance in the mutant and wild-type mice. The Cre in the transgenic mouse [33]. In that mouse model skm-/- amount of glucose in the blood was measured before (0 (Pparδ ), a reduction in the oxidative capacity and minutes) and 15, 30, 60 and 90 minutes after intraperi- muscle fiber-type switching are also detected, whereas toneal injection of 2 g/kg glucose (Figures 6C and 6D). such changes are not evident in the Pparδ-cKO mouse Again, young mice showed no differences in the rate of model used in our study. The discrepancy between insulin-stimulated glucose uptake as measured by AUC these observations may be due to the genetic back- analysis of the glucose tolerance curves (Figure 6E). ground and nutrition status (that is, diet composition) Strikingly, older mutant mice showed a reduced rate of of the mice used and the different techniques used for glucose uptake; their blood glucose levels spiked higher fiber typing. The previous study used qPCR analysis to than those of the wild-typemiceand took longer to show increases in MyHC1 and MyHC2b transcripts in clear from the blood (Figure 6D), resulting in significant the gastrocnemius muscle [33]. As many muscle fibers increases in the AUC in every time period measured express multiple MyHC transcripts, small changes in (Figure 6F). These results suggest that the older mice these transcripts may not be sufficient to induce fiber- had developed glucose intolerance and an obese pheno- type switching [46]. In our current study, we used type even when fed a normal diet. MyHC isoform-specific mAbs to unambiguously identify fiber types, and we examined all fibers within the entire Discussion muscle to eliminate bias due to regional clustering of The results produced by our Pparδ-cKO model establish specific fiber types within the same muscle. We have a previously unappreciated role of PPARδ in skeletal therefore provided strong evidence that Pparδ-cKO does muscle progenitor cell proliferation. The specificity of notleadtoanovert switch of muscle fibertypein the Pparδ-cKO mouse model is demonstrated by young animals based on MyHC isoform protein reduced Pparδ mRNA expression in both the skeletal expression. muscle and BAT, known to be derived from Myf5 line- We show herein that the expression levels of Pax7 are age cells. In contrast, Pparδ expression was unchanged significantly reduced in the whole muscle of Pparδ-cKO in WAT, which is derived from a Myf5-independent mice. Because Pax7 is a satellite cell-specific marker [10], this observation suggests a reduction in satellite lineage [20]. The residual Pparδ expression detected in Angione et al. Skeletal Muscle 2011, 1:33 Page 12 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 6 Age-dependent increases in body weight and glucose tolerance in Pparδ-cKO mice. (A) and (B) Body weights of female wild- type mice and mutant littermates at two and nine months old (N = 6). (C) and (D) Blood glucose concentration at different time points following intraperitoneal injection of glucose (2 g/kg). The mice were fasted for three hours prior to glucose injection. Glucose concentration was measured using an ACCU-CHEK Active blood glucose meter system (Roche Diagnostics, Indianapolis, IN, USA) (N = 3 pairs of female littermates). (E) and (F) Area under the curve (AUC) analysis the of glucose tolerance test results shown in parts (C) and (D), respectively. population doubling time based on the cell growth cell number, which we confirmed by our later results showing that Pparδ-cKO mice have fewer satellite cells curve. Conversely, the PPARδ agonist GW501516 stimu- in vivo. Our single-fiber culture model that mimics lates myoblast proliferation. These results provide com- satellite cell activation in vivo further demonstrates that pelling evidence that PPARδ acts to facilitate satellite there are fewer proliferating satellite cells and increased cell proliferation. numbers of differentiating cells in Pparδ-cKO mice. In Recent studies have shown that PPARδ promotes the vitro growth and proliferation of Pparδ-cKO satellite proliferation of several cell types. PPARδ activation has cell-derived primary myoblasts were also reduced, as been shown to increase the proliferation and migration shown by the decreased number of cells expressing the of endothelial progenitors as well as the number of cell proliferation marker Ki67 and the increased hematopoietic stem cells in the bone marrow [34,47,48]. Angione et al. Skeletal Muscle 2011, 1:33 Page 13 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 In addition, PPARδ has been shown to be essential for incidence of sarcopenia (age-related muscle loss) has high glucose-stimulated proliferation of embryonic stem been shown to correlate with reduced levels of PPARδ, cells [35]. Moreover, PPARδ is an important regulator of and pharmacological activation of PPARδ has been the proliferation of several cancer cell types [49,50] shown to reduce the incidence of sarcopenia by increas- (older references are reviewed by Peters and Gonzalez ing nuclear accretion in myofibers [57]. These results [36]). The PPARδ antagonists SR13904 and GSK0660, support the notion that PPARδ is important for proper skeletal muscle regeneration in response to injury or which block PPARδ activation, have been shown to daily wear and tear (maintenance) by increasing satellite reduce cell proliferation [50,51]. The role of PPARδ in cell proliferation and promoting fusion of differentiated cell proliferation has been shown to be due to its activa- tion of the phosphatidylinositol 3-kinase and AKT path- myocytes in vivo. ways [34,38], which has been shown to be important in We examined the expression levels of several pre- preventing apoptosis and enhancing cell proliferation by viously identified PPARδ target genes to understand triggering cells to overcome cell-cycle arrest and initiat- how PPAR functions in skeletal muscle and satellite ing cell proliferation [52,53]. Importantly, AKT signaling cells. Our results show that the expression of both plays critical roles in skeletal muscle progenitor cell mCPT1b and FoxO1 is reduced in mature uninjured function and muscle hypotrophy [4,5,54]. muscles of the Pparδ-cKO mice, suggesting that they A number of other studies have shown that PPARδ are molecular targets of PPARδ under resting condi- promotes differentiation and inhibits (or has no effect tions. Since PPARδ has been shown by many groups to on) proliferation in many cell types, particularly kerati- be a regulator of oxidative capacity in skeletal muscle nocytes, smooth muscle cells, cardiac fibroblasts and [21], it is not surprising that key players in the lipid oxi- several cancer cell lines (extensively reviewed by Peters dation pathway would be downregulated after removal and Gonzalez [36] and Foreman et al. [55]). Therefore, of PPARδ. Interestingly, mCPT1b expression is not sig- whether PPARδ promotes terminal differentiation or cell nificantly reduced in Pparδ-cKO muscle at day 5 during proliferation is highly cell type-specific [36]. Intriguingly, regeneration. At this time point, degenerated muscle investigators in a recent study demonstrated that PPARδ fibers have not been replaced by new fibers and myo- can exert its transcriptional activation or repression blast proliferation is at the peak stage [8]. This result both ligand-dependently and ligand-independently in a seems to suggest that mCPT1b is a target of PPARδ in target-specific manner [56]. Such variations in the mode mature resting muscles, but not in proliferating myo- of PPARδ action probably account for the paradoxical blasts. Of all the PPARδ target genes we examined, effects of PPARδ in different cell types or even in the FoxO1 was the only gene whose expression was downre- same cell type. More research is needed to elucidate gulated in both resting and regenerating muscles of how PPARδ regulates satellite cell proliferation and Pparδ-cKO mice. The downregulation of FoxO1 in whether it also functions at later stages of myogenesis Pparδ-cKO regenerating muscle indicates that FoxO1 (see discussion below). may act at downstream of PPARδ in regulating myoblast Pparδ-cKO animals showed delayed muscle regenera- proliferation and inhibiting its differentiation. Indeed, it tion after injury with CTX. We found that, compared to has been shown that constitutive activation of FoxO1 their wild-type littermates, transgenic mice lacking inhibits myoblast differentiation [44]. However, FoxO1 PPARδ in the skeletal muscle had a reduced proportion also acts at later stages of myogenesis, during the fusion of proliferating myoblasts and that the regenerated of differentiated myocytes into myotubes [58]. These fibers were smaller in caliber at both five days and two previous studies and our present results suggest that weeks after CTX injury. These results are consistent PPARδ acts through FoxO1 in both satellite cells and with our observations that these animals had fewer mature muscle fibers but has distinct functions in these satellite cells and that Pparδ-deficient satellite cells have cell types. It regulates the proliferation of satellite cells reduced proliferative potential in vitro. Because satellite and fusion of muscle fibers. These combined effects of cells are responsible for skeletal muscle regeneration, PPARδ’s acting through FoxO1 explain the regenerative reductions in satellite cell number and defective satellite defects seen in the Pparδ-cKO muscles. cell proliferation may have resulted in the observed PPARδ has also been implicated in the treatment of delay in muscle regeneration. In addition to decreased degenerative muscle diseases such as Duchenne muscu- satellite cell proliferation, loss of PPARδ in myofibers lar dystrophy (DMD), which is caused by the mutation may have contributed to the observed defects in muscle of the dystrophin gene [59,60]. One research group regeneration. Moreover, other signaling mechanisms showed that PPARδ activation by the agonist mayhavecompensated forthe loss of PPARδ in our GW501516 increased the expression of utrophin A, a conditional mutant, leadingtocompletionofmuscle key member of the dystrophin-associated protein com- regeneration at a later time. Coincidentally, the plex, in the C C myoblast cell line [61]. PPARδ has 2 12 Angione et al. Skeletal Muscle 2011, 1:33 Page 14 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 been suggested to be a direct transcriptional regulator of wild-type primary myoblasts at 24 hours after control (dimethyl sulfoxide utrophin A in vivo [43]. Intriguingly, utrophin A overex- vehicle) and 100 nM PPARδ agonist GW501516 treatments (N = 10). pression can improve the integrity of the sarcolemma, Additional file 6: Figure S5 NADH-tetrazolium reductase staining showing the relative abundance of oxidative fibers in Pparδ-cKO protect muscles from contraction induced damage and and wild-type skeletal muscles. Staining shows the three different fiber help to alleviate muscle wasting and slow down the dis- types. Oxidative fibers (type 1) are darkly stained, intermediate fibers ease progression of DMD [62]. Therefore, understanding (type 2a) are moderately stained and glycolytic fibers (types 2b and 2x) are unstained. (A) and (B) Representative images of fast muscles how PPARδ functions in skeletal muscle tissue and its (extensor digitorum longus (EDL)). (C) and (D) Representative images of progenitor cells has important implications for muscle slow muscles (soleus (SOL)). (E) and (F) Relative nicotinamide adenine regeneration and the treatment of degenerative muscle dinucleotide, reduced (NADH), intensity levels between peroxisome proliferator-activated receptor δ (PPARδ) wild-type and PPARδ-conditional diseases. knockout (Pparδ-cKO) animals at six weeks of age (N = 3). Conclusions Our in vivo and in vitro analyses of myogenic lineage Abbreviations specific Pparδ gene knockouts demonstrate a critical BSA: bovine serum albumin; DMEM: Dulbecco’s modified Eagle’s medium; H role of PPARδ in satellite cell proliferation and postnatal & E: hematoxylin and eosin; mAb: monoclonal antibody; PBS: phosphate- regeneration of skeletal muscles. In addition, we provide buffered saline; PCR: polymerase chain reaction. evidence for a role of PPARδ in regulating muscle insu- Acknowledgements lin resistance, as indicated by the glucose tolerance test We thank Jun Wu for mouse colony maintenance and other members of results. However, we were unable to detect overt the Kuang laboratory for their comments and technical assistance. This project is partially supported by funding from the Muscular Dystrophy changes in skeletal muscle fiber types by MyHC iso- Association, the National Institutes of Health and the United Sates form-specific antibody labeling. These results not only Department of Agriculture (to SK). The authors declare no conflict of support the previously established role of PPARδ in interests. muscle energy metabolism and insulin sensitivity but Author details also demonstrate a novel role of PPARδ in muscle pro- Department of Animal Sciences, Purdue University, 901 West State Street, genitor cell function. These results have implications for West Lafayette, IN 47907, USA. Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical the treatment of muscular dystrophies and muscle-wast- School, 364 Plantation Street, Worcester, MA 01605, USA. Center for Cancer ing conditions by targeting PPARδ signaling at the stem Research, Purdue University, 201 S. University Street, West Lafayette, IN cell level. 47907, USA. Authors’ contributions Additional material AA carried out the mouse and cell culture studies, analyzed the data and drafted the manuscript. CJ carried out part of the muscle regeneration and immunofluorescence labeling studies. DP conducted the Western blot Additional file 1: Table S1 Primer sequences for quantitative PCR. analysis. YW provided the Pparδ-floxedmice, analyzed the data and revised Additional file 2: Figure S1 Pparδ-cKO strategy. (A) Peroxisome the manuscript. SK conceived, designed and coordinated the study; analyzed proliferator-activated receptor δ (Pparδ) gene structure with exons the data; and revised the manuscript. All authors read and approved the numbered sequentially. Note that LoxP sequences are inserted before final manuscript. and after exon 4 encoding the DNA-binding domain of PPARδ. In the presence of Cre (driven by Myf5 locus in this study), exon 4 is excised, Competing interests resulting in premature stop in translation and generation of a short, The authors declare that they have no competing interests. truncated protein without a DNA-binding domain. (B) Representative Western blot showing the relative expression of PPARδ protein in the Received: 19 May 2011 Accepted: 1 November 2011 wild-type (WT) and Pparδ-conditional knockout (Pparδ-cKO) Published: 1 November 2011 gastrocnemius muscles. The upper nonspecific band serves as an indicator of the relative amount of total protein loaded onto the gel. References Additional file 3: Figure S2 Relative expression of PPARδ target 1. Zierath JR, Hawley JA: Skeletal muscle fiber type: influence on contractile genes in mature noninjured muscles. RNA samples isolated from the and metabolic properties. PLoS Biol 2004, 2:e348. tibialis anterior (TA) muscles of six-week old mice were used for 2. 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PPARδ regulates satellite cell proliferation and skeletal muscle regeneration

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Copyright © 2011 by Angione et al; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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10.1186/2044-5040-1-33
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22040534
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Abstract

Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that play important roles in development and energy metabolism. Whereas PPARδ has been shown to regulate mitochondrial biosynthesis and slow-muscle fiber types, its function in skeletal muscle progenitors (satellite cells) is unknown. Since constitutive mutation of Pparδ leads to embryonic lethality, we sought to address this question by conditional knockout (cKO) flox/flox of Pparδ using Myf5-Cre/Pparδ alleles to ablate PPARδ in myogenic progenitor cells. Although Pparδ-cKO mice were born normally and initially displayed no difference in body weight, muscle size or muscle composition, they later developed metabolic syndrome, which manifested as increased body weight and reduced response to glucose challenge at age nine months. Pparδ-cKO mice had 40% fewer satellite cells than their wild-type littermates, and these satellite cells exhibited reduced growth kinetics and proliferation in vitro. Furthermore, regeneration of Pparδ-cKO muscles was impaired after cardiotoxin-induced injury. Gene expression analysis showed reduced expression of the Forkhead box class O transcription factor 1 (FoxO1) gene in Pparδ-cKO muscles under both quiescent and regenerating conditions, suggesting that PPARδ acts through FoxO1 in regulating muscle progenitor cells. These results support a function of PPARδ in regulating skeletal muscle metabolism and insulin sensitivity, and they establish a novel role of PPARδ in muscle progenitor cells and postnatal muscle regeneration. Keywords: Cre/LoxP, skeletal muscle, stem cell, proliferation, differentiation, self-renewal Background other physiological signaling pathways [2-4]. In addition, Skeletal muscle is the most abundant tissue in mam- skeletal muscle mass is always in a state of hypertrophy mals, making up 45% to 55% of total body mass in or wasting, based on relative use or disuse, respectively humans, and plays important roles in body movement [5,6]. Skeletal muscle has superior capacity to regenerate and metabolic regulation. Muscle is made up of different itself upon injury [7]. fiber types which have different metabolic requirements Skeletal muscle plasticity is mainly maintained by a that affect the whole body energy homeostasis of the subset of cells known as satellite cells [8,9]. These animal [1]. Type 1 fibers are classified as slow fibers and cells, located beneath the basal lamina of the muscle use oxidative metabolism as a fuel source, making them fiber, are normally maintained in a quiescent state. highly fatigue-resistant. Conversely, type 2 fibers are Satellite cells become activated when the muscle classified as fast fibers, use mainly glycolytic metabolism becomes damaged through injury or normal activity. and are less resistant to fatigue. Type 2 fibers are further Once activated the cells will reenter the cell cycle and broken down into three subtypes, known as types 2a, 2x undergo a few rounds of division, then differentiate and fuse with existing muscle fibers to rebuild the and 2b, that express corresponding myosin heavy chain (MyHC) isoforms and have decreasing resistance to fati- damaged area. Satellite cells in the quiescent state gue. Notably, skeletal muscles are plastic, and fiber-type express paired-box transcription factor 7 (Pax7) [10]. switching occurs in response to changes in activity and After activation the cells will express Pax7 and myo- genic differentiation antigen 1 (MyoD) concurrently while the cells undergo a few rounds of division (pro- * Correspondence: skuang@purdue.edu liferation). These proliferating cells eventually with- Department of Animal Sciences, Purdue University, 901 West State Street, draw from the cell cycle and either return to West Lafayette, IN 47907, USA Full list of author information is available at the end of the article © 2011 Angione et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Angione et al. Skeletal Muscle 2011, 1:33 Page 2 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 quiescence (self-renewal) through downregulation of cell types and animal species [28,29]. These receptors MyoD or differentiate through downregulation of Pax7 must form heterodimers with the retinoid X receptor and upregulation of myogenin. Thus expression of (RXR) before they can bind to specific recognition Pax7 and MyoD distinguishes the status of a cell, sequences, called PPAR response elements (PPREs), + - whether it is self-renewing (Pax7 /MyoD ), proliferat- which are located in the promoter and intron regions of + + - + ing (Pax7 /MyoD ) or differentiating (Pax7 /MyoD ), a wide variety of target genes [21,30]. The PPARs acti- vate or repress transcription through the recruitment of respectively [11-13]. Notably, researchers in several coactivators and corepressors. recent studies have demonstrated that the choice PPARδ has been shown to be important for the proper between self-renewal and differentiation of newly divided satellite cells is dynamically regulated [14-16]. function of skeletal muscle in gain- and loss-of-function Whereas most proliferating myoblasts divide symmetri- studies. Synthetic compound-mediated activation or cally, a subpopulation of cells can divide asymmetri- overexpression of the Pparδ gene in mice causes an cally to give rise to both self-renewal and increase in the oxidative capacity of the muscle resulting differentiating progenies [14-16]. from an increase in the number of type 1 oxidative Myogenic factor 5 (Myf5) is also important for skeletal fibers and a decrease in the number of type 2 glycolytic muscle development and satellite cell function. Myf5 fibers [31,32]. These increases in oxidative capacity have transcripts can first be detected at E8 in the developing been shown to contribute to an organism’s overall exer- embryo and are important for specifying the cells of the cise endurance. Along with increases in oxidative capa- muscle lineage [17]. Myf5-Cre lineage tracing labels a city, transgenic mice also show a decrease in overall majority of satellite cells, which will have become com- body fat content and individual adipocyte size. Mice mitted to the myogenic lineage. However, 10% of satel- with constitutively active Pparδ can maintain a normal lite cells remain Myf5-negative and are thought to be a body weight even when challenged with a high-fat diet, population of more primitive, uncommitted stem cells whereas their wild-type littermates become obese when that can give rise to committed cells through asym- fed the same diet. By contrast, skeletal muscle-specific metric cell division [15]. Myf5-null satellite cells are knockout of Pparδ seems to cause a decrease in oxida- defective in transient proliferation prior to differentia- tive capacity and makes mice prone to obesity and tion [18,19]. Intriguingly, investigators in recent studies metabolic disorders [33]. This loss of oxidative capacity have demonstrated that brown adipocytes, but not white could be due in part to reduced expression of PGC1a, whichisknownto playaroleintype1fiberformation adipocytes, are derived from Myf5 lineage progenitors and maintenance. These results suggest that PPARδ [20]. Therefore, Myf5-Cre-mediated conditional knock- out can be used to knock out floxed target genes in plays a role in preventing obesity and the development committed myogenic progenitor cells and their descen- of metabolic disorders. dants (mature skeletal muscles) as well as in brown Until now all the studies on PPARδ in skeletal muscle adipocytes. have focused on mature muscle fibers. It remains to be Peroxisome proliferator-activated receptors (PPARs) determined whether PPARδ plays a role in myogenic are members of the nuclear estrogen receptor superfam- satellite cells and postnatal muscle regeneration. PPARδ ily and have been shown to be important for the proper has been shown to regulate the proliferation and/or metabolism of fatty acids [21]. There are three PPAR maturation of several cell types, including mouse isoforms (a, δ and g), and each plays a specific role in embryonic stem cells, oligodendrocytes, keratinocytes, metabolism [22-24]. The PPARs are expressed in a wide endothelial progenitors and cancer cells [34-38]. How- range of adult tissues, but each has its own tissue-speci- ever, whether PPARδ positively or negatively regulates fic expression patterns. PPARa is highly expressed in proliferation is highly cell type-specific, and the evidence the liver and heart, PPARδ (alsoreferred toasPPARb, presented in the literature has sometimes been contra- PPARb/δ or NR1C2) is highly expressed in the intestine dictory [36]. In the current study, we used a Cre/LoxP- and liver, and PPARg is highly expressed in both brown based conditional mutation approach to remove Pparδ and white adipose tissues [21-23,25,26]. The three PPAR from the satellite cells to examine its function in muscle isoforms are ligand-activated receptors that are activated progenitor cells. We show herein that PPARδ is an by fatty acids, fatty acid derivatives and a variety of syn- important regulator of satellite cell proliferation in vitro thetic compounds [21]. The ligand-binding domains of and of muscle regeneration in vivo. PPARs vary slightly, resulting in specific affinity for fatty acids and synthetic compounds [27]. For example, Materials and methods GW501516 is one synthetic compound that has been Animals shown to specifically activate PPARδ with 60- to 1, 000- All experimental procedures involving the use of mice fold selectivity over the other isoforms, depending on were carried out in accordance with Purdue University’s Angione et al. Skeletal Muscle 2011, 1:33 Page 3 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Animal Care and Use Committee. C57BL/6J mice con- minutes using 25 ml of DMEM containing 2% fetal taining LoxP sites flanking exon 4, which encodes the bovine serum (FBS) and 10 mM 4-(2-hydroxyethyl)-1- N-terminal zinc finger of the DNA-binding domain of piperazineethanesulfonic acid, then passed through a f/f the Pparδ gene (Pparδ ), had been generated previously 100-μm filter. After filtration, the cells and muscle deb- [39]. These mice were crossed with Myf5-Cre to gener- ris were pelleted at 250 × g for five minutes and the f/f ate Myf5-Cre/Pparδ offspring for ablation of Pparδ in DMEM was removed. The cells and muscle debris were Myf5 lineage cells (called “Pparδ-cKO” hereinafter) [40]. then plated on noncoated plates with Ham’scomplete +/f f/f Pparδ or Pparδ littermates that did not inherit the medium containing 20% FBS, 4 ng/ml basic fibroblast Myf5-Cre allele were used as controls (called “wild type” growth factor and 1% penicillin/streptomycin (p/s) (10, hereinafter, as these mice express Pparδ normally). Gen- 000 U penicillin/g/ml, 10 mg streptomycin/ml). The otyping was done by PCR to confirm the presence of cells and muscle debris were maintained in an incubator Crealong withthepresenceof floxedand wild-type at 37°C with 5% CO for three days, and 5 ml of Ham’s Pparδ alleles as described by The Jackson Laboratory medium were added each day. On the third day, all cells under mouse stock numbers 007893 and 005897 (Bar and muscle debris (with cells attached) were collected Harbor, ME, USA). into a 15-ml conical tube and digested with 1 ml of 0.025% trypsin for five minutes at 37°C. Dissociated cells Cardiotoxin injection were resuspended in 10 ml of Ham’s complete medium Tibilais anterior (TA) muscles taken from six-week-old and passed through a 30-μm filter, then plated on a col- C57BL/6J mice were injured by injection of cardiotoxin lagen-coated plate. Myoblasts were maintained in Ham’s (CTX) (C3987; Sigma-Aldrich, St Louis, MO, USA) to medium and passed through the filter several times induce muscle regeneration. The animals were first before they became senescent and were discarded. To anesthetized by intraperitoneal injection of 0.2 ml keta- differentiate myoblasts, Ham’s medium was switched to mine cocktail/20 g body weight. Ketamine cocktail con- DMEM containing 5% horse serum with 1% p/s when tains 0.9 ml of ketamine (100 mg/ml), 0.1 ml of xylazine cultures reach 80% confluence. For cell-growth analysis, (100 mg/ml) and 9.0 ml of saline. The hind limbs were primary myoblasts from both Pparδ-cKO and wild-type then shaved to expose the belly of the TA and wiped mice were counted, and 50, 000 cells were seeded into with 70% ethanol. Next we injected 50 μlof10 μM each well of a six-well plate. Cells were removed from CTX into the belly of the TA muscle. The mice were the plate with trypsin and counted with a hemocyt- allowed to recover on a heating pad for about one hour. ometer on days 3, 6 and 9 after the initial plating. The mice were then harvested at days 5 and 14 after injection, and their TA muscles were removed for RNA Isolation and culture of single myofibers extraction and histological examination after being fixed Single myofiber-carrying satellite cells were isolated as in 4% paraformaldehyde. previously described [41]. Fibers were harvested from the soleus (SOL) and extensor digitorum longus (EDL) mus- Glucose challenge cles of mice that were six weeks of age. Whole muscles Glucose tolerance tests were performed on two- and were removed from the hind limbs by careful handling of nine-month-old mice. All mice were fasted for three the tendons only. The SOL and EDL muscles were first hours prior to the start of testing. Blood was collected placed in 5 ml of DMEM containing 0.2% collagenase I from the tip of the tail and tested before (0 minutes) and then into the water bath at 37°C for 40 minutes and 15, 30, 60 and 90 minutes after intraperitoneal (EDL muscle) or 80 minutes (SOL muscle). The fibers injection of glucose (2 g/kg). Blood glucose levels were were then placed into a 6-cm plate with 5 ml of DMEM measured using an ACCU-CHEK Active blood glucose and separated from the tendons by careful manipulation meter system (Roche Diagnostics, Indianapolis, IN, using heat-polished Pasteur pipettes coated with horse USA). Body weight data were collected prior to glucose serum to prevent sticking. After the fibers were sepa- tolerance testing. rated, they were fixed immediately for staining or trans- ferred onto a new plate containing 5 ml of DMEM with Cell culture 20% FBS and 2% chick embryo extract, then placed in the Primary myoblasts were harvested from the limb mus- incubator at 37°C with 5% CO for three days. At the end cles of mice that were six weeks of age. Limb muscles of the three days, the fibers were collected, fixed with 2% were digested in a solution containing 1% collagenase B paraformaldehyde and prepared for staining. (Roche Diagnostics) and 2.4 U/ml Dispase II (neutral protease, grade II; Roche Applied Science) for 30 min- Immunocytochemistry utes in an incubator at 37°C with 5% CO and triturated Primary myoblasts were growninchamber slides for every 15 minutes. The digestion was stopped after 30 staining. The cells were fixed with 2% paraformaldehyde Angione et al. Skeletal Muscle 2011, 1:33 Page 4 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 for ten minutes, then washed three times with PBS covered with a coverslip and allowed to dry overnight. before the primary antibody was added. The primary For nicotinamide adenine dinucleotide, reduced antibodies used were Pax7 (Developmental Studies (NADH) staining, sections were washed briefly in PBS Hybridoma Bank, Department of Biology, University of to remove excess O.C.T. compound. The slides were Iowa,IowaCity, IA USA),MyoD(sc-20; SantaCruz then incubated in a solution containing 100 mg/ml Biotechnology, Santa Cruz, CA, USA), and mAb clones NADH and 0.1 g/ml nitroblue tetrazolium (NBT) for 30 HB287, HB277 and HB283 for MyHC1, MyHC2a and minutes at 37°C. The slides were then washed three MyHC2b, respectively (American Type Culture Collec- times with deionized water. Unbound NBT was removed tion, Manassas, VA, USA). Cells were incubated with from the sections by washing the slides three times each the primary antibody for one hour at room temperature with 30%, 60% and 90% acetone. The sections were then on a shaker. After incubation with the primary antibody, washed with deionized water and mounted. the cells were washed three times with PBS before the secondary antibody was added. The secondary antibo- Gene expression dies used were goat anti-mouse immunoglobulin G2b All gene expression data were gathered by real-time (IgG2b) conjugated with Alexa Fluor 647 dye, goat anti- PCR. RNA was extracted from cell culture, whole-mus- mouse IgG1 conjugated with Alexa Fluor 568 dye, goat cle and adipose tissue using the RNeasy RNA extraction anti-mouse IgM conjugated with Alexa Fluor 488 dye kit (QIAGEN, Germantown, MD, USA). RNA was con- (Molecular Probes/Life Technologies, Carlsbad, CA, verted into cDNA using Moloney murine leukemia virus USA) and daylight Alexa Fluor 488-conjugated goat reverse transcriptase and random hexamer primers. The anti-rabbit (Jackson ImmunoResearch Laboratories, Inc, real-time PCR oligonucleotides are listed in Additional West Grove, PA, USA). The cells were incubated with file 1, Table S1. Samples were then run in a LightCycler secondary antibody and Hoechst dye (a DNA dye) for 480 System (Roche Applied Science) for 40 cycles. Fold -ΔΔCt 30 minutes. The cells were again washed three times changes were calculated using the 2 method. with PBS before being mounted with Dako Fluorescence Mounting Medium (Glostrup, Denmark) and covered Western blot analysis with a coverslip. Images were recorded on a Leica Expression of PPARδ protein in Pparδ-cKO and wild- DMI6000 B inverted fluorescence microscope (Leica type muscles was examined by Western blot analysis as Microsystems, Mannheim, Germany). previously described [32]. Briefly, equal amounts of nuclear proteins extracted under identical conditions Histology were run on SDS-PAGE gels and transferred onto poly- The muscles harvested from the hind limbs were vinylidene difluoride membrane. PPARδ was detected embedded in optimal cutting temperature (O.C.T.) com- with rabbit anti-PPARδ antibody from Santa Cruz Bio- pound (Sakura Finetek USA Inc, Torrance, CA, USA) technology (SC-7197). and quickly frozen in dry ice-cooled isopentane. Cryo- sections were cut to 10-μm thickness and placed on Statistical analysis glass slides. For immunohistochemistry, slides were The number of mice used for each experiment is listed blocked with blocking buffer (5% horse serum, 2% BSA, in the figure legend. Every experiment was performed in 0.2% Triton X-100 and 0.1% sodium azide in PBS) for triplicate. For the glucose tolerance tests, statistical ana- two hours prior to antibody staining. The slides were lysis was based on area under the curve (AUC) using incubated with primary antibodies for one hour at room the trapezoidal rule. All data are presented as means ± temperature, then washed three times with PBS before SEM. P-values were calculated using a two-tailed Stu- incubation with secondary antibody for 30 minutes. dent’s t-test unless otherwise indicated. All values equal After three more washes with PBS, the slides were to 0.05 or less were considered significant and are mounted with Dako Fluorescence Mounting Medium denoted by asterisks in the figures. and covered with a coverslip. Results H & E and NADH staining Characterization of the Pparδ-cKO model Sections (10-μm thickness) were first washed with PBS To generate tissue-specific knockout of Pparδ,weused to remove excess O.C.T. compound, then incubated in mice bearing a floxed exon 4 in the Pparδ gene [39]. hematoxylin for five minutes, washed and incubated in The presence of Cre will excise exon 4, corresponding 1% eosin for 30 seconds. The sections were then dehy- to the N-terminal zinc finger of the DNA binding drated in increasing concentrations of ethanol and domain of PPARδ, and lead to premature stop of trans- mounted with CytoSeal Mounting Medium (Electron lation and abolish the transcriptional activity of the Microscopy Sciences, Fort Washington, PA, USA), resulting truncated PPARδ (Additional file 2, Figure Angione et al. Skeletal Muscle 2011, 1:33 Page 5 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 f/f S1A). The Pparδ mice were bred to mice expressing To confirm the tissue-specific knockout, we collected Cre recombinase under the control of the endogenous RNA samples from adult skeletal muscle, BAT and Myf5 promoter Myf5-Cre (007893; The Jackson Labora- white fat (WAT) and conducted quantitative RT-PCR tory) to generate the Pparδ-cKO mice. Since Myf5 is (qPCR) analysis. The levels of Pparδ in the Pparδ-cKO expressed in the mesoderm during development, we mice compared to their wild-type littermates were expected that PPARδ would be selectively ablated in reduced by 7-fold in TA muscle and by 11-fold in BAT, several mesodermal tissues that express Myf5, including buttheywerenot changedinWAT (Figure1A).These skeletal muscle and brown fat (BAT) [20]. results confirm the tissue-specific knockout and are Figure 1 Characterization of the PPARδ-cKO model. Blue and green bars represent wild-type (WT) and peroxisome proliferator-activated receptor δ conditional knockout (Pparδ-cKO) (MUT) tissues, respectively. (A) Relative expression levels of Pparδ in tibialis anterior (TA) muscle, brown fat and white fat tissues (N = 8 pairs of wild-type and mutant mice) for each tissue type. (B) Quantitative RT-PCR showing the relative expression levels of Pparδ in whole-muscle tissue, proliferating myoblasts, white fat and brown fat in wild-type mice (N = 8 for each tissue type and N = 2 for myoblasts). (C) and (D) Expression levels of Ppara in TA muscle (C) and Pparg in TA, brown fat and white fat tissues (N = 8 for each tissue type) (D). (E) and (F) Expression levels of carnitine palmitoyltransferase 1b (mCPT1b) and Forkhead box class O transcription factor 1 (FoxO1) genes in TA muscles (N = 3). Angione et al. Skeletal Muscle 2011, 1:33 Page 6 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 consistent with the notion that skeletal muscle and BAT observation, qPCR analysis indicated that satellite cell- derive from the Myf5 lineage and WAT derives from a specific Pax7 gene expression was reduced by about Myf5-independent lineage [20]. We further confirmed 40% in the mutant compared to the wild-type TA mus- by Western blot analysis that PPARδ protein levels are cles (Figure 2D). Interestingly, normal satellite cell num- reduced by 60% to 80% in the gastrocnemius muscles of bers were detected at three weeks of age, and a nearly Pparδ-cKO mice compared to those of wild-type mice 20% reduction was detected at age five weeks in the (Additional file 2, Figure S1B). Interestingly, analysis of Pparδ-cKO compared to the wild-type EDL fibers relative Pparδ expression in various wild-type tissues (Additional file 4, Figure S3). Furthermore, the number of 4’, 6-diamidino-2-phenylindole-positive nuclei per indicated that both proliferating myoblasts (derived from satellite cells) and WAT expressed much higher myofiber was reduced in the Pparδ-cKO mice at five levels of Pparδ compared to whole muscle and BAT weeks old but not at three weeks old (Additional file 4, (Figure 1B). Specifically, Pparδ mRNA levels in primary Figure S3). The gradual reduction in satellite cells and myoblasts were about 15-fold greater than those in the differentiated myonuclei in the Pparδ-cKO muscle dur- TA muscles, suggesting a specific role of PPARδ in ing postnatal growth suggests that PPARδ is important myoblast proliferation. In addition, we found that the for satellite cell proliferation and maintenance. levels of Pparg and Ppara remained unchanged in Next we examined whether the satellite cells asso- Pparδ-cKO compared to wild-type tissues (Figures 1C ciated with the isolated fibers were able to proliferate and 1D), confirming that Pparδ-cKO did not elicit com- and differentiate normally using a single-myofiber cul- pensation by other PPAR isoforms. Therefore, any ture paradigm to mimic satellite cell activation in vivo observed phenotypes are due to specific knockout of [13,15]. We cultured isolated fibers for three days in Pparδ in our mouse model. vitro and fixed and stained clusters of myoblasts that To understand how PPARδ functions in muscle, we had proliferated on single fibers with antibodies to Pax7 compared the expression of a number of known PPARδ and MyoD (Figure 2B). Previous studies have established + - + + - + target genes in wild-type and mutant TA muscles from that Pax7 /MyoD ,Pax7 /MyoD and Pax7 /MyoD adult mice. The expression of carnitine palmitoyltrans- cells represent self-renewing, proliferating and differen- ferase 1b (mCPT1b), whose protein regulates the rate- tiating progenies, respectively [11-13]. The relative per- limiting step for b-oxidation of long-chain fatty acids centage of cells in these three categories was examined. [21,24], and Forkhead box class O transcription factor 1 Compared to the wild type, mutant SOL fibers had half + + (FoxO1), a PPARδ target that regulates skeletal muscle as many proliferating cells (Pax7 /MyoD ) and twice as metabolism and progenitor cell function [42-44], were many differentiating cells (MyoD ), whereas the propor- significantly decreased in Pparδ-cKO muscle (Figures 1E tion of self-renewing cells (Pax7 )remainedthe same and 1F). The expression of several other known target between wild-type and mutant fibers (Figure 2E). genes of PPARδ, including Sirt1, UCP1 and PGC1a, was Together, these results show that PPARδ is important not altered in the mutant muscles (Additional file 3, Fig- for maintaining the proliferation of activated myoblasts ure S2). These results confirm that Pparδ-cKO affects and that loss of PPARδ leads to accelerated myogenic candidate target gene expression and identify mCPT1b differentiation. and FoxO1 as potential target genes regulated by PPARδ To further characterize the proliferative defects of in resting skeletal muscles. Pparδ-null myoblasts, we examined the expression of Ki67, a cell proliferation marker, in cultured primary Satellite cell and myoblast proliferation and myoblasts from mutant mice and wild-type littermates differentiation (Figure 3A-B). Real-time PCR analysis confirmed a 60% Adult Pparδ-cKO mice have a reduced number of satel- reduction in Pparδ expression in newly established cul- lite cells, and Pparδ-null myoblasts exhibited reduced tures of Pparδ-cKO compared to wild-type myoblasts proliferation and increased differentiation kinetics. (Additional file 5, Figure S4A). The percentage of Ki67 Because proliferating myoblasts expressed high levels of cells and the Ki67 immunofluorescence intensity of the Pparδ compared to whole muscles (Figure 1B), we Pparδ-mutant myoblasts were threefold less than those investigated the effect of Pparδ mutation on the satellite of wild-type plates (Figures 3C). Conversely, a PPARδ cells in vivo and in myoblasts in culture. Intact single agonist, GW501516, significantly increased wild-type fibers were isolated from the representative slow (SOL) myoblast proliferation (Additional file 5, Figure S4B). and fast (EDL) muscles and stained with Pax7 antibody We also plotted the growth curve of wild-type and to label satellite cells (Figure 2A). At two to three mutant myoblasts at days 3, 6 and 9 after they were pla- months old, the mutant mice had, on average, a 40% ted in culture. Myoblasts from Pparδ-cKO muscle reduction in satellite cell numbers in both EDL and showed a reduced growth rate compared to myoblasts SOL muscles (Figure 2C). Consistent with this from wild-typemuscle(Figure3D). Theseresults Angione et al. Skeletal Muscle 2011, 1:33 Page 7 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 2 Reduced satellite cell number and proliferation in Pparδ-cKO mice. (A) Freshly isolated extensor digitorum longus (EDL) muscle fibers were stained with paired-box transcription factor 7 (Pax7) antibody to label satellite cells. Nuclei were counterstained with 4’, 6-diamidino- 2-phenylindole (DAPI). Scale bar = 25 μm for all panels in parts (A) and (B). (B) Soleus (SOL) muscle fibers were cultured for 72 hours, and the proliferating satellite cells on the fibers were fixed and stained with Pax7 and myogenic differentiation antigen 1 (MyoD) antibodies. Nuclei were + + + ), proliferating cells (Pax7 /MyoD ) and differentiating cells counterstained with DAPI. Satellite cell status is classified as self-renewed cells (Pax7 + + (MyoD ). (C) Quantification of the number of Pax7 satellite cells per single fiber isolated from the EDL and SOL muscles (N = 360 fibers each from the SOL and EDL muscles). **P < 0.0001. (D) Relative expression of the Pax7 gene in tibialis anterior (TA) muscles by quantitative PCR (N = 3). (E) Quantification of the number of self-renewed, proliferating and differentiating cells on SOL fibers after culture for 72 hours (N = 20 fibers for each group). provide compelling evidence that PPARδ positively regu- 14 days, the mice were killed and the TA muscles were lates myoblast growth and proliferation. collected for gene expression and histological analysis. We examined the regenerating regions under a micro- Muscle regeneration after injury is impaired in Pparδ-cKO scope, and the number of regenerating fibers (small, animals centrally located nucleus) and regenerated fibers (large, To examine whether the reduced rates of proliferation eccentrically located nucleus) were counted. Both wild- in the Pparδ-cKO myoblasts result in defects in muscle type and Pparδ-cKO TA muscles underwent regenera- regeneration in vivo, we injured the TA muscles of tion, but they exhibited marked differences (Figures 4A mutant and wild-type animals at six weeks of age. After and 4B). The mutant muscles had much larger areas of Angione et al. Skeletal Muscle 2011, 1:33 Page 8 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 3 Defective growth and proliferation of primary myoblast cells derived from Pparδ-cKO muscles. (A) and (B) Representative images of Ki67 staining that specifically labeled proliferating primary myoblasts: Ki67 staining (red) and 4’, 6-diamidino-2-phenylindole (DAPI) staining (blue). Scale bar = 50 μm. (C) Relative Ki67 signal intensity in wild-type and mutant myoblast cultures. The intensity of Ki67 and DAPI staining and the number of pixels were measured using Photoshop software (Adobe Systems Inc, San Jose, CA, USA). The ratio of Ki67 intensity values to DAPI intensity values was also quantified using Photoshop (N = 3). (D) Growth curve of wild-type and mutant myoblasts after nine days in culture (N = 3). - + small-caliber regenerating fibers that covered almost the differentiated Pax7 /MyoD myocytes in the Pparδ-cKO whole muscle sections, whereas the wild-type TA mus- muscle, together with reduced total myoblast number cles had reduced regenerating areas that were mostly per unit area and decreased size of newly regenerated confined to the center of the section (Figures 4A and fibers (Figures 4F to 4I). In addition, we confirmed by 4B). The number of small regenerating fibers was 30% qPCR that Myogenin gene expression was upregulated more, and the number of large regenerating fibers was in regenerating Pparδ-cKO muscles at this stage (Figure 20% fewer, in Pparδ-cKO muscles compared to wild- 4J). By contrast, Myf5 was expressed at similar levels in type muscles (Figure 4C). In addition, the regenerating the mutant and wild-type muscles. These results indi- Pparδ-cKO TA muscles expressed reduced levels of cate that the observed regenerative defects of Pparδ- MyHC 2b mRNA, the most abundant MyHC isoform in cKO may be due to reduced proliferation and increased the TA muscle (Figure 4D), which further confirms the differentiation kinetics of activated myoblasts. regenerative defects. The Pparδ-cKO muscles were We also examined the expression of mCPT1b and eventually able to regenerate after 30 days, suggesting FoxO1 genes during muscle regeneration at day 5 after that Pparδ-cKO mainly causes a delay in muscle CTX treatment, when damaged muscles are not yet regeneration. regenerated and satellite cell proliferation peaks [8]. Because Pparδ-cKO myoblasts are defective in prolif- Interestingly, expression of mCPT1b was unchanged but eration in vitro, we sought to determine whether such FoxO1 expression was reduced in the Pparδ-cKO mus- defects exist in vivo during muscle regeneration. We cles compared to wild-type muscles during muscle analyzed Pax7 and MyoD expression (Figures 4F and regeneration (Figure 4E), suggesting that PPARδ may 4G) at days 3 to 5 post-CTX treatment, at which stage activate FoxO1 in proliferating myoblasts and that myoblast proliferation peaks. Consistent with our in mCPT1b is a PPARδ target only in mature muscle vitro results, we observed a reduction of proliferating fibers. Together with our earlier observation that Pparδ + + Pax7 /MyoD myoblasts and an increase in is expressed at much higher levels in proliferating Angione et al. Skeletal Muscle 2011, 1:33 Page 9 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 4 Defective regeneration of skeletal muscles and gene expression patterns in Pparδ-cKO mice after injury with cardiotoxin. (A) and (B) Tibialis anterior (TA) muscles from six-week-old wild-type and peroxisome proliferator-activated receptor δ conditional knockout (Pparδ- cKOmice were injected with cardiotoxin (CTX) to induce injury. The mice were allowed to recover for 14 days before their muscles were harvested and processed for staining. Small and large fibers (indicating regenerating and nonregenerating fibers, respectively) were counted. Scale bars = 50 μm for parts (A) and (B) and for parts (F) and (G). (C) Quantification of regenerating and nonregenerating fibers after injury with CTX (N = 3 pairs of mice, with five sections taken from each mouse). (D) Relative mRNA expression level showing reduced myosin heavy chain isoform 2b (MyHC-2b) in mutant muscle after injury with CTX (N = 4). (E) Relative mRNA expression levels of Pparδ target genes in the TA muscle after injury with CTX (N = 4). (F) and (G) Cryosections of regenerating wild-type (F) and mutant (G) mice at day 5 after injury with CTX. The sections are labeled with paired-box transcription factor 7 (Pax7) (red), myogenic differentiation antigen 1 (MyoD) (green) and 4’,6- + - - + + + diamidino-2-phenylindole (DAPI) (blue). Arrows, arrowheads and asterisks indicate examples of Pax7 /MyoD , Pax7 /MyoD and Pax7 /MyoD myoblasts, respectively. (H) The number of total myoblasts labeled by Pax7 and/or MyoD per unit area counted from 8 to 13 areas. (I) + - + + - + Percentage distribution of Pax7 /MyoD , Pax7 /MyoD and Pax7 /MyoD myoblasts in regenerating wild-type and mutant TA muscles at day 5 after injury with CTX. (J) Relative mRNA expression levels of Myf5 and Myogenin in TA muscles at day 3 after injury with CTX (N = 3). *P < 0.05 by single-tailed Student’s t-test. Angione et al. Skeletal Muscle 2011, 1:33 Page 10 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 myoblasts compared to mature muscles (Figure 1B), mAbs (Figures 5A to 5D). We chose the EDL and SOL these results suggest a critical role of PPARδ in satellite muscles to represent fast and slow muscle types, respec- cell function in vivo. tively. To overcome potential bias associated regiona- lized distribution of fiber types within a muscle [45], we Pparδ-cKO does not alter myosin heavy chain isoforms enumerated all muscle fibers in the entire muscle (Fig- Because Pparδ overexpression increases oxidative mus- ures 5A to 5D). No difference in the percentage of each cle fiber types [32], we sought to determine whether fiber type was found between mutant and wild-type ani- Pparδ-cKO causes any changes in fiber-type distribution. mals in either the EDL or SOL muscle at six weeks old We labeled MyHC protein isoforms with a panel of (Figure 5E). To further determine whether there are Figure 5 Normal fiber-type distributions in extensor digitorum longus and soleus muscles of six-week-old Pparδ-cKO mice. (A) through (D) Immunostained sections showing the distribution of fiber types in the extensor digitorum longus (EDL) and soleus (SOL) ("C-” denotes wild- type littermate control and “M-” denotes Pparδ-cKO): myosin heavy chain isoform 1 (MyHC-1) (red), MyHC-2a (blue), MyHC-2b (green), MyHC-2x (black) and laminin (white). The basal lamina surrounding each fiber is shown. Scale bar = 1 mm. (E) Percentage distribution of each fiber type in the EDL and SOL muscles (N = 4 pairs of littermates. Angione et al. Skeletal Muscle 2011, 1:33 Page 11 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 changes in muscle oxidative metabolism without chan- Pparδ-cKO whole muscle and BAT (10% to 20% of ging MyHC isoform expression, we carried out NADH- wild-type levels) is probably due to non-Myf5 lineage tetrazolium reductase (NADH-TR) staining of the SOL cells’ residing in these tissues, such as nerve cells (neural and EDL muscles. NADH-TR staining marks ATPase branches and Schwann cells), vessel-associated cells activity that is correlated to oxidative capacity (type 1 (blood, lymph, endothelial and smooth muscle cells) and fibers arehighlyoxidative anddarklystained,type 2a interstitial connective tissue-associated cells (fibroblasts fibers are intermediate fibers and lightly stained and and white adipocytes). In addition, because Myf5-Cre is type 2b fibers are glycolytic and unstained) (Additional expressed in about 90% of satellite cells [15], a small file 6, Figures S5A to S5D). In agreement with our fraction of Myf5 satellite cells would still have normal MyHC isoform staining experiments, there were no dif- levels of Pparδ expression. We nevertheless observed ferences in NADH activity between the Pparδ-cKO and proliferative defects in satellite cells in vitro and muscle wild-type mice in either the EDL or the SOL muscles regeneration in vivo. Further studies using other Cre (Additional file 6, Figures S5E and S5F). These results lines with improved efficiency of Pparδ deletion in satel- demonstrate that Pparδ-cKO does not lead to changes lite cells would help to better address the muscle-regen- in muscle fiber type or oxidative capacity in young erative defects. animals. Mice lacking PPARδ in their skeletal muscles are born at normal ratios and are viable. These animals do not Old Pparδ-cKO mice become obese and have impaired appear to have any noticeable defects in body weight or glucose clearance response to glucose early in life. There was no difference PPARδ is an important regulator of muscle energy in total fiber number or percentage of each fiber type in metabolism. Therefore, we tested the body weight and representative slow (SOL) and fast (EDL) muscles. How- insulin sensitivity in young and old mutant mice. Young ever, old mice (eight to nine months old) were signifi- Pparδ-cKO mice (two months of age) did not differ sig- cantly fatter than their wild-type littermates and were nificantly in body weight compared to their sex-matched found to have developed glucose intolerance. This phe- wild-type littermates (Figure 6A). However, older notype in the aged mouse is similar to that reported in mutant mice (nine months of age) were about 25% hea- another transgenic mouse model in which conditional vier on average than their wild-type littermates (Figure mutation of Pparδ in mature skeletal muscles is 6B). Next we measured the rate of insulin-mediated glu- mediated by human a-skeletal actin promoter-driven cose clearance in the mutant and wild-type mice. The Cre in the transgenic mouse [33]. In that mouse model skm-/- amount of glucose in the blood was measured before (0 (Pparδ ), a reduction in the oxidative capacity and minutes) and 15, 30, 60 and 90 minutes after intraperi- muscle fiber-type switching are also detected, whereas toneal injection of 2 g/kg glucose (Figures 6C and 6D). such changes are not evident in the Pparδ-cKO mouse Again, young mice showed no differences in the rate of model used in our study. The discrepancy between insulin-stimulated glucose uptake as measured by AUC these observations may be due to the genetic back- analysis of the glucose tolerance curves (Figure 6E). ground and nutrition status (that is, diet composition) Strikingly, older mutant mice showed a reduced rate of of the mice used and the different techniques used for glucose uptake; their blood glucose levels spiked higher fiber typing. The previous study used qPCR analysis to than those of the wild-typemiceand took longer to show increases in MyHC1 and MyHC2b transcripts in clear from the blood (Figure 6D), resulting in significant the gastrocnemius muscle [33]. As many muscle fibers increases in the AUC in every time period measured express multiple MyHC transcripts, small changes in (Figure 6F). These results suggest that the older mice these transcripts may not be sufficient to induce fiber- had developed glucose intolerance and an obese pheno- type switching [46]. In our current study, we used type even when fed a normal diet. MyHC isoform-specific mAbs to unambiguously identify fiber types, and we examined all fibers within the entire Discussion muscle to eliminate bias due to regional clustering of The results produced by our Pparδ-cKO model establish specific fiber types within the same muscle. We have a previously unappreciated role of PPARδ in skeletal therefore provided strong evidence that Pparδ-cKO does muscle progenitor cell proliferation. The specificity of notleadtoanovert switch of muscle fibertypein the Pparδ-cKO mouse model is demonstrated by young animals based on MyHC isoform protein reduced Pparδ mRNA expression in both the skeletal expression. muscle and BAT, known to be derived from Myf5 line- We show herein that the expression levels of Pax7 are age cells. In contrast, Pparδ expression was unchanged significantly reduced in the whole muscle of Pparδ-cKO in WAT, which is derived from a Myf5-independent mice. Because Pax7 is a satellite cell-specific marker [10], this observation suggests a reduction in satellite lineage [20]. The residual Pparδ expression detected in Angione et al. Skeletal Muscle 2011, 1:33 Page 12 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 Figure 6 Age-dependent increases in body weight and glucose tolerance in Pparδ-cKO mice. (A) and (B) Body weights of female wild- type mice and mutant littermates at two and nine months old (N = 6). (C) and (D) Blood glucose concentration at different time points following intraperitoneal injection of glucose (2 g/kg). The mice were fasted for three hours prior to glucose injection. Glucose concentration was measured using an ACCU-CHEK Active blood glucose meter system (Roche Diagnostics, Indianapolis, IN, USA) (N = 3 pairs of female littermates). (E) and (F) Area under the curve (AUC) analysis the of glucose tolerance test results shown in parts (C) and (D), respectively. population doubling time based on the cell growth cell number, which we confirmed by our later results showing that Pparδ-cKO mice have fewer satellite cells curve. Conversely, the PPARδ agonist GW501516 stimu- in vivo. Our single-fiber culture model that mimics lates myoblast proliferation. These results provide com- satellite cell activation in vivo further demonstrates that pelling evidence that PPARδ acts to facilitate satellite there are fewer proliferating satellite cells and increased cell proliferation. numbers of differentiating cells in Pparδ-cKO mice. In Recent studies have shown that PPARδ promotes the vitro growth and proliferation of Pparδ-cKO satellite proliferation of several cell types. PPARδ activation has cell-derived primary myoblasts were also reduced, as been shown to increase the proliferation and migration shown by the decreased number of cells expressing the of endothelial progenitors as well as the number of cell proliferation marker Ki67 and the increased hematopoietic stem cells in the bone marrow [34,47,48]. Angione et al. Skeletal Muscle 2011, 1:33 Page 13 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 In addition, PPARδ has been shown to be essential for incidence of sarcopenia (age-related muscle loss) has high glucose-stimulated proliferation of embryonic stem been shown to correlate with reduced levels of PPARδ, cells [35]. Moreover, PPARδ is an important regulator of and pharmacological activation of PPARδ has been the proliferation of several cancer cell types [49,50] shown to reduce the incidence of sarcopenia by increas- (older references are reviewed by Peters and Gonzalez ing nuclear accretion in myofibers [57]. These results [36]). The PPARδ antagonists SR13904 and GSK0660, support the notion that PPARδ is important for proper skeletal muscle regeneration in response to injury or which block PPARδ activation, have been shown to daily wear and tear (maintenance) by increasing satellite reduce cell proliferation [50,51]. The role of PPARδ in cell proliferation and promoting fusion of differentiated cell proliferation has been shown to be due to its activa- tion of the phosphatidylinositol 3-kinase and AKT path- myocytes in vivo. ways [34,38], which has been shown to be important in We examined the expression levels of several pre- preventing apoptosis and enhancing cell proliferation by viously identified PPARδ target genes to understand triggering cells to overcome cell-cycle arrest and initiat- how PPAR functions in skeletal muscle and satellite ing cell proliferation [52,53]. Importantly, AKT signaling cells. Our results show that the expression of both plays critical roles in skeletal muscle progenitor cell mCPT1b and FoxO1 is reduced in mature uninjured function and muscle hypotrophy [4,5,54]. muscles of the Pparδ-cKO mice, suggesting that they A number of other studies have shown that PPARδ are molecular targets of PPARδ under resting condi- promotes differentiation and inhibits (or has no effect tions. Since PPARδ has been shown by many groups to on) proliferation in many cell types, particularly kerati- be a regulator of oxidative capacity in skeletal muscle nocytes, smooth muscle cells, cardiac fibroblasts and [21], it is not surprising that key players in the lipid oxi- several cancer cell lines (extensively reviewed by Peters dation pathway would be downregulated after removal and Gonzalez [36] and Foreman et al. [55]). Therefore, of PPARδ. Interestingly, mCPT1b expression is not sig- whether PPARδ promotes terminal differentiation or cell nificantly reduced in Pparδ-cKO muscle at day 5 during proliferation is highly cell type-specific [36]. Intriguingly, regeneration. At this time point, degenerated muscle investigators in a recent study demonstrated that PPARδ fibers have not been replaced by new fibers and myo- can exert its transcriptional activation or repression blast proliferation is at the peak stage [8]. This result both ligand-dependently and ligand-independently in a seems to suggest that mCPT1b is a target of PPARδ in target-specific manner [56]. Such variations in the mode mature resting muscles, but not in proliferating myo- of PPARδ action probably account for the paradoxical blasts. Of all the PPARδ target genes we examined, effects of PPARδ in different cell types or even in the FoxO1 was the only gene whose expression was downre- same cell type. More research is needed to elucidate gulated in both resting and regenerating muscles of how PPARδ regulates satellite cell proliferation and Pparδ-cKO mice. The downregulation of FoxO1 in whether it also functions at later stages of myogenesis Pparδ-cKO regenerating muscle indicates that FoxO1 (see discussion below). may act at downstream of PPARδ in regulating myoblast Pparδ-cKO animals showed delayed muscle regenera- proliferation and inhibiting its differentiation. Indeed, it tion after injury with CTX. We found that, compared to has been shown that constitutive activation of FoxO1 their wild-type littermates, transgenic mice lacking inhibits myoblast differentiation [44]. However, FoxO1 PPARδ in the skeletal muscle had a reduced proportion also acts at later stages of myogenesis, during the fusion of proliferating myoblasts and that the regenerated of differentiated myocytes into myotubes [58]. These fibers were smaller in caliber at both five days and two previous studies and our present results suggest that weeks after CTX injury. These results are consistent PPARδ acts through FoxO1 in both satellite cells and with our observations that these animals had fewer mature muscle fibers but has distinct functions in these satellite cells and that Pparδ-deficient satellite cells have cell types. It regulates the proliferation of satellite cells reduced proliferative potential in vitro. Because satellite and fusion of muscle fibers. These combined effects of cells are responsible for skeletal muscle regeneration, PPARδ’s acting through FoxO1 explain the regenerative reductions in satellite cell number and defective satellite defects seen in the Pparδ-cKO muscles. cell proliferation may have resulted in the observed PPARδ has also been implicated in the treatment of delay in muscle regeneration. In addition to decreased degenerative muscle diseases such as Duchenne muscu- satellite cell proliferation, loss of PPARδ in myofibers lar dystrophy (DMD), which is caused by the mutation may have contributed to the observed defects in muscle of the dystrophin gene [59,60]. One research group regeneration. Moreover, other signaling mechanisms showed that PPARδ activation by the agonist mayhavecompensated forthe loss of PPARδ in our GW501516 increased the expression of utrophin A, a conditional mutant, leadingtocompletionofmuscle key member of the dystrophin-associated protein com- regeneration at a later time. Coincidentally, the plex, in the C C myoblast cell line [61]. PPARδ has 2 12 Angione et al. Skeletal Muscle 2011, 1:33 Page 14 of 16 http://www.skeletalmusclejournal.com/content/1/1/33 been suggested to be a direct transcriptional regulator of wild-type primary myoblasts at 24 hours after control (dimethyl sulfoxide utrophin A in vivo [43]. Intriguingly, utrophin A overex- vehicle) and 100 nM PPARδ agonist GW501516 treatments (N = 10). pression can improve the integrity of the sarcolemma, Additional file 6: Figure S5 NADH-tetrazolium reductase staining showing the relative abundance of oxidative fibers in Pparδ-cKO protect muscles from contraction induced damage and and wild-type skeletal muscles. Staining shows the three different fiber help to alleviate muscle wasting and slow down the dis- types. Oxidative fibers (type 1) are darkly stained, intermediate fibers ease progression of DMD [62]. Therefore, understanding (type 2a) are moderately stained and glycolytic fibers (types 2b and 2x) are unstained. (A) and (B) Representative images of fast muscles how PPARδ functions in skeletal muscle tissue and its (extensor digitorum longus (EDL)). (C) and (D) Representative images of progenitor cells has important implications for muscle slow muscles (soleus (SOL)). (E) and (F) Relative nicotinamide adenine regeneration and the treatment of degenerative muscle dinucleotide, reduced (NADH), intensity levels between peroxisome proliferator-activated receptor δ (PPARδ) wild-type and PPARδ-conditional diseases. knockout (Pparδ-cKO) animals at six weeks of age (N = 3). Conclusions Our in vivo and in vitro analyses of myogenic lineage Abbreviations specific Pparδ gene knockouts demonstrate a critical BSA: bovine serum albumin; DMEM: Dulbecco’s modified Eagle’s medium; H role of PPARδ in satellite cell proliferation and postnatal & E: hematoxylin and eosin; mAb: monoclonal antibody; PBS: phosphate- regeneration of skeletal muscles. In addition, we provide buffered saline; PCR: polymerase chain reaction. evidence for a role of PPARδ in regulating muscle insu- Acknowledgements lin resistance, as indicated by the glucose tolerance test We thank Jun Wu for mouse colony maintenance and other members of results. However, we were unable to detect overt the Kuang laboratory for their comments and technical assistance. This project is partially supported by funding from the Muscular Dystrophy changes in skeletal muscle fiber types by MyHC iso- Association, the National Institutes of Health and the United Sates form-specific antibody labeling. These results not only Department of Agriculture (to SK). The authors declare no conflict of support the previously established role of PPARδ in interests. muscle energy metabolism and insulin sensitivity but Author details also demonstrate a novel role of PPARδ in muscle pro- Department of Animal Sciences, Purdue University, 901 West State Street, genitor cell function. These results have implications for West Lafayette, IN 47907, USA. Program in Gene Function and Expression and Program in Molecular Medicine, University of Massachusetts Medical the treatment of muscular dystrophies and muscle-wast- School, 364 Plantation Street, Worcester, MA 01605, USA. Center for Cancer ing conditions by targeting PPARδ signaling at the stem Research, Purdue University, 201 S. University Street, West Lafayette, IN cell level. 47907, USA. Authors’ contributions Additional material AA carried out the mouse and cell culture studies, analyzed the data and drafted the manuscript. CJ carried out part of the muscle regeneration and immunofluorescence labeling studies. DP conducted the Western blot Additional file 1: Table S1 Primer sequences for quantitative PCR. analysis. YW provided the Pparδ-floxedmice, analyzed the data and revised Additional file 2: Figure S1 Pparδ-cKO strategy. (A) Peroxisome the manuscript. SK conceived, designed and coordinated the study; analyzed proliferator-activated receptor δ (Pparδ) gene structure with exons the data; and revised the manuscript. All authors read and approved the numbered sequentially. Note that LoxP sequences are inserted before final manuscript. and after exon 4 encoding the DNA-binding domain of PPARδ. In the presence of Cre (driven by Myf5 locus in this study), exon 4 is excised, Competing interests resulting in premature stop in translation and generation of a short, The authors declare that they have no competing interests. truncated protein without a DNA-binding domain. (B) Representative Western blot showing the relative expression of PPARδ protein in the Received: 19 May 2011 Accepted: 1 November 2011 wild-type (WT) and Pparδ-conditional knockout (Pparδ-cKO) Published: 1 November 2011 gastrocnemius muscles. The upper nonspecific band serves as an indicator of the relative amount of total protein loaded onto the gel. References Additional file 3: Figure S2 Relative expression of PPARδ target 1. Zierath JR, Hawley JA: Skeletal muscle fiber type: influence on contractile genes in mature noninjured muscles. RNA samples isolated from the and metabolic properties. PLoS Biol 2004, 2:e348. tibialis anterior (TA) muscles of six-week old mice were used for 2. 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Skeletal Muscle 2011 1:33. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Nov 1, 2011

References