PPARδ regulates satellite cell proliferation and skeletal muscle regeneration

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) of Pparδ using Myf5-Cre/Pparδflox/flox 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.

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 Pparα in TA muscle (C) and Pparγ in TA, brown fat and white fat tissues (N = 8 for each tissue type) (D). (E) and (F) Expression levels of carnitine palmitoyltransferase 1β (mCPT1β) and Forkhead box class O transcription factor 1 (FoxO1) genes in TA muscles (N = 3).

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 counterstained with DAPI. Satellite cell status is classified as self-renewed cells (Pax7⁺), proliferating cells (Pax7⁺/MyoD⁺) and differentiating cells (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).

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).

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.

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.

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.