Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Apr 1;21(7):770-83.
doi: 10.1101/gad.1525107.

PGC-1alpha Regulates the Neuromuscular Junction Program and Ameliorates Duchenne Muscular Dystrophy

Affiliations
Free PMC article

PGC-1alpha Regulates the Neuromuscular Junction Program and Ameliorates Duchenne Muscular Dystrophy

Christoph Handschin et al. Genes Dev. .
Free PMC article

Abstract

The coactivator PGC-1alpha mediates key responses of skeletal muscle to motor nerve activity. We show here that neuregulin-stimulated phosphorylation of PGC-1alpha and GA-binding protein (GABP) allows recruitment of PGC-1alpha to the GABP complex and enhances transcription of a broad neuromuscular junction gene program. Since a subset of genes controlled by PGC-1alpha and GABP is dysregulated in Duchenne muscular dystrophy (DMD), we examined the effects of transgenic PGC-1alpha in muscle of mdx mice. These animals show improvement in parameters characteristic of DMD, including muscle histology, running performance, and plasma creatine kinase levels. Thus, control of PGC-1alpha levels in skeletal muscle could represent a novel avenue to prevent or treat DMD.

Figures

Figure 1.
Figure 1.
PGC-1α regulates NMJ gene expression. (A) C2C12 myotubes were infected with adenoviral vectors encoding GFP and PGC-1α, respectively, on day 3 after differentiation. RNA was harvested 48 h later and relative gene expression levels were measured by real-time PCR. (*) p < 0.05 between GFP- and PGC-1α-infected cells. (B) Gastrocnemius muscle was isolated from wild-type and PGC-1α muscle-specific transgenic animals (MCK-PGC-1α). After RNA isolation, relative gene expression was analyzed by real-time PCR. (*) p < 0.05 between control and transgenic animals. (C) Control and PGC-1α muscle-specific knockout animals (PGC-1α MKO) were sacrificed, RNA was isolated from gastrocnemius, and relative gene expression levels were measured by real-time PCR. (*) p < 0.05 between control and muscle-specific knockout animals. The bars depict average gene expression normalized to 18S rRNA levels, with the error bars representing standard deviations.
Figure 2.
Figure 2.
AChR clustering induced by PGC-1α. (A) C2C12 myotubes were infected with adenoviral GFP and PGC-1α, respectively, for 48 h. Sixteen hours before harvesting, the myotubes were treated with vehicle (PBS) or 10 ng/mL recombinant agrin. Subsequently, cells were fixed and incubated with rhodamine-labeled α-BTX, and AChR clusters were visualized by fluorescence microscopy. (B,C) The average number of NMJ counted in 20 fields per genotype was normalized to the number of nuclei in these fields, and relative acetylcholine cluster numbers were expressed as percentage of control mice. (*) p < 0.05 between muscle fibers from MCK-PGC-1α and controls as well as fibers from MKO and controls, respectively.
Figure 3.
Figure 3.
PGC-1α and GABP activate NMJ gene promoters. (A) C2C12 myoblasts were transfected with wild-type and N-box mutated promoter constructs, respectively, together with expression plasmids for PGC-1α, GABPA, and GABPB1 (the combination of GABPA and GABPB1 is referred to as GABP). The cells were subsequently differentiated for 2 d and harvested, and luciferase activity was measured. (B) C2C12 myoblasts were transfected with NMJ promoter constructs together with expression plasmids for PGC-1α, GABP, PGC-1α 3A, and GABPA as well as GABPB1 alleles (collectively called GABP P-mut) with mutated phosphorylation sites. The cells were subsequently differentiated for 2 d and harvested, and luciferase activity was measured. (C) C2C12 myoblasts were transfected with NMJ promoter constructs together with expression plasmids for PGC-1α and GABP. The cells were subsequently differentiated for 2 d. Simultaneously, cells were treated with vehicle (PBS) 5 nM recombinant NRG-1. Cells were harvested and luciferase activity was measured. (D) C2C12 myoblasts were transfected with NMJ promoter constructs together with expression plasmids for PGC-1α, GABP, PGC-1α 3A, and GABPA as well as GABPB1 alleles (collectively called GABP P-mut) with mutated phosphorylation sites. The cells were subsequently differentiated for 2 d. Simultaneously, cells were treated with vehicle (PBS) 5 nM recombinant NRG-1. Cells were harvested and luciferase activity was measured. The bars depict average luciferase expression normalized to β-galactosidase activity, with the error bars representing standard deviations.
Figure 4.
Figure 4.
Physical interaction between PGC-1α and GABP requires phosphorylation and HCF. (A) BOSC cells were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, constitutively active MEKK (MEKK*), and HCF as indicated. Cells were harvested 48 h after transfection, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (B) BOSC cells were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, the phosphorylation-deficient alleles Flag-PGC-1α 3A, myc-GABPA P-mut, and myc-GABPB1 P-mut, respectively, constitutively active MEKK (MEKK*), and HCF as indicated. Cells were harvested 48 h after transfection, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (C) C2C12 myotubes were transfected with expression plasmids for Flag-tagged PGC-1α, myc-tagged GABPA and GABPB1, constitutively active MEKK (MEKK*), and HCF as indicated. The cells were differentiated for 2 d and simultaneously treated with 5 nM recombinant NRG-1. Cells were harvested, 1 mg of total protein from the cell lysates was incubated with anti-myc beads, and the bound proteins as well as input (10 μg protein) were blotted with anti-Flag and anti-myc antibodies. (D) GABPA and GABPB1 bind as heterotetramers and upon phosphorylation of GABPB1 recruit phosphorylated PGC-1α and HCF. These phosphorylation events are induced by NRG-1 signaling or constitutive MAPK activation.
Figure 5.
Figure 5.
Specificity of NRG-1-induced NMJ gene expression via PGC-1α. (A,B) Primary muscle cells were isolated from control and PGC-1α knockout animals. After differentiation into myotubes, cells were treated with vehicle (PBS) or 5 nM recombinant NRG-1 for 48 h. RNA was isolated from harvested cells and relative gene expression levels of NMJ genes (A) and other PGC-1α target genes (B) were determined by real-time PCR. (*) p < 0.05 between vehicle and NRG-1 treated cells. The bars depict average gene expression normalized to 18S rRNA levels, with the error bars representing standard deviations. (C,D) C2C12 myoblasts were transfected with a reporter plasmid containing the ERRα promoter and expression plasmids for PGC-1α, GABPA and GABPB1 (called GABP), ERRα, and the phosphorylation-deficient PGC-1α 3A. The cells were subsequently differentiated for 2 d and harvested, and luciferase activity controlled by PGC-1α and GABP (C) and PGC-1α and ERRα (D) was measured. The bars depict average luciferase expression normalized to β-galactosidase activity, with the error bars representing standard deviations.
Figure 6.
Figure 6.
PGC-1α ameliorates muscle dystrophy in sedentary mdx mice. (A) Five-week-old control, PGC-1α muscle-specific transgenic (MCK-PGC-1α), mdx, and PGC-1α muscle-specific transgenic animals in the mdx background (MCK-PGC-1α/mdx) were sacrificed and serum creatine kinase activity was determined. The bars depict the average serum creatine kinase levels, and error bars represent standard deviations. (B) H&E-stained histology of gastrocnemius from control, PGC-1α muscle-specific transgenic (MCK-PGC-1α), mdx, and PGC-1α muscle-specific transgenic animals in the mdx background (MCK-PGC-1α/mdx). (C) Five-week-old control, PGC-1α muscle-specific transgenic (MCK-PGC-1α), mdx, and PGC-1α muscle-specific transgenic animals in the mdx background (MCK-PGC-1α/mdx) were injected i.p. with 1% v/w Evans blue solution and sacrificed 16 h later. Dye incorporation into muscle fibers was visualized by fluorescence microscopy.
Figure 7.
Figure 7.
PGC-1α improves muscle function in exercised mdx mice. (A) Twelve-week-old control, PGC-1α muscle-specific transgenic (MCK-PGC-1α), mdx, and PGC-1α muscle-specific transgenic animals in the mdx background (MCK-PGC-1α/mdx) were run for 1 h on a treadmill at a 15° downhill angle (first run). This exercise protocol was repeated after a resting period of 24 h (second run). The bars depict average running times, with the error bars representing standard deviations. (B) Two hours after the first run, blood was drawn from the mice and serum creatine kinase activity was determined. The bars depict average serum creatine kinase levels, with the error bars representing standard deviations. (C) Mice were sacked after the second run and gastrocnemius sections were stained with H&E. (D) Before sacking, Evans blue was injected i.p. and histological sections from diaphragms were analyzed by fluorescence microscopy.

Similar articles

See all similar articles

Cited by 146 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback