A PGC-1α isoform induced by resistance training regulates skeletal muscle hypertrophy

Abstract

PGC-1α is a transcriptional coactivator induced by exercise that gives muscle many of the best known adaptations to endurance-type exercise but has no effects on muscle strength or hypertrophy. We have identified a form of PGC-1α (PGC-1α4) that results from alternative promoter usage and splicing of the primary transcript. PGC-1α4 is highly expressed in exercised muscle but does not regulate most known PGC-1α targets such as the mitochondrial OXPHOS genes. Rather, it specifically induces IGF1 and represses myostatin, and expression of PGC-1α4 in vitro and in vivo induces robust skeletal muscle hypertrophy. Importantly, mice with skeletal muscle-specific transgenic expression of PGC-1α4 show increased muscle mass and strength and dramatic resistance to the muscle wasting of cancer cachexia. Expression of PGC-1α4 is preferentially induced in mouse and human muscle during resistance exercise. These studies identify a PGC-1α protein that regulates and coordinates factors involved in skeletal muscle hypertrophy.

Figure 1. Cloning and characterization of novel PGC-1α isoforms

(A) Schematic representation of the conservation between human and mouse PGC-1α gene (www.dcode.org). Two promoters that can drive expression of the PGC-1α gene. (Ex) indicates exons seen in the depicted region. Structure of the different PGC-1α isoform mRNA is shown. / indicates partial conservation. * stop codon. (B) PGC-1α protein domain conservation. Amino acid numbers refer to mouse PGC-1α (hereafter PGC-1α1). Numbers in brackets indicate the number of amino acids for each isoform. Red boxes indicate new N- and C-terminal amino acid sequences. (C) Three different exon1 coding sequences result in different N-terminal amino acid sequences. PGC-1α2 and α4 share the same alternative exon1, and therefore the same first 12 amino acids. All isoforms share exon2. (D) Differential promoter usage and splicing options result in proteins with different molecular weights. The different PGC-1α isoforms were expressed in HEK293 cells. Whole-cell extracts were resolved by SDS PAGE followed by immunoblotting using an anti-PGC-1α antibody (Zhang et al., 2009) that we have found to recognize all isoforms described here. See also Figure S1.

Figure 2. Gene expression profiling of PGC-1α isoforms and their target genes

(A) Tissue-specific PGC-1α isoform expression patterns. Absolute quantification of gene expression in mouse tissues (n=6) by qRT-PCR using isoform-specific primers. (B) Heat map summary of relative changes in gene expression by each PGC-1α isoform. Gene expression was analyzed (affymetrix) in myotubes expressing GFP alone (control), or together with each PGC-1α isoform. Experiments were performed in triplicate and results were analyzed with dChip software. (C) Venn diagram represents the number of genes regulated by PGC-1α1, PGC-1α4, and in common between both isoforms. (D, E, and F) From the Affymetrix results, gene sets were validated by qRT-PCR using specific primers. RNA was prepared as described in (B). Bars depict mean values and error bars represent standard deviation. *, p< 0.05 between indicated group and control. ^*,#, p< 0.05 between all groups. See also Figure S2.

Figure 3. Myotubes expressing PGC-1α4 show cellular hypertrophy

(A) Fluorescence microscopy analysis of myotubes expressing GFP alone, or together with PGC-1α1 or PGC-1α4. Fully differentiated myotubes were transduced with the different adenovirus and observed under a fluorescence microscope 36 hours later with a 10X objective. (B) Protein accumulation normalized by genomic DNA content in myotubes expressing GFP control, PGC-1α1 or PGC-1α4. Experiments were performed as described in (A) but cells were processed for either total protein or DNA quantification. Graphs show the total protein/genomic DNA ratio. (C) Protein accumulation normalized by genomic DNA content in myotubes expressing GFP control, PGC-1α1 or PGC-1α4 and treated with the IGF1R inhibitor BMS-754807. Myotubes were transduced as described in (A) and treated with 5 nM BMS-754807. At the end of 36 hours cells were processed for total protein or DNA quantification. (D) Analysis of gene expression for markers of myogenic differentiation. Experiments were performed as described in (A) and gene expression analyzed by qRT-PCR using primers specific to the indicated genes. (E) PGC-1α4 loss-of-function blunts clenbuterol-induced myotube hypertrophy. Fully differentiated primary myotubes were treated with 500 nM Clenbuterol (or vehicle) and transduced with adenovirus expressing PGC-1α4-specific or scrambled control shRNAs. 48 hours later, cells were processed for fluorescence microscopy or analysis of protein/DNA content. (F) Chromatin immunoprecipitation (ChIP) of DNA regions associated with Acetyl-H3K9. Myotubes expressing GFP alone or with PGC-1α4 were processed for chromatin immunoprecipitation. Purified DNA fragments were identified and quantified by PCR using primers targeting the IGF1 gene at 1 kb intervals. Graph shows enrichment relative to input after normalized by IgG. (G) ChIP of regions associated with di/trimethyl-H3K9. Myotubes were processed as described above. Purified DNA fragments were identified and quantified by PCR using primers targeting the Myostatin gene at 1 kb intervals. Graph shows enrichment relative to input normalized by IgG. Bars depict mean values and error bars represent standard deviation. *, p< 0.05 between indicated group and control. ^*,#, p< 0.05 between all groups. See also Figure S3.

Figure 4. In vivo expression of PGC-1α4 induces skeletal muscle hypertrophy

(A) Adenovirus-mediated expression of PGC-1α4 in mouse skeletal muscle. Cross-section of the gastrocnemius muscle, seven days after intramuscular injection of adenovirus expressing GFP alone or with PGC-1α4. (B) Cross-sectional area frequency distribution (sections from 6 mice per group). (C) PGC-1α4 and NT-PGC-1α mRNA expression levels in electroporated tibialis anterior analyzed by qRT-PCR using primers targeting exon 2 (present in both isoforms). (D) PGC-1α4 and NT-PGC-1α protein expression levels in electroporated muscle. (E, F) Electroporation-mediated delivery of plasmids into the tibialis anterior (TA). Each mouse (n=6 per group) received a control plasmid in one limb (pCI-neo), and in the contralateral limb the plasmid encoding PGC-1α4 or NT-PGC-1α. Bars depict mean values and error bars represent standard error. *, p< 0.05 between indicated group and control. See also Figure S4.

Figure 5. PGC-1α4 expression increases during muscle hypertrophy and resistance training

(A,B) PGC-1α4 expression increases during the hypertrophy phase of a suspension/reloading protocol. C57Bl/6 mice were divided into groups (n=4 each): control, 10 days hindlimb suspension (Suspension), or 10 days suspension plus 24 hours of reloading (Reloading). The soleus muscles were harvested and processed for gene expression analysis by qRT-PCR using primers specific for the indicated genes. (C–F) Analysis of gene expression in skeletal muscle biopsies from human volunteers. Percutaneous vastuls lateralis biopsies were obtained at baseline and eight weeks later, ~48h after the last training session. Gene expression was analyzed by qRT-PCR using primers specific to the indicated genes. (G) Increase in PGC-1α4 expression correlates with improvement in leg press exercise performance. Graph shows the percent change in the number of leg press repetitions observed between baseline and after 8 weeks of training. (H) Morphology of hindlimbs from wild-type and PGC-1α4 transgenic mice (Myo-PGC-1α4). Immunoblot using an anti-PGC-1α antibody (α-PGC-1α) shows PGC-1α4 protein levels in the gastrocnemius muscle. (I) Total PGC-1α and PGC-1α4 mRNA levels in the Myo-PGC-1α4 mouse line were determined by qRT-PCR and compared to the wild-type littermate controls. Bars depict mean values and error bars represent standard deviation. *, p< 0.05 between indicated group and control. ^*,#, p< 0.05 between all groups.

Figure 6. Myo-PGC-1α4 skeletal muscle transgenics have increased muscle mass and strength

(A) Determination of muscle wet weight from PGC-1α4 transgenics and wild-type controls (n=6). Muscle weights are normalized by tibia length. (B) Fiber cross-sectional area in the gastrocnemius muscle of wild-type and PGC-1α4 transgenics. (C) Immunohistochemical analysis of gastrocnemius muscle from wild-type (WT) and Myo-PGC-1α4 animals using antibodies against different myosin heavy chain (MyHC) types. (D) Determination of Epididimal (EAT) and Peri-renal (PRAT) adipose tissue wet weight from PGC-1α4 transgenics and wild-type controls (n=6). (E) Maximal force measurements of the gastrocnemius muscle of wild-type and PGC1α4 transgenics (Myo-PGC-1α4). (F) Muscle fatigue test performed under the same experimental settings as in (E). (G) Changes in normalized muscle mass with hindlimb suspension/reloading. N= 4 per group. (H) Exercise tolerance test. Muscle-specific PGC-1α1 (MCK-PGC-1α) and PGC-1α4 (Myo-PGC-1α4) transgenics ran to exhaustion on a treadmill. Data is normalized by values obtained with wild-type animals (n=6 per group). Bars depict mean values and error bars represent standard error. *, p< 0.05 between indicated group and control. ^*,#, p< 0.05 between all groups. See also Figure S6 and Table S1.

Figure 7. PGC-1α4 transgenic mice show resistance to muscle wasting during experimental cancer cachexia

(A) Representative images of hindlimb muscle muscles from wild-type, and tumor bearing (+LLC) wild-type or Myo-PGC-1α4 mice. (B) Gastrocnemius muscle mass in wild-type and Myo-PGC-1α4 tumor bearing mice normalized to their own genotype non-tumor bearing control. (C) Muscle force production in wild-type and Myo-PGC-1α4 tumor bearing mice normalized to their own genotype non tumor bearing control. (D) Myostatin and IGF-1 mRNA expression in wild-type and Myo-PGC-1α4 with or without LLC tumor. (E) Physical activity throughout the progression of tumor load. (F) Glucose tolerance test. (G) Quantification of glucose clearance. Bars depict mean values and error bars represent standard error. *, p< 0.05 between indicated group and its genotype control. #, p< 0.05 between indicated group and group receiving same treatment across genotypes. †, p< 0.05 between indicated group and wild-type mice. ‡, p< 0.05 between indicated group and wild-type + LLC mice. See also Figure S7.