Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle

Abstract

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.

Keywords: angiogenesis; endurance exercise responses; oxidative metabolism; skeletal muscle plasticity.

Figure 1.

AAV1-mediated expression of Perm1 in TA muscle. AAV1 vectors expressing LacZ or FLAG-Perm1 were injected in the frontal area of TA muscles. After 4 wk, muscles were harvested. A) Perm1 protein levels were determined in muscle lysates by Western blotting, using antibodies against Perm1 (detecting endogenous and AAV1-expressed protein) and anti-FLAG (detecting only AAV1-expressed Perm1). The arrow on the right indicates the over-expressed Perm1 protein; arrowheads on the left indicate the 2 major protein forms encoded by endogenous Perm1 (32). Anti-tubulin antibodies were used to control for loading. B) Weights of TA muscles expressing LacZ or Perm1. Data are the means ± sd of 6 mice. C, D) Representative images of cross-sections of LacZ and Perm1 TA muscles stained with H&E (C) or immunostained with anti-laminin antibody (D). Scale bars, 100 μm. E) Distribution of fiber cross-sectional areas of LacZ and Perm1 TA muscles, calculated from laminin-stained images. Data are the means ± sd (n = 4 mice per group).

Figure 2.

Perm1 enhances mitochondrial biogenesis and oxidative activity in skeletal muscle. A) The relative mtDNA content was determined as the ratio of mitochondrial (CoxII) to genomic (Dio3) DNA copy numbers, and expressed relative to the ratio seen in control (LacZ) TA muscles. B) mRNA levels for the indicated mitochondrial genome-encoded oxphos genes were determined by RT-quantitative PCR, normalized to GAPDH levels, and expressed relative to levels of each gene in control (LacZ) TA muscles. A, B) Data are the means ± se (n = 14). **P < 0.01; ***P < 0.001. C, D) The levels of oxphos complex proteins in TA muscles were determined by Western blot analysis, using total protein lysates and the Total OxPhos Complex antibody cocktail (C). The intensity of the bands was quantified using ImageJ software, and the values are expressed relative to the signal intensity in control (LacZ) TA muscles (D). E, F) Enzymatic activities of oxphos complexes I–IV (E) and of CS (F) in total muscle lysates are shown. Data are the means ± sd, expressed relative to enzymatic activity in control (LacZ) TA muscles (n = 4). *P < 0.05; **P < 0.01. G) Representative transmission electron micrographs of LacZ and Perm1 TA muscles. Arrowheads indicate mitochondria. Scale bar, 1 μm. H) Representative images of cross-sections of LacZ and Perm1 TA muscles stained for SDH activity. Scale bar, 100 μm.

Figure 3.

Perm1 increases spare respiratory capacity in muscle. FDB muscles were injected with AAV1 vectors expressing LacZ (−) or Perm1 (+) and dissected 4 wk later. A) Protein levels of Perm1 and oxphos complexes in FDB muscles were determined by Western blotting, as in Figs. 1 and 2. B) A representative image of intact single FDB fibers cultured in a Seahorse Bioscience XFe96 plate. Scale bar, 600 μm. C) OCRs of intact single FDB fibers were measured in the absence and presence of 1 µM oligomycin and/or 800 nM FCCP. Rates are normalized by the number of fibers, corrected for nonmitochondrial oxygen consumption, and expressed as OCR per 20 fibers. The rates are the means ± sd of FDB fibers from 6 mice. **P < 0.01.

Figure 4.

Perm1 does not alter fiber-type composition. A) MHC isoforms of control (LacZ) or Perm1 TA muscles were separated by SDS-PAGE and visualized by silver staining (125 ng skeletal muscle myosins per lane). Std, standard sample containing soleus (50%) and gastrocnemius (50%) muscle myosins was used as control for the migration pattern of all isoforms. B) The relative abundance of MHC isoforms in control (LacZ) and Perm1 TA muscles, based on quantification of myosin bands in (A) by ImageJ software. Data are expressed as percentages (%) of total MHCs and are the means ± sd (n = 4 mice per group). **P < 0.01. C) Representative images of cross-sections show the midportion of LacZ and Perm1 TA muscles stained with antibodies against MHC isoforms, as indicated. Scale bar, 200 μm. D) The relative abundance of different fiber types in TA, based on quantitation of an entire TA section. Data are expressed as percentages (%) of total fibers and are the means ± sd (n = 4 mice per group). E) mRNA levels for the indicated MHC-encoding genes in TA muscles were determined by RT-quantitative PCR, normalized to GAPDH levels, and expressed relative to the levels of each gene in control (LacZ) muscle. Data are the means ± se (n = 14).

Figure 5.

Increased expression of Perm1 enhances skeletal muscle vascularization. A) Representative images of cross-sections of LacZ and Perm1 TA muscles stained with anti-CD31 and anti-laminin antibodies. Scale bars, 50 μm. B) Capillary density of LacZ and Perm1 TA muscles, quantified from CD31-stained muscles, using ImageJ software. Data are the means ± se (n = 5). *P < 0.05. C) Vegfa mRNA levels were determined by RT-quantitative PCR, normalized to GAPDH levels, and expressed relative to the levels in control (LacZ) muscle. Data are the means ± se (n = 14). **P < 0.01.

Figure 6.

Increased expression of Perm1 enhances the expression of genes important for mitochondrial biogenesis and oxidative function. Total RNA was harvested from whole LacZ and Perm1 TA muscles. A–C) RNA levels for the indicated genes were determined by RT-quantitative PCR, normalized to GAPDH levels, and expressed relative to the levels of each gene in control (LacZ) muscles. Note that the primers used to quantify PGC-1α expression detect all PGC-1α isoforms, and it is thus unclear whether Perm1 affects differentially the different isoforms (–49). Data are the means ± se (n = 14). *P < 0.05; **P < 0.01. D) Protein levels of PGC-1α, ERRα, Sirt3, and myoglobin (Mb) were determined by Western blot analysis of whole TA muscles from 4 mice, expressing LacZ (L1–L4) or Perm1 (P1–P4, with numbers indicating contralateral muscles from the same mouse). The intensity of the bands was quantified using ImageJ software, normalized to the loading control (tubulin), and is indicated below each lane [expressed relative to the mean signal intensity in control (LacZ) muscles]. Note that the anti-PGC-1α antibody detects a protein isoform of ∼115 kDa, corresponding to full-length PGC-1α (also called PGC-1α1) (48, 49).

Figure 7.

Perm1 modulates the active state of p38 MAPK. A, B) The levels of total and phospho-p38 MAPK in control (LacZ) and Perm1 TA muscles dissected at 11 am [day/rest phase (A)] or 11 pm [night/active phase (B)] were determined by Western blot analysis. To the right of each Western blot, the graph presents the relative levels of phospho-p38/total p38 in day/rest phase (based on 11 am and 3 pm) and in night/active phase (based on 11 pm and 3 am), quantified from the Western blots and Supplemental Fig. 4A, and using ImageJ software. Data are the means ± sd and expressed relative to the signal intensity in control (LacZ) muscles (n = 6). **P < 0.01; ***P < 0.001. C) The levels of total and phospho-p38 MAPK in primary myotubes infected with adenoviruses expressing control (LacZ) or Perm1 for 24 h were determined by Western blot analysis. A–C) Ponceau staining is shown as control for loading.

Figure 8.

Increased Perm1 expression enhances fatigue resistance in skeletal muscle. A) Specific force generated at different frequency stimulations of EDL muscles injected with AAV1-LacZ (control) or AAV1-Perm1. Other contractile properties of the muscles are shown in Table 2. B) Relative force generation of LacZ and Perm1 EDL muscles during repeated stimulation for 3 min (with rest intervals between stimulations of 4 s for the first minute, 3 s for the second minute, and 2 s for the third minute). Fatigue curves were obtained from control (LacZ) or Perm1 EDL muscles. Force is expressed relative to force generated in the first contraction for each muscle. C) Time to fatigue is defined here as time taken for each muscle to reach 40% of the force generated in the first contraction. Data are the means ± se (n = 8). *P < 0.05.