Activation of PPARδ Leads to Muscle Fiber Transformation

A role of PPARδ in muscle fiber was suggested by its enhanced expression—at levels 10-fold and 50-fold greater than PPARα and γ isoforms, respectively (unpublished data). An examination of PPARδ in different muscle fibers reveals a significantly higher level in type I muscle (soleus) relative to type II–rich muscle (extensor digitorum longus) or type I and type II mixed muscle (gastrocnemius) (Figure 1A); this expression pattern closely resembles that of PGC-1α (Lin et al. 2002). A similar pattern but with more pronounced differences was found at the protein level (Figure 1B).

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larger image TIFF original image Download: Figure 1. Expression of Endogenous PPARδ and VP16-PPARδ Transgene in Muscle (A) Pooled RNA isolated from various muscles of five wild-type male C57B6 mice was hybridized with indicated probes. EDL, extensor digitorum longus; Gastro, gastrocnemius. (B) Pooled nuclear proteins (15 μg/lane) isolated from muscles of five wild-type male C57B6 were probed with anti-PPARδ antibody. RNA polymerase II (Pol II) is shown as a loading control. (C) Expression of the VP16-PPARδ transgene in various tissues. 10 μg of total RNA from each tissue was hybridized with a VP16 cDNA probe. Gastrocnemius muscle was used here. (D) Nuclear proteins (15 μg/lane) isolated from gastrocnemius muscle of the transgenic mice (TG) and the wild-type littermates (WT) were probed with indicated antibodies. The upper, nonspecific band that cross-reacted with the anti-PPARδ antibody serves a loading control. https://doi.org/10.1371/journal.pbio.0020294.g001

To directly assess the role of activation of PPARδ in control of muscle fiber plasticity and mitochondrial biogenesis, we generated mice expressing a transgene in which the 78-amino-acid VP16 activation domain was fused to the N-terminus of full-length PPARδ, under control of the 2.2-kb human α-skeletal actin promoter. In agreement with the previous characterization of this promoter (Brennan and Hardeman 1993; Clapham et al. 2000), the VP16-PPARδ transgene was selectively expressed in skeletal muscle, with 10-fold less in the heart (Figure 1C). Among different types of muscle fibers, the levels of VP16-PPARδ expression appeared to be similar (unpublished data). As shown in Figure 1D for gastrocnemius muscle, VP16-PPARδ fusion protein was produced at a level similar to that of endogenous PPARδ in wild-type littermates. Interestingly, the level of endogenous muscle PPARδ protein in the transgenic mice was much higher than in the control littermates. The substantial increase of endogenous PPARδ may have been caused by a switch to type I fiber (see below), which intrinsically expresses higher levels of PPARδ (Figure 1A and 1B).

Type I muscle can be readily distinguished from type II or mixed muscle by its red color, because of its high concentration of myoglobin, a protein typically expressed in oxidative muscle fibers. We found that muscles in the transgenic mice appeared redder (Figure 2A), which is particularly evident in the mixed type I/II fibers of the hindlimb (Figure 2B). Indeed, metachromatic staining revealed a substantial muscle fiber transformation (Figure 2C). In gastrocnemius muscle, we estimated that there was a 2-fold increase of type I fibers. A diagnostic component of oxidative fibers is their high myoglobin and mitochondrial content, which is supported by the mRNA analysis shown in Figure 3A. In addition to myoglobin, mitochondrial components for electron transfer (cytochrome c and cytochrome c oxidase [COX] II and IV) and fatty-acid β-oxidation enzymes were elevated (Figure 3A; unpublished data). These effects appear to be direct consequences of PPARδ activation, as levels of PGC-1α, a coactivator involved in muscle fiber switch and mitochondrial biogenesis (Wu et al. 1999; Lehman et al. 2000; Lin et al. 2002), remained unchanged. Southern blot analysis detected a substantially higher copy number of the mitochondrial genome–encoded COXII DNA in the transgenic mice (Figure 3B). Mitochondrial DNA was increased 2.3-fold in gastrocnemius muscle of the transgenic mice (Figure 3C). These results reveal a marked stimulation of mitochondrial biogenesis and further support the idea that there is a muscle fiber switch. This conclusion was also confirmed by Western blot analysis. As shown in Figure 3D, the characteristic type I fiber proteins, such as myoglobin and cytochrome c and b, were significantly increased. More importantly, the specialized contraction protein troponin I (slow) of type I fiber was robustly induced; this was accompanied by a marked reduction of the specialized contraction protein troponin I (fast) of type II fiber, indicating a high degree of fiber transformation. We next examined whether acute activation of endogenous PPARδ would induce similar target genes. In agreement with the chronic effects in the transgenic mice, we found that, after treatment of wild-type C57B6J mice with the PPARδ-specific agonist GW501516 for only 10 d, genes for slow fiber contractile proteins, mitochondrial biogenesis, and β-oxidation were all upregulated (Figure 3E). This indicates that rapid, systematic, and coordinated changes of muscle fiber properties toward type I can be achieved by activation of the endogenous PPARδ pathway.

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larger image TIFF original image Download: Figure 2. Increased Oxidative Type I Fibers in the Transgenic Mice (A and B) Muscles in transgenic mice (TG) are redder than those in wild-type mice (WT). (C) Metachromatic staining of the type II plantaris muscle. Type I fibers are stained dark blue. https://doi.org/10.1371/journal.pbio.0020294.g002