Activation of the PPAR/PGC-1alpha pathway prevents a bioenergetic deficit and effectively improves a mitochondrial myopathy phenotype

Abstract

Neuromuscular disorders with defects in the mitochondrial ATP-generating system affect a large number of children and adults worldwide, but remain without treatment. We used a mouse model of mitochondrial myopathy, caused by a cytochrome c oxidase deficiency, to evaluate the effect of induced mitochondrial biogenesis on the course of the disease. Mitochondrial biogenesis was induced either by transgenic expression of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator alpha (PGC-1alpha) in skeletal muscle or by administration of bezafibrate, a PPAR panagonist. Both strategies successfully stimulated residual respiratory capacity in muscle tissue. Mitochondrial proliferation resulted in an enhanced OXPHOS capacity per muscle mass. As a consequence, ATP levels were conserved resulting in a delayed onset of the myopathy and a markedly prolonged life span. Thus, induction of mitochondrial biogenesis through pharmacological or metabolic modulation of the PPAR/PGC-1alpha pathway promises to be an effective therapeutic approach for mitochondrial disorders.

Figure 1. Longer life span and delayed onset of the myopathy coincide with a rescued COX activity muscle homogenates of PGC-1αΔCOX10 mice compared to ΔCOX10 mice

A: Survival curve of female PGC-1αΔCOX10 mice in comparison to ΔCOX10 mice (n=20 for each group). No mice of the PGC-1α and wild-type control groups died in the observed timeframe. B: Treadmill performance test at different ages for female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice (n= 6 for each group). One way ANOVA, followed by Post-Hoc Tukey analyses showed significance between ΔCOX10 and each of the other groups. C: Cytochrome c oxidase (COX) activity of female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice at different ages comparing muscle homogenates and muscle mitochondria (n=3 for each group). D: Western blot of COXI, SDH and tubulin in muscle homogenates and mitochondria of 3 months old female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice. COXI levels were below the detection limit of the assay in some samples. E: Histology of the biceps femoris muscle from female mice at different ages (20x magnification) showing the development of the COX deficiency in PGC-1αΔCOX10 mice in comparison to a myopathic ΔCOX10 and to PGC-1α and wild-type mice as controls. Shown are succinate dehydrogenase (SDH), cytochrome c oxidase (COX) and combined COX/SDH staining.

Figure 2. Increased mitochondrial mass in mice expressing PGC-1α

A: Citrate synthase activity as a mitochondrial marker protein in muscle homogenates and muscle mitochondria from female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice at different ages. B: Electron micrograph from longitudinal section taken from biceps femoris muscle from 3 months old female PGC-1αCOX10, ΔCOX10, PGC-1α and wild-type mice. Mitochondria are indicated by black arrows. Scale bare: 2 µm. C: Relative quantification of mitochondrial DNA versus nuclear DNA by signal intensity of mtDNA and 18S bands in a southern blot of DNA isolated from skeletal muscle from 3 months old female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice (n=3 for each group). D: Quantification of ATP in the biceps femoris muscle from 3 months old female PGC-1αΔCOX10, ΔCOX10, PGC-1α and wild-type mice (n=4 for each group).

Figure 3. Bezafibrate administration to ΔCOX10 mice induces PGC-1α expression in skeletal muscle resulting in delayed onset of the myopathy

A: Survival curve of female ΔCOX10 on the bezafibrate diet mice in comparison to ΔCOX10 mice on a regular diet (n=7 for each group). No mice of in wild-type groups (bezafibrate diet or regular diet) died in the observed timeframe. B: Treadmill performance test at different ages for ΔCOX10 and wild-type mice on a bezafibrate diet. ΔCOX10 and wild-type mice on a regular diet were used as reference (n= 3 for each group). At the 3 months time point, statistical significance was reached between ΔCOX10 on the standard diet and each of the other groups. At the 6 months time point, significance was reached between ΔCOX10 on the bezafibrate diet and each of the other groups. C: Cytochrome c oxidase (COX) activity and citrate synthase activity in muscle homogenates of 3 months old female ΔCOX10 and wild-type mice on a bezafibrate and regular diet (n=3 for each group). D: Histology of the biceps femoris muscle from 3 months old female mice showing ΔCOX10 and wild-type mice on a bezafibrate and regular diet. SDH staining is shown to indicate the mitochondrial proliferation. COX and combined COX/SDH staining highlight the degree of COX deficiency. E: Quantification of ATP in the biceps femoris muscle from 3 months old ΔCOX10 and wild-type mice on a bezafibrate and regular diet (n=2 for each group). F: Relative expression of PGC-1α, PGC-1β and PPARα/δ and γ in skeletal muscle of 3 months old female ΔCOX10 and wild-type mice on a bezafibrate diet. ΔCOX10 and wild-type mice on a regular diet were used as reference.

Figure 4. Model for the potential role of PGC-1α for the treatment of mitochondrial disease

A: Animal model of mitochondrial myopathy: Muscle fibers in the ΔCOX10 mice have different degrees of COX10 deletion and mitochondria with variable OXPHOS capacity. PGC-1α expression induced mitochondrial biogenesis enriches all mitochondria populations resulting in an increased number of OXPHOS competent units per muscle fiber and increased ATP levels. B: Mitochondrial disease patient with mutation in mtDNA: Muscle fibers are heteroplasmic for the mtDNA mutation resulting in mitochondria with different levels of OXPHOS activity. During red ragged fiber (RRF) formation, mitochondria carrying the mutation are enriched. PGC-1α expression induced mitochondrial biogenesis might enrich all mitochondria populations resulting in an increased number of OXPHOS competent units per muscle fiber and increased ATP levels. C: Mitochondrial disease patient with mutation in nDNA: Muscle fibers have mitochondria with residual OXPHOS activity. PGC-1α expression induced mitochondrial biogenesis might increase the mitochondrial mass resulting in an increased OXPHOS capacity per muscle fiber and increased ATP levels.