Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Abstract

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations.

Figure 1.

MCKPGC-1αMut mice have increased PGC-1α levels in the skeletal muscle. (A) Gene expression of PGC-1α relative to WT in the quadriceps of 10-month-old male mice. mRNA levels are normalized to GAPDH. (B) Western blot showing PGC-1α protein levels in the quadriceps of 10-month-old male mice with loading control actin. (C) Quantification of western blot in (B) showing PGC-1α protein levels normalized to actin. n = 4/group; Student's t-test: *P < 0.05 and ***P < 0.001. Error bars represent the SEM.

Figure 2.

Increased PGC-1α expression increases mitochondrial biogenesis and function in the skeletal muscle of Mut mice. (A) Western blot showing the levels of mitochondrial proteins in the quadriceps of mice and loading control actin. ATP synthase subunit 5α (ATP5A; subunit of complex V), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2; subunit of complex III), mitochondrial cytochrome c oxidase subunit 1 (MTCO1; subunit of complex IV), succinate dehydrogenase subunit A and B, or SDHA and SDHB (subunits of complex II) and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8; subunit of complex I; n = 4/group). (B) Quantification of western blot in (A) showing protein levels normalized to actin. (C) Histology of the quadriceps showing COX (complex IV) and SDH (complex II) activity staining (n = 3/group). (D) Complex I and III activity, (E) complex IV activity and (F) CS activity in total quadricep homogenate (n = 4/group). (G) MtDNA levels relative to WT in the quadriceps based on the ND1 copy number (subunit of complex I) normalized to GAPDH (n = 4/group). Analyzed 10-month-old male mice. Student's t-test: *P< 0.05, **P< 0.01 and ***P< 0.001. Error bars represent the SEM.

Figure 3.

Increased PGC-1α expression has no effect on skeletal muscle weight but improves skeletal muscle function of Mut mice. (A) Weight of quadricep and gastrocnemius of 10-month-old male mice (n = 4–5/group). Student's t-test: *P< 0.05 and **P< 0.01. Error bars represent the SEM. (B) The number of falls of mice when put to run on a treadmill for 3 min at 9 m/min (n = 5–10/group). *P< 0.05 represents the difference between Mut and MCKPGC-1αMut; ^##P< 0.01 and ^###P< 0.001 represents the difference between WT and Mut; ⁺⁺P< 0.01 and ⁺⁺⁺P< 0.001 represents the difference between MCKPGC-1αWT and Mut; ^φP< 0.05 represents the difference between WT and MCKPGC-1αMut. Difference between MCKPGC-1αWT and MCKPGC-1αMut is significant at the 9-month time point only and the difference between WT and MCKPGC-1αWT is not significant at any time point. Statistics represent two-way ANOVA followed by the Bonferroni post-tests. Error bars represent the SEM.

Figure 4.

Increased PGC-1α expression stabilizes mtDNA levels and improves mitochondrial function and ejection fraction in the heart of Mut mice. (A) Gene expression of PGC-1α in the heart relative to WT (n = 4/group). mRNA levels are normalized to GAPDH. (B) CS activity and (C) COX (complex IV) activity in total heart homogenate (n = 4/group). (D) MtDNA levels in the heart relative to WT based on the ND1 copy number (subunit of complex I) normalized to GAPDH (n = 4/group). We analyzed 10-month-old male mice. (E) Percent heart ejection fraction based on echocardiogram of the mouse heart (n = 11–12, 10-month-old male and female mice per group). Student's t-test: *P< 0.05 and ***P< 0.001. Error bars represent the SEM.

Figure 5.

Increased PGC-1α expression increases the average abundance of somatic mtDNA point mutations in the skeletal muscle of Mut mice. (A) Representative plot showing the first minor allele frequency of variants throughout the entire mtDNA from the quadricep of WT and Mut mice. (B) Data as in (A) for variants between positions 5300 and 7800 of the mtDNA [which spans MTCO1, tRNAs1 (Trns1), tRNAd (Trnd), MTCO2 and tRNAk (Trnk) genes] for three different mice (numbered 1–3) for each group. (C) Graph showing abundance versus rank abundance for first minor allele variants in the quadriceps. (D) Average abundance of rare (rank #100–#10 000) variants in the quadriceps. n = 3/group 10-month-old male mice. Student's t-test: *P< 0.05 and **P< 0.01. Error bars represent the SEM.