Generation and bioenergetic analysis of cybrids containing mitochondrial DNA from mouse skeletal muscle during aging

Abstract

Mitochondrial respiratory chain defects have been associated with various diseases and normal aging, particularly in tissues with high energy demands including skeletal muscle. Muscle-specific mitochondrial DNA (mtDNA) mutations have also been reported to accumulate with aging. Our understanding of the molecular processes mediating altered mitochondrial gene expression to dysfunction associated with mtDNA mutations in muscle would be greatly enhanced by our ability to transfer muscle mtDNA to established cell lines. Here, we report the successful generation of mouse cybrids carrying skeletal muscle mtDNA. Using this novel approach, we performed bioenergetic analysis of cells bearing mtDNA derived from young and old mouse skeletal muscles. A significant decrease in oxidative phosphorylation coupling and regulation capacity has been observed with cybrids carrying mtDNA from skeletal muscle of old mice. Our results also revealed decrease growth capacity and cell viability associated with the mtDNA derived from muscle of old mice. These findings indicate that a decline in mitochondrial function associated with compromised mtDNA quality during aging leads to a decrease in both the capacity and regulation of oxidative phosphorylation.

Figure 1.

Generation of cybrids containing mtDNA from skeletal muscle. Flowchart of how mtDNA from the skeletal muscle was transferred to ρ⁰ cells to produce cybrids.

Figure 2.

Validation of transfer of mtDNA from muscle to cybrids. Representative chromatograms of signature sequences of mtDNA at positions 9461 and 9818 and surrounding areas of DBA/2-muscle sample (A), cybrids derived from fusion of mitochondria from DBA/2 muscle and ρ⁰ cells (B), C57BL/6-muscle (C) and cybrids derived from fusion of mitochondria from C57BL/6 muscle and ρ⁰ cells (D). An arrow shows the base difference at 9461, and the different lengths of the A-stretch are also indicated.

Figure 3.

Oxygen consumption measurement. Six and 26 months represent 24 and 22 cybrids per age group, respectively. Endogenous respiration was measured in intact cells (A), after treatment with the ATP synthase inhibitor oligomycin at 2.5 µg/ml (B), and with the uncoupler FCCP at 0.5 µM (C). The activities were determined with about 5 × 10⁶ cells. Three determinations were made for each cybrid and the average values are shown. Ratios of oligomycin-resistant to native endogenous respiration rates (D) and FCCP-treated fully uncoupled to oligomycin-resistant respiration rates (E) of each age group were also calculated.

Figure 4.

Growth capacity measurement. Six and 26 months represent 24 and 25 cybrids per age group, respectively. Cells were grown in glucose-containing DMEM for 24 h, then in glucose medium (A) or in galactose medium (B) for another 72 h. Doubling times were calculated as: 72 h × Log (2)/[Log (total cell number at the end)-Log (total cell number when media were changed)]. Three determinations were made for each cybrid. Growth index was calculated as the ratio of doubling time in galactose-containing medium to that in glucose medium (C).

Figure 5.

Cell viability analysis. Six and 26 months represent 24 and 25 cybrids per age group, respectively. Cells were grown in glucose-containing DMEM for 24 h, then in glucose medium (A) or in galactose medium (B) for another 72 h. Cell viabilities were analyzed with a Vi-cell^TM XR cell viability analyzer (Beckman Coulter). Three determinations were made for each cybrid. Viability index was calculated as the ratio of percentage of cell viability in galactose-containing medium to that in glucose medium (C).

Figure 6.

mtDNA copy number analysis. (A) Six and 26 months represent 24 and 20 cybrids per age group. Relative mtDNA copy number (mtDNA/nDNA) was the mtDNA content normalized by nuclear DNA encoded β-actin gene. (B) Relationship between the relative mtDNA copy number and the maximal respiration as measured by oxygen consumption with uncoupler FCCP in 40 individual transformants of both age groups. (C) Relationship between the relative mtDNA copy number and the ratio of doubling time (in galactose/in glucose media) in 40 individual transformants of both age groups. (D) Relationship between the relative mtDNA copy number and the ratio of viability (in galactose/in glucose media). In (B–D), n includes 22 cybrids of 6-month-young mice, and 18 cybrids of 26-month-old mice.