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. 2016 Aug;38(4):323-333.
doi: 10.1007/s11357-016-9930-1. Epub 2016 Jul 21.

Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle

Dustin R Masser  1   2   3   4 , Nicholas W Clark  1 , Holly Van Remmen  4   5 , Willard M Freeman  6   7   8   9
Affiliations

Loss of the antioxidant enzyme CuZnSOD (Sod1) mimics an age-related increase in absolute mitochondrial DNA copy number in the skeletal muscle

Dustin R Masser et al. Age (Dordr). 2016 Aug.

Abstract

Mitochondria contain multiple copies of the circular mitochondrial genome (mtDNA) that encodes ribosomal RNAs and proteins locally translated for oxidative phosphorylation. Loss of mtDNA integrity, both altered copy number and increased mutations, is implicated in cellular dysfunction with aging. Published data on mtDNA copy number and aging is discordant which may be due to methodological limitations for quantifying mtDNA copy number. Existing quantitative PCR (qPCR) mtDNA copy number quantification methods provide only relative abundances and are problematic to normalize to different template input amounts and across tissues/sample types. As well, existing methods cannot quantify mtDNA copy number in subcellular isolates, such as isolated mitochondria and neuronal synaptic terminals, which lack nuclear genomic DNA for normalization. We have developed and validated a novel absolute mtDNA copy number quantitation method that uses chip-based digital polymerase chain reaction (dPCR) to count the number of copies of mtDNA and used this novel method to assess the literature discrepancy in which there is no clear consensus whether mtDNA numbers change with aging in skeletal muscle. Skeletal muscle in old mice was found to have increased absolute mtDNA numbers compared to young controls. Furthermore, young Sod1 -/- mice were assessed and show an age-mimicking increase in skeletal muscle mtDNA. These findings reproduce a number of previous studies that demonstrate age-related increases in mtDNA. This simple and cost effective dPCR approach should enable precise and accurate mtDNA copy number quantitation in mitochondrial studies, eliminating contradictory studies of mitochondrial DNA content with aging.

Keywords: Aging; Digital PCR; Mitochondria; Sod1; Synaptosomes; mtDNA; mtDNA copy number.

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Conflict of interest statement

Compliance with ethical standards All procedures were approved by the Institutional Animal Care and Use Committee at the Oklahoma Medical Research Foundation. Competing interests The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Absolute mtDNA quantitation dPCR workflow. a Total genomic DNA is isolated from tissue/cells and mixed with proper assay components. The reactions are distributed across a chip with 20,000 856-pl wells. Template is diluted to a point where there is either 0 or 1 copies per well. Reactions are then cycled to end-point and fluorescence is read in each well. Those wells that were loaded with template are positive wells, while those wells that were filled without template are negative. Based on the count of fluorescent positive and negative wells and using a Poisson distribution, the number of target copies can be calculated per microliter. b Representative raw fluorescence data from a mtDNA assay (left) and a nuclear DNA reference assay (right) showing chips with positive and negative wells (top) and corresponding well counts of fluorescent intensities showing a bimodal distribution of positive and negative wells
Fig. 2
Fig. 2
mtDNA standard validation of dPCR. Plasmids containing either human, rat, or mouse mtDNA were diluted to 600, 1000, or 1600 copies per microliter based on mass. Each of these plasmid dilutions were subjected to dPCR. Measured copies per microliter versus expected copies per microliter are plotted. Points represent 2–3 chips per dilution. Error bars = SEM
Fig. 3
Fig. 3
mtDNA absolute quantitation from rat retinal synaptosomes. a Somatic and synaptic retinal cell fractions were used to determine the absolute quantitation of mtDNA. Somatic cell fraction contained both nuclear and mtDNA molecules. Synaptic fraction only contained mtDNA molecules with an insignificant amount of nuclear. b Representative western blots on nuclear, cytoplasmic, and synaptosome fractions probed for Lamin B1 (66–70 kDa), a nuclear marker, Synaptophysin (38 kDa), a pre-synaptic terminal marker, and COXIV (15–16 kDa), a mitochondrial marker
Fig. 4
Fig. 4
Mouse heart muscle contains more mtDNA compared to neural (retina) tissue. dPCR was used to absolutely quantify mtDNA copies in mouse heart muscle and retinal neural tissue. Data is represented as either normalized per nanogram input DNA (a) or normalized on a per haploid genome basis (b). Both demonstrate that heart muscle contains more mtDNA when compared to retinal neural tissue (***p < 0.001, parametric t test, n = 5–7/tissue). Break interval—30,000–100,000 (right)
Fig. 5
Fig. 5
Increase in mtDNA copies from skeletal muscle but not brain from aged wild type and Sod1 −/− mice. Absolute mtDNA copy numbers in a skeletal muscle and b brain from young (8 months) , old (28 months), and young (8 months) Sod1 −/− mice. Aging and deletion of Sod1 −/− result in an increase in mtDNA copies compared to young animals in skeletal muscle, but not brain. c Skeletal muscle has higher mtDNA copies compared to brain (*p < 0.05, **p < 0.01, one-way ANOVA on group, SNK post hoc, n = 5–7/group; ***p < 0.001, two-way ANOVA on group and tissue, n = 5–7/group). Relative mtDNA copy number from young, old, and Sod1 −/− mouse skeletal muscle (d) and brain (e). (F) Relative mtDNA copy number is higher in the muscle compared to the brain (***p < 0.001, two-way ANOVA on group and tissue, SNK post hoc, n = 5–6/group)
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