Somatic mitochondrial DNA deletions accumulate to high levels in aging human extraocular muscles

Abstract

PURPOSE. Mitochondrial function and the presence of somatic mitochondrial DNA (mtDNA) defects were investigated in extraocular muscles (EOMs) collected from individuals covering a wide age range, to document the changes seen with normal aging. METHODS. Cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemistry was performed on 46 EOM samples to determine the level of COX deficiency in serial cryostat muscle sections (mean age, 42.6 years; range, 3.0-96.0 years). Competitive three-primer and real-time PCR were performed on single-fiber lysates to detect and quantify mtDNA deletions. Whole-genome mitochondrial sequencing was also performed to evaluate the contribution of mtDNA point mutations to the overall mutational load. RESULTS. COX-negative fibers were seen in EOMs beginning in the third decade of life, and there was a significant age-related increase: <30 years, 0.05% (n = 17); 30 to 60 years, 1.94% (n = 13); and >60 years, 3.34% (n = 16, P = 0.0001). Higher levels of COX deficiency were also present in EOM than in skeletal muscle in all three age groups (P < 0.0001). Most of the COX-negative fibers harbored high levels (>70%) of mtDNA deletions (206/284, 72.54%) and the mean deletion level was 66.64% (SD 36.45%). The mutational yield from whole mitochondrial genome sequencing was relatively low (1/19, 5.3%), with only a single mtDNA point mutation identified among COX-negative fibers with low deletion levels < or =70%. CONCLUSIONS. The results show an exponential increase in COX deficiency in EOMs beginning in early adulthood, which suggests an accelerated aging process compared with other postmitotic tissues.

Figure 1.

Mean percentage of COX-negative fibers found in EOM and skeletal muscle samples. Error bars, SEM.

Figure 2.

Distribution of COX-deficient EOM fibers with age. The simulated curve models an exponential increase in COX deficiency with normal aging (r² = 0.5753); dotted lines: predicted upper and lower 95% confidence range (n = 46).

Figure 3.

Level of mtDNA deletion in single COX-positive and COX-negative EOM fibers. (A) Initial group; (B) total group. Horizontal line: mean deletion value. (A) COX-positive, 14.71% (SD 24.13%, n = 103); COX-negative, 66.46% (SD 36.66%, n = 109); (B) COX-positive, 14.86% (SD 22.0%, n = 135); COX-negative, 66.64% (SD 36.45%, n = 284). There was no significant difference in the distribution of mtDNA deletion levels for both COX-positive (P = 0.9581) and COX-negative fibers (P = 0.9634), when comparing the initial and total groups.

Figure 4.

Proportion of COX-positive and COX-negative EOM fibers with different mtDNA deletion load.

Figure 5.

(A) Total and (B) wild-type mtDNA copy number/μm² in single 20 μm–thick COX-positive (n = 32) and COX-negative (n = 30) EOM fiber sections (***P < 0.0001).