Accumulation of mitochondrial DNA deletion mutations in aged muscle fibers: evidence for a causal role in muscle fiber loss

Abstract

Although mitochondrial mutation abundance has been recognized to increase in an age-dependent manner, the impact of mutation has been more difficult to establish. Using quantitative polymerase chain reaction, we measured the intracellular abundance of mutant and wild-type mitochondrial genomes along the length of individual laser-captured microdissected muscle fibers from aged rat quadriceps. Aged muscle fibers possessed segmental, clonal intracellular expansions of unique somatically derived mitochondrial DNA (mtDNA) deletion mutations. When the mutation abundance surpassed 90% of the total mitochondrial genomes, the fiber lost cytochrome c oxidase activity and exhibited an increase in succinate dehydrogenase activity. In addition to the mitochondrial enzymatic abnormalities, some fibers displayed abnormal morphology such as fiber splitting, atrophy, and breakage. Deletion mutation accumulation was linked to these aberrant morphologies with more severe cellular pathologies resulting from higher deletion mutation abundance. In summary, our measurements indicate that age-induced mtDNA deletion mutations expand within individual muscle fibers, eliciting fiber dysfunction and breakage.

Figure 1

Typical quantitative PCR data from wild-type primer pair RM13645F- RM13927R. (A) Amplification curves for standards and mtDNA from laser-captured muscle fibers. The baseline curves are no-template control reactions. RFU is Relative Fluorescence Units. (B) Standard Curve generated by amplifying cloned wild-type standards. Log starting quantity of the mtDNA from the laser captured sample is interpolated from the equation of the best-fit line. (C) Melt-curve analysis of real-time amplification products illustrating the specificity of melting temperature and confirming product identity. Plot of the first derivative of fluorescence with regard to temperature.

Figure 2

Standard curves generated by amplifying serial dilutions of wild-type or deletion-specific plasmids.

Figure 3. Fiber One

(A) Serial micrographs showing enzymatic staining for COX and SDH. The scale bar represents 25μm. (B) Morphometric digital reconstruction of fiber one. The color denotes the ETS abnormal phenotype, red is COX⁻/SDH⁺⁺ and orange is COX⁻/SDH^normal. (C) Percentage of mtDNA genomes that are mutant along the length of the abnormal fiber.

Figure 4. Fiber Two

(A) Serial micrographs showing enzymatic staining for COX and SDH. The scale bar is 25μm. (B) Morphometric digital reconstruction of fiber two. The color denotes the ETS abnormal phenotype, red is COX⁻/SDH⁺⁺ and orange is COX⁻/SDH^normal. (C) Percentage of mtDNA genomes that are mutant along the length of the abnormal fiber.

Figure 5. Fiber Three

(A) Serial micrographs staining for morphology with H & E and enzymatic activities for COX and SDH. The muscle fiber splits into three distinct sub-fibers before fusing back together. The deletion mutation was detected throughout 1080μm. The scale bar is 25μm. (B) Morphometric digital reconstruction of fiber three. The color denotes the ETS abnormal phenotype, red is COX⁻/SDH⁺⁺ and orange is COX⁻/SDH^normal. (C) Percentage of mtDNA genomes that are mutant along the length of the abnormal fiber. The unique mtDNA deletion mutation was found at high levels within different “branches” of the same fiber.

Figure 6. Fiber Four

(A) Serial micrographs staining for morphology with H & E and enzymatic activities for COX and SDH. The ETS abnormal region extends for over 700μm and includes a 250μm region where the fiber has ruptured and cannot be detected. The scale bar is 25μm. (B) Morphometric digital reconstruction of fiber four. The color denotes the ETS abnormal phenotype, red is COX⁻/SDH⁺⁺ and orange is COX⁻/SDH^normal. (C) Percentage of mtDNA genomes that are mutant along the length of the abnormal fiber. The same mtDNA deletion mutation is detected across the broken fiber region and the highest levels (>99%) of mutation are found immediately flanking the fiber break.

Figure 7

Absolute quantification of mtDNA genomes. (A) Fiber one. (B) Fiber two. (C) Fiber three. Each branch of the split fiber is represented by a different color line. (D) Fiber four.

Figure 8

Distribution of the mtDNA deletion breakpoints. Red lines represent the 5′ breakpoint. The blue lines represent the 3′ breakpoint. O_L, O_H, light and heavy strand origins of replication respectively. (A) Deletion mutations from individual ETS abnormal fibers. Breakpoints occur within the major arc of the mitochondrial genome adjacent to the light and heavy strand origins of replication. (B) Deletion mutations from tissue homogenates. Breakpoints are scattered throughout the mitochondrial genome, occasionally removing the light strand origin, but never removing the heavy strand origin.