Authors: 
J.W. Pak, J.M. Aiken
Category: 
Poster
Conference: 
Abstract: 

Mitochondrial DNA (mtDNA) deletion mutations can accumulate to levels that result in electron transport system (ETS) abnormal regions (cytochrome c oxidase negative, succinate dehydrogenase hyperreactive) in skeletal muscle fibers of aged rats. This ETS abnormal phenotype was primarily observed in type II fibers. MtDNA sequence analysis of microdissected portions of single fibers containing these ETS abnormal regions identified deletion mutational hotspots near cyt b and COX subunit genes in the major arc region of the mitochondrial genome.

Deletion mutations were identified in ETS normal fibers, albeit at low abundance. In ETS normal type II fibers, the deletion mutations were exclusively localized to the major arc region of the mitochondrial genome. In type I fibers, however, deletion mutations were also identified which removed the light strand origin. These large deletions were more prominent in soleus, which is comprised mainly of type I fibers and rarely displays ETS abnormal phenotypes.

Real-time quantitative PCR of mtDNA in single cells showed very low levels (0.2 - 2%) of deletion mutations in aged normal fibers regardless of the fiber type, while over 80% mutation load in ETS abnormal fibers. Interestingly, there was no significant difference in the level of the wild-type mtDNA genome between type I and type II fibers. In spite of the similar wild-type DNA content, histochemical staining for COX and SDH enzyme activities displayed higher enzymatic activities in type I/IIa fibers than in type IIb fibers. Immunofluorescence analysis for COX and SDH protein subunits confirmed that the higher enzymatic activities in type I/IIa fibers were due to higher protein levels. The elevated cellular activities of type I fibers may be preventing or delaying the ETS abnormal phenotypic expression of mtDNA deletion mutations, resulting in fiber type-specific distributions of the ETS abnormality.

Keywords (Optional): 
mitochondrial DNA
deletion mutations
ETS abnormality
fiber type