High mutational burden in the mtDNA control region from aged muscles: a single-fiber study
Introduction
Human mitochondrial DNA (mtDNA) is a circular double-stranded DNA of 16,569 bp in size. The genome contains genes coding for 13 polypeptides involved in respiration and oxidative phosphorylation, 2 rRNAs and a set of 22 tRNAs that are essential for protein synthesis of the mitochondria. In contrast to the nuclear DNA, mtDNA is a naked compact DNA molecule without introns and is replicated at a much higher rate without an efficient DNA repair mechanism. Therefore, mtDNA is more vulnerable to attacks by reactive oxygen species and free radicals that are generated by electron leak of the respiratory chain of mitochondria [38]. In the past few years a number of point mutations, deletions and insertions have been found in mtDNA from patients with specific neuromuscular diseases [4]. Several mtDNA deletions have also been identified in various tissues of old humans [12], [13], [14]. Since mutant mtDNA, or mitochondria containing high levels of mutant mtDNA, may, in certain situations, have a replicative advantage within the cell, they might, with time, cause a specific biochemical effect [44]. Somatic point mutations and deletions in mtDNA have been established to increase in an age-dependent manner in humans: the accumulation of deleterious alterations in the mitochondrial genome has been proposed to be the major cause of the respiratory function decline during human ageing process [15], [41].
Several reports show that skeletal muscle develops a histochemical mosaic during the process of normal ageing; in particular, the frequency of cytochrome c oxidase (COX) deficient muscle fibers is highly dependent on the age of the subject [6], [31]. It has been hypothesised that multiple mtDNA rearrangements occurring in muscle tissue from aged individuals in a generally low frequency (<1% of total mtDNA) might correlate with the observed progressive decrease in COX activity [25] although these low levels have raised questions about their direct functional significance.
The main control region of mtDNA (the D-loop and adjacent transcription promoters (DLP)) [22] represents the most variable portion of the human mitochondrial genome and may contain several somatic heteroplasmic point mutations [8], [23], [30]. Previous findings demonstrate the presence of specific control region mtDNA mutations in skin fibroblasts from normal individuals above 65 years of age and from adult Down’s syndrome subjects several years in advance [17], [30]. In particular, the T to G transversion at nucleotide 414 shows a striking age-related accumulation reaching high levels of heteroplasmy (up to 50%) in some fibroblast cell lines. Using a very sensitive method, low levels of T414G mutation have been described to accumulate also in skeletal muscles but not in brain [32], [40]. Recently, a large-scale analysis of mtDNA from biopsied or autopsied muscle samples has shown the ageing-dependent accumulation of two other mutations, A189G and T408A, having high levels of heteroplasmy (up to 64% of muscle mtDNA molecules) [43]. These mutations have not been found in fibroblasts from aged subjects (T408A) or were present only in three subjects in very low amounts (A189G). At present the role and the phenotypic effects linked to all these somatic age-related mutations are unknown.
With the aim to investigate the relationship between the presence of control region mutations and the muscle fiber COX activity, we assessed the muscle tissue distribution of somatic point mutations and the total mutational burden in the mtDNA control region from four unrelated elderly individuals without muscle disease.
Section snippets
Histochemistry
We have analysed four biceps brachii muscle specimens from individuals deemed to be free of neuro-muscular disorders aged 81, 94, 96 and 97 years. Indeed, they had no neuromuscular signs or symptoms, which was confirmed by neurological examination. Also, serum creatine kinase levels were normal. The skeletal muscle specimens, obtained during orthopaedic surgery after informed consent, showed no evidence of neuro-muscular pathology.
Eight-micrometer-thick cryostat cross-sections were stained for
Results
Using a histochemical double-staining for COX and SDH we isolated muscle fibers from 4 unrelated aged subjects. A very low percentage of COX negative fibers was present in all biopsy specimens (range 2–6 COX negative fibers per 1000 fibers counted); an abnormal proliferation of mitochondria reflected by increased SDH staining, was evident in a few fibers: double staining showed that all ragged-red fibers were COX negative. A total of 24 single fibers were included in this study: we collected 9
Discussion
Ageing is a very complex physiological phenomenon characterized by energy deficits. Among the different theories about its origin, the “mitochondrial theory of ageing” hypothesises that the accumulation of somatic mutations during life due to the cellular oxidative stress on mitochondrial DNA (mtDNA) might be considered a primary cause in the ageing process [20]. MtDNA has a high mutation rate; several heteroplasmic mtDNA defects, including point mutations, deletions and duplications have been
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G. Scarlato recently died.