Digital PCR Quantitation of Muscle Mitochondrial DNA: Age, Fiber Type, and Mutation-Induced Changes

Abstract

Definitive quantitation of mitochondrial DNA (mtDNA) and mtDNA deletion mutation abundances would help clarify the role of mtDNA instability in aging. To more accurately quantify mtDNA, we applied the emerging technique of digital polymerase chain reaction to individual muscle fibers and muscle homogenates from aged rodents. Individual fiber mtDNA content correlated with fiber type and decreased with age. We adapted a digital polymerase chain reaction deletion assay that was accurate in mixing experiments to a mutation frequency of 0.03% and quantitated an age-induced increase in deletion frequency from rat muscle homogenates. Importantly, the deletion frequency measured in muscle homogenates strongly correlated with electron transport chain-deficient fiber abundance determined by histochemical analyses. These data clarify the temporal accumulation of mtDNA deletions that lead to electron chain-deficient fibers, a process culminating in muscle fiber loss.

Mitochondrial DNA alterations in aged macrophage migration inhibitory factor-knockout mice

“…Frozen muscle samples were powdered under liquid nitrogen using a mortar and pestle. Approximately 25mg of powdered muscle was used in DNA isolation as previously described (Herbst et al, 2017). Quality control of the DNA was performed with UV spectrometry and gel electrophoresis.…”

“…Quality control of the DNA was performed with UV spectrometry and gel electrophoresis. A 5-prime nuclease cleavage assay and chip-based digital PCR (dPCR) was used to quantitate copy numbers for nuclear DNA (nDNA), total mtDNA, and mtDNA deletions with specific primer/probe sets for each as previously described (Herbst et al, 2017). Samples were diluted to the manufacturer's recommended target range (200 to 2,000 copies per microliter) in 17.5 microliter reactions using QuantStudio 3D Digital PCR Master Mix v2 (ThermoFisher; Waltham, MA) and loaded onto Quantstudio 3D digital PCR 20K Chip (Version 2, ThermoFisher; Waltham, MA).…”

Regulation of skeletal muscle recovery

“…Data on TEE in COPD are scarce and sometimes contradicting, which may be related to the use of different methodology or patient characteristics. Based on the available evidence, it seems that in particular the emphysematous phenotype is prone to increased TEE, as high values are more often observed in patients with low carbon monoxide diffusing capacity [117][118][119]. This is in line with a positive effect of lung volume-reduction surgery on body weight in emphysema, although surgical intervention might not only decrease energy requirements but also improve dietary intake by alleviating dyspnea [120].…”

“…Numerous studies have shown that REE is raised [121][122][123]. This is more prevalent in emphysema [124,125], during acute exacerbations [126], and appears inversely correlated with forced expiratory volume in 1 s when comparing different studies [118,119,122,127]. Highest values are found among weight-losing patients [122].…”

“…Importantly, mtDNA is more sensitive than nDNA to oxidative stress [118], which is a well-known driver of muscle pathology in COPD. Furthermore, impaired mtDNA quality has been linked to a decrease in oxidative phenotype, a change in fiber type, and sarcopenia [119][120][121], which are all characteristic of COPD-induced skeletal muscle pathology. Previous reports suggest that satellite cells support the restoration of mtDNA quality [122,123], which may potentially occur via the direct donation of satellite cell mtDNA to the muscle fiber [124,125], and may be particularly important for muscle maintenance in COPD patients.…”