Purine Nucleotide Regulation in Mitochondrial Dysfunction and Muscle Wasting

Abstract

Purine nucleotides are critical to cellular energetics and information storage. Changes in the relative amounts of purine (adenine and guanine) nucleotides are mutagenic for DNA in simple model organisms. In mammals, long-standing theories have attributed the onset of mitochondrial DNA (mtDNA) mutations that occur with aging to reactive oxygen species (ROS) based DNA damage, but this has not been shown experimentally. This investigation tested the hypothesis that changes in purine nucleotide content during skeletal muscle atrophy in mice is mutagenic for mtDNA. Our investigation required new analytical techniques to precisely quantitate purine nucleotides. We developed a new ultra performance liquid chromatography (UPLC) method compatible with mass spectrometry (MS). Using this method, we measured guanine and adenine nucleotides after overexpression of AMPD3 and IMPDH2 in C2C12 myotubes and showed substantial changes in the GTP:ATP ratios. Having demonstrated these two enzymes coordinate to control relative nucleotide pools, we then demonstrated using wild-type and AMPD3-deficient C57Bl/6J mice that denervation-induced muscle atrophy results in an AMPD3-dependent increase in GTP. Thus, we established denervation as model of muscle atrophy capable of purine nucleotide dysregulation. To accurately determine the rate to which muscle atrophy was mutagenic to mtDNA, we utilized young POLG mutator mice, which lack the ability to repair mtDNA insults because of a mutation in the mitochondrially restricted DNA polymerase. We demonstrated that denervation of muscles of POLO mice exacerbated mitochondrial dysfunction as measured by decreased NAD+: NADH ratio. This was associated with an increased number of unique mtDNA variants, demonstrating an increased mutational burden of the denervated leg compared to sham. Critically, the vast majority of the variants predicted to be most deleterious were localized in genes encoding NADHdehydrogenase. This investigation demonstrates that AMPD3 and IMPDH2 coordinately control adenine and guanine nucleotide pools in skeletal muscle. Denervation atrophy results in an upregulation of these enzymes, resulting in a shift of the purine nucleotide pool, favoring OTP. Finally, denervation atrophy induces mtDNA mutations in skeletal muscle. Ultimately, this suggests an alternative framework to explain mtDNA-based ageing in skeletal muscle whereby atrophying muscle is a mutagenic environment for mitochondria, leading to accelerated mtDNA insults.