Enhanced ROS Production in Mitochondria from Prematurely Aging mtDNA Mutator Mice

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Irina G. Shabalina^1,a, Daniel Edgar^1,b, Natalia Gibanova^1,c, Anastasia V. Kalinovich^1,d, Natasa Petrovic^1,e, Mikhail Yu. Vyssokikh^1,f, Barbara Cannon^1,g, and Jan Nedergaard^1,h*

¹Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, SE-106 91 Sweden

Received November 17, 2023; Revised January 20, 2024; Accepted January 21, 2024

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An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process (“the mitochondrial hypothesis of aging”). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower – not higher – than in wildtype, thus apparently invalidating the “mitochondrial hypothesis of aging”. We have here re-examined ROS production rates in mtDNA-mutator mice mitochondria. Using traditional conditions for measuring ROS (succinate in the absence of rotenone), we indeed found lower ROS in the mtDNA-mutator mitochondria compared to wildtype. This ROS mainly results from reverse electron flow driven by the membrane potential, but the membrane potential reached in the isolated mtDNA-mutator mitochondria was 33 mV lower than that in wildtype mitochondria, due to the feedback inhibition of succinate oxidation by oxaloacetate, and to a lower oxidative capacity in the mtDNA-mutator mice, explaining the lower ROS production. In contrast, in normal forward electron flow systems (pyruvate (or glutamate) + malate or palmitoyl-CoA + carnitine), mitochondrial ROS production was higher in the mtDNA-mutator mitochondria. Particularly, even during active oxidative phosphorylation (as would be ongoing physiologically), higher ROS rates were seen in the mtDNA-mutator mitochondria than in wildtype. Thus, when examined under physiological conditions, mitochondrial ROS production rates are indeed increased in mtDNA-mutator mitochondria. While this does not prove the validity of the mitochondrial hypothesis of aging, it may no longer be said to be negated in this respect. This paper is dedicated to the memory of Professor Vladimir P. Skulachev.

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KEY WORDS: mtDNA mutator mice, ROS production, aging, succinate, membrane potential, oxidative phosphorylation

DOI: 10.1134/S0006297924020081

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