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Abstract

Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.
Author summary Mammalian mitochondria contain multiple copies of the mitochondrial genome (mtDNA), which encodes genes that are essential for the oxidative phosphorylation system. An important feature of mtDNA is that it is evenly distributed throughout the mitochondrial network. Dynamin-related GTPase proteins help control the size and shape of mitochondria by fusion and fission events and are intimately linked to maintenance and distribution of mtDNA. Certain human mutations in mitofusin 2 (MFN2) and optic atrophy protein 1 (OPA1) cause disease phenotypes, such as peripheral neuropathy and optic atrophy, which are often also associated with mtDNA depletion. However, the mechanism whereby MFNs and OPA1 are involved in maintenance of mtDNA is unclear. In this study, we demonstrate that rapid mtDNA synthesis in proliferating tissue-culture cells or cardiomyocytes during post-natal heart development requires mitochondrial fusion. However, the absence of mitochondrial fusion in mouse heart is not associated with mtDNA integrity defects but instead affects the replication of mtDNA. These findings provide direct evidence for the importance of mitochondrial fusion in maintaining mtDNA replication.