LSP Methylation in Skeletal Muscle and Cross-Tissue Candidate NUMT Signals Reveal Complementary Features of Mitonuclear Aging in Mice

Aging disrupts communication between mitochondria and the nucleus, but compact molecular readouts of this interface remain limited. Here, we examined mitochondrial DNA light-strand promoter (LSP) methylation signals in skeletal muscle across natural-aging and mitochondrial-background models, and independently profiled candidate nuclear mitochondrial DNA insertion (NUMT) junction signals in brain and skeletal muscle across age groups. Natural aging was associated with reduced motor performance, altered skeletal-muscle mitochondrial status, and lower LSP methylation signals. Reciprocal F1 crosses and conplastic mice showed coordinated associations among mitochondrial background, LSP methylation signals, bioenergetic parameters, nuclear mitochondrial-gene expression, and physical performance. In parallel, short-read whole-genome sequencing revealed a non-monotonic candidate NUMT signal pattern, with the highest mean signal at 12 months. A Ptprc-associated candidate mitochondrial-nuclear junction was supported by split-read inference and junction-specific droplet digital PCR in a candidate-positive sample. Together, these findings identify LSP methylation signals and candidate NUMT junction signals as complementary but independently measured molecular features of mitonuclear aging.