Hypothesis

Mitochondrial succinate exported via SLC25A1 acts as a retrograde signal that directly alters the activity of α‑ketoglutarate‑dependent dioxygenases in the nucleus, driving super‑enhancer remodeling in aged muscle stem cells.

• In young MuSCs, low mitochondrial succinate keeps KDM5B and TET2 active, maintaining balanced H3K4me2/3 and 5‑hmC at myogenic super‑enhancers.
• With age, heteroplasmic mtDNA mutations increase electron‑leak, raising matrix succinate that is exported through SLC25A1.
• Elevated nucleoplasmic succinate competitively inhibits KDM5B/TET2, causing local H3K4me3 accumulation and loss of 5‑hmC at enhancer regions.
• This shifts the binding of pioneer factors such as Pax7 and FOXO3, weakening CTCF‑mediated TAD borders and allowing ectopic enhancer‑promoter contacts at Myf5/Myf6 loci, as observed in aged cells [PMC12740102].
• The same succinate‑driven epigenetic shift has been linked to inflammatory gene programs in other stem‑cell contexts [ELife 62250], and systemic succinate levels rise with age in humans [PubMed 39371074].

Testable predictions

1. Introducing the pathogenic m.5024C>T mtDNA mutation into young MuSCs via cytoplast transfer will increase cytosolic succinate, reduce KDM5B/TET2 activity (measured by α‑KG‑dependent demethylase assays), and reproduce the aged super‑enhancer signature (gain of H3K27ac, loss of TAD insulation) without altering nuclear DNA sequence.
2. Pharmacological blockade of succinate export with SLC25A1 inhibitor (e.g., glutamine analog) or supplementation with cell‑permeable α‑KG will restore KDM5B/TET2 activity, normalize enhancer chromatin, and improve myogenic differentiation in old MuSCs.
3. Conversely, overexpressing mitochondrial succinate dehydrogenase (SDHA) to lower matrix succinate should prevent age‑related enhancer rewiring even in the presence of heteroplasmic mtDNA load.

Falsifiable outcomes

• If succinate levels do not rise in aged MuSCs, or if manipulating succinate export fails to alter KDM5B/TET2 activity or enhancer maps, the hypothesis is refuted.
• If rescuing nuclear α‑KG or inhibiting KDM5B/TET2 phenocopies the aged enhancer state without mitochondrial manipulation, the causal direction would be questioned.

This framework shifts the focus from viewing mtDNA as a passive source of ROS to recognizing specific mitochondrial metabolites as direct epigenetic regulators that can rewrite the nuclear regulatory landscape in stem‑cell aging.