OSK-mediated epigenetic rejuvenation selects for low-heteroplasmy mitochondria, positioning mtDNA quality as the driver of methylation clock reversal

OSK selects for low-heteroplasmy mitochondria

Hypothesis: OSK-induced partial reprogramming drives epigenetic age reversal by eliminating cells harboring high mtDNA heteroplasmy, thereby linking mitochondrial genome quality to methylation clock read‑outs.

Mechanistic rationale

• OSK triggers a transient pluripotent state that activates mitophagy and mitochondrial biogenesis pathways [3].
• Cells with high heteroplasmy produce excess ROS and reduced ATP, activating stress‑sensitive kinases (e.g., AMPK, p38 MAPK) that favor apoptosis or differentiation rather than reprogramming [4].
• Consequently, the surviving iPSC‑like population becomes enriched for mitochondria with low mutation load, shifting the bulk heteroplasmy distribution downward [2].
• Reduced heteroplasmy normalizes TCA‑cycle metabolites (acetyl‑CoA, α‑ketoglutarate) that serve as cofactors for TET enzymes and HDACs, allowing the methylation clock to reset [1].

Testable predictions

1. In OSK-treated aged mice, single‑cell mtDNA heteroplasmy will show a bimodal shift: a loss of cells >60% heteroplasmy and a gain of cells <20% heteroplasmy, correlating with regional epigenetic age reduction measured by bisulfite sequencing.
2. Pharmacological inhibition of mitophagy (e.g., with Mdivi‑1) during OSK expression will block heteroplasmy clearing and attenuate epigenetic age reversal, despite normal OSK transduction.
3. Forced expression of a ROS‑scavenging enzyme (mito‑CAT) in high‑heteroplasmy fibroblasts will rescue their reprogramming efficiency to levels seen in low‑heteroplasmy cells, indicating ROS as the selection barrier.

Experimental approach

Generate heteroplasmic mouse models carrying a pathogenic mtDNA point mutation (e.g., mtDNA^Mut) at defined loads [6]. Administer inducible OSK via AAV9 in tissues (brain, liver, muscle) and sort cells by heteroplasmy using droplet‑digital PCR or single‑cell mtDNA sequencing. Perform parallel epigenetic clock profiling (e.g., Horvath mouse clock) on sorted fractions. Include control groups: OSK + mitophagy inhibitor, OSK + mito‑CAT, and OSK alone.

Falsifiability

If OSK rejuvenation occurs without a measurable reduction in high‑heteroplasmy cells, or if blocking mitophagy fails to impair epigenetic clock reversal, the hypothesis would be falsified, indicating that OSK acts primarily through nuclear chromatin remodeling independent of mtDNA quality.