Hypothesis

Transient OSK expression administered at the circadian peak of NAD+ and SIRT1 activity (approximately ZT6 in mice) will produce a stronger mesenchymal-to-epithelial transition (MET) and lower treatment‑induced apoptosis than the same pulse given at the trough (ZT18), leading to greater epigenetic resetting and improved functional outcomes.

Rationale

Partial reprogramming restores youthful epigenetic states while preserving cell identity, a process linked to MET and improved mitochondrial oxidative phosphorylation (OXPHOS) https://www.liebertpub.com/doi/10.1089/cell.2023.0072. SIRT1, a NAD+-dependent deacetylase, peaks during the early active phase and promotes epithelial programs by deacetylating SNAI1 and β‑catenin, thereby inhibiting mesenchymal drivers https://pmc.ncbi.nlm.nih.gov/articles/PMC10909732/. NAD+ levels themselves oscillate with the circadian clock, influencing SIRT1 activity and downstream histone acetylation at mesenchymal gene promoters. By aligning OSK pulses with high NAD+/SIRT1, we hypothesize that the repressive chromatin environment at mesenchymal loci is reinforced, enhancing MET while limiting the stress‑response pathways that trigger apoptosis.

Predictions

1. Mice receiving a 4‑day OSK pulse at ZT6 will show a ≥30 % increase in epithelial markers (E‑cadherin, Krt8/18) and a ≥30 % decrease in mesenchymal markers (Vimentin, α‑SMA) compared with ZT18‑treated controls, measured by immunofluorescence and qPCR.
2. Apoptosis, assessed by cleaved caspase‑3 staining, will be ≤10 % of total cells in ZT6 groups versus ≥20 % in ZT18 groups.
3. Epigenetic age, estimated by liver and heart Horvath clocks, will be reduced by an additional 10‑15 % relative to baseline in ZT6 mice.
4. Mitochondrial spare respiratory capacity will be higher in ZT6 tissues, reflecting synergistic OXPHOS upregulation.

Experimental Design

• Use aged (20‑month‑old) C57BL/6J mice with doxycycline‑inducible OSK AAV9 system (systemic delivery).
• Cohorts: (a) ZT6 OSK pulse (4 days doxycycline), (b) ZT18 OSK pulse, (c) vehicle controls at each time point.
• Collect liver and heart tissue 24 h after pulse completion for flow cytometry, immunofluorescence, RNA‑seq, and metabolomics.
• Perform Seahorse XF analysis on isolated cardiomyocytes and hepatocytes to assess basal respiration, ATP production, and spare capacity.
• Statistical analysis: two‑way ANOVA (time × treatment) with post‑hoc Tukey; n = 8 per group to achieve 80 % power for detecting a 20 % difference.

Potential Confounds

• Circadian variability in doxycycline metabolism could affect OSK expression levels; we will measure OSK transgene mRNA at each time point to normalize.
• Stress from handling at different zeitgeber times may independently influence apoptosis; we will include handling‑only controls.
• Sex differences in circadian NAD+ rhythms; we will run parallel experiments in male and female mice and analyze separately.

If the ZT6 cohort fails to show superior MET or reduced apoptosis relative to ZT18, the hypothesis would be falsified, indicating that NAD+/SIRT1 timing does not modulate partial reprogramming outcomes.