Hypothesis

In aged skeletal muscle, persistent low‑grade inflammation sustains calcineurin activity, keeping NFATc4 predominantly nuclear. Rather than driving atrophy genes, this NFATc4 state redirects transcription toward a set of low‑complexity, amyloid‑prone proteins that undergo regulated phase separation and mature into functional amyloid‑like aggregates. These assemblies act as a controlled depot for irreversibly damaged polypeptides, converting proteotoxic disorder into a thermodynamically stable, inert state. Dissolving these aggregates removes a protective sink, releasing sequestered toxic species and accelerating proteostatic collapse.

Mechanistic Rationale

1. Inflammation‑calcineurin‑NFATc4 axis – Chronic TNF‑α/IL‑6 signaling elevates intracellular Ca²⁺, activating calcineurin. In aged muscle, NFATc4 remains nuclear (see constitutive nuclear NFATc4 in fast‑twitch fibers) but does not induce classic atrophy programs because co‑factor availability shifts (e.g., reduced FOXO, increased HDAC).
2. Transcriptional reprogramming – Nuclear NFATc4 partners with PGC‑1α and AP‑1 to up‑regulate genes encoding amyloid‑ogenic substrates such as cystatin C, α‑synuclein‑like low‑complexity domains, and specific RNA‑binding proteins (see NFAT isoform control of activity‑dependent fiber‑type genes). Their promoters contain NFAT response elements validated in muscle.
3. Phase separation to amyloid – The expressed proteins contain prion‑like domains that undergo stress‑induced phase separation, forming membraneless condensates. Oxidative stress in aging promotes cross‑β maturation within these condensates, yielding functional amyloids (see amyloid as a storage depot for misfolded proteins).
4. Protective sequestration – Functional amyloids bind irreversibly oxidized, ubiquitinated proteins, reducing soluble oligomer load. This aligns with the seed idea that aggregation represents the proteome’s last ordered state rather than random garbage.
5. Consequences of dissolution – Pharmacological or genetic disruption of amyloid formation (e.g., Congo red, CRISPR knockout of the amyloid‑ogenic substrate) releases sequestered cargo, increasing soluble toxic species and exacerbating muscle weakness.

Testable Predictions

• Prediction 1: In aged mouse tibialis anterior, NFATc4 nuclear intensity correlates positively with Thioflavin‑T‑positive amyloid signal and inversely with soluble ubiquitin‑positive oligomers.
• Prediction 2: Muscle‑specific NFATc4 knockout in 24‑month‑old mice will reduce amyloid‑like deposits (filter‑trap assay) and elevate soluble toxic oligomers, leading to greater decline in grip strength and treadmill endurance compared with wild‑type aged controls.
• Prediction 3: Constitutively active NFATc4 (caNFATc4) expressed in young mice will induce amyloid formation without hypertrophy; these mice will show resistance to induced proteotoxic stress (e.g., arsenite treatment) as measured by reduced protein carbonyls and preserved contractile force.
• Prediction 4: Treating aged mice with an amyloid‑disaggregase (e.g., Hsp104 overexpression) will diminish NFATc4‑dependent amyloid deposits and precipitate functional decline, confirming the adaptive role of the aggregates.

Experimental Approach

1. Generate MCK‑Cre;NFATc4^fl/fl (knockout) and MCK‑Cre;caNFATc4 (overexpressor) lines; assess at 6, 18, and 24 months.
2. Quantify nuclear NFATc4 (immunofluorescence), amyloid load (Thioflavin‑T, filter‑trap), soluble oligomers (dot‑blot for ubiquitin), and proteasome activity.
3. Perform functional assays: grip strength, in‑situ tetanic force, voluntary wheel running.
4. Rescue experiments: administer amyloid‑stabilizing peptide (e.g., KLVFF) to knockouts to test whether restoring amyloid deposition rescues function.

Falsifiability

If NFATc4 loss or gain does not alter amyloid burden, or if modulating amyloid levels fails to affect soluble toxin levels and muscle function in the predicted directions, the hypothesis is refuted. Conversely, confirmation would reposition NFATc4 from a purely hypertrophic regulator to a central node in adaptive proteostasis during sarcopenia.

References

• Constitutive nuclear NFATc4 in fast‑twitch fibers: https://pmc.ncbi.nlm.nih.gov/articles/PMC2726382/
• NFAT isoform control of activity‑dependent fiber‑type genes: https://pmc.ncbi.nlm.nih.gov/articles/PMC4234965/
• Amyloid as a storage depot for misfolded proteins: https://doi.org/10.1073/pnas.0812911106
• Inflammation‑driven calcineurin signaling in muscle: https://doi.org/10.1101/gr.240093.118
• miR‑23a downregulation attenuates calcineurin/NFAT during atrophy: https://pubmed.ncbi.nlm.nih.gov/24336651/
• Recent preprint on NFAT‑dependent transcriptional remodeling in aged muscle: https://doi.org/10.1101/2025.05.06.650963/ }