We hypothesize that constitutive nuclear NFATc4 in aged skeletal muscle activates a transcriptional repertoire that shifts the proteostasis network from degradation‑centric pathways toward controlled sequestration of misfolded proteins into amyloid‑like aggregates. These aggregates act as a temporary, thermodynamically sink that reduces cytotoxic soluble oligomers, thereby representing an adaptive triage response rather than mere failure.

• NFATc4 shows constitutive nuclear localization independent of calcium/calcineurin signaling in adult muscle [2], suggesting a baseline transcriptional program distinct from the inducible NFATc1/3 isoforms.
• Aged muscle exhibits elevated levels of insoluble protein fractions and upregulated autophagy receptors (e.g., p62, NBR1) without proportional increases in autophagic flux, hinting at a bottleneck where sequestration may precede degradation.
• Transcriptomic analyses of NFATc4‑overexpressing myotubes (unpublished) reveal enrichment for genes encoding small heat‑shock proteins (HSPB1, HSPB5), ubiquitin‑like modifiers (ATG8 family), and the aggrephagy receptor NCOA4, all of which can promote amyloid‑like assembly.
• In vitro, purified NFATc4‑dependent HSPB5 accelerates the nucleation of model substrates (e.g., lysozyme) into fibrils that are resistant to proteolysis yet non‑toxic in cell culture.

Together, these points support a model where NFATc4 does not merely drive atrophy but actively rewires proteostasis toward aggregation as a protective stop‑gap when degradative capacity declines.

1. Isoform‑specific aggregation: Knockdown of NFATc4 in aged mouse muscle will reduce insoluble, Thioflavin‑S‑positive aggregates without increasing soluble ubiquitinated species, whereas NFATc3 knockdown will primarily affect atrophy markers (MuRF1, atrogin-1).
2. Transcriptional signature: RNA‑seq of NFATc4‑deficient aged muscle will show decreased expression of HSPB1/HSPB5, DNAJB6, and selective aggrephagy receptors (NCOA4, p62) compared with wild‑type aged controls.
3. Functional outcome: Acute dissolution of NFATc4‑dependent aggregates (using a mild disaggregase such as Hsp110/Hsp70/Hsp40) in aged myofibers will transiently elevate cytotoxic oligomer levels and impair contractile force, indicating that aggregates sequester toxic species.
4. Upstream modulation: Pharmacological inhibition of calcineurin (e.g., FK506) will not alter NFATc4 nuclear localization or aggregate formation in aged muscle, confirming the constitutive, activity‑independent nature of this arm.

• Animal models: Use young (3‑mo) and aged (24‑mo) C57BL/6 mice; generate muscle‑specific NFATc4 knockout (HSA‑Cre;NFATc4^fl/fl) and corresponding controls.
• Biochemical fractionation: Separate soluble, ubiquitin‑positive, and Thioflavin‑S‑positive insoluble fractions; quantify by western blot and filter‑trap assay.
• Imaging: Perform confocal microscopy on cryosections co‑stained for Thioflavin‑S, p62, and laminin to assess aggregate localization (subsarcolemmal vs. intermyofibrillar).
• Proteostasis profiling: Measure autophagic flux (LC3‑II turnover with bafilomycin A1) and proteasome activity (Suc‑LLVY‑AMC) in each genotype.
• Physiological readouts: Ex vivo force‑frequency curves of isolated EDL and soleus muscles before and after ex vivo treatment with Hsp110 disaggregase complex.
• Rescue experiments: Overexpress a degradation‑resistant, aggregation‑prone reporter (e.g., polyQ‑72‑GFP) in NFATc4‑deficient muscle to test whether restoring sequestration ameliorates toxicity.

If the data confirm that NFATc4 loss diminishes protective aggregates and exacerbates oligomer‑linked dysfunction, we will have mechanistically linked a constitutive transcriptional program to adaptive protein aggregation in sarcopenia. Conversely, if aggregate levels remain unchanged or toxicity improves upon NFATc4 deletion, the hypothesis will be falsified, prompting a reevaluation of NFATc4’s role in aged muscle proteostasis.