Hypothesis

We propose that chronic low‑grade inflammation in aging skeletal muscle activates the IKKβ/NF‑κB axis, leading to IKKβ‑mediated phosphorylation of NFATc4 on serine residues within its regulatory domain. This phosphorylation promotes CRM1‑dependent nuclear export and cytoplasmic sequestration of NFATc4, overriding the RSK2‑driven stabilization that normally retains NFATc4 in the nucleus under resting conditions. Consequently, nuclear NFATc4‑dependent repression of the miR‑23a/atrogene axis is lost, miR‑23a levels drop, and atrogenes such as atrogin‑1 and MuRF1 are derepressed, accelerating muscle atrophy.

Predictions

1. In aged mouse muscle, phospho‑specific antibodies will detect increased IKKβ‑dependent phosphorylation of NFATc4 compared with young muscle.
2. Pharmacological inhibition of IKKβ or expression of a non‑phosphorylatable NFATc4 mutant (Ser→Ala) will restore nuclear NFATc4 localization in aged fibers.
3. Restoring nuclear NFATc4 will elevate miR‑23a expression, suppress atrogene transcription, and attenuate atrophy‑induced loss of muscle mass and force.
4. Conversely, overexpression of a phosphomimetic NFATc4 (Ser→Asp) in young muscle will mimic the aged phenotype: cytoplasmic NFATc4, reduced miR‑23a, and heightened atrogene expression.

Experimental Approach

• Animal models: Young (3‑mo) and aged (24‑mo) C57BL/6 mice; optionally a TNF‑α knockout or IKKβ muscle‑specific knockout.
• Readouts: Western blot for total and phospho‑NFATc4 (custom phospho‑serine antibody), subcellular fractionation followed by immunoblot for NFATc4 in nuclear vs cytoplasmic fractions, immunofluorescence microscopy to quantify nuclear/cytoplasmic NFATc4 ratio.
• miR‑23a and atrogene levels: qRT‑PCR for miR‑23a, atrogin‑1, MuRF1.
• Functional assays: grip strength, in situ tetanic force, muscle cross‑sectional area via histology.
• Interventions: Treat aged mice with IKKβ inhibitor (e.g., BMS‑345541) or deliver AAV9‑NFATc4‑S/A or NFATc4‑S/D constructs; assess rescue.

Falsifiability

If aged muscle shows no increase in IKKβ‑dependent NFATc4 phosphorylation, or if blocking IKKβ fails to restore nuclear NFATc4 and miR‑23a levels, the hypothesis would be refuted. Likewise, if phosphomimetic NFATc4 does not reproduce the aged atrophy phenotype, the proposed mechanism would be insufficient.

Significance

Linking inflammatory signaling to the RSK2‑NFATc4 axis reveals a novel node where cytokine‑driven kinases override basal protective NFATc4 activity, providing a mechanistic bridge between inflammaging and sarcopenia. Targeting this interaction could preserve NFATc4‑mediated miR‑23a repression and mitigate age‑related muscle loss.