Hypothesis

The selective, hierarchical nature of autophagy is dictated by a mechanosensitive phosphorylation cycle of the autophagy receptor p62/SQSTM1. In young, mechanically active tissues (e.g., skeletal muscle), low‑tension conditions promote p62 dephosphorylation at Ser403 (via PP2A), increasing its affinity for ubiquitin‑tagged, mechanically unfolded cytoskeletal proteins (FLNC, CAPZB) and favoring their recruitment into BAG3‑dependent chaperone‑assisted selective autophagy (CASA). Conversely, elevated mechanical tension or stress‑activated kinases (TBK1, IKKε) phosphorylate p62‑Ser403, reducing ubiquitin binding but enhancing LC3 interaction via exposed LIR domains, thereby biasing p62 toward organellar cargos (ER, mitochondria) and promoting ER‑phagy/mitophagy. With age, chronic low‑grade inflammation and reduced phosphatase activity shift the p62 equilibrium toward a hyper‑phosphorylated state that sequesters ubiquitin chains without efficiently recruiting LC3, creating a “dead‑end” sink that starves CASA of receptor availability while sustaining organellar autophagy flux. This misrouting explains the paradox of increased BAG3 expression yet impaired CASA clearance in aged muscle and predicts that restoring p62 dephosphorylation will re‑establish the CASA‑first hierarchy and ameliorate age‑related Z‑disk pathology.

Mechanistic Reasoning

1. Phosphorylation regulates p62’s dual affinity – Structural studies show Ser403 phosphorylation induces a conformational change that weakens the ubiquitin‑associated (UBA) domain while stabilizing the LC3‑interacting region (LIR) (1). Mechanical stretch activates PP2A at the Z‑disk, locally dephosphorylating p62 to favor ubiquitin capture of tension‑exposed FLNC/CAPZB.
2. Competitive receptor limitation – p62 is a shared scaffold for CASA, ER‑phagy (2), and mitophagy (3). When phosphorylated p62 accumulates, it binds ubiquitinated cargo but fails to nucleate autophagosomes, effectively titrating away ubiquitin chains from CASA while still permitting LC3 recruitment via alternative receptors (NBR1, OPTN) for organelles.
3. Age‑related phosphatase decline – Aging reduces PP2A activity in skeletal muscle (4), shifting the p62 phospho‑state toward the inactive, ubiquitin‑sequestering form. This aligns with observed BAG3 upregulation but persistent aggregate accumulation.
4. Pathological read‑out – Failure to clear FLNC/CAPZB triggers myofibrillar myopathy and dilated cardiomyopathy (5), whereas ER‑phagy/mitophagy remain active, contributing to the observed selectivity shift during atrophy.

Testable Predictions

• Prediction 1: In young mouse tibialis anterior, mechanical stretch (in vivo overload) decreases p62‑Ser403 phosphorylation at the Z‑disk and increases co‑immunoprecipitation of p62 with FLNC; phosphatase inhibition (okadaic acid) abolishes this effect.
• Prediction 2: Aged mice (24 mo) show elevated p62‑Ser403‑p signal, reduced p62‑FLNC binding, and increased p62‑ER‑phagy adaptor (FAM134B) interaction; transgenic expression of a phospho‑dead p62‑S403A rescues FLNC clearance without altering total BAG3 levels.
• Prediction 3: Pharmacological activation of PP2A (FTY720 low dose) in aged muscle restores p62 dephosphorylation, improves CASA flux (measured by mCherry‑GFP‑LC3‑FLNC reporter), and reduces cardiomyopathy markers (fibrosis, ejection fraction decline) relative to vehicle.
• Prediction 4: Expressing a phospho‑mimetic p62‑S403D in young muscle reproduces the aged phenotype: impaired FLNC degradation, compensatory increase in ER‑phagy/mitophagy flux (mt‑Keima, ER‑phagy reporter), and induction of myopathic histology.

Experimental Outline

1. In vivo mechanical manipulation – Overload synergist ablation or treadmill running in WT and PP2A‑heterozygous mice; collect muscle for western blot (p62‑Ser403‑p), proximity ligation (p62‑FLNC), and immunofluorescence (LC3 colocalization).
2. Genetic rescue – AAV9‑mediated muscle‑specific expression of p62‑S403A or S403D in young and aged mice; assess autophagic flux (lysosomal inhibition with chloroquine), organelle turnover (mito‑Keima, ER‑phagy reporter), and functional outcomes (grip strength, echocardiography).
3. Pharmacological modulation – Treat aged mice with FTY720 (0.3 mg/kg/day) or vehicle for 4 weeks; measure p62 phosphorylation state, CASA substrate load (Ub‑FLNC immunoblot), and cardiac pathology.
4. Human relevance – Analyze biopsied human sarcopenic vs. control muscle for p62‑Ser403‑p, BAG3, and FLNC aggregation; correlate with clinical measures of muscle strength and cardiac function.

If restoring p62 dephosphorylation selectively reinstates CASA precedence and alleviates Z‑disk pathology without globally inhibiting autophagy, the hypothesis is supported. Conversely, if manipulating p62 phosphorylation fails to shift substrate preference or rescue pathology, the hypothesis is falsified, indicating that other mechanisms (e.g., receptor expression changes, ubiquitin chain topology) dominate the age‑related hierarchy breakdown.