Hypothesis: Mechanosensitive protein phosphatase 2A (PP2A) directly dephosphorylates BAG3 in response to integrin‑mediated tension, thereby promoting its incorporation into the CASA complex and driving selective autophagy of damaged sarcomeric proteins (see BAG3-mediated CASA). This phosphatase activity is modulated by estrogen receptor‑α (ERα) signaling, which sustains PP2A activity in female cardiomyocytes and skeletal muscle fibers, explaining the preserved BAG3‑CASA function observed in females with dilated cardiomyopathy (Sex‑specific BAG3 preservation). Loss of integrin‑PP2A coupling or ERα‑dependent PP2A regulation leads to hyperphosphorylated BAG3, impaired CASA flux, and accumulation of ubiquitinated filamin‑C, contributing to proteostatic collapse during aging (Aging shift to macroautophagy).

Predictions

1. In primary human myotubes subjected to cyclic stretch, pharmacological inhibition of PP2A (e.g., with okadaic acid at low nM concentrations) will block exercise‑induced BAG3 dephosphorylation and reduce LC3‑II conversion, whereas PP2A activation (e.g., via FTY720) will enhance CASA flux even without mechanical stimulus (Exercise triggers BAG3 dephosphorylation).
2. Co‑immunoprecipitation will reveal a ternary complex of integrin β1, PP2A catalytic subunit, and BAG3 that forms within 5 min of strain application; this interaction will be absent in cells expressing a BAG3 mutant lacking the predicted PP2A docking motif (S/T‑X‑X‑R).
3. Female mice lacking ERα specifically in skeletal muscle will show male‑like phenotypes: decreased BAG3 dephosphorylation after resistance‑type training, increased p‑BAG3 levels, and accelerated accumulation of insoluble myofilament proteins with age.
4. Overexpressing a phospho‑deficient BAG3 (S/T→A) in male mdx mice will rescue CASA‑dependent clearance of dystrophin‑associated protein aggregates and improve contractile force, mimicking the protective effect seen in females.

Experimental Approaches

• Use phospho‑specific antibodies against BAG3 serine/threonine residues identified by mass spectrometry to monitor dephosphorylation dynamics in live‑cell FRET reporters.
• Apply proximity ligation assay (PLA) to detect integrin‑PP2A‑BAG3 complexes in fixed tissue sections from young and old male/female hearts (Sarcomere localization of BAG3).
• Conduct autophagy flux assays (mCherry‑GFP‑LC3) in isolated fibroblasts treated with PP2A modulators and subjected to substrate stretch using Flexcell plates.
• Perform sex‑stratified proteomics of soluble versus insoluble sarcomeric fractions from aged human donor myocardium to correlate BAG3 phosphorylation status with aggregate load.

Falsifiability If PP2A inhibition does not alter BAG3 phosphorylation status under mechanical load, or if ERα loss fails to reproduce the male‑specific decline in CASA activity, the hypothesis would be refuted. Likewise, absence of a detectable integrin‑PP2A‑BAG3 ternary complex would challenge the proposed mechanosensor mechanism.