Abstract Body: Introduction
Barlow disease (BD), a severe clinical manifestation of mitral valve prolapse, is marked by extensive myxomatous transformation and pronounced mitral regurgitation. Despite its high prevalence, no effective pharmacological strategies exist. A defining feature is the emergence of senescent myofibroblasts, derived from quiescent valvular interstitial cells (VICs), which degrade the extracellular matrix through the senescence-associated secretory phenotype (SASP). Autophagy, an evolutionarily conserved catabolic mechanism integral to cellular homeostasis, remains insufficiently explored in BD pathophysiology.

Hypothesis
We sought to investigate the role of autophagy in modulating VIC senescence in the pathogenesis of BD.

Methods and Results
Primary VICs were isolated from normal mitral valves (n=6) and myxomatous BD valves (n=12). Autophagic flux and autophagosome–lysosome fusion were evaluated via mCherry-eGFP-LC3B reporter. Senescent VICs exhibited marked reductions in autophagosomes (p=0.0038), autolysosomes (p=0.0015), and autolysosome ratios (p=0.0156). Scanning transmission electron microscopy corroborated these data, revealing fewer double-membrane autophagosomes (p=0.0216) and single-membrane autolysosomes (p=0.0378) in senescent cells. Mechanistic analyses (immunoblotting, qRT-PCR, immunofluorescence) indicated that mTOR-dependent autophagy induction and ATG gene overexpression mitigated senescence, evidenced by lower CDKN1A/p21^CIP1 (p=0.0049) and CDKN2A/p16^INK4A (p=0.0167), decreased senescence-associated β-galactosidase activity (p=0.034), and reduced secretion of key SASP mediators (TGFβ1, IL6, TNFα). In contrast, ATG knockdown upregulated CDKN1A/CDKN2A and amplified SASP, whereas reintroduction of ATG genes reversed senescence. Immunofluorescence colocalization confirmed that CDKN1A and CDKN2A were selectively sequestered within autophagosomes and lysosomes upon mTOR inhibition or proteasomal blockade. Co-immunoprecipitation implicated SQSTM1/p62 as the principal receptor facilitating autophagic clearance of these cell cycle inhibitors. Single-molecule super-resolution imaging verified robust aggrephagy-mediated degradation of cytoplasmic CDKN1A/CDKN2A aggregates.

Conclusions
These findings provide unprecedented evidence that autophagy antagonizes senescence-driven myxomatous VIC degeneration, thereby identifying a promising therapeutic avenue for BD and other age-related degenerative cardiac pathologies.