Proteomic analysis of early SASP-associated responses in doxorubicin-treated cardiac fibroblasts

Anthracycline-induced cardiotoxicity is associated with progressive cardiac dysfunction and fibrotic remodeling, processes in which cardiac fibroblast senescence and the senescence-associated secretory phenotype have been implicated. However, the early secretory responses that precede established senescence following doxorubicin (DOX) exposure remain poorly defined. Primary cardiac fibroblasts isolated from healthy rat hearts were treated with varying concentrations of DOX (0-3 M) for 24 hours. Media collected at Day 1 were analyzed by untargeted proteomics, while cell viability, proliferation, and senescence-associated {beta}-galactosidase activity were assessed at Days 1 and 7. Acute DOX exposure reduced cell viability and proliferation at Day 1 without significantly increasing senescence-associated {beta}-galactosidase activity. At Day 7, we observed persistent proliferative arrest and increased {beta}-galactosidase activity with DOX exposure, consistent with delayed senescence. Proteomic analysis of Day 1 conditioned media identified 35 proteins significantly associated with DOX treatment after false discovery rate correction (22 positive and 13 negative associations; P-FDR < 0.05). Functional annotation identified proteins related to extracellular matrix (ECM) regulation, mitochondrial function, cellular stress, cytoskeletal organization, calcium signaling, protease activity, and cell-cycle regulation. Network analysis identified a collagen-centered network (COL1A1, COL1A2, and COL3A1) associated with reduced abundance of ECM proteins and a second network centered on GAPDH, HSPA8, and HSPA9 that included proteins associated with mitochondrial proteostasis, oxidative stress, metabolic adaptation, and structural remodeling.