PROJECT SUMMARY Dermal injury leads to fibrosis and scar formation, which can be a significant source of morbidity. Interestingly, humans respond to identical skin injuries with different degrees of scar formation that range from low to high scaring phenotypes. Understanding the mechanisms that drive heterogeneous scarring outcomes will allow us to design strategies to direct wound healing toward regeneration and reduced scarring. Biomechanical forces are known to influence how the skin heals. Biomechanical tension signals fibroblast proliferation, migration, inflammatory functions and production of extracellular matrix (ECM). These responses, in conjunction with oxidative stress in the wound, place a high energy demand on fibroblasts. Typically, metabolic requirements of cells are met via mitochondrial oxidative phosphorylation (OXPHOS) under homeostatic conditions or via glycolysis when oxygen is limited. Recent studies have shown that increase in mechanical cues can alter energy metabolism by promoting glycolysis. Notably, the phenomenon of a metabolic shift towards ‘aerobic glycolysis’ (Warburg effect) was mainly described in progression of fibrotic diseases, but our data showed that fibroblasts from uninjured skin of healthy patients with high scarring phenotype (HS) have higher OXPHOS and glycolysis than those from low scarrers, and demonstrated changes in mitochondrial function that suggest a higher energy state at baseline. Expression of PKM2, a key rate-limiting enzyme of aerobic glycolysis, was also higher in HS fibroblasts, with increased PKM2 phosphorylation/dimerization shunting metabolites toward increased ATP production and promoting aerobic glycolysis and pro-fibrotic pathways under TGF-β stimulation. HS fibroblasts also had an exaggerated response to mechanical tension, with an increase in total and phosphorylated PKM2. These data support the concept that PKM2-mediated aerobic glycolysis in fibroblasts under tension may influence the magnitude of fibrosis. Consistently, we also noted an exaggerated increase in phosphorylation of Hsp27 in HS fibroblast under tension. To our knowledge, this is the first evidence of differential aerobic glycolysis and biomechanical tension responses being linked to opposing scar outcomes in physiologic wounds, which could explain wound healing heterogeneity. We hypothesize that patient-specific scarring responses are due to PKM2/Hsp27-dependent alterations in fibroblast aerobic glycolysis that are influenced by wound biomechanical forces. In Aim 1, we designed in vitro and in vivo experiments with low and high scar-derived patient fibroblasts to investigate differences in PKM2 and Hsp27 phosphorylation/activation and their effect on metabolic pathways, energy metabolism, and ECM production. In Aim 2, we will utilize in vitro and human skin xenotransplant wound models to examine how biomechanical tension alters PKM2/Hsp27 mediated energy metabolism to drive patient scarring responses and then develop...