Thursday, May 2, 2024
5/2/2024
Project Summary After myocardial infarction (MI), fibroblasts differentiate to a hypersecretory phenotype. Attesting to the scale of their extracellular matrix (ECM)-manufacturing capacity, a single fibroblast can produce >500,000 procollagen chains per hour. Such large demands for collagen production must be met by changes in the metabolic network to drive carbon for glycine and proline synthesis, which constitute a large proportion of collagen. Previous studies have shown that this demand is met by de novo amino acid synthesis, which requires metabolic remodeling to coordinate substrate utilization with biosynthetic pathway activity. Understanding mechanisms that facilitate metabolic changes to promote fibroblast-mediated ECM deposition could provide actionable targets to improve cardiac remodeling after injury. Previous studies indicate that metabolism plays critical roles in fibroblast function, with glycolysis and glutaminolysis playing important roles in fibroblast activation and collagen secretion. We find that fibrogenic stimuli upregulate enzymes known to participate in a unique metabolic cycle that could uphold rapid and voluminous collagen synthesis after MI. Fibroblasts isolated from infarcted hearts, as well as naïve fibroblasts treated with TGF-ß, upregulate pyruvate kinase M2 (Pkm2) and phosphoenolpyruvate carboxykinase 2 (Pck2), which play essential roles in the phosphoenolpyruvate (PEP) cycle. In our working model, Pkm acts as a rheostat in the PEP cycle: catalytically slower Pkm2 promotes a PEPSLOW cycle that functions to augment biosynthesis, while catalytically faster Pkm1 promotes a futile PEPFAST cycle that diminishes biosynthetic pathway activity and collagen production. The PEP cycle is completed via Pck2, which utilizes additional carbon from anaplerosis to form non-glycolytic PEP, which provides additional triose precursors for glycine and collagen synthesis. Thus, we hypothesize that fibroblast collagen secretion is amplified by an extant PEPSLOW cycle, which functions to accelerate glycine synthesis and can be targeted to diminish fibrotic burden. Consistent with a role of the PEP cycle in fibrosis, our data suggest that induction of a PEPFAST cycle via fibroblast-specific Pkm2¿Pkm1 splice variant switching improves cardiac remodeling and diminishes collagen deposition after MI. Our preliminary data also suggest that promoting Pkm1-like activity by pharmacologically activating Pkm2 decreases collagen abundance in isolated fibroblasts. Similarly, deletion of Pck2 influences fibroblast phenotype and could play a major role in regulating collagen synthesis. Thus, in this project, we will thoroughly examine the role of the PEP cycle in cardiac fibroblasts and assess the mechanisms by which it influences cardiac fibrosis. To test our general hypothesis, we will: (1) elucidate the role of Pkm alternative splicing on cardiac fibroblast metabolism and cardiac fibrosis; and (2) delineate how the PEP cycle influences cardiac fibrosis after MI. Results of these studies will delineate the mechanisms by which the PEP cycle influences ECM synthesis and modulates the response of the heart to MI. This project could lead to the development of actionable therapies to diminish collagen biosynthesis and cardiac fibrosis, for which there are no currently approved therapies.
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