Creatine phosphagen system regulates energy metabolism and growth of trophoblasts

Abstract

The creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system maintains ATP homeostasis in tissues with high energy demands, but its role in early human placental development remains poorly understood. We characterized Cr-CK-PCr system components across gestation and investigated its function in trophoblast development. Proteins involved in Cr biosynthesis, transport, and utilization were examined in first-trimester, second-trimester, and term human placentae (n=3-6/group). Components showed gestational age- and compartment-specific localization. The biosynthetic enzymes AGAT and GAMT localized predominantly to fetal villous mesenchyme and broadly across villous compartments, respectively. CKB was enriched in fetal mesenchyme, whereas CKMT1 was broadly expressed across trophoblast subtypes. The main Cr transporter SLC6A8 was abundant in syncytiotrophoblast early in gestation but shifted predominantly to fetal blood vessels at term. To determine functional importance, primary human trophoblast stem cells (hTSCs) were treated with a creatine analog, cyclocreatine (cCr; 0-40 mM), for 48 h. Treatment with 20 mM cCr reduced proliferation (P<0.001), decreased EGFR expression (P<0.05), induced cell-cycle arrest (P<0.0001), and increased apoptosis (P<0.0001). cCr also impaired mitochondrial and glycolytic function (P<0.01) with morphological evidence of mitochondrial stress. Consistently, treatment of primary human trophoblast organoids with cCr reduced organoid growth and EGFR expression (P<0.05). Together, these findings demonstrate that the Cr-CK-PCr system is dynamically expressed across human placental development and that disruption of Cr/PCr metabolism compromises trophoblast bioenergetics, proliferation, and survival, supporting its importance in placental development and providing a foundation for investigating its contribution to pregnancy disorders associated with altered placental energy metabolism.