(30) Creatine Metabolism in the Human Placenta is Essential for Placental Development

Creatine Metabolism in The Human Placenta is Essential for Placental Development.

Nirvay Sah¹, Eshanya Gupta¹, Jaroslav Slamecka¹, Claire Zheng², Donal Pizzo¹, Francesca Soncin¹

¹Department of Pathology, University of California San Diego, San Diego, CA.

²School of Biological Sciences, University of California San Diego, San Diego, CA.

Abstract Text:

The placenta is a highly energy-demanding organ that must generate sufficient adenosine triphosphate (ATP) to support both its own bioenergetic and biosynthetic needs as well as those of the rapidly growing fetus. Key cellular processes such as trophoblast proliferation, migration, and fusion require substantial energy, particularly in the first trimester, to ensure proper placental development, and adequate nutrient and gas exchange later in gestation. Given these high ATP demands, the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system, which buffers ATP levels in metabolic tissues like muscle and brain, may play a critical role in placental function. However, the involvement of this system in placental bioenergetics and development remains largely unexplored. Previous studies showed that the term-placenta can synthesize creatine locally, and first-trimester placentae express genes involved in creatine and phosphocreatine biosynthesis. However, prior studies have primarily analyzed whole tissue lysates, leaving the specific roles in different trophoblast compartments unclear. To address this, we characterized the spatial and temporal expression of key components of the Cr-CK-PCr system throughout gestation. Immunolocalization was performed on paraffin-embedded placental tissue samples (n=3 per group) from early first trimester (6–8 weeks), late first trimester (11–14 weeks), second trimester (16–24 weeks), and term (male and female). We assessed the expression of proteins involved in creatine biosynthesis (AGAT, GAMT), transport (SLC6A8), and ATP buffering (CKB, CKMT1). AGAT expression was restricted to the fetal villous mesenchyme across all gestational stages. GAMT was detected in cytotrophoblasts (CTB), syncytiotrophoblasts (STB), and extra-villous trophoblasts (EVT) during the first and second trimesters and to the STB of term-placenta. GAMT was also consistently expressed in the fetal villous mesenchyme throughout gestation. CKB localized predominantly to the fetal villous mesenchyme, with stronger expression in term placentae compared to first trimester. CKMT1A had strong immunoreactivity in all trophoblast subpopulations (CTB, STB, EVT) throughout gestation. SLC6A8 was present in trophoblasts during the first and second trimesters but was undetectable in term trophoblasts. Additionally, SLC6A8 expression in the fetal villous endothelium increased with gestational age. No fetal sex-related differences were observed in creatine metabolism protein expression in term placentae. The dynamic expression patterns of Cr-CK-PCr system components suggest compartment-specific regulation of this phosphagen system to adapt to placental development and functional demands for ATP homeostasis across gestation. Mining published single-cell RNA sequencing data from diseased placentae revealed decreased expression of CKMT1 and SLC6A8 in CTB, GAMT in EVT, and CKMT1 in STB in gestational diabetes mellitus cases, while preeclampsia cases showed reduced CKMT1 expression in CTB. To further investigate the role of the Cr-CK-PCr system in early placental development, we treated human trophoblast stem cells (hTSCs) derived from first-trimester placentae with cyclocreatine, an inhibitor of CK activity. This treatment significantly reduced hTSC proliferation (P< 0.05) in a dose-dependent manner, indicating that the Cr-CK-PCr system is essential for trophoblast growth. Overall, our findings demonstrate that the human placenta metabolizes creatine and phosphocreatine throughout gestation, and this system is critical for proper trophoblast development. Future studies will confirm alterations in Cr-CK-PCr system components in diseased placentae and manipulate CKMT1, GAMT, and SLC6A8 expression in hTSC and organoids to further dissect their roles in differentiation. This research was supported by the Lalor Foundation and the California Institute for Regenerative Medicine.