ABSTRACT: Background: Cardiac fibroblasts (CFs) play a vital role in the physiological and pathological processes of the heart. Previous studies have demonstrated that high glucose stimulation induces the transformation of CFs into myofibroblasts, contributing to cardiac fibrogenesis. However, in vivo experiments have predominantly utilized adult animals, whereas most in vitro studies have focused on CFs derived from neonatal animals. The responses of CFs from different age groups to high glucose levels remain unclear. This study aimed to investigate transcriptional alterations in CFs at distinct developmental stages in response to high glucose exposure. Methods: CFs were isolated from neonatal (S1, 0–3 days), juvenile (S2, 3–4 weeks), adult (S3, 10–13 weeks), and aged (S4, 20 months) rats. CFs were exposed to normal (5.5 mM, NG) or high glucose (33 mM, HG). The cellular RNA was extracted for sequencing and analysis. Differentially expressed genes (DEGs) were validated by quantitative real-time PCR. Results: After NG treatment, fibrosis and inflammation-related gene expression in CFs (e.g., Col8a1, Col8a2, IL-6, Ccl2, Ccl20, Mmp2 and Mmp9) increased with age, while proliferation-related genes (MCM family, Sox10, Sox11) decreased. HG treatment most affected S3-CFs, showing 228 DEGs; it suppressed growth-related genes (Adra1d, Htr2b) and enhanced inflammatory genes (IL-6, Olr1). In S1-CFs, 197 inflammation-rich genes were upregulated under HG. S4-CFs displayed 166 DEGs, mostly metabolic downregulation (G6pc). S2-CFs had the fewest DEGs (112), focusing on cell metabolism. Conclusions: Fibrosis- and inflammation-associated gene expression in CFs showed an age-dependent stepwise elevation. CFs from distinct developmental stages responded differently to HG stimulation, with S3-CFs exhibiting the most pronounced response. These findings highlight the developmental characteristics of CFs and provide implications for the selection of appropriate CFs to investigate diabetes-associated cardiac fibrosis.
