title: Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes.
abstract: Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20-60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3-65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes.
authors.affiliation: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida; and.
authors.forename: Xiefan
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Biology, University of Florida, Gainesville, Florida.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Mei
authors.affiliation: Department of Biology, University of Florida, Gainesville, Florida.
authors.forename: William B
authors.initials: WB
authors.lastname: Barbazuk
authors.affiliation: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida; and.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Rivkees
authors.affiliation: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, Florida; and cwendler@ufl.edu.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Wendler
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D005865:Gestational Age; D006321:Heart / Q000187:drug effects* / Q000196:embryology; D018811:Maternal Exposure; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; D014157:Transcription Factors; D002110:Caffeine
keywords: caffeine; cardiac development; cardiomyocyte; differential exon usage; differential gene expression