Ethanol-induced activation of BMP signaling and reprogramming of cardiomyocytes' transcriptome

Abstract

Congenital heart disease (CHD) comprises a diverse group of structural heart defects present at birth due to complex interactions between genetic and environmental factors. Prenatal alcohol exposure (PAE) is a known environmental factor that disrupts fetal cardiogenesis and increases the risk of CHD. However, the molecular mechanisms behind ethanol (EtOH)-induced CHD remain obscure. This study investigated the effects of EtOH on bone morphogenetic protein (BMP) signaling and transcriptomic reprograming in HL-1 cardiomyocytes. HL-1 cells were treated with varying concentrations of EtOH (25, 50, and 100 mM) for 24 h. 100 mM of EtOH exposure significantly enhanced SMAD1/5 phosphorylation and upregulated BMP-responsive genes, namely Id1, Gata4, Mef2c, and Nkx2.5. Increased histone acetyltransferase activity further validated activation of BMP signaling through histone hyperacetylation. These effects were reversed by the BMP pathway inhibitor LDN-193189, confirming pathway-specific activation. Further, transcriptome analysis following 100 mM EtOH treatment identified 3,876 differentially expressed genes. KEGG enrichment analysis revealed significant dysregulation of cardiogenic pathways, including TGF-{beta}, Hedgehog, PI3K-Akt, Notch, FoxO, and calcium signaling pathways, along with extracellular matrix-receptor interaction and focal adhesion pathways. Gene Ontology analysis highlighted disturbances in heart development, cellular differentiation, apoptosis, extracellular matrix (ECM) organization, and chromatin regulation. Network analysis identified key hub genes, viz. Kras, Fn1, Col1a1, Prkaca, Fbn1, Col6a1, Col6a2, Ccnd1, Col1a2 and Myc which are upregulated and Hsp90aa1, Mdm2, Jun, Hras, Il6, Hsp90ab1, Pdgfra, Cdkn1a, Pparg, Fos and Hspa8 are downregulated which were subsequently validated by qRT-PCR. Collectively, these findings provide novel insights into the molecular basis of EtOH-induced CHD and identify potential biomolecule candidates for future therapeutic investigation.