Loss of circular EYA3 attenuates formaldehyde-induced cardiomyocyte pyroptosis and congenital heart defects by regulating Smad5 stability.

Formaldehyde (FA) is a significant risk factor that impacts fetal heart development. Circular RNAs (circRNAs) have been recognized as potent regulators of heart development and disease. We discovered a particular circRNA, circEYA3 (rno_circ_009926), which is upregulated in cardiomyocytes and fetal rat heart tissue exposed to FA. Additionally, circEYA3 shows elevated expression in the serum of pregnant women who have been exposed to FA and whose fetuses have congenital heart disease (CHD). While circEYA3 knockdown inhibited FA-induced cardiomyocyte pyroptosis, circEYA3 overexpression exhibited the opposite effect. Our results showed that circEYA3 interacts with SMAD family member 5 (Smad5) protein, serving as a scaffold to enhance the association between Smad5 and the Smad5 ubiquitination regulatory factor 1 (Smurf1). CircEYA3 knockdown stabilised Smad5 at the post-translational level by upregulating Smad5 and promoting Smurf1-mediated Smad5 ubiquitination. In addition, we found that circEYA3 knockdown in an FA-exposed foetal rat heart model inhibited foetal rat cardiomyocyte pyroptosis and ameliorated abnormal heart development in vivo. Overall, our work links circEYA3 to the onset of fetal CHDs induced by maternal FA exposure and provides potential therapeutic and diagnostic strategies targeting circEYA3 for CHDs.