Abstract

ALPK3 , a gene associated with pediatric cardiomyopathy, plays a pivotal role in cardiac development, yet the regulatory mechanisms governing its involvement in cardiomyocyte apoptosis are still not well understood. Detect the expression changes of ALPK3 in embryonic and neonatal mouse heart tissues at various developmental stages (E11.5, E12.5, E14.5, and P3). Establish ALPK3 knockdown cell lines to evaluate phenotypic changes in cardiomyocyte apoptosis. At the cellular level, validate the mechanism by which ALPK3 regulates cardiomyocyte apoptosis through the Notch1 signaling pathway. Transcriptional analysis of ALPK3 in embryonic mouse hearts revealed a progressive increase in expression during development, peaking in the perinatal period. In vitro experiments using ALPK3-knockdown AC16 cardiomyocytes demonstrated a 50% reduction in ALPK3 mRNA and protein levels, accompanied by significant apoptosis induction. RNA sequencing indicates that Notch1 signaling is significantly inhibited. ALPK3 deficiency suppressed Notch1 signaling, as evidenced by reduced levels of Hes-1 and NICD proteins; however, treatment with the Notch1 ligand Jagged1 reversed these effects. Furthermore, ALPK3 knockdown elevated pro-apoptotic BAX and decreased anti-apoptotic BCL-2 expression, whereas Jagged1 restored their levels. The findings indicate that ALPK3 regulates cardiomyocyte apoptosis and viability via the Notch1 pathway, offering mechanistic insights into ALPK3-associated cardiomyopathy and highlighting potential therapeutic targets for heart failure. Graphical abstract