Beclin1 deficiency unlocks cardiac lineage commitment through convergent Wnt and BMP signaling activation

Background: Embryonic stem cell (ESC)-derived cardiomyocytes are a key resource for studying cardiac development and advancing regenerative therapies. Beclin1 (Becn1), a core regulator of Autophagy and cardiac morphogenesis, has an undefined role in cardiomyocyte lineage specification. This study aims to investigate the regulatory function of Becn1 during cardiac differentiation from both mouse and human ESCs.

Methods: Mouse and human ESCs were differentiated into cardiomyocytes through established embryoid body (EB) formation or monolayer differentiation protocols. Stable Becn1 knockdown was achieved using short hairpin RNA (shRNA). Cardiomyocyte differentiation efficiency was evaluated by flow cytometry, immunocytochemistry, and contraction assays. Differentiation cardiomyocyte function was evaluated by sarcomere arrangement, calcium transients, and microelectrode array (MEA). Transcriptomic profiling was conducted by bulk RNA Sequencing, and pathway dynamics were analyzed using qPCR and western blotting.

Results: Becn1 expression declined over the course of differentiation. Knockdown of Becn1 significantly enhanced cardiomyocyte yield and promoted earlier onset of contractile activity, accompanied by increased expression of cardiac-specific markers. Mechanistically, Becn1 deficiency elicited a biphasic Wnt signaling response, characterized by early activation during mesodermal induction followed by suppression at later stages of differentiation. This shift was accompanied by sustained BMP pathway activation. Notably, Becn1-deficient ESCs underwent efficient cardiac differentiation in the absence of exogenous VEGF or FGF, with BMP signaling compensating for their omission. These findings were recapitulated in human ESCs, where BECN1 knockdown supported Wnt modulators-independent cardiomyocyte differentiation through coordinated modulation of Wnt and BMP pathways.

Conclusions: Becn1 serves as a negative regulator of cardiac lineage commitment by orchestrating stage-specific Wnt/BMP signaling dynamics. Silencing Becn1 enhances cardiomyocyte differentiation and enables growth factor-independent lineage progression. These findings offer a novel approach to improve the efficiency and scalability of stem cell-derived cardiomyocyte production for regenerative applications.

BMP signaling pathway; Becn1; Cardiomyocyte differentiation; Embryonic stem cells; Wnt signaling pathway.