Bufalin alleviates myocardial ischemia/reperfusion injury by restoring autophagic flux via the RNF216/Beclin-1 axis

Myocardial ischemia/reperfusion (I/R) injury is partially attributed to impaired autophagic clearance and pyroptotic inflammation. This study investigated whether bufalin protects the myocardium by preserving Beclin-1-dependent autophagic flux. In mice subjected to myocardial I/R, bufalin preserved cardiac function, reduced infarct size and myocardial injury, and inhibited NLRP3/GSDMD-associated pyroptotic signaling. In neonatal mouse primary cardiomyocytes exposed to oxygen-glucose deprivation/reoxygenation (OGD/R), bufalin enhanced cell viability, mitigated oxidative stress and pyroptotic signaling, and restored autophagic flux and lysosomal homeostasis, evidenced by increased Beclin-1 and LC3B levels, decreased P62 accumulation, improved tandem mCherry-GFP-LC3 flux, and preserved lysosomal proteins and cathepsin compartmentalization. Similar cellular effects were observed in H9c2 cells. Pharmacological disruption of early autophagosome formation or late autophagosome–lysosome turnover compromised bufalin-mediated protection. Mechanistically, bufalin increased the stability of Beclin-1 protein without elevating its mRNA levels, weakened the RNF216–Beclin-1 association, and reduced K48-linked ubiquitination of Beclin-1. In primary cardiomyocytes, RNF216 overexpression restored Beclin-1 ubiquitination and attenuated bufalin-mediated protection, whereas RNF216 knockdown generated a similar protective profile and limited the additional response to bufalin. Together, these findings identify the RNF216/Beclin-1 axis as a regulator of cardiomyocyte autophagic competence and support bufalin as a potential strategy for limiting pyroptotic injury during myocardial reperfusion.