PTBP1 promotes coronary artery calcification by regulating CARD8 alternative splicing and NLRP3 inflammasome...

Coronary artery calcification (CAC) serves as a significant predictor of cardiovascular events; however, its underlying molecular mechanisms remain incompletely elucidated. PTBP1, an RNA-binding protein with profound regulatory functions, plays a critical role in post-transcriptional regulation. Nevertheless, its function and mechanism in vascular calcification have yet to be fully explored.

RNA sequencing analysis revealed decreased skipping of exon 4 in CARD8 within human aortic smooth muscle cells (HASMCs) calcification model. RNA immunoprecipitation and minigene reporter assays were employed to validate the effect of PTBP1 on CARD8 splicing patterns. Functional experiments were conducted to untangle the regulatory role of CARD8 in HASMCs calcification. A HASMC calcification model and a CAC mouse model were established, and techniques including RNA interference and overexpression were utilized to systematically investigate the mechanistic role of the PTBP1/CARD8 axis in CAC.

In vitro experiments, PTBP1 directly bound to CARD8 pre-mRNA and regulated its alternative splicing process. Functional studies demonstrated that knock down of PTBP1 significantly inhibited the calcification of HASMCs, while concurrent knock down of CARD8 reversed the protective effect. Mechanistic studies revealed that CARD8 deficiency promoted NLRP3 inflammasome assembly, enhanced Caspase-1 activation and IL-1β and IL-18 secretion, thereby driving the osteogenic-like transdifferentiation and calcification process of HASMCs. Animal experiments further verified that PTBP1 knock down significantly alleviated the degree of CAC in mice, and the effect was dependent on the expression level of CARD8.

This study untangles a novel mechanism whereby PTBP1 promotes CAC by regulating CARD8 alternative splicing to activate the NLRP3 inflammasome. These findings provide new insights into the pathophysiology of CAC and suggest potential targets for developing novel therapeutic strategies.