Abstract Body (Do not enter title and authors here): Calcific aortic valve disease (CAVD) is one of the most common valvular heart diseases worldwide. In recent years, the rise of minimally invasive treatment concepts, primarily Transcatheter aortic valve replacement (TAVR), and innovations in prosthetic valve development have led to substantial breakthroughs in CAVD treatment. However, effective pharmacotherapeutic targets to delay CAVD progression are still lacking. Therefore, uncovering novel molecular mechanisms underlying the initiation and progression of aortic valve calcification and improving CAVD management remain urgent clinical challenges.
Single-cell RNA sequencing of human aortic valve tissues revealed a significantly increased proportion of a specific Valvular interstitial cell (VIC) subpopulation in calcified aortic valves (Figure 1A). Functional enrichment analysis of differentially expressed genes in this subpopulation showed high enrichment for pathways such as ubiquitination. Subsequently, bulk RNA sequencing was performed on human calcified aortic valves, revealing a set of significantly upregulated genes,which were cross-referenced with the marker genes of disease-specific VIC subpopulations identified through single-cell RNA sequencing, the MKRN1 gene—associated with protein ubiquitination—was identified as a key candidate gene. (Figure 1B). Validation by Western blot confirmed that MKRN1 protein levels were significantly higher in calcified human aortic valves compared to normal tissues (Figure 1C), consistent with the transcriptomic sequencing results. This finding was further corroborated by immunohistochemistry on human valve tissues (Figure 2C).
In vivo experiments, we generated MKRN1^-/-/ApoE^-/-mice. These 10-week-old mice were fed a high-fat diet for 6 months. Cardiac ultrasound examination at 24 weeks (Figure 2A/B), along with Hematoxylin and Eosin (H&E) staining (Figure 2C) and von Kossa (VK) staining (Figure 2D) of the aortic valves, demonstrated that MKRN1 knockout delayed the onset of calcific lesions in the mouse aortic valves.
STRING protein-protein interaction (PPI) network analysis predicted proteins interacting with MKRN1, suggesting a direct interaction with IGF2BP3 (Figure 3A). This interaction was confirmed by co-immunoprecipitation(Co-IP) assays (Figure 3B).
This study reveals that MKRN1 exacerbates CAVD progression by regulating IGF2BP3 expression, providing a novel potential intervention target for CAVD treatment.