The levels of Twist1 homodimerization over heterodimerization with E proteins control osteogenic differentiation and calcification in the adult porcine aortic cusps

Millions of people are affected by calcific aortic valve disease (CAVD) every year globally, and the only effective therapy is limited to surgical or transcatheter aortic valve replacement. While molecular signaling pathways in CAVD are well characterized, Twist1, a bHLH transcription factor, is reported to be upregulated in adult diseased calcific aortic valves with limited functional characterization. Twist1 regulates gene expression by forming homodimers and heterodimers, influencing cell differentiation and disease processes. In calcific aortic valve disease, its elevated expression is linked to valvular pathological extracellular matrix (ECM) remodelling, but the function of its dimerization remains unclear. In this study, we used an adult porcine aortic valve model to investigate Twist1 expression and dimerization status under osteogenic induction. Whole aortic cusps and isolated valvular interstitial cells (VICs) were cultured with calcifying media. Under osteogenic conditions, VICs showed decreased Twist1-E protein (T/E) association and increased Inhibitor of DNA binding 1 (ID1) -E protein interactions, suggesting a shift toward Twist1 homodimer formation. Likewise, Harmine treatment induced calcification, marked by elevated osteogenic markers Bmp2, Osteopontin, and Osteocalcin, and was associated with a shift in Twist1 dimerization toward homodimer formation. Conversely, Cannabidiol (CBD), an ID1 inhibitor, reduced ID1-E protein co-localization and increased Twist1-E protein association, which promotes heterodimerization and inhibits calcification. Overall, these findings supported a model in which Twist1 dimerization dynamics regulate osteogenic differentiation in aortic valves. The shift from homodimers toward heterodimers of Twist1 inhibited valvular calcification, presenting a potential therapeutic target for CAVD intervention.