Stanley J Stachelek, Emily J Hauber, Xiaoqiu Yang, Devin W Laurence, Vitali Rusinkevich, Peiran Lu, Estibaliz Castillero, Benjamin B Pressly, Claire Brady, Nikia T Toomey, Ilia Fishbein, Alexis Martinez, Alireza Mohammad Karim, Ephraim Jacobson, Dov Levine, Shawnaleh Cada, Kenneth B Margulies, Kenneth C Bedi, George M Anderson, Abba M Krieger, Liming Pei, Giovanni Ferrari, Robert J Levy
Our findings have identified a novel link between mechano-transduction, 5-HT production and receptor signaling, and collagen synthesis, suggesting PIEZO1, PIEZO2, and downstream 5-HT pathways as potential therapeutic targets for mitigating MR progression.
AIMS: Mitral regurgitation (MR) pathophysiology involves increased serotonin (5-HT) receptor (HTR) expression and signaling, together with reduced 5-HT transporter (SLC6A4) expression in mitral valve interstitial cells (MVIC). The mechanosensitive calcium channels, PIEZO1 and PIEZO2, regulate cellular responses to a variety of mechanical conditions; however, PIEZO1 and PIEZO2 have not been studied in MR. We investigated the hypothesis that PIEZO1 and PIEZO2 mediate, through 5-HT mechanisms both homeostasis in normal mitral valves (nMV) and in the pathophysiology of MR.
METHODS AND RESULTS: Immunofluorescence and Western blots demonstrated the presence of PIEZO1 and PIEZO2 in samples of explanted human nMV and MR. MVIC derived from nMV and MR leaflets also expressed PIEZO1 and PIEZO2. Single-cell RNA sequencing (scRNA-seq) analyses of nMV and MR leaflet samples from 9 MR and 5 nMV cases identified 7 different cell types: Endothelial cells, smooth muscle cells, T-cells, macrophages, and three distinct MVIC phenotypes, VIC1, VIC2, and VIC3. MVIC together comprised 97% of all valve cells, irrespective of valve pathology. In addition, scRNA-seq of nMV and MR leaflets demonstrated MVIC PIEZO1 and PIEZO2 expression, together with HTR2B and SLC6A4. MVIC cultures, with no added 5-HT, demonstrated that PIEZO1 activation with its agonist, Yoda1, increased MVIC Ca2+ uptake. This was inhibited by PIEZO1-siRNA, and Dooku1, a Yoda1 antagonist, but not PIEZO2-siRNA or noncoding-RNA. Additional MVIC studies revealed that in static MVIC cultures, PIEZO1 activation with Yoda1 downregulated SLC6A4, upregulated HTR2B, and increased 5-HT levels and protein synthesis. Cyclic tension MVIC bioreactor studies showed that either PIEZO1-siRNA or PIEZO2-siRNA mitigated tension induced SLC6A4 downregulation and increased collagen synthesis; only PIEZO2-siRNA mitigated both static and cyclic tension-induced MVIC 5-HT production.
CONCLUSIONS: Our findings have identified a novel link between mechano-transduction, 5-HT production and receptor signaling, and collagen synthesis, suggesting PIEZO1, PIEZO2, and downstream 5-HT pathways as potential therapeutic targets for mitigating MR progression.