Chondrocyte mechanotransduction is not well understood, but recently, it has been proposed that mechanically activated ion channels such as transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 are of functional importance in chondrocyte mechanotransduction. The aim of this study was to distinguish the potential contributions of TRPV4, Piezo1, and Piezo2 in transducing different intensities of repetitive mechanical stimulus in chondrocytes. To study this, TRPV4-, Piezo1-, or Piezo2-specific siRNAs were transfected into cultured primary chondrocytes to knock down (KD) TRPV4, Piezo1, or Piezo2 expression, designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells. Then we used Flexcell® Tension System to apply cyclic tensile strains (CTS) of 3% to 18% at 0.5 Hz for 8 h to the knockdown and control siRNA-treated cells. Finally, using a Ca2+ imaging system, stretch-evoked intracellular Ca2+ ([Ca2+] i) influx in chondrocytes was examined to investigate the roles of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different intensities of repetitive mechanical stretch stimulation. The characteristics of [Ca2+] i in chondrocytes evoked by stretch stimulation were stretch intensity dependent when comparing unstretched cells. In addition, stretch-evoked [Ca2+] i changes were significantly suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, indicating that any channel essential for Ca2+ signaling induced by stretch stimulation in chondrocytes. Of note, they played different roles in calcium oscillation induced by different intensities of stretch stimulation. More specifically, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to physiologic levels of strain (3% and 8% of strain), while Piezo2-mediated Ca2+ signaling played a central role in the response of chondrocytes to injurious levels of strain (18% of strain). These results provide a basis for further examination of mechanotransduction in cartilage and raise a possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of cartilage disease induced by repetitive mechanical forces. Impact statement Chondrocytes in cartilage are constantly subjected to load-induced stimuli and regulate their metabolic activities in order to maintain cartilage homeostasis. Therefore, mechanotransduction is important in chondrocytes and is vital for their role in cartilage function. Our results indicate that chondrocytes might sense and distinguish the different intensities of repetitive mechanical stimulus by using different mechanosensitive ion channels. Specifically, TRPV4 is mainly responsible for sensing physiologic levels of repetitive CTS stimulus, while Piezo2 mainly contributes to chondrocyte sensing noxious levels of repetitive CTS loading. These results provide a basis for further examination of mechanotransduction in cartilage and raise the possibility of therapeutically targeting Piezo2-mediated mechanotransduction for the treatment of OA which is induced by injurious and repetitive mechanical stimulation.
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