Poster D9 - Differential expression and functional roles of Piezo1 in articular condyle medial and lateral cartilage in situ

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Xiang Tang

Research Technician
University of Rochester
Rochester, New York, United States

Co-Author(s)

SL

Suk Hee Lee

Post Doctoral Associate
University of Rochester Medical Center, United States
YZ

Yaxin Zhang

Graduate Student
University of Rochester, United States
Johann Kintzel (he/him/his)

Research Scientist/Lab Technician
University of Rochester
Rushville, New York, United States

Primary Investigator(s)

Whasil Lee, PhD

Assist Professor
University of Rochester, NY
Rochester, New York, United States

Introduction: Mechanical injury is a major risk factor for osteoarthritis (OA), a debilitating condition characterized by cartilage breakdown and joint pain without disease-modifying therapeutics [1]. Mechanical factors influence cartilage matrix degradation and cellular death via chondrocyte mechanotransduction [2, 3]. In human, condyle medial is exerted significantly higher mechanical loading than lateral [4]. Correspondingly, significantly higher OA vulnerability was found in femoral condyle medial than lateral [5]. We have discovered essential roles of Piezo1 mechanosensitive (MS) Ca2+-permeating channels in chondrocytes sensing injurious loading and inflammation [6, 7]. We question if chondrocytes express Piezo1 channels heterogeneously and exhibit differential Ca2+ homeostasis functionality. Here, we hypothesize higher mRNA and functional expression patterns of Piezo1 in condyle medial than lateral. In superficial zones of cartilage harvested from condyle medial and lateral regions (Fig.1A), we quantified mRNA, basal cytosolic Ca2+ ([Ca2+]o), and intra-cellular Ca2+ responses to Yoda1 and GsMTx4, a Piezo1-specific agonist and a non-specific Piezo1 inhibitor respectively.

Materials and

Methods: Cartilage plugs were harvested from femoral condyle and trochlea regions of mature pig knee joints using a 4 mm biopsy punch (Fig.1A). Ratiometric Ca2+ Imaging Cartilage plugs loaded with Fura-2 (Invitrogen) are imaged at 340nm/380nm with 0.5 fps using ratiometric Ca2+ imaging system (Intracellular Imaging, Inc.). Basal cytosolic Ca2+ levels ([Ca2+]o) were obtained by averaging 1 min before treatments (Fig.1C,D). Cartilage plugs were treated separately 40 min with Yoda1 (40 µM) and 24 hr with GsMTx4 (10 µM) and imaged again for 1 min (Fig.1E,F). Piezo 1 IHC and mRNA Cartilage plugs were immunofluorescence stained and intensities were averaged for differential Piezo1 expressions (Fig.1E,F). Cartilage superficial were homogenized for Piezo1 mRNA expressions (Fig.1I).

Results, Conclusions, and Discussions:

Results: First, chondrocytes in condyle medial cartilage exhibit higher resting [Ca2+]o versus chondrocytes in condyle lateral regions (Fig. 1C,D). Second, Yoda1 and GsMTx4 provoked functional Ca2+ responses were significant among condyle medial and lateral regions (p < 0.05), suggesting functional roles of Piezo1 in condyle medial and lateral (Fig.1E,F). Third, condyle medial cartilage exhibit higher Piezo1 expression levels than condyle lateral (p < 0.01) (Fig. 1G-I).

Conclusions: Intracellular Ca2+ imaging data shows higher resting [Ca2+]o in chondrocytes in superficial zones of condyle medial than lateral. Interestingly, IHC and mRNA also show higher Piezo1 expressions in condyle medial than lateral, suggesting regional differential Piezo1 expressions may play a role in regulating basal cytosolic Ca2+ homeostasis. Yoda1-mediated [Ca2+] increase and GsMTx4-mediated [Ca2+] decrease suggest active functional roles of [Ca2+]-regulating Piezo1 in articular chondrocytes from condyle regions. Future work includes the influence of other mechano-transducing ion channels. We will also investigate roles of cellular morphology on [Ca2+] regulation.

Acknowledgements (Optional): Acknowledgement:
We thank Dr. Thomas Suchyna for the GsMTx4 peptides and the Center for Musculoskeletal Research (CMSR) Histology Core for their technical assistance. This work was supported by NIH/NIAMS R01AR082349, NIH/NIGMS R35GM147054, and URMC/CMSR Discover award.