Chondroprotective role of the osmotically sensitive ion channel transient receptor potential vanilloid 4: age- and sex-dependent progression of osteoarthritis in Trpv4-deficient mice

Abstract

Objective: Mechanical loading significantly influences the physiology and pathology of articular cartilage, although the mechanisms of mechanical signal transduction are not fully understood. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(++)-permeable ion channel that is highly expressed by articular chondrocytes and can be gated by osmotic and mechanical stimuli. The goal of this study was to determine the role of Trpv4 in the structure of the mouse knee joint and to determine whether Trpv4(-/-) mice exhibit altered Ca(++) signaling in response to osmotic challenge.

Methods: Knee joints of Trpv4(-/-) mice were examined histologically and by microfocal computed tomography for osteoarthritic changes and bone structure at ages 4, 6, 9, and 12 months. Fluorescence imaging was used to quantify chondrocytic Ca(++) signaling within intact femoral cartilage in response to osmotic stimuli.

Results: Deletion of Trpv4 resulted in severe osteoarthritic changes, including cartilage fibrillation, eburnation, and loss of proteoglycans, that were dependent on age and male sex. Subchondral bone volume and calcified meniscal volume were greatly increased, again in male mice. Chondrocytes from Trpv4(+/+) mice demonstrated significant Ca(++) responses to hypo-osmotic stress but not to hyperosmotic stress. The response to hypo-osmotic stress or to the TRPV4 agonist 4α-phorbol 12,13-didecanoate was eliminated in Trpv4(-/-) mice.

Conclusion: Deletion of Trpv4 leads to a lack of osmotically induced Ca(++) signaling in articular chondrocytes, accompanied by progressive, sex-dependent increases in bone density and osteoarthritic joint degeneration. These findings suggest a critical role for TRPV4-mediated Ca(++) signaling in the maintenance of joint health and normal skeletal structure.

Figure 1

Fluorescence imaging of [Ca⁺⁺]_i showing different channels for (A) Fluo 4, (B) Fura Red, and (C) the composite ratio image. (D) A portion of the ratio image has been enlarged to demonstrate resolution. Lower panel shows the percentage of chondrocytes from (E) wild type and (F) Trpv4^−/− femoral condyles responding with single or multiple calcium signals as a function of final osmolarity (starting osmolarity = 300mOsm for all cases) or (G) as a function of the presence or absence (control) of 4α-PDD. **p<0.01 versus control; *p<0.05 versus control, Chi-square analysis.

Figure 2

Representative frontal (left) and sagittal (right) μCT views of the intact knee of male wildtype (A,B) and Trpv4^−/− (C,D) mice. (A,C) 4 month old mice. (B,D) 12 month old mice. Scale bar = 1mm. Significant enlargement of the calcified regions of the menisci was observed, as well as that of the patella and condylar sesamoid bones (arrows), in Trpv4^−/− mice at 12 months.

Figure 3

Calcified meniscal bone volume (A) and density (B) as a function of age, sex and genotype. Calcified meniscal bone volume (C) as a function of site (lateral, medial), position (anterior, posterior) and genotype. Vertical bars represent 0.95 confidence intervals and significance is defined as p<0.05. ^asignificant genotype difference between equivalent data points, ^bsignificant difference from all 4 month data points, ^csignificant difference from 6 month equivalent data points, ^dsignificant difference from all other data points, and ^esignificant difference between anterior and posterior equivalent data points.

Figure 4

Subchondral bone (A) volume and (B) density and (D) trabecular bone density as a function of age, sex, and genotype. Subchondral bone volume (C) and trabecular bone density (E) as a function of site: lateral (lat), medial (med), bone (femoral condyle/tibial plateau) and genotype. Vertical bars represent 0.95 confidence intervals and significance is defined as p<0.05. ^asignificant genotype difference between equivalent data points, ^bsignificant sex difference between equivalent data points, ^csignificant difference from all 4 month data points, ^dsignificant difference from 4 month equivalent data points, ^esignificant difference from all other data points except 4 and 9 month equivalents, ^fsignificant difference between femoral and tibial equivalent data points and ^gsignificant difference between lateral and medial equivalent data points.

Figure 5

Total histological score (A) as a function of age, sex and genotype, and (B) as a function of site: lateral (lat), medial (med), bone (femoral condyle/tibial plateau) and genotype. Vertical bars represent 0.95 confidence intervals and significance is defined as p<0.05. ^asignificant genotype difference between equivalent data points, ^bsignificant sex difference between equivalent data points, ^csignificant difference from all 4 month data points, ^dsignificant difference from all other data points, and ^esignificant difference between femoral and tibial equivalent data points.

Figure 6

Representative sagittal histology sections through the central region of the medial condyle of male wildtype (A-D) and Trpv4^−/− (E-H) mice. (A,B,E,F) 4 month old mice. (C,D,G,H) 12 month old mice. Sections were stained with Hematoxylin, Fast Green and Safranin-O and imaged at 40× (A,C,E,G scale bar = 500μm) or 200× (B,D,F,H, scale bar = 100μm). Note the enlarged menisci, thickened subchondral bone and severe loss of articular cartilage (arrow) in Trpv4^−/− mice at 12 months.