Increased susceptibility of Trpv4-deficient mice to obesity and obesity-induced osteoarthritis with very high-fat diet

Abstract

Objective: To test the hypotheses that: (1) the transient receptor potential vanilloid 4 (TRPV4) ion channel is protective in the obesity model of osteoarthritis (OA), resulting in more severe obesity-induced OA in Trpv4 knockout (Trpv4(-/-)) mice; and (2) loss of TRPV4 alters mesodermal stem cell differentiation.

Methods: Male Trpv4(-/-) and wild-type (Trpv4(+/+)) mice were fed a control or high-fat diet (10% kcal and 60% kcal from fat, respectively) for 22 weeks, at which time spontaneous cage activity and severity of knee OA were evaluated. In addition, the adipogenic, osteogenic and chondrogenic potential of bone marrow-derived (MSC) and adipose-derived (ASC) stem cells from Trpv4(-/-) and Trpv4(+/+) mice were compared.

Results: A high-fat diet significantly increased knee OA scores and reduced spontaneous cage activity in Trpv4(-/-) mice, while also increasing weight gain and adiposity. MSCs from Trpv4(-/-) mice had decreased adipogenic and osteogenic differentiation potential versus Trpv4(+/+) MSCs. ASCs from Trpv4(-/-) mice had increased adipogenic and osteogenic and reduced chondrogenic differentiation potential versus Trpv4(+/+) ASCs.

Conclusions: Pan-Trpv4(-/-) mice develop more severe OA with high-fat feeding, potentially due to more severe diet-induced obesity. The altered differentiation potential of Trpv4(-/-) progenitor cells may reflect the importance of this ion channel in the maintenance and turnover of mesodermally-derived tissues.

Figure 1. Trpv4^−/− mice show increased adiposity in response to high-fat feeding

A. High-fat fed mice weighed significantly more than control diet mice by week 7. By week 10, knockout (KO) 60% mice weigh significantly more than all other groups (p=0.022) & Trpv4 KO >WT (p<0.05), *60% diet >10% diet (p<0.05), ^60% KO >all other groups (p<0.05), +60% KO >10% WT (p<0.05). B, Wild-type (WT) 60% mice gained (insignificantly) more weight after 22 weeks of high-fat feeding compared to WT 10% mice (p=0.0837), whereas KO 60% mice gained more weight than all other groups (p=0.003). C, Post-diet, mice did not differ in the amount of lean body mass. WT 60% mice had significantly more body fat than WT 10% mice, but KO 60% had more body fat than all other groups (p<0.001). D, Histological sections of subcutaneous adipose tissue from 10-week-old mice, with KO adipocytes appearing larger than WT adipocytes, Scale bar=100μm. E, When fed a high-fat diet, Trpv4^−/−mice were 40% as active during the dark cycle as Trpv4^+/+ mice (diet: p=0.833, genotype: p=0.003, genotype*diet: p=0.091). Data are shown as mean ±SEM. Data not sharing a common superscript letter indicate a significant difference (p<0.05).

Figure 2. Trpv4^−/− mice have more severe diet-induced osteoarthritis and altered chondrocyte histomorphology

A. Representative histological images, scale bar = 500 μm. B. KO 60% mice have more severe joint degeneration than WT 10% mice (genotype: p=0.057, diet: p=0.049, genotype*diet: p=0.779). C. Trpv4^−/− joints have less chondrocyte cloning and chondrocyte hypertrophy (p<0.001). Data are shown as mean±SEM. Data not sharing a common superscript letter indicate a significant difference (p<0.05).

Figure 3. Adult stem cells from Trpv4^−/− mice exhibit altered differentiation potential

MSCs and ASCs were purified and expanded as described previously (17,18). Data for adipogensis and osteogenesis were normalized to DNA content and to the staining of cells cultured in control media. A. 1. MSCs and ASC from Trpv4^−/− mice expanded equally rapidly in hypoxic culture. A2–4. A: WT MSC B: WT ASC C: KO MSC D: KO ASC. A2. Cell morphology at day 7 of adipogenic differentiation. A3. Alizarin Red staining at day 14 osteogenesis. A4. Chondrogenically induced cell pellets (Alcian Blue/Nuclear Fast Red). B. Bone marrow derived MSCs have a reduced ability to differentiate when cultured in adipogenic media (p=0.008), while ASCs show a large increase (p<0.001). C. Bone marrow derived MSCs have a reduced ability to differentiate when cultured in osteogenic media (p<0.001), while ASCs show an increased ability (p<0.001). D. Trpv4 deficiency does not affect in vitro MSC chondrogenesis. However, Trpv4^−/− ASCs have decreased GAG accumulation (p=0.036) and Alcian Blue staining. D. Data are shown as mean±SEM. * indicates significant difference (p<0.05).