Skeletal dysplasia-causing mutations in TRPV4 alter the chondrocyte transcriptomic response to mechanical loading

Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel highly expressed in chondrocytes that supports cartilage development and homeostasis. Mutations in the channel can cause skeletal dysplasias, including the gain-of-function mutations V620I and T89I, which lead to brachyolmia and metatropic dysplasia, respectively. These mutations suppress hypertrophic differentiation, but the mechanisms by which they alter chondrocyte response to mechanical load remain to be elucidated. To determine the effect of these mutations on chondrocyte mechanotransduction, tissue-engineered cartilage was derived from differentiated clustered regularly interspaced short palindromic repeats (CRISPR)-edited human-induced pluripotent stem cells (hiPSCs) harboring the moderate V620I or severe T89I TRPV4 mutations. Wild-type and mutant tissue-engineered hiPSC-derived cartilage contructs were subjected to compressive mechanical loading at physiological levels, and transcriptomic signatures were assessed by RNA-sequencing. Our results demonstrate that the V620I and T89I mutations diminish the mechanoresponsiveness of chondrocytes, as evidenced by reduced gene expression downstream of TRPV4 activation, including those involved in endochondral ossification. Changes in the expression of genes involved in extracellular matrix production and organization were found to contribute toward the phenotype in V620I mutant chondrocytes, whereas dysregulated retinoic acid signaling was linked to T89I, and disrupted proliferation was common to both. Our findings suggest that dysfunctional mechanotransduction due to V620I and T89I mutations in TRPV4 contribute to the developmental phenotypes, supporting TRPV4 modulation as a potential pharmacologic target.<b>NEW & NOTEWORTHY</b> Gain-of-function mutations in TRPV4, a mechano- and osmosensitive ion channel, are linked to skeletal dysplasias, but their effects on chondrocyte mechanotransduction remain unknown. Using human iPSCs harboring skeletal dysplasia-causing mutations, we developed and mechanically loaded tissue-engineered cartilage. Our findings show that V620I and T89I mutations reduce chondrocyte mechanoresponsiveness, evidenced by decreased gene expression downstream of TRPV4 activation, providing insight into TRPV4-related skeletal disorders and potential pharmacological targets.