siRNA‐mediated knockdown of caldesmon expression inhibits the assembly of actin and intermediate‐sized filaments in the cytoskeleton of cultured bladder smooth muscle cells

This study was conducted to determine the effect of silencing the expression of caldesmon (CaD), a smooth muscle thin filament protein, on actin filament assembly and cytoskeletal structure in smooth muscle. A stable rabbit bladder smooth muscle cell (BSM) line was transfected with siRNA and their inhibitory efficiency was tested by western blotting. Untreated and GFPsiRNA‐treated BSM cultures served as controls. The western analysis indicated a 92% knocked down (KO) of CaD. Cultures were processed for microscopy after antibody and/or phalloidin staining. Our results show that the suppression of CaD expression induced disassembly of F‐actin filaments in BSM cells. Despite the F‐actin disassembly in CaD KO cells, there was no change in the actin expression compared to controls. Furthermore, CaD knockdown changed the distribution of intermediate filament proteins, vimentin and desmin. CaD‐KO cells revealed perinuclear vimentin aggregates. The pull‐down assay using CaD antibody showed binding of vimentin and desmin to CaD. This is the first report of an effect of knockdown of CaD gene expression on the actin filament assembly and the intermediate filaments in cells. This finding opens up the possibility of modulating CaD gene expression to alter smooth muscle cytoskeletal structure and function. Supported by RO1 DKO69898 & P50 DK52620