The goal of this proposal is to develop and characterize two tools to study the physiological function for caldesmon (CaD), an actin/calmodulin-binding protein associated with smooth muscle thin filaments and thought to play a role in the maintenance of smooth muscle contractile apparatus and the regulation of contraction. These tools are 1) a knockout (KO) mouse model with disruption of the gene that encodes CaD and 2) a rabbit bladder smooth muscle cell line (BSM) which maintains the smooth muscle phenotype, including the ability to contract in response to agonists. We have been successful in making a CaD mouse KO model by homologous recombination. This CaD KO model lacks the C-terminal functional domains, important for inhibition of actin-activated ATP hydrolysis by myosin, actin-and tropomyosin-binding and tethering of myosin to actin. This model requires further characterization with respect to the effect of deletion of functional domains on the structure and function of CaD. Using myocytes dissociated from urinary bladders, muscle strips and whole bladders, we have generated preliminary data, which reveal changes in the structural and functional phenotypes in the KO mouse when compared with that of the wild-type (WT). Similarly, our preliminary studies on the BSM cell line show that the overexpression of smooth muscle specific CaD (h-CaD) in h-CaD-deficient myocytes favors the assembly of cytoplasmic filaments, including myosin filaments. The effect of deletion of CaD C-terminal functional domains on force, ATPase, and organization of cytoplasmic filaments will be studied using myocytes from CaD KO and the BSM cells after silencing the expression of CaD with siRNA or after over- or under-expression of intact or truncated CaD by transfection with CaD cDNA. Characterization of these two models (the transgenic mouse and bladder myocyte cell models) and the data from proposed studies would help to establish a role for CaD in the maintenance of the contractile apparatus and contraction. This is particularly important, since CaD is present in all urologic smooth muscles and its expression is altered in diseases associated with contractile dysfunction. The expected data would also show what effects the deletion of the CaD functional domains has on the differentiated state of the myocyte and the function of organs which depend on smooth muscle contraction. Loss of the differentiated state of the myocyte and alterations in function are correlated with decreased smooth muscle contraction in diseases such as bladder outlet obstruction, urinary incontinence, and erectile dysfunction. Furthermore, this reagent mouse and cell line would be beneficial to other investigators to study not only the role of CaD in bladder smooth muscle but also to study the signal transduction, structure and contractile function in various regions of the urogenital system. ? ? ? ?
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