Disruption of Caldesmon Gene Expression in Bladder Myocytes

描述（由申请人提供）：本提案的目标是开发和表征两种工具来研究caldesmon （CaD）的生理功能，caldesmon是一种与平滑肌细丝相关的肌动蛋白/钙调素结合蛋白，被认为在平滑肌收缩装置的维持和收缩调节中发挥作用。这些工具是：1)敲除（KO）小鼠模型，其编码CaD的基因被破坏；2)兔膀胱平滑肌细胞系（BSM），其维持平滑肌表型，包括对激动剂的收缩能力。我们成功地用同源重组法制备了CaD小鼠KO模型。该CaD - KO模型缺少c端功能域，这对于抑制肌动蛋白激活的ATP水解、肌动蛋白与原肌动蛋白结合以及肌动蛋白与肌动蛋白的结合至关重要。该模型需要进一步表征功能域的缺失对CaD结构和功能的影响。利用从膀胱、肌条和整个膀胱分离的肌细胞，我们获得了初步数据，揭示了与野生型（WT）相比，KO小鼠结构和功能表型的变化。同样，我们对BSM细胞系的初步研究表明，在h-CaD缺陷的肌细胞中，平滑肌特异性CaD （h-CaD）的过表达有利于细胞质丝的组装，包括肌球蛋白丝。CaD c端功能域的缺失对细胞质丝的力、atp酶和组织的影响将在用siRNA沉默CaD表达或用CaD cDNA转染完整或截断的CaD过表达或过低表达后，使用CaD KO和BSM细胞的肌细胞进行研究。这两种模型（转基因小鼠和膀胱肌细胞模型）的特性以及拟议研究的数据将有助于确定CaD在维持收缩装置和收缩中的作用。这一点尤其重要，因为CaD存在于所有泌尿系统平滑肌中，并且在与收缩功能障碍相关的疾病中其表达发生改变。预期的数据还将显示CaD功能域的缺失对肌细胞分化状态和依赖平滑肌收缩的器官功能的影响。在膀胱出口梗阻、尿失禁和勃起功能障碍等疾病中，肌细胞分化状态的丧失和功能的改变与平滑肌收缩减少有关。此外，该试剂小鼠和细胞系将有助于其他研究者研究CaD在膀胱平滑肌中的作用，以及研究泌尿生殖系统各区域的信号转导、结构和收缩功能。

DESCRIPTION (provided by applicant): 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.