Disruption of Caldesmon Gene Expression in Bladder Myocytes

描述(由申请人提供)：这项建议的目标是开发和表征两种工具来研究钙调蛋白(CAD)的生理功能，钙调蛋白是一种肌动蛋白/钙调素结合蛋白，与平滑肌细丝相关，被认为在维持平滑肌收缩装置和调节收缩中发挥作用。这些工具是1)基因敲除(KO)小鼠模型，编码CAD的基因被破坏；2)兔膀胱平滑肌细胞系(BSM)，它保持平滑肌的表型，包括对激动剂的反应收缩的能力。我们已经成功地通过同源重组制作了CAD小鼠KO模型。这种CAD KO模型缺乏C-末端功能域，对于肌球蛋白、肌球蛋白和原肌球蛋白结合以及肌球蛋白与肌球蛋白的结合以及肌球蛋白与肌动蛋白的结合抑制肌动蛋白激活的ATP水解酶是重要的。这一模型需要进一步表征功能结构域的缺失对CAD结构和功能的影响。使用从尿囊、肌条和整个膀胱分离的肌细胞，我们产生了初步数据，揭示了与野生型(WT)相比，KO小鼠在结构和功能表型上的变化。同样，我们对BSM细胞系的初步研究表明，在h-CAD缺陷的心肌细胞中，平滑肌特异性CAD(h-CAD)的过度表达有利于细胞质细丝的组装，包括肌球蛋白细丝。通过用siRNA沉默CAD的表达，或通过转染完整或截短的CAD来研究CAD C末端功能域的缺失对细胞内力、ATPase和细胞质细丝组织的影响。这两个模型(转基因小鼠和膀胱肌细胞模型)的特征和拟议研究的数据将有助于确立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存在于所有的泌尿系平滑肌肉中，其表达在与收缩功能障碍相关的疾病中发生变化。预期的数据还将显示，CAD功能结构域的缺失对心肌细胞的分化状态和依赖于平滑肌收缩的器官的功能有什么影响。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不仅有利于研究CAD在膀胱平滑肌中的作用，而且有助于进一步研究其在泌尿生殖系统不同区域的信号转导、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.