Titin in the pregnant uterus – a loss of function approach to smooth muscle biomechanics and mechanosignaling

Titin是哺乳动物中已知的最大的蛋白质。它的功能主要在心脏和骨骼肌中被分析，在那里它决定了肌节（横纹肌的主要收缩单位）的适当组装和间距，以及肌丝的弹性特性。titin基因的突变被认为是遗传性心脏病的最常见原因。在平滑肌中，研究人员一直在努力表征titin的表达、定位和功能。虽然初步数据表明与α-肌动蛋白和肌球蛋白丝相互作用，但肌动蛋白在平滑肌中的作用尚不清楚。最近，我们产生了平滑肌特异性的titin敲除小鼠，并在缺陷母亲中发现了胚胎植入缺陷。由此产生的错误定位和拥挤模拟了溶血磷脂酸受体3和磷脂酶A2 α缺陷小鼠的表型，这些缺陷与g蛋白偶联受体信号、钙动员和花生四烯酸途径有关。因此，我们认为平滑肌titin决定了子宫肌壁的机械、代谢和信号特性。我们的初步分析表明，平滑肌titin有助于怀孕子宫的蠕动运动，这是正确植入的先决条件。在这里，我们将研究表型的分子、细胞和功能基础，并在RNA和蛋白质水平上表征平滑肌titin的表达，包括其定位和平滑肌titin相互作用组。我们将确定野生型和缺乏titin的子宫环的弹性和收缩特性，并研究titin信号是否有助于植入表型。这项工作可能为异位妊娠和植入缺陷的发展提供新的见解。

Titin is the largest known protein in mammals. Its function has been primarily analyzed in heart and skeletal muscle, where it determines the proper assembly and spacing of the sarcomere - the main contractile unit of striated muscle - as well as the elastic properties of the myofilament. Mutations in the titin gene have been recognized as the most common cause of inherited heart disease.In smooth muscle, researchers have struggled to characterize titin expression, localization, and function. Although preliminary data suggests an interaction with α-actinin and myosin filaments, the role of titin in smooth muscle is only poorly understood.Recently, we have generated smooth muscle specific titin knockout mice and uncovered an embryo implantation defect in deficient mothers. The resulting mislocalization and crowding mimics the phenotype of mice with a defect in the lysophosphatidic acid receptor 3 and phospholipase A2 alpha, which link to G-protein coupled receptor signaling, calcium mobilization, and the arachidonic acid pathway. Accordingly, we propose that smooth muscle titin determines the mechanical, metabolic, and signaling properties of the muscular wall of the uterus. Our preliminary analysis suggests that smooth muscle titin contributes to the peristaltic movement of the pregnant uterus as a prerequisite for proper implantation. Here, we will study the molecular, cellular, and functional basis of the phenotype and characterize smooth muscle titin expression on the RNA and protein level, including its localization and the smooth muscle titin interactome. We will determine the elastic and contractile properties of wildtype and titin-deficient uterus rings and investigate if titin based signaling contributes to the implantation phenotype. The proposed work could provide novel insights into the development of ectopic pregnancies and implantation defects.