Role of Obscurin and Obscurin-MLCK in myofibrillogenesis

描述（由申请人提供）：在发育期间、肥大生长期间和对损伤的反应中，心脏在组织和细胞水平上经历深度重塑。 新肌原纤维组装和整合的缺陷可导致肌原纤维紊乱、肌细胞死亡和充血性心力衰竭。 Obscurin是一种新型的巨大肌节蛋白，通过与肌联蛋白和锚蛋白相互作用将肌节连接到肌质膜上。 因此，它可能在肌原纤维组装和强直相关离子通道相对于收缩器官的正确定位中起重要作用。 因此，本研究的具体目的是1）使用免疫组织化学分析和心脏功能的视频评估来检查反义吗啉代介导的obscurin抑制对斑马鱼胚胎中体内肌原纤维组装以及心脏和骨骼肌发育的影响2）使用酵母双杂交和体外结合研究来表征obscurin和其他肌节蛋白之间的物理相互作用，并通过竞争性抑制培养的搏动心肌细胞中的相互作用来确定它们的生理意义 3)确定锚蛋白-obscurin相互作用是否被病理过程破坏，例如慢性心律失常（使用心房颤动的犬模型）或缺氧（在培养的心肌细胞中），并检查锚蛋白和/或obscurin磷酸化在调节相互作用中的潜在作用 4)使用条件基因靶向以组织和阶段特异性方式在小鼠中抑制暗蛋白Rho鸟嘌呤核苷酸交换因子（RhoGEF）信号传导，确定其在适应性心脏肥大（继发于主动脉收缩）和骨骼肌修复期间对肌原纤维组装的特异性贡献。 表征obscurin在新的肌原纤维组装和肌节-肌浆细胞组织中的作用，可能会发现治疗充血性心力衰竭和预防缺血期间肌原纤维损伤的新策略。

DESCRIPTION (provided by applicant): During development, during hypertrophic growth and in response to injury, the heart undergoes profound remodeling at both the tissue and cellular level. Defects in the assembly and integration of new myofibrils can lead to myofibril disarray, myocyte death, and congestive heart failure. Obscurin is a novel giant sarcomeric protein links the sarcomere to the sarcoplasmic membrane through interactions with titin and ankyrin. As such, it may have vital roles in myofibril assembly and in proper positioning of ankyrin-associated ion channels relative to the contractile apparatus. The specific aims of this study will therefore be to 1) examine the effects of antisense morpholino-mediated obscurin inhibition on in vivo myofibril assembly and cardiac and skeletal muscle development in zebrafish embryos using immunohistochemical analysis and videographic assessment of cardiac function 2) characterize physical interactions between obscurin and other sarcomeric proteins using yeast two hybrid and in vitro binding studies, and determine their physiologic significance through competitve inhibition of the interaction in beating cardiac myocytes in culture 3) determine if the ankyrin-obscurin interaction is disrupted by pathologic processes such as chronic arrhythmia (using a canine model of atrial fibrillation) or hypoxia (in cultured cardiac myocytes) and examine the potential role of ankyrin and/or obscurin phosphorylation in regulating the interaction 4) using conditional gene targeting to inhibit the obscurin Rho guanine nucleotide exchange factor (RhoGEF) signaling in a tissue- and stage-specific manner in mice, determine its specific contributions to myofibril assembly during adaptive cardiac hypertrophy (secondary to aortic constriction) and skeletal muscle repair. Characterizing obscurin's roles in new myofibril assembly and in sarcomere-sarcoplasma organization may identify new strategies to treat congestive heart failure and prevent myofibril damage during ischemia.