In vivo titin cleavage by AAV9-mediated gene transfer in a genetic mouse model to determine the link between titin-based mechanical function and cardiac performance

在遗传小鼠模型中通过 AAV9 介导的基因转移进行体内肌联蛋白裂解，以确定基于肌联蛋白的机械功能和心脏性能之间的联系

• 批准号: 529713662
• 负责人: Professor Dr. Wolfgang Linke, Ph.D.
• 金额: --
• 依托单位: Institut für Physiologie II (Vegetative Physiologie)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/529713662?language=en
• 关键词: vivo; titin; cleavage; AAV9; mediated

The giant sarcomere protein titin encoded by TTN is important for cardiomyocyte structure and function and is pathologically altered in heart disease, including heart failure and cardiomyopathies. Titin contains an elastic segment in the sarcomeric I-band, which is thought to determine most of the viscoelasticity of a cardiomyocyte and be responsible, in part, for myocardial passive stiffness, as well as regulate active contraction. However, the role of the titin springs in healthy and diseased hearts is difficult to assess directly. We have developed a unique genetic mouse model, in which a tobacco etch virus (TEV) protease-recognition site and a HaloTag are cloned into elastic titin. This titin cleavage (TC) model allows the specific and acute severing of the titin springs in situ by application of TEV protease (TEVp), while the HaloTag can be used for titin visualization. Here, I hypothesize that the titin-based spring forces critically determine both the diastolic and the systolic function of the heart, which will be revealed by in vivo titin cleavage using the TC mouse model. To this end, titin will be cleaved in a graded manner in heterozygous and homozygous mutant TC mice via adeno-associated virus (AAV)9 mediated overexpression of TEVp in the cardiomyocytes. Changes to cardiac mechanical properties caused by in vivo titin cleavage will be measured in the living heart by echocardiography and cardiac magnetic resonance imaging and in isolated cardiomyocytes or cardiac fibers by sarcomere length-tension recordings and atomic force microscopy-based nanoindentation. Specific aims include the quantification of: (1) changes to the running activity, heart function of the living animal, and cardiac proteomic composition, upon graded in vivo titin cleavage; (2) associated changes to the mechanical and structural properties of the cardiac fibers and cardiomyocytes, including the stretch-dependent passive tension in both the longitudinal and the transverse direction, the Ca2+-dependent active tension, and the (sub)cellular organization as detected by confocal and electron microscopy; and (3) the extent of titin degradation, aggregation, and turnover in native cardiomyocytes following titin cleavage. Findings expected from this project should firmly establish the link between titin-based cellular functions and in vivo performance of the heart. Crucial insight into the interplay between passive and active tension generation as well as diastolic and systolic heart function can be gained and much learned about sarcomeric protein homeostasis. Results will also aid in our mechanistic understanding of myocardial remodeling in various types of heart disease.

TTN 编码的巨型肌节蛋白肌联对于心肌细胞的结构和功能很重要，并且在心脏病（包括心力衰竭和心肌病）中发生病理改变。肌联蛋白在肌节 I 带中含有一个弹性片段，该片段被认为决定了心肌细胞的大部分粘弹性，并在一定程度上负责心肌被动僵硬以及调节主动收缩。然而，肌动蛋白弹簧在健康和患病心脏中的作用很难直接评估。我们开发了一种独特的遗传小鼠模型，其中将烟草蚀刻病毒 (TEV) 蛋白酶识别位点和 HaloTag 克隆到弹性肌联中。该肌动蛋白裂解 (TC) 模型允许通过应用 TEV 蛋白酶 (TEVp) 对肌动蛋白弹簧进行原位特异性和急性切断，而 HaloTag 可用于肌动蛋白可视化。在这里，我假设基于肌动蛋白的弹簧力关键地决定心脏的舒张和收缩功能，这将通过使用 TC 小鼠模型的体内肌动蛋白裂解来揭示。为此，肌联蛋白将通过腺相关病毒 (AAV)9 介导的心肌细胞中 TEVp 的过度表达，在杂合子和纯合子突变 TC 小鼠中以分级方式裂解。将通过超声心动图和心脏磁共振成像在活体心脏中测量由体内肌动蛋白裂解引起的心脏机械特性的变化，并通过肌节长度张力记录和基于原子力显微镜的纳米压痕在分离的心肌细胞或心肌纤维中测量。具体目标包括量化：（1）根据体内肌联蛋白裂解分级，跑步活动、活体动物心脏功能和心脏蛋白质组组成的变化； (2) 心肌纤维和心肌细胞的机械和结构特性的相关变化，包括纵向和横向上的拉伸依赖性被动张力、Ca2+依赖性主动张力以及检测到的（亚）细胞组织通过共焦和电子显微镜； (3)肌联蛋白裂解后天然心肌细胞中肌联蛋白降解、聚集和周转的程度。该项目的预期结果应牢固地建立基于肌联蛋白的细胞功能与心脏体内性能之间的联系。可以获得对被动和主动张力产生以及舒张和收缩心脏功能之间相互作用的重要见解，并了解有关肌节蛋白稳态的更多信息。结果还将有助于我们了解各种类型心脏病中心肌重塑的机制。