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

由TTN编码的巨肌节蛋白titin对心肌细胞的结构和功能很重要，并在心脏病（包括心力衰竭和心肌病）中发生病理改变。Titin在肌体i带中包含一个弹性段，它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，titin弹簧在健康和患病心脏中的作用很难直接评估。我们建立了一种独特的遗传小鼠模型，将烟草蚀刻病毒（TEV）蛋白酶识别位点和HaloTag克隆到弹性titin中。这种titin切割（TC）模型允许使用TEV蛋白酶（TEVp）在原位特异性和急性切断titin弹簧，而HaloTag可用于titin可视化。在这里，我假设基于titin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titin切割来揭示。为此，将通过腺相关病毒（AAV）9介导心肌细胞中TEVp的过表达，在杂合子和纯合子突变体TC小鼠中以分级方式切割titin。在活体心脏中，通过超声心动图和心脏磁共振成像，以及在分离的心肌细胞或心脏纤维中，通过肌节长度张力记录和基于原子力显微镜的纳米压痕，可以测量体内titin切割引起的心脏力学特性的变化。具体目标包括量化：(1)活体动物的跑步活动、心脏功能和心脏蛋白质组学组成的变化，根据体内titin的分级切割；(2)心肌纤维和心肌细胞的机械和结构特性的相关变化，包括纵向和横向拉伸依赖的被动张力，Ca2+依赖的主动张力，以及通过共聚焦和电子显微镜检测到的（亚）细胞组织；(3) titin切割后天然心肌细胞中titin降解、聚集和转换的程度。这个项目的预期结果应该牢固地建立以titin为基础的细胞功能和心脏的体内表现之间的联系。对被动和主动张力产生以及心脏舒张和收缩功能之间的相互作用的重要见解可以获得，并对肌合成蛋白稳态有更多了解。结果也将有助于我们对各种类型心脏病的心肌重构的机制理解。

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.