Titin-based adaptations of cardiac function

说明(申请人提供)：基于Titin的心脏功能适应性Titin的I带区域起到分子弹簧的作用，与细胞外基质(ECM)一起调节被动心肌硬度，这对舒张期充盈至关重要。先前的工作表明，出生后发育过程中广泛的差异剪接可以将巨大的胎儿心脏肌动蛋白亚型转化为更小、更坚硬的成体肌动蛋白亚型。出生后异型转换的力学意义和ECM变化的力学重要性需要深入研究。目的1在左心室(LV)和右心室(RV)都聚焦于这一主题。在这项工作中，我们制作了一个小鼠模型，其中负责剪接僵硬的成人乳头的剪接因子(RBM20)已经被删除，并且在成人心肌中表达的乳头大小类似于巨大的胎儿心脏亚型。该模型模拟具有RBM20突变的扩张型心肌病(DCM)患者，其中胎儿心脏大小的Tiins在成人中表达。将研究Titin的差异剪接和翻译后修饰，以及细胞外基质的变化，并将利用多学科实验在广泛的水平上研究被动心肌僵硬。顺应性肌动蛋白亚型上调也可能影响向心性和偏心性重构中的心肌肥厚(目标2)。最近的研究表明，拉伸感应信号小体与肌动蛋白结合，在应变增加时触发肥大。这一重要概念需要进行关键的测试和深入的研究 学习。在我们的新的KO小鼠模型中，顺应性Tiins的表达预示着肥大减轻(因为Titin的应变减少)，这有助于DCM的表型。目的2将通过研究后负荷增加(横断性主动脉缩窄(TAC)模型)或前负荷增加(主动脉腔瘘(ACF)手术模型)的肥厚来验证这一点，这两种不同的模型分别导致明显的向心性或偏心性肥厚表型。肌动蛋白亚型转换也可能影响收缩，因为基于肌动蛋白的被动张力在心脏的Frank-Starling机制(FSM)中发挥着重要作用，即随着长度的增加收缩能力增加。尽管FSM对于调整每搏输出量以匹配静脉回流至关重要，但FSM背后的机制(S)仍然难以捉摸。目的3将使用在成人心脏中表达巨型肌动蛋白异构体的RBM20小鼠模型来测试肌动蛋白所起的作用，并提供一个令人兴奋的机会来研究在生理条件下减少基于肌动蛋白的被动张力对FSM的影响。实验将在细胞、组织和整个心脏水平上进行。为了这一应用，开发了一种新的小鼠模型，开发了新的试剂，成立了一个强大的团队，初步研究的结果支持了该提案的指导性假设。拟议的研究将为肌动蛋白和细胞外基质在舒张期充盈、收缩功能、 以及肥大信号，这是基础科学观点中非常感兴趣的话题，对儿童心脏病、成人心力衰竭和肥厚型扩张型心肌病具有很高的临床意义。

DESCRIPTION (provided by applicant): Titin-based adaptations of cardiac function Titin's I-band region functions as a molecular spring that together with the extracellular matrix (ECM) regulates the passive myocardial stiffness that is critically important for diastolic filling. Prevous work has shown that extensive differential splicing during postnatal development converts the giant fetal cardiac titin isoform into smaller and stiffer adult titin isoforms. The mechanical significance of postnatal isoform switching and the mechanical importance of changes in the ECM require in-depth study. Aim 1 focuses on this topic in both the left ventricle (LV) and right ventricle (RV). For this work we made a mouse model in which a splice factor (RBM20) that is responsible for splicing stiff adult titins has been deleted and that expresses in adult myocardium titins similar in size to the giant fetal cardiac isoforms. This model mimics dilated cardiomyopathy (DCM) patients with RBM20 mutations in which fetal cardiac sized titins are expressed in the adult. Differential splicing and posttranslational modifications of titin will be studied, as well as changes in the ECM, and passive myocardial stiffness will be studied using multi-disciplinary experiments at a wide range of levels. Upregulating compliant titin isoforms might also affect cardiac hypertrophy in both concentric and eccentric remodeling (Aim 2). Recent work indicated that a stretch-sensing signalosome binds to titin that triggers hypertrophy in response to an increase in strain. This important concept requires critical testing and in-depth study. The expression of compliant titins in our new KO mouse model predict that hypertrophy is attenuated (because titin's strain is reduced) and that this contributes to the DCM phenotype. Aim 2 will test this by studying hypertrophy in response to increased afterload (transverse aortic constriction (TAC) model) or increased preload (aortocaval fistula (ACF) surgery model), two contrasting models that result in the distinct concentric or eccentric hypertrophy phenotypes, respectively. Titin isoform switching is also likely to affect systole as titin- based passive tenson has been proposed to play an important role in the Frank-Starling mechanism (FSM) of the heart, i.e., the increase in contractility with length. Although the FSM is critical for adjusting stroke volume to match venous return, the mechanism(s) underlying the FSM have remained elusive. Aim 3 will test the role that titin plays using the RBM20 mouse model that expresses giant titin isoforms in the adult heart and that provides an exciting opportunity to study the effet of a reduction in titin-based passive tension on the FSM under physiological conditions. Experiments will be performed at the cell, tissue, and whole heart levels. A novel mouse model was made for this application, novel reagents were developed, a strong team is in place, and results from pilot studies support the proposal's guiding hypotheses. The proposed studies will provide insights in the importance of titin and the ECM in diastolic filling, systolic performance, and hypertrophy signaling, topics of great interest from a basic science viewpoint and that have high clinical significance for pediatric heart disease, heart failure in adults, and hypertrophic ad dilated cardiomyopathy.