Titin-based adaptations of cardiac function

描述（由申请人提供）：基于Titin的心脏功能适应Titin的I带区域作为分子弹簧发挥作用，与细胞外基质（ECM）一起调节被动心肌硬度，这对舒张充盈至关重要。先前的工作表明，在出生后发育过程中广泛的差异剪接将巨大的胎儿心脏肌联蛋白亚型转化为更小和更硬的成人肌联蛋白亚型。出生后亚型转换的机械意义和ECM变化的机械重要性需要深入研究。目标1集中在左心室（LV）和右心室（RV）中的这个主题。在这项工作中，我们制作了一个小鼠模型，其中负责剪接刚性成人titins的剪接因子（RBM 20）已被删除，并在成人心肌中表达大小与巨大胎儿心脏同种型相似的titins。该模型模拟具有RBM 20突变的扩张型心肌病（DCM）患者，其中胎儿心脏大小的titins在成人中表达。将研究肌联蛋白的差异剪接和翻译后修饰，以及ECM的变化，并将使用多学科实验在广泛的水平上研究被动心肌硬度。上调顺应性肌联蛋白亚型也可能影响向心性和离心性重塑中的心脏肥大（目的2）。最近的研究表明，牵张感应信号体与肌联蛋白结合，肌联蛋白在应变增加时触发肥大。这一重要概念需要进行严格的测试和深入研究。 study.顺应性肌联蛋白在我们的新KO小鼠模型中的表达预测肥大减弱（因为肌联蛋白的应变减少），并且这有助于DCM表型。目的2将通过研究响应于增加的后负荷（横向主动脉缩窄（TAC）模型）或增加的前负荷（下腔静脉瘘（ACF）手术模型）的肥大来测试这一点，这两种对比模型分别导致不同的向心性或离心性肥大表型。肌联蛋白同种型转换也可能影响收缩，因为已经提出基于肌联蛋白的被动张力在心脏的Frank-Starling机制（FSM）中起重要作用，即，收缩力随长度的增加。尽管FSM对于调节每搏输出量以匹配静脉回流至关重要，但FSM的潜在机制仍然难以捉摸。目的3将使用RBM 20小鼠模型测试肌联蛋白所起的作用，该模型在成人心脏中表达巨大的肌联蛋白同种型，并且提供了一个令人兴奋的机会来研究生理条件下基于肌联蛋白的被动张力对FSM的减少的影响。实验将在细胞、组织和整个心脏水平进行。为此应用制作了一种新型小鼠模型，开发了新型试剂，建立了一支强大的团队，初步研究的结果支持了该提案的指导假设。拟议的研究将提供肌联蛋白和ECM在舒张期充盈、收缩期性能， 和肥大信号传导，从基础科学的观点来看是非常感兴趣的主题，并且对于小儿心脏病、成人心力衰竭和肥大性和扩张型心肌病具有高度的临床意义。

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.