Function of Giant Sarcomere Matrix Proteins in Muscle

肌联蛋白是已知的最大的蛋白质，其功能是作为一种复杂的分子弹簧， 被动性心肌僵硬重要的是，肌联蛋白的刚度可以在转录后调节（通过改变蛋白质的结构）。 刚性N2 B和更柔顺的N2 BA肌联蛋白同种型的表达比率）和后分化（例如，经由 肌联蛋白的蛋白激酶G（PKG）磷酸化的变化）。心力衰竭时肌联蛋白的硬度增加， 射血分数保留（HFpEF），由于肌联蛋白磷酸化紊乱，特别是低磷酸化 PKG网站目前还没有针对HFpEF的有效疗法。本申请研究潜在的基于肌联蛋白的 治疗方案。对于这项工作，我们有几种HFpEF的动物模型： 其中Titin的弹性区域被延伸到更高的程度并且被动刚度相应地增加， 压力超负荷（TAC/DOCA）诱导的具有舒张功能障碍的小鼠和豚鼠模型， 错乱的肌联蛋白磷酸化。现有的药物改善肌联蛋白为基础的舒张硬化的潜力， HFpEF将在目标1和2中解决。二甲双胍是一种胰岛素增敏药物， 在动物和人类研究中改善舒张功能。我们的初步研究表明，二甲双胍挽救舒张期 功能障碍和正常化肌联蛋白为基础的僵硬。磷酸二酯酶PDE 9A最近被证明可以减少 已知肌联蛋白的PKG位点的低磷酸化发生在HFpEF中。因此，我们也 研究PDE 9A抑制（PDE 9Ai）是否改善舒张功能障碍。 最近的研究表明，肌联蛋白突变是扩张型心肌病（DCM）的病因， 以进行性左心室（LV）扩张和收缩功能障碍为特征的HF。目标3旨在提高 了解肌联蛋白引起扩张型心肌病的机制。通过基因打靶，我们产生了第一个titin- 基于小鼠模型，在基线条件下发展DCM，N2 BA-PEVK KO。在该模型中，PEVK 特异于N2 BA肌联蛋白同种型的序列被删除，初步研究表明，这会导致严重的 扩张和射血分数降低。将在临床前阶段研究DCM的致病机制（2 月）和心脏扩张后（6个月），使用无偏方法和候选方法， 对N2 BA同种型的独特机制的研究。救援实验包括其中的压力， 通过靶向肌联蛋白剪接因子RBM 20将降低肌联蛋白的弹簧。 该提案以其基础和临床重要研究及其深入和综合的方法，寻求 继续我们的铁蛋白前沿研究我们预计，这些研究将大大改善 理解肌联蛋白在HFpEF中的作用，并提供治疗选择的新见解。我们还将研究 一种新的基于肌联蛋白的DCM小鼠模型，并研究肌联蛋白可引起DCM的机制， 这也需要紧急研究。我们有支持性的试点数据，并拥有一支优秀的研究团队。

Titin, the largest protein known, functions as a complex molecular spring that is a dominant contributor to passive myocardial stiffness. Importantly, titin’s stiffness can be tuned post-transcriptionally (by varying the expression ratio of the stiff N2B and more compliant N2BA titin isoforms) and post-translationally (e.g., via changes in protein kinase G (PKG) phosphorylation of titin). Titin’s stiffness is increased in heart failure with preserved ejection fraction (HFpEF), due to deranged titin phosphorylation, in particular hypo-phosphorylation of PKG sites. Currently no effective therapies for HFpEF exist. This application studies potential titin-based treatment options. For this work we have available several animal models of HFpEF: a genetic mouse model in which titin’s spring region is extended to a higher degree and passive stiffness is increased accordingly and pressure-overload (TAC/DOCA) induced mouse and guinea pig models that have diastolic dysfunction and deranged titin phosphorylation. The potential of existing drugs to ameliorate titin-based diastolic stiffening in HFpEF will be addressed in Aims 1 and 2. Metformin is an insulin sensitizing drug that has been shown to improve diastolic function in animal and human studies. Our pilot studies show that metformin rescues diastolic dysfunction and normalizes titin-based stiffness. The phosphodiesterase PDE9A was recently shown to reduce PKG activity and it is known that hypo-phosphorylation of titin’s PKG sites occurs in HFpEF. Hence we also study whether PDE9A inhibition (PDE9Ai) ameliorates diastolic dysfunction. Recent studies showed that titin mutations are causative for dilated cardiomyopathy (DCM), a prevalent form of HF characterized by progressive left ventricular (LV) dilation and systolic dysfunction. Aim 3 seeks to boost understanding of mechanisms by which titin can cause DCM. Through gene targeting we generated the first titin- based mouse model that under baseline conditions develops DCM, the N2BA-PEVK KO. In this model, PEVK sequences that are specific to the N2BA titin isoform were deleted and pilot studies show that this causes severe dilation and a reduction in ejection fraction. DCM-causing mechanisms will be studied at a preclinical stage (2 mo) and after the heart dilates (6 mo), using both an unbiased approach and a candidate approach that focusses on mechanisms that are unique to the N2BA isoform. A rescue experiment is included in which the stress on titin’s spring will be reduced by targeting the titin splicing factor RBM20. With its basic and clinically important research and its in-depth and integrative approach, this proposal seeks to continue our track record of cutting edge titin research. We anticipate that these studies will greatly improve understanding of the roles of titin in HFpEF and provide novel insights in therapeutic options. We also will study a novel mouse model with titin-based DCM and investigate mechanisms by which titin can cause DCM, an area that also needs urgent study. We have supportive pilot data and have an excellent research team in place.