Myofilament-based mechanisms of diastolic dysfunction in HFpEF

描述（由申请人提供）：射血分数保留性心力衰竭（HFpEF）是一种主要的医疗保健问题，目前尚无已知的可改善长期结局的治疗方法。大多数患者有高血压（HTN）和向心性左心室（LV）重构的病史，我们称之为高血压性心脏病（HHD）。绝大多数患者还患有LV舒张功能障碍（DD），导致心腔僵硬度增加。DD恶化与从向心性重塑进展为症状性HFpEF平行。该多PI应用程序旨在使用对照组和非衰竭（HHD-NF）或衰竭（HFpEF）HHD患者的术中活检获得的心肌阐明肌丝水平的变化，这些变化导致DD。我们专注于肌联蛋白（Granzier）和肌动球蛋白动力学（LeWinter），HFpEF舒张期僵硬的两个主要决定因素。肌联蛋白是一种巨大的弹性肌丝，与细胞外基质（ECM）一起决定被动心肌僵硬度。最近的研究揭示了HFpEF患者肌联蛋白的改变可能导致DD。目的1和2侧重于测量皮肤心肌条中基于肌联蛋白的硬度。(An其他结果将是HFpEF中基于ECM的刚度的首次评价。）为了解决肌联蛋白为基础的机制，我们专注于亚型表达，PKA/PKG磷酸化肌联蛋白的N2 B元件，降低被动刚度和新发现的PKCa磷酸化的PEVK元件，增加被动刚度。将在野生型（WT）小鼠和具有遗传改变的肌联蛋白顺应性的小鼠（不具有和具有实验HFpEF）上进行平行实验。肌动球蛋白跨桥动力学将研究使用正弦长度扰动在皮肤心肌条和测量力松弛动力学在单个肌原纤维。初步数据显示，HHD患者在次最大[Ca 2 +]下的跨桥附着时间（ton）延长，心肌肌钙蛋白I（cTnI）和肌球蛋白结合蛋白C（cMyBPC）磷酸化降低。Ton是弛豫速率的关键决定因素。指导性假设是cTnI和/或cMyBP-C上PKA位点的低磷酸化导致Ton延长，并导致HHD中的DD，在HFpEF患者中最严重。我们将确定延长的ton是否与肌原纤维松弛动力学减慢、向心性重构的严重程度、DD恶化和进展为HFpEF相关。机制研究包括用磷酸模拟cTnI突变体取代天然cTnI。将研究cTnI和cMyBPC PKA磷酸化位点置换的转基因小鼠（有和没有实验诱导的HFpEF），以确定磷酸化状态是否以我们的假设预测的方式改变ton，以及模拟完全磷酸化的置换是否挽救ton表型。拟议的工作是综合性的，将人体组织研究与遗传小鼠模型相结合。这些方法被最近的NHLBI工作组确定为HF预防的优先事项。这项工作的长期目标是通过对HFpEF的肌丝基础的机械理解提供新的治疗靶点。

DESCRIPTION (provided by applicant): Heart failure with preserved ejection fraction (HFpEF) is a major health care problem for which there are no known treatments that improve long-term outcomes. Most patients have a history of hypertension (HTN) and concentric left ventricular (LV) remodeling, a combination we term hypertensive heart disease (HHD). The vast majority of patients also have LV diastolic dysfunction (DD) resulting in increased chamber stiffness. Worsening DD parallels progression from concentric remodeling to symptomatic HFpEF. This multi-PI application is designed to elucidate the changes at the myofilament level that contribute to DD using myocardium obtained by intra-operative biopsy from controls and patients with HHD who are either non-failing (HHD-NF) or failing (HFpEF). We focus on titin (Granzier) and actomyosin dynamics (LeWinter), two major determinants of diastolic stiffness in HFpEF. Titin is a giant elastic myofilament that together with the extracellular matrix (ECM) determines passive myocardial stiffness. Recent studies reveal alterations in titin in HFpEF patients that might contribute to DD. Aims 1 and 2 focus on measuring titin-based stiffness in skinned myocardial strips. (An additional outcome will be the first evaluation of ECM-based stiffness in HFpEF.) To address titin-based mechanisms we focus on isoform expression, PKA/PKG phosphorylation of titin's N2B element that decreases passive stiffness and the newly discovered PKCa phosphorylation of the PEVK element that increases passive stiffness. Parallel experiments will be carried out on wild-type (WT) mice and mice with genetically altered titin compliances, without and with experimental HFpEF. Actomyosin cross-bridge dynamics will be studied using sinusoidal length perturbation in skinned myocardial strips and measurement of force relaxation kinetics in single myofibrils. Preliminary data reveal prolonged cross-bridge attachment time (ton) at submaximal [Ca2+] and reduced phosphorylation of cardiac troponin I (cTnI) and myosin binding protein C (cMyBPC) in HHD patients. Ton is a key determinant of relaxation rate. The guiding hypothesis is that hypo- phosphorylation of PKA sites on cTnI and/or cMyBP-C causes prolonged ton and contributes to DD in HHD and is most severe in HFpEF patients. We will determine if prolonged ton is associated with slowed myofibrillar relaxation kinetics, severity of concentric remodeling, worsening DD and progression to HFpEF. Mechanistic studies include replacing native cTnI with phospho-mimetic cTnI mutants. Transgenic mice with cTnI and cMyBPC PKA phosphorylation site substitutions with and without experimentally induced HFpEF will be studied to determine if phosphorylation state alters ton in the way our hypothesis predicts and whether substitutions simulating complete phosphorylation rescue the ton phenotype. The proposed work is integrative, combining studies in human tissue with genetic mouse models. These approaches were identified by recent NHLBI working groups as priorities for HF prevention. The long-term goal of this work is to provide novel therapeutic targets through a mechanistic understanding of the myofilament basis of HFpEF.