Myofilament-based mechanisms of diastolic dysfunction in HFpEF

描述（由申请人提供）：保留射血分数的心力衰竭（HFpEF）是一个主要的医疗保健问题，目前还没有已知的治疗方法可以改善长期预后。大多数患者有高血压（HTN）和同心性左心室（LV）重构史，我们称之为高血压性心脏病（HHD）。绝大多数患者还伴有左室舒张功能障碍（DD），导致腔室硬度增加。DD的恶化与从同心重构到症状性HFpEF的进展平行。这项多pi应用旨在通过术中活检获得的心肌，阐明肌丝水平的变化对DD的影响，这些心肌来自对照组和HHD非衰竭（HHD- nf）或衰竭（HFpEF）患者。我们关注的是凝血蛋白（Granzier）和肌动球蛋白动力学（LeWinter），这是HFpEF舒张刚度的两个主要决定因素。肌凝素是一种巨大的弹性肌丝，与细胞外基质（ECM）一起决定被动心肌刚度。最近的研究表明，HFpEF患者中titin的改变可能导致DD。Aims 1和2的重点是测量剥皮心肌条带中titin的硬度。（另一个结果将是对HFpEF中基于ecm的刚度的首次评估。）为了解决基于titin的机制，我们重点研究了同种异构体的表达，PKA/PKG磷酸化的titin的N2B元件降低被动刚度，以及新发现的PKCa磷酸化的PEVK元件增加被动刚度。平行实验将在野生型（WT）小鼠和基因改变的titin依从性小鼠中进行，不含实验性HFpEF和具有实验性HFpEF。肌动球蛋白过桥动力学研究将使用蒙皮心肌条的正弦长度扰动和测量单个肌原纤维的力松弛动力学。初步数据显示，HHD患者在亚最大[Ca2+]下的过桥附着时间（吨）延长，心肌肌钙蛋白I （cTnI）和肌球蛋白结合蛋白C （cMyBPC）的磷酸化降低。Ton是松弛速率的关键决定因素。指导假设是cTnI和/或cMyBP-C上的PKA位点的低磷酸化导致HHD的持续ton并导致DD，在HFpEF患者中最为严重。我们将确定延长的ton是否与肌纤维松弛动力学减慢、同心重构的严重程度、DD恶化和进展为HFpEF有关。机制研究包括用拟磷cTnI突变体替代天然cTnI。我们将对cTnI和cMyBPC PKA磷酸化位点替换的转基因小鼠进行研究，以确定磷酸化状态是否按照我们的假设预测的方式改变，以及模拟完全磷酸化的替换是否挽救了ton表型。拟议的工作是综合的，将人类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肌丝基础的机制理解来提供新的治疗靶点。

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.