Calcium-independent enhancement of contractility in normal and failing cardiomyoc

描述(由申请人提供)：心脏病是美国的主要死亡原因，并且在世界各地急剧上升。这是一种探索性应用，旨在通过靶向心肌细胞肌节的收缩装置来开发改善心脏性能的策略。虽然目前的应用主要集中在体外研究，但长期目标是在不影响心脏舒张期功能的情况下增强收缩功能，并仍允许对肾上腺素能刺激的反应。我们将直接针对心肌细丝激活和肌动蛋白-肌球蛋白‘交叉桥’循环，从而在不需要增加细胞内[Ca~(2+)]的情况下增强心肌细胞的收缩(而不是松弛)。为了在给定的[Ca~(2+)]下增加细丝的激活，我们将用具有增强的钙结合特性的突变体TNC(L48Q)取代肌丝中的天然肌钙蛋白C(目标1)。为了加强跨桥循环，我们将通过增加心肌细胞中将ATP转化为dATP的酶(核糖核苷酸还原酶；RR)的表达来增加细胞2脱氧-ATP(DATP)的产量(目标2)。我们提供的重要数据表明，这些方法改善了去膜(去皮)心肌组织的收缩能力，而不影响松弛动力学或静息僵硬。在拟议的研究中，我们将使用病毒转染策略来确定在培养的完整成年心肌细胞中是否存在类似的心肌收缩能力增加。我们还将确定这些方法是否会影响肌小球收缩蛋白的异构体和磷酸化特征，或者在刺激激活和松弛过程中细胞内[Ca+]的水平。在提案的第二年，我们将开始为扩展提案开发动物模型，以研究这些操作在正常心脏和病理条件下如何在原位和体外影响整个心脏功能。公共卫生相关性：心力衰竭的基础是心肌肌丝收缩能力的降低。目前的大多数治疗方法都集中在收缩过程中增强细胞内钙离子的机制，其中包括影响舒张期功能的机制。为了避免这一点，我们的建议直接针对肌丝，以增强收缩，而不需要增加细胞内钙离子。

DESCRIPTION (provided by applicant): Heart disease is the leading cause of death in the United States and has been rising dramatically around the world. This is an exploratory application to develop strategies for improving cardiac performance by targeting the contractile apparatus of cardiomyocyte sarcomeres. While the current application focuses on in vitro studies, the long term goal is to enhance systolic function without compromising diastolic function of the heart, and still allow responsiveness to adrenergic stimulation. We will target the cardiac thin filament activation and the actin-myosin `crossbridge' cycle directly, such that cardiomyocyte contraction (but not relaxation) is enhanced without the need for increased intracellular [Ca2+]. To increase thin filament activation at a given [Ca2+] we will replace native troponin C in myofilaments with a mutant TnC (L48Q) that has enhanced Ca2+ binding properties (aim 1). To enhance crossbridge cycling we will increase the cellular production of 2 deoxy-ATP (dATP) via increased expression of the enzyme that converts ATP to dATP in cardiomyocytes (ribonucleotide reductase; RR) (aim 2). We have provided significant data demonstrating these approaches improve contractility in demembranated (skinned) cardiac tissue, without affecting relaxation kinetics or resting stiffness. For the proposed studies we will use a viral transfection strategy to determine if similar increases in cardiac contractility occur in intact adult cardiomyocytes in culture. We will also determine whether these approaches affect sarcomere contractile protein isoform and phosphorylation profiles or the level of intracellular [Ca2+] during stimulated activation and in relaxation. In the second year of the proposal we will begin development of animal models for an expanded proposal to study how these manipulations influence whole heart function in situ and in vitro in normal hearts and under pathological conditions. PUBLIC HEALTH RELEVANCE: Heart failure at its base is a reduction in cardiac myofilament contractility. Most current therapies focus on mechanisms that enhance intracellular Ca2+ during systole which, among other things, can affect diastolic function. To avoid this, our proposal targets myofilaments directly to enhance contraction without the need for increased intracellular Ca2+.