Calcium-independent enhancement of contractility in normal and failing cardiomyoc

描述（由申请人提供）：心脏病是美国人死亡的主要原因，并且在世界范围内急剧上升。这是一项探索性应用，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功能的情况下增强心脏的收缩功能，并仍然允许对肾上腺素能刺激的反应。我们将直接针对心脏细丝激活和肌动蛋白-肌球蛋白“过桥”周期，这样心肌细胞收缩（而不是松弛）得到增强，而不需要增加细胞内[Ca2+]。为了在给定的[Ca2+]下增加细丝的激活，我们将用具有增强Ca2+结合特性的突变TnC （L48Q）取代肌丝中的天然肌钙蛋白C（目的1）。为了增强过桥循环，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2脱氧ATP （dATP）的细胞生成（目的2）。我们提供的重要数据表明，这些方法可以改善脱膜（剥皮）心脏组织的收缩性，而不影响松弛动力学或静息刚度。对于拟议的研究，我们将使用病毒转染策略来确定在培养的完整成人心肌细胞中是否发生类似的心脏收缩性增加。我们还将确定这些方法是否会影响肌节收缩蛋白异构体和磷酸化谱，或在刺激激活和放松期间细胞内[Ca2+]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，以研究这些操作如何在正常心脏和病理条件下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纤维收缩性降低。目前的大多数治疗方法都集中在收缩期间增强细胞内Ca2+的机制上，这可以影响舒张功能。为了避免这种情况，我们的建议直接针对肌丝来增强收缩，而不需要增加细胞内Ca2+。

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+.