Phenotypic characterization of nitric oxide regulation of cardiac function by MRI

描述（由申请人提供）：兴奋-收缩（EC）偶联和钙（Ca2+）循环在调节心脏收缩力和心脏疾病的发展中起重要作用。Ca2+处理障碍在心力衰竭中发生在多个水平，并与病理表现密切相关。然而，在体内评估EC偶联改变的方法有限。最近的研究表明，神经元NOS （nNOS）在调节EC偶联中的作用，但nNOS调节心肌收缩力的几个方面及其在心脏病中的作用仍知之甚少。在心脏，有报道称nNOS与肌膜、肌浆网（SR）和线粒体有关。nNOS与其效应蛋白的共定位已被认为是心肌控制的重要机制。然而，采用全局nNOS敲除模型NOS1-/-小鼠的研究无法通过空间限制来解决NO作用的复杂性。因此，本研究的目标是：1)开发锰增强磁共振成像（MEMRI）方法，用于在体内表征心肌中Ca2+摄取，这是Ca2+循环的第一步；2)应用最新的MRI技术研究nNOS在心功能调控和心肌病发展中的差异作用。我们将描述两种不同的nNOS破坏小鼠模型，即全局nNOS敲除小鼠和1- strobrevin敲除小鼠，这两种小鼠模型只导致细胞膜上的nNOS破坏。通过将心脏表型（如功能和Ca2+摄取）的体内MRI表征与体外肌细胞收缩性和Ca2+循环的分子/细胞分析相结合，对具有不同nNOS破坏模式的新型小鼠模型进行系统比较研究，该方法为剖析nNOS在不同亚细胞区室调节心功能中的作用提供了独特的机会。nNOS对心肌收缩和疾病进展影响的机制阐明将使nNOS成为心血管疾病的治疗靶点。

DESCRIPTION (provided by applicant): Excitation-contraction (EC) coupling and calcium (Ca2+) cycling play an important role in regulating cardiac contractile force and in the development of cardiac diseases. Disturbance of Ca2+ handling occurs at multiple levels in heart failure and is closely related to pathological performance. However, there are limited means to evaluate alterations in EC coupling in vivo. Recent studies have indicated the role of neuronal NOS (nNOS) in regulating EC coupling, with several aspects of nNOS modulation of myocardial contractility and its role in cardiac diseases still poorly understood. In the heart, nNOS has been reported to be associated with the sarcolemma, sarcoplasmic reticulum (SR), and mitochondria. Co-localization of nNOS with its effector proteins has been suggested to be important mechanisms in myocardial control. However, studies that employ a global nNOS knockout model, the NOS1-/- mouse, cannot address the complexities of NO action through spatial confinement. Therefore, the objectives of the proposed research are 1) to develop manganese-enhanced magnetic resonance imaging (MEMRI) methods for in vivo characterization of Ca2+ uptake in myocardium, the first-step in Ca2+ cycling; 2) to apply state-of-the-art MRI technology to the investigation of the differential roles of nNOS in the regulation of cardiac function and the development of cardiomyopathy. We will characterize two mouse models that differ in nNOS disruption, i.e., the global nNOS knockout mouse and the 1-dystrobrevin knockout mouse, which leads to the disruption of nNOS in cell membrane only. By combining in vivo MRI characterization of cardiac phenotypes such as function and Ca2+ uptake with in vitro molecular/cellular analysis of myocyte contractility and Ca2+ cycling in a systematic comparative study of novel mouse models with distinctive modes of nNOS disruption, this approach offers unique opportunity for dissecting the roles of nNOS in regulating cardiac function in distinct subcellular compartments. The mechanistic elucidation of the effects of nNOS on myocardial contraction and disease progression will allow nNOS to be a therapeutic target in cardiovascular diseases. PUBLIC HEALTH RELEVANCE: Excitation-contraction (EC) coupling and calcium cycling play an important role in regulating cardiac contractile force and in the development of cardiac diseases. Neuronal nitric oxide synthase (nNOS) regulates several key processes in EC coupling and is abnormal in heart failure. Pharmacological intervention that targets nNOS may be effective treatment for heart failure. The goal of the proposed research is to develop in vivo imaging method that is sensitive to altered calcium cycling, and to apply this method to elucidate the role of nNOS in EC coupling and cardiac function in mouse models of nNOS disruption.