Phenotypic characterization of nitric oxide regulation of cardiac function by MRI

说明(申请人提供)：兴奋-收缩(EC)偶联和钙(Ca~(2+))循环在调节心脏收缩力量和心脏疾病的发展中起着重要作用。心衰患者存在多水平的钙离子调节障碍，且与心衰的病理表现密切相关。然而，在活体内评估EC偶联改变的手段有限。最近的研究表明，神经元型一氧化氮合酶(NNOS)在调节EC偶联中的作用，但nNOS对心肌收缩功能的调节及其在心脏病中的作用尚不清楚。在心脏中，nNOS被报道与肌膜、肌浆网(SR)和线粒体有关。NNOS与其效应蛋白的共存被认为是心肌控制的重要机制。然而，使用全球nNOS基因敲除模型NOS1/-小鼠的研究不能通过空间限制来解决NO作用的复杂性。因此，这项研究的目标是：1)建立体内表征心肌钙摄取的锰增强磁共振成像(MEMRI)方法，这是钙循环的第一步；2)应用最先进的MRI技术来研究nNOS在心功能调节和心肌病发生发展中的不同作用。我们将描述两种不同的nNOS基因敲除小鼠模型，即全局nNOS基因敲除小鼠和1-dystrobrevin基因敲除小鼠，后者仅导致细胞膜上nNOS的破坏。通过将心脏表型(如功能和钙摄取)的在体MRI表征与体外心肌细胞收缩和钙循环的分子/细胞分析相结合，对具有独特nNOS干扰模式的新型小鼠模型进行了系统的比较研究，该方法为剖析nNOS在不同亚细胞分区中调节心脏功能的作用提供了独特的机会。从机制上阐明nNOS对心肌收缩和疾病进展的影响，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.