Physiological Effects of Autonomous CaMKII Activation in Cardiac Myocytes

项目摘要/摘要 钙/钙调蛋白依赖的蛋白激酶II(CaMKII)是一种关键的调节蛋白，具有多种功能。 心肌细胞包括钙调节、收缩和转录调控。当Cam绑定和 激活CaMKII，低的基础Ca/CaM亲和力允许CaMKII快速开启和关闭。CaMKII 然而，众所周知的T287位点上的自动磷酸化可以通过减缓关闭速率来延长活性状态 ~100倍并“捕获”结合态的Ca/CaM，或使CaMKII“自主”--或部分激活 即使在钙/钙调素解离之后。CaMKII活性和慢性自主的这种延长的“记忆”效应 CaMKII过度激活与心力衰竭(HF)和心律失常等心脏病理有关。 最近，我们和其他人发现了四种新的翻译后修饰(PTM)(氧化在 MM281/282，S280上的O-GlcN酰化，C273和C290上的S亚硝化)，延长CaMKII的活性和 可能被更广泛的病理应激源激活，如氧化应激(ROS)、一氧化氮 合酶(NOS)激活与糖尿病高血糖。尽管公认的重要性和潜在的 重要的临床意义，关于这些新的PTM对CaMKII的影响，人们知之甚少 自主性或其对心肌细胞生理学的特定影响。此外，关键的职能作用是 CaMKII在许多心脏病理中发挥作用，因此有必要准确地了解CaMKII到底是如何 在成年心肌细胞中被调节。因此，这项拟议研究的总体目标是调查如何 这四种新的PTM整合在一起，调节CaMKII记忆并影响心肌细胞的生理。目标1将 验证所有四个调控结构域PTM都会相对地增加CaM亲和力的假设。所有的PTM 被预测促进自主激活，尽管C273上的S-亚硝化被预测抑制激活 按Ca/Cam。目标2将检验这样的假设，即认为更大的CaM亲和力和更长的自主性 CaMKII激活时间对心肌细胞生理有较大影响。为了达到这些目的， 使用分子成像技术(例如，FRET)涉及新的荧光标记蛋白质(例如， GFP-CaMKII、AF-CaM、CFP-CaMKII-YFP(Camui))及其PTM抗性突变体膜片钳 电生理学和生化方法将用于通透性和完整的心室肌细胞。 来自兔子或耐PTM突变小鼠。拟议的研究预计将提供大量和 对心肌细胞中CaMKII激活和记忆的基本机制的高度原创性见解，以及 临床相关的PTM之间的协同作用。阐明CaMKII活性的这些机制是关键和及时的， 并可能产生治疗心脏病的新的治疗靶点。

PROJECT SUMMARY/ABSTRACT Calcium/calmodulin dependent protein kinase II (CaMKII) is a key regulator of a wide range of functions in cardiac myocytes including Ca regulation, contraction, and transcriptional control. When CaM binds and activates CaMKII, the low basal Ca/CaM affinity allows for CaMKII to be turned on and off rapidly. CaMKII autophosphorylation at the well-known T287 site, however, can prolong the active state by slowing the off-rate ~100-fold and “trapping” Ca/CaM in the bound state, or by making CaMKII “autonomous” – or partially active even after Ca/CaM dissociation. This prolonged “memory” effect of CaMKII activity and chronic autonomous CaMKII over-activation are implicated in cardiac pathologies such as heart failure (HF) and arrhythmias. Recently, we and others have discovered four novel post-translational modifications (PTMs) (oxidation at MM281/282, O-GlcNAcylation at S280, and S-nitrosylation at C273 and C290) that prolong CaMKII activity and are likely to be activated by a broader range of pathological stressors such as oxidative stress (ROS), nitric oxide synthase (NOS) activation, and diabetic hyperglycemia. Despite the recognized importance and potentially significant clinical implications, little is known about these new PTMs with respect to their effects on CaMKII autonomy or their specific influence on cardiac myocyte physiology. Moreover, the key functional role that CaMKII plays in numerous cardiac pathologies makes it essential to understand exactly how CaMKII is really regulated in adult cardiac myocytes. The overall goal of this proposed study, therefore, is to investigate how these four novel PTMs integrate to modulate CaMKII memory and affect cardiac myocyte physiology. Aim 1 will test the hypothesis that all four regulatory domain PTMs would comparably increase CaM affinity. All of the PTMs are predicted to promote autonomous activation, though S-nitrosylation at C273 is predicted to inhibit activation by Ca/CaM. Aim 2 will test the hypothesis that PTMs that impute greater CaM affinity and longer autonomous CaMKII activation durations would have greater impact on cardiac myocyte physiology. To achieve these ends, experiments using molecular imaging techniques (e.g. FRET) involving novel fluorescently-labeled proteins (e.g. GFP-CaMKII, AF-CaM, CFP-CaMKII-YFP (Camui)) and their PTM-resistant mutant variants, patch clamp electrophysiology, and biochemical approaches will be utilized in permeabilized and intact ventricular myocytes from rabbits or PTM-resistant mutant mice. The proposed studies are anticipated to provide tremendous and highly original insights into fundamental mechanisms of CamKII activation and memory in myocytes, and into the synergy among clinically relevant PTMs. Clarifying these mechanisms of CaMKII activity is critical and timely, and may yield new therapeutic targets for treating cardiac disease.