Physiological Effects of Autonomous CaMKII Activation in Cardiac Myocytes

项目总结/摘要 钙/钙调蛋白依赖性蛋白激酶II（CaMKII）是一种重要的调节因子，在细胞内具有广泛的功能。 心肌细胞包括Ca调节、收缩和转录控制。当钙调素结合， 激活CaMKII，低基础Ca/CaM亲和力允许CaMKII快速开启和关闭。CaMKII 然而，在众所周知的T287位点的自磷酸化可以通过减慢解离速率来延长活性状态 ~100倍并“捕获”结合状态的Ca/CaM，或通过使CaMKII“自主”或部分活化 即使在Ca/CaM解离之后。这种CaMKII活性的长期“记忆”效应和慢性自主性 CaMKII过度激活与心脏病如心力衰竭（HF）和心律失常有关。 最近，我们和其他人发现了四种新的翻译后修饰（PTM）（氧化在 MM 281/282，S280处的O-GlcNAc酰化，以及C273和C290处的S-亚硝基化），其延长CaMKII活性， 可能被更广泛的病理应激源激活，如氧化应激（ROS）、一氧化氮（NO） 合成酶（NOS）激活和糖尿病性高血糖。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的影响知之甚少 自主性或其对心肌细胞生理学的特定影响。此外， CaMKII在许多心脏病中起作用，因此必须确切了解CaMKII是如何在心脏病中发挥作用的。 在成人心肌细胞中调节。因此，这项拟议研究的总体目标是调查如何 这四种新的PTM整合以调节CaMKII记忆并影响心肌细胞生理学。目标1将 检验所有四种调节结构域PTM都将不可避免地增加CaM亲和力的假设。所有PTM 虽然C273的S-亚硝基化被预测为抑制激活，但预测为促进自主激活 Ca/CaM目的2将检验假设，归因于更大的CaM亲和力和更长的自主性的PTM CaMKII激活持续时间对心肌细胞生理学有更大的影响。为了实现这些目标， 使用分子成像技术（例如FRET）的实验涉及新的荧光标记的蛋白质（例如， GFP-CaMKII、AF-CaM、CFP-CaMKII-YFP（Camui））及其PTM抗性突变体变体，膜片钳 电生理学和生物化学方法将用于透化和完整的心室肌细胞 来自兔子或PTM抗性突变小鼠。预计拟议的研究将提供巨大的， 对肌细胞中CamKII激活和记忆的基本机制的高度原创性见解， 临床相关PTM之间的协同作用。澄清CaMKII活性的这些机制是关键和及时的， 并且可能产生用于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.