InsP3 and CaMKII Isoforms Signaling

InsP3 和 CaMKII 同工型信号转导

• 批准号: 8114850
• 负责人: Joan Heller Brown
• 金额: $ 38.85万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8114850
• 关键词: Adult; Affect; Agonist; Attenuated; Biochemical; Biological Assay; Body Weight; Calcineurin; Calcium/calmodulin-dependent protein kinase; Cardiac; Cardiac Myocytes; Cell Extracts; Cell Fractionation; Cell Nucleus; Cells; Coupling; Cytoplasm; Dependence; Detection; Development; Dominant-Negative Mutation; Endothelin-1; Enzymes; Failure; Feedback; Fluorescence Resonance Energy Transfer; Fractionation; Functional disorder; Gene Expression; Hand; Heart; Heart Atrium; Heart Hypertrophy; Heart failure; High Pressure Liquid Chromatography; Histone Deacetylase; Hypertrophy; Immunoblotting; Infusion procedures; Inositol; Intervention; Knock-out; Knockout Mice; Lead; Left ventricular structure; Ligands; Link; Measures; Mediating; Metabolism; Methods; Mus; Muscle Cells; Myocardium; Nature; Neonatal; Nuclear; Nuclear Localization Signal; Pathway interactions; Pattern; Phosphatidylinositols; Phospholipase C; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Preparation; Protein Isoforms; Proteins; RNA Splicing; Regulation; Reporter; Reporting; Reticulum; Role; Sarcoplasmic Reticulum; Signal Transduction; Stimulus; Techniques; Testing; Time; Tissues; Transcriptional Regulation; Variant; Ventricular; Ventricular Dysfunction; Work; base; calmodulin-dependent protein kinase II; constriction; in vivo; myo-inositol-trisphosphate 3-kinase; protein E; receptor; research study; response; sensor; spatiotemporal; tripolyphosphate

The Ca/calmodulin-dependent protein kinase II (CaMKII) is regulated by and involved in control of Ca cycling in the myocardium and has been implicated in transcriptional regulation leading to cardiac hypertrophy. CaMKIIdelta is the predominant cardiac isoform and splice variants lacking (delta-c) or containing (delta-B) a nuclear localization signal are both expressed in cardiomyocytes. The functional significance of the existence of both cytoplasmic (delta-c) and nuclear (delta-B) CaMKII subunits in the heart, and the nature of the Ca signals that activate them, remain unknown. The Ca involved in E-C coupling is released from the sarcopiasmic reticulum (SR), but cardiomyocytes also contain inositol 1,4,5-triphosphate (InsP3) receptor regulated Ca stores. Ligands for receptors that activate phospholipase C (PLC)-mediated InsP3 formation are established activators of hypertrophy, but a signaling role for InsP3 has not been demonstrated. We hypothesize that local Ca release from perinuclear InsP3 sensitive stores activates CaMKII and regulates gene expression and hypertrophy through effects on HDAC/MEF2 or other transcriptional pathways. Studies proposed in Aim #1 examine the effects of deletion of the type 2 and type 3 InsP3 receptor (InsP3Rs) or CaMKIIdelta on the development of physiological and pathological hypertrophy and failure induced by transverse aortic constriction (TAG) or agonist infusion, assessing changes in echocardiographic parameters, left ventricle to body weight ratio, ventricular myocyte size, gene expression and protein phosphorylation. In Aim #2 we determine whether localized Ca release from InsP3 sensitive Ca stores preferentially activates nuclear CaMKIIdelta while stimuli that lead to global increases in cytosolic Ca via release from the SR activate the cytoplasmic CaMKIIdelta. CaMKII activation in these compartments and the activation of delta-B versus delta-c CaMKII will be examined by cell fractionation and immunoblotting, as well as by development of a FRET based CaMKII activity reporter (CaMKAR). In Aim #3 we examine a further corollary to this hypothesis i.e, that the nuclear vs. cytosolic localization of CaMKIIdelta targets different substrates. Proposed studies are directed at establishing whether there are distinct roles for nuclear and cytoplasmic CaMKII in activating MEF2/SRF gene expression, HDAC kinase activity, HDAC phosphorylation and cardiomyocyte hypertrophy, versus in phosphorylation of RyR and other SR proteins. This hypothesis is also tested in vivo using mice in which delta-B and delta-c subunits are individually targeted for knockout. Aim #4 considers an intriguing but unexplored possibility for feedback regulation i.e, in the ability of CaMKII to regulate both an InsP3 kinase and an InsP3 5' phosphatase involved in metabolism of InsP3. The possibility that CaMKII regulates these enzymes in cardiomyocytes will be examined by direct expression and phosphorylation assays, and by measuring InsP3 accumulation. Biochemical assays and a FRET based InsP3 sensor will be utilized to examine changes in InsP3 metabolism. Aim #4 also uses these methods to compare agonist induced InsP3 formation in atrial versus ventricular cardiomyocytes and alterations that accompany heart failure, and considers the question of whether and how InsP3 levels increase in the nuclear compartment.

CA/钙调蛋白依赖性蛋白激酶II（CAMKII）受心肌中CA循环的控制并参与了导致心脏肥大的转录调节。 Camkiidelta是缺少缺乏（Delta-C）或包含（Delta-B）核定位信号的主要心脏同工型和剪接变体。心脏中细胞质（Delta-C）和核（Delta-B）Camkii亚基的功能意义，以及激活它们的CA信号的性质，仍然未知。与E-C耦合相关的CA是从肌张力网状（SR）中释放出来的，但心肌细胞还含有肌醇1,4,5-三磷酸（INSP3）受体调节的Ca商店。激活受体的配体 磷脂酶C（PLC）介导的INSP3形成是肥大的激活剂，但尚未证明INSP3的信号传导作用。我们假设局部CA从核周INSP3敏感存储中释放会激活CAMKII，并通过对HDAC/MEF2或其他转录途径的影响来调节基因表达和肥大。目标＃1中提出的研究检查了2型和3型INSP3受体（INSP3R）或Camkiidelta对生理和病理肥大的发展以及横向主动脉膜收缩（TAG）或激动剂输液引起的失败的影响，评估体重的尺寸，分别构造，分别是体重率的尺寸，分别构成了体重率，评估了横向的变化。在AIM＃2中，我们确定局部CA是否从INSP3敏感的CA存储中释放优先激活 核Camkiidelta而导致通过从SR释放激活细胞质Camkiidelta的刺激会导致全球胞质CA的全球增加。这些隔室中的CAMKII激活以及Delta-B与Delta-C Camkii的激活将通过细胞分馏和免疫印迹以及通过开发基于FRET的CAMKII活动报告（CAMKAR）来检查。在AIM＃3中，我们研究了该假设的进一步推论，即Camkiidelta的核与胞质定位靶向不同的底物。拟议的研究旨在确定核和细胞质CAMKII在激活MEF2/SRF基因表达，HDAC激酶活性，HDAC磷酸化和心肌细胞肥大，在RYR和其他SR蛋白的磷酸化中是否存在明显的作用。该假设还使用小鼠在体内进行了检验 昏死。 AIM＃4考虑了反馈调节的有趣但未探索的可能性，即CamkII调节INSP3激酶和INSP3 5'磷酸酶参与INSP3代谢的能力。 CAMKII在心肌细胞中调节这些酶的可能性将通过直接表达和磷酸化测定以及测量INSP3积累来检查。生化测定和基于FRET的INSP3传感器将用于检查INSP3代谢的变化。 AIM＃4还使用这些方法比较了动力学诱导的心房与心室心肌细胞的INSP3形成，以及伴随心力衰竭的改变，并考虑了核腔中INSP3水平是否以及如何增加核腔中的问题。