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 循环的调节并参与其控制，并且与导致心脏肥大的转录调节有关。 CaMKIIdelta 是主要的心脏亚型，缺乏 (delta-c) 或含有 (delta-B) 核定位信号的剪接变体均在心肌细胞中表达。心脏中细胞质 (δ-c) 和核 (δ-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。参与 E-C 耦合的 Ca 从肌网 (SR) 释放，但心肌细胞也含有肌醇 1,4,5-三磷酸 (InsP3) 受体调节的 Ca 储存。激活受体的配体 磷脂酶 C (PLC) 介导的 InsP3 形成是肥大的既定激活剂，但 InsP3 的信号传导作用尚未得到证实。我们假设核周 InsP3 敏感库释放的局部 Ca 会激活 CaMKII，并通过影响 HDAC/MEF2 或其他转录途径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 (InsP3Rs) 或 CaMKIIdelta 的缺失对横主动脉缩窄 (TAG) 或激动剂输注引起的生理和病理性肥大和衰竭发展的影响，评估了超声心动图参数、左心室与体重之比、心室肌细胞大小、基因表达和蛋白质磷酸化。在目标 #2 中，我们确定 InsP3 敏感 Ca 存储的局部 Ca 释放是否优先激活 核 CaMKIIdelta，而通过 SR 释放导致胞浆 Ca 整体增加的刺激激活细胞质 CaMKIIdelta。这些区室中的 CaMKII 激活以及 delta-B 与 delta-c CaMKII 的激活将通过细胞分级和免疫印迹以及基于 FRET 的 CaMKII 活性报告基因 (CaMKAR) 的开发进行检查。在目标 #3 中，我们研究了该假设的进一步推论，即 CaMKIIdelta 的核定位与胞质定位针对不同的底物。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中是否具有不同的作用，与 RyR 和其他 SR 蛋白的磷酸化相比。这一假设也在小鼠体内进行了测试，其中 delta-B 和 delta-c 亚基分别针对 昏死。目标 #4 考虑了反馈调节的一种有趣但尚未探索的可能性，即 CaMKII 调节参与 InsP3 代谢的 InsP3 激酶和 InsP3 5' 磷酸酶的能力。 CaMKII 调节心肌细胞中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。生化检测和基于 FRET 的 InsP3 传感器将用于检查 InsP3 代谢的变化。 目标 #4 还使用这些方法来比较心房心肌细胞和心室心肌细胞中激动剂诱导的 InsP3 形成以及伴随心力衰竭的改变，并考虑核区室中 InsP3 水平是否以及如何增加的问题。