Pathophysiological Regulation of Cardiac Myocyte RyR Channel

心肌细胞 RyR 通道的病理生理调节

• 批准号: 9258465
• 负责人: Donald M Bers
• 金额: $ 64.91万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2019-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258465
• 关键词: 3-Dimensional; Address; Adult; Affect; Affinity; American; Arrhythmia; Binding; Binding Proteins; Biochemistry; Biological Assay; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cardiac; Cardiac Myocytes; Catecholaminergic Polymorphic Ventricular Tachycardia; Cell Nucleus; Complement; Coupling; Dantrolene; Diastole; Disease; Drug Receptors; Environment; FKBP1B gene; Felis catus; Flecainide; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Functional disorder; Funding; Genetic Transcription; Glutathione Disulfide; Health; Heart; Heart failure; Hereditary Disease; Human; Hydrogen Peroxide; Image; Ions; Ischemia; Kinetics; Measurement; Measures; Mediating; Methods; Minor; Modeling; Molecular; Molecular Biology; Molecular Conformation; Muscle Cells; Mutation; Oryctolagus cuniculus; Pathologic; Pathology; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Physiological; Physiology; Positioning Attribute; Post-Translational Protein Processing; Proteins; Regulation; Ryanodine Receptor Calcium Release Channel; Saponins; Sarcoplasmic Reticulum; Signal Transduction; Site; Structural Models; Structure; Tacrolimus Binding Proteins; Technical Expertise; Testing; Therapeutic; Therapeutic Agents; Ventricular; Vesicle; Work; base; calmodulin-dependent protein kinase II; carvedilol; high throughput screening; improved; inhibitor/antagonist; insight; interest; mitochondrial dysfunction; mutant; novel; novel strategies; novel therapeutics; oxidation; prevent; protein E; public health relevance; receptor function; receptor structure function; reconstruction; sensor; small molecule; sorcin; therapeutic development; therapeutic target

DESCRIPTION (provided by applicant): Mutation and dysregulation of the cardiac ryanodine receptor (RyR2) or sarcoplasmic reticulum (SR) calcium release channel contributes directly to catecholaminergic polymorphic ventricular tachycardia (CPVT) and heart failure (HF) in humans. Affected RyRs are more active than normal, leaking Ca from the SR during diastole causing arrhythmias and dysfunction. FKBP12.6 and calmodulin (CaM) are proteins that bind tightly with and may stabilize RyR2. We have developed novel quantitative methods to assess (in adult cardiac myocytes) how FKBP, CaM and other peptides bind to and modulate RyR2 gating, and are structurally positioned on the myocyte RyR2. While FKBP12.6 binds RyR2 with high affinity, it is not a critical RyR2 regulator. Here we examine how CaM binding and altered domain-domain interaction in RyR2 are involved in disease-related RyR2 dysfunction, using fluorescent tagged proteins and novel targeted sensors in adult ventricular myocyte confocal imaging to address 4 aims. We will: 1) Test whether HF-related RyR alterations decrease CaM binding and increase the access of the structural unzipping peptide DPc10. 2) Test whether known RyR inhibitors work on pathological RyR2 by altering FKBP12.6, CaM or DPc10 binding. 3) Measure Cleft [Ca] using novel targeted Ca sensors. 4) Enhance RyR2 structural model by mapping sites of S100A1, CaMKII, IPTx and Sorcin. This is a highly collaborative project between two groups with shared interests in cardiac calcium regulation in HF and arrhythmias and with complementary technical expertise in physiology, pharmacology, biochemistry, molecular biology, fluorescence spectroscopy and confocal imaging. This will greatly enhance our understanding of RyR2 structure and function in cardiac myocytes in health and disease and provide novel strategies for the development of therapeutics for pathologically modulated RyR2 in the heart.

描述（由申请人提供）：心脏兰尼碱受体（RyR2）或肌浆网（SR）钙释放通道的突变和失调直接导致人类儿茶酚胺能多形性室性心动过速（CPVT）和心力衰竭（HF）。受影响的 RyR 比正常情况更活跃，在舒张期从 SR 中渗漏 Ca，导致心律失常和功能障碍。 FKBP12.6 和钙调蛋白 (CaM) 是与 RyR2 紧密结合并可稳定 RyR2 的蛋白质。我们开发了新的定量方法来评估（在成人心肌细胞中）FKBP、CaM 和其他肽如何结合和调节 RyR2 门控，以及在结构上定位在心肌细胞 RyR2 上。虽然 FKBP12.6 以高亲和力结合 RyR2，但它并不是关键的 RyR2 调节剂。在这里，我们研究了 RyR2 中 CaM 结合和改变的域-域相互作用如何参与疾病相关的 RyR2 功能障碍，在成人心室肌细胞共聚焦成像中使用荧光标记蛋白和新型靶向传感器来实现 4 个目标。我们将： 1) 测试 HF 相关的 RyR 改变是否会减少 CaM 结合并增加结构解链肽 DPc10 的接近。 2) 测试已知的 RyR 抑制剂是否通过改变 FKBP12.6、CaM 或 DPc10 结合对病理性 RyR2 起作用。 3) 使用新型目标 Ca 传感器测量裂缝 [Ca]。 4) 通过绘制S1​​00A1、CaMKII、IPTx和Sorcin位点增强RyR2结构模型。这是两个团队之间的高度合作项目，两个团队对心力衰竭和心律失常的心脏钙调节有着共同的兴趣，并且在生理学、药理学、生物化学、分子生物学、荧光光谱和共聚焦成像方面具有互补的技术专长。这将极大地增强我们对健康和疾病中心肌细胞中 RyR2 结构和功能的理解，并为开发心脏中病理调节 RyR2 的治疗方法提供新策略。