Pathophysiological Regulation of Cardiac Myocyte RyR Channel

心肌细胞 RyR 通道的病理生理调节

• 批准号: 8842176
• 负责人: Donald M Bers
• 金额: $ 63.76万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8842176
• 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; Cell Nucleus; Cleaved cell; Complement; Coupling; Dantrolene; Diastole; Disease; Environment; FKBP1B gene; Flecainide; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Functional disorder; Funding; Glutathione Disulfide; Health; Heart; Heart failure; Hereditary Disease; Human; Hydrogen Peroxide; Image; Ions; Ischemia; Kinetics; Maps; 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; Relative (related person); RyR2; Ryanodine Receptor Calcium Release Channel; Saponin; Saponins; Sarcoplasmic Reticulum; Signal Transduction; Site; Structural Models; Structure; Tacrolimus Binding Proteins; Technical Expertise; Testing; Therapeutic; Therapeutic Agents; Ventricular; Ventricular Tachycardia; 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; 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ryanodine受体（RYR2）或肌质网（SR）钙释放通道的突变和失调直接导致人类中的儿茶酚胺能多态性心室心动过心（CPVT）和心脏失败（HF）。受影响的RYR比正常人更活跃，在舒张期间从SR泄漏Ca，导致心律不齐和功能障碍。 FKBP12.6和钙调蛋白（CAM）是与RYR2紧密结合并可能稳定的蛋白质。我们已经开发了新颖的定量方法来评估（在成人心肌细胞中）FKBP，CAM和其他肽如何结合并调节RYR2门控，并在结构上位于肌细胞RYR2上。虽然FKBP12.6与高亲和力结合RYR2，但它不是关键的RYR2调节剂。在这里，我们使用荧光标记的蛋白质和成人心室心肌共焦成像中的荧光标记蛋白和新的靶向传感器来解决RYR2中的CAM结合和改变的域域相互作用与疾病相关的RYR2功能障碍如何解决4个目标。我们将：1）测试与HF相关的RYR改变是否会减少CAM结合并增加结构性解动肽DPC10的访问。 2）测试已知的RYR抑制剂是否通过改变FKBP12.6，CAM或DPC10结合在病理RYR2上起作用。 3）使用新型靶向CA传感器测量裂缝[Ca]。 4）通过映射S100A1，CAMKII，IPTX和Sorcin的位点来增强RYR2结构模型。这是两个小组之间在HF和心律不齐中具有共同兴趣的两组之间的高度协作项目，并具有生理学，药理学，生物化学，分子生物学，荧光光谱和共聚焦成像方面的互补技术专业知识。这将大大增强我们对健康和疾病心脏肌细胞中RYR2结构和功能的理解，并为心脏中病理调节的RYR2的疗法提供新的策略。