Molecular mechanisms of load-induced t-tubule regulation in the mammalian heart

哺乳动物心脏负荷诱导 T 管调节的分子机制

• 批准号: 10664338
• 负责人: Michael Ibrahim
• 金额: $ 16.67万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664338
• 关键词: Address; Adenovirus Vector; Adult; Affect; Architecture; Binding; Biomechanics; Biomimetics; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular system; Cells; Cellular biology; Chronic Disease; Coupling; Custom; Disease; Dissection; Elastomers; Electrophysiology (science); Event; Heart; Heart Abnormalities; Heart failure; Hour; Human; Impairment; In Vitro; Length; Location; Maps; Mechanics; Mediating; Mediator; Membrane; Mentors; Mentorship; Methodology; Methods; Microtubules; Modeling; Molecular; Muscle; Myocardial; Myocardium; Nature; Pathologic; Pathway interactions; Phenotype; Preparation; Process; Proteins; Protocols documentation; Pump; Rattus; Regulation; Research; Rodent; Role; Ryanodine Receptor Calcium Release Channel; Scientist; Slice; Stress; Structure; Surgeon; System; Techniques; Testing; Thoracic aorta; Time; Training; Variant; Viral Vector; Work; adenoviral mediated; career; elastomeric; experimental study; flexibility; gain of function; genetic manipulation; improved; in vivo; insight; junctophilin; live cell imaging; loss of function; mechanical load; novel; overexpression; post-doctoral training; preservation; pressure; response; superresolution imaging; superresolution microscopy; targeted treatment; tool

Project Summary Heart failure is most commonly associated with poor contractile function due to multi-level pathologic remodeling, including excitation-contraction coupling (ECC). This depends upon the proximity between membrane-bound L-type Ca2+ channels (LTCC) within the transverse (t)-tubule network and intracellular ryanodine receptors (RyR), which are normally very tightly colocalized. The PI and others have shown that abnormal mechanical load in vivo damages the t-tubule network, which results in uncoupling of LTCC and RyR. Junctophilin (JPH2), BIN 1 and Telethonin (TCAP), in interaction with the microtubule network, regulate t- tubule structure, but how they do so in response to load variation is not known. Prior experimental strategies have been unable to assess the effect of direct mechanical loading upon isolated cardiomyocytes, nor have they had the experimental flexibility to allow facile genetic manipulation of the pathways involved. Using new methods to directly modulate mechanical load on isolated cardiomyocytes and intact human myocardium in vitro, this K99/R00 seeks to test the hypothesis that t-tubule structure is normally regulated by a microtubule dependent JPH2, BIN1 and TCAP pathway, which in conditions of direct mechanical overload is deranged by microtubule mediated redistribution of JPH2, and reduced expression of JPH2, BIN 1 and TCAP. In Aim 1, using a novel magnetorheological elastomer (MRE) culture system, isolated cardiomyocytes will be subjected to pathological overload and undergo comprehensive characterization of ECC and t-tubule structure to test the hypothesis that cardiomyocyte-autonomous mechanisms are sufficient to mediate the load-dependent remodeling of the t-tubule system observed in heart failure. Because the phenotype arises in 48 hours, comprehensive dissection of the underlying molecular mechanisms will be performed by combined live cell imaging and adenoviral mediated genetic manipulations. Second, the novel but well-validated cardiac slice method will be used to specifically control pre-load and after-load in order to vertically integrate insights from cardiomyocyte-autonomous experiments in understanding the role of mechanical load regulation of the t- tubule system at the level of the isolated myocardium, including in human control and diseased myocardium. Mechanical unloading of failing hearts in vivo rescues t-tubule structure and ECC, which has been associated with significant contractile improvements. Using the tools developed in Aims 1 and 2, failing cells and slices will undergo mechanical unloading to determine the biomechanical and molecular mediators of this reverse remodeling. The completion of this work will significantly add to the PI's post-doctoral training in cellular electrophysiology, advanced super-resolution imaging and translational cardiovascular research and will be essential for his transition to independence.

项目摘要 心力衰竭通常与由于多层次病理而导致的收缩功能不良 重塑，包括激发反应耦合（ECC）。这取决于 横向（t） - 微管网络和细胞内的膜结合的L型Ca2+通道（LTCC） ryanodine受体（RYR）通常非常紧密地共定位。 PI和其他人表明 体内的异常机械负载损害了t-pubule网络，这导致LTCC和 里尔。与微管网络相互作用，枢纽素（JPH2），bin 1和望索蛋白（TCAP）调节T- 小管结构，但是他们如何响应负载变化而做到这一点。先前的实验策略 一直无法评估直接机械负荷对孤立的心肌细胞的影响，也没有 他们具有实验灵活性，可以允许对所涉及的途径的轻松遗传操纵。使用新 在孤立的心肌细胞上直接调节机械载荷的方法和完整的人体心肌 体外，此K99/R00试图检验以下假设，即T型结构通常受微管调节 依赖性JPH2，BIN1和TCAP途径，在直接机械过载条件下 微管介导的JPH2的重新分布，以及JPH2，BIN 1和TCAP的表达降低。在AIM 1中， 使用新型的磁性弹性体（MRE）培养系统，将对孤立的心肌细胞进行 对病理超负荷并经历了ECC和T型管结构的全面表征，以测试 假设心肌自动机制足以介导载荷依赖性 在心力衰竭中观察到的T管系统的重塑。因为表型在48小时内出现，所以 基础分子机制的全面解剖将通过合并的活细胞进行 成像和腺病毒介导的遗传操作。第二，小说但验证了心脏片 方法将用于专门控制预载和后加载，以垂直整合见解 从心肌自动实验中，在理解T-机械负荷调节的作用中 小管系统处于孤立的心肌水平，包括人类控制和患病的心肌。 体内失败的心脏的机械卸载T-Tubule结构和ECC，这已经关联 有重大的收缩改进。使用AIMS 1和2中开发的工具，故障单元格和切片将 进行机械卸载以确定此反向的生物力学和分子介质 重塑。这项工作的完成将大大增加PI的细胞后培训 电生理学，高级超分辨率成像和转化心血管研究，将是 他向独立过渡至关重要。