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 心力衰竭最常与收缩功能低下有关，这是由于多层次的病理。 重塑，包括兴奋-收缩耦合(ECC)。这取决于两者之间的接近程度 横(T)小管网络和细胞内膜结合型L钙通道 Ryanodine受体(RyR)，通常是紧密共存的。公安党和其他人已经表明 体内异常的机械负荷破坏了T小管网络，导致LTCC和TTCC的解偶联 RyR。JPH2、BIN1和TCAP与微管网络相互作用，调节t-半乳糖苷酶活性。 小管结构，但它们如何对负荷变化作出反应尚不清楚。先期实验策略 一直无法评估直接机械负荷对分离的心肌细胞的影响，也没有 他们有实验上的灵活性，可以方便地对所涉及的途径进行基因操作。使用新的 方法直接调节体外培养的人心肌细胞和完整人心肌的机械负荷 在体外，K99/R00试图测试t-小管结构通常由微管调节的假设 依赖JPH2、BIN1和TCAP途径，在直接机械超负荷条件下被 微管介导JPH2的再分布，降低JPH2、BIN-1和TCAP的表达。在目标1中， 使用一种新的磁流变弹性体(MRE)培养系统，分离的心肌细胞将受到 病理超负荷，并进行ECC和T小管结构的综合表征，以测试 心肌自主机制足以调节负荷依赖的假说 在心力衰竭中观察到的t-微管系统重构。因为表型在48小时内出现， 联合活细胞将对潜在的分子机制进行全面的剖析 成像和腺病毒介导的基因操作。第二，新的但经过充分验证的心脏切片 方法将用于具体控制预加载和后加载，以便垂直整合洞察力 从心肌细胞自主实验中了解机械负荷调节在心肌细胞中的作用 在离体心肌水平上的小管系统，包括在人类对照和病变心肌中。 在体心脏衰竭的机械卸载挽救了t-小管结构和ECC，这已经被认为是 有显著的收缩改善。使用AIMS 1和AIMS 2中开发的工具，故障单元和切片将 进行机械卸载以确定这一逆转的生物力学和分子介质 改建。这项工作的完成将大大增加PI在细胞方面的博士后培训 电生理学、先进的超分辨率成像和翻译心血管研究，并将 对于他向独立的过渡至关重要。