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 心力衰竭最常见与由于多水平病理性心脏病导致的收缩功能差相关。 重构，包括兴奋-收缩偶联（ECC）。这取决于之间的接近程度 膜结合L型钙通道（LTCC）在横（t）-小管网络和细胞内 Ryanodine受体（RyR），其通常非常紧密地共定位。PI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，这导致LTCC的解偶联， RyR。嗜接线蛋白（JPH 2）、BIN 1和Telethonin（TCAP）与微管网络相互作用，调节t- 微管结构，但他们如何这样做，以响应负载变化是未知的。先前的实验策略 尚不能评估直接机械负荷对分离的心肌细胞的影响， 他们有实验的灵活性，可以对相关的途径进行简单的遗传操作。使用新 直接调节分离的心肌细胞和完整的人心肌上的机械负荷的方法， 在体外，这个K99/R 00试图检验t-小管结构通常由微管调节的假设 依赖性JPH 2、BIN 1和TCAP通路，在直接机械过载条件下， 微管介导的JPH 2再分布，并降低JPH 2、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 病理性超负荷，并进行ECC和t-小管结构的综合表征，以测试 假设心肌细胞自主机制足以介导负荷依赖性 在心力衰竭中观察到的T-小管系统的重塑。因为在48小时内出现表型， 将通过联合活细胞进行潜在分子机制的全面解剖， 成像和腺病毒介导的遗传操作。第二，新颖但经过验证的心脏切片 将使用一种方法专门控制预加载和后加载，以便垂直整合见解 从心肌细胞自主实验中了解机械负荷调节的作用， 在分离的心肌水平上，包括在人对照和患病心肌中，微管系统。 体内衰竭心脏的机械卸载挽救了t-小管结构和ECC，这与 收缩性显著改善。使用目标1和2中开发的工具，失败的单元和切片将 进行机械卸载，以确定这种逆转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的博士后培训细胞 电生理学，先进的超分辨率成像和转化心血管研究，并将 这对他走向独立至关重要。