Examining the Function of a Novel Protein in the Cardiac Junctional Membrane Complex

检查心脏连接膜复合体中新型蛋白质的功能

• 批准号: 10749672
• 负责人: Grace Ciampa
• 金额: $ 3.29万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749672
• 关键词: Ablation; Age Months; Amino Acids; Animal Model; Binding; Binding Proteins; Biological Assay; Calcium; Calcium Signaling; Calpain; Cardiac; Cardiac Muscle Contraction; Cardiac Myocytes; Cardiomyopathies; Cell Nucleus; Cell surface; Cells; ChIP-seq; Characteristics; Co-Immunoprecipitations; Compensation; Complex; Coupling; Data; Deformity; Detection; Disease; Down-Regulation; Event; Fibrosis; Functional disorder; Gene Expression; Genes; Genetic; Genomics; Goals; Heart; Heart Diseases; Heart Hypertrophy; Heart Ventricle; Heart failure; Human; Image; Impairment; In Vitro; Incidence; Ion Channel; Knock-in; Knock-out; Knockout Mice; L-Type Calcium Channels; Length; Link; Longevity; Maps; Mass Spectrum Analysis; Mechanics; Membrane; Messenger RNA; Modeling; Molecular; Molecular Target; Mus; Myocardial Contraction; Myocardial dysfunction; Myocardium; N-terminal; Nuclear Translocation; Operative Surgical Procedures; Outcome; Pathogenicity; Patients; Peptide Hydrolases; Physiology; Prevention; Process; Protein Family; Protein Isoforms; Proteins; Proteolysis; Proteomics; Recombinants; Regulation; Repression; Research; Rodent Model; Role; RyR2; Sampling; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Sodium-Calcium Exchanger; Stress; Structure; Testing; Tissues; Translating; Transmission Electron Microscopy; Treatment Failure; United States; Western Blotting; Work; cardiogenesis; comparison control; conditional knockout; experimental study; heart function; in vivo; insight; junctophilin; loss of function; mouse model; novel; overexpression; postnatal development; pressure; protein complex; protein expression; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Incidence of heart failure (HF) remains on the rise in the United States despite advances in treatment and prevention. On a cellular level during HF, excitation-contraction coupling and calcium signaling in the heart becomes severely impaired. These mechanisms occur in junctional membrane complexes (JMCs) or cardiac dyads in cardiomyocytes. Cardiac JMCs are the ultrastructure juxtaposed between the transverse tubules (T- tubules) and the sarcoplasmic reticulum (SR) and are integral for facilitating excitation-contraction coupling and calcium signaling. Junctophilin family proteins (JPs) are critical in establishing and modulating JMCs. There are four isoforms of JPs found in excitable tissues (JP1-4). While JP2 has been established as the primary JP isoform expressed in cardiac muscle, our lab has recently found that JP1, normally in skeletal muscle, is also expressed in cardiac muscle. Through examining mouse cardiac tissue, I found that JP1 protein is expressed primarily in the ventricles of the heart and increases during postnatal development until maturity. I have also found that JP1 localizes with JP2 and RyR2, the primary calcium release channel, in the Z-disc. Using a cardiomyocyte specific JP1 knockout mouse model, I found that JP1 knockout mice have a shortened lifespan, reduced cardiac function, and increased incidence of fibrosis and HF compared to control mice. Despite genetic ablation of JP1 in these mice, JP2 protein expression does not change indicating that JP2 is not sufficient to overcome the loss of JP1. To examine whether JP1 expression changes during cardiac stress I used a model of pressure overload-induced cardiac hypertrophy in mice and found that JP1 protein expression decreases with increased cardiac stress. To investigate whether this downregulation could be due to proteolysis by calpain, a stress induced, calcium-dependent protease, I subjected purified recombinant JP1 protein to an in vitro calpain cleavage assay and found JP1 cleaved between amino acid 505 and 510. My primary hypothesis is that JP1 is functionally distinct from JP2 in cardiomyocytes within the cardiac dyad at baseline and in the nucleus after stress-induced cleavage. To test the hypothesis, I will use two novel mouse models developed in our lab: a cardiomyocyte specific knockout of JP1 (JP1cKO), and a global knock-in of 3xHA-tagged JP1. In Aim 1, I plan to examine the functional role of JP1 in cardiac muscle by (1) determining the consequences of depleting JP1 from JMCs by analyzing cardiomyocytes from JP1cKO mice and (2) examining JP1-interacting molecular targets and the potential signaling pathways using proteomics-based approaches and RNA-sequencing. Aim 2 will explore the mechanism of downregulation of JP1 during cardiac stress by further investigating the molecular consequence of calpain proteolysis of JP1. This work will have broad implications for the mechanism of cardiac muscle contraction and calcium signaling in the heart by characterizing a previously unrecognized protein, JP1, in cardiac muscle.

项目摘要/摘要 尽管治疗进展和 预防。在HF期间的细胞水平上，心脏中的激发反应耦合和钙信号传导 变得严重受损。这些机制发生在交界膜复合物（JMC）或心脏中 心肌细胞中的二元。心脏JMC是横向小管之间并置的超微结构（t- 小管）和肌浆网（SR），并且是促进激发反应耦合和 钙信号传导。封口素家族蛋白（JPS）对于建立和调节JMC至关重要。 在令人兴奋的组织中发现了四种JP的同工型（JP1-4）。而JP2已被确定为 我们的实验室在心肌中表达的主要JP同工型，最近发现JP1通常在骨骼中 肌肉也以心肌表达。通过检查小鼠心脏组织，我发现JP1 蛋白质主要在心脏的心室中表达，并在产后发育期间增加直到 到期。我还发现JP1与JP2和RYR2（主要钙释放通道）本地化，在 Z式。使用心肌细胞特异性JP1敲除鼠标模型，我发现JP1敲除小鼠有一个 与对照相比 老鼠。尽管在这些小鼠中JP1的遗传消融，但JP2蛋白表达并未改变，表明 JP2不足以克服JP1的损失。检查JP1表达是否在心脏中发生变化 压力我使用了小鼠压力超负荷引起的心肥大的模型，发现JP1蛋白 表达随着心脏应激的增加而降低。调查该下调是否可能到期 对于钙蛋白酶的蛋白水解，应力诱导的钙依赖性蛋白酶，我对纯化的重组JP1进行 蛋白质至体外钙蛋白酶裂解测定法，发现JP1在氨基酸505和510之间裂解。 主要假设是JP1在功能上与心肌二元组中心肌细胞的JP2不同 应力诱导的切割后的基线和核中。 为了检验假设，我将使用我们实验室中开发的两个新型小鼠模型：心肌细胞特异性 JP1（JP1CKO）的淘汰赛和3XHA标签的JP1的全球敲门。在AIM 1中，我计划检查 通过（1）通过（1）确定从JMC耗尽JP1的后果的功能作用 分析来自JP1CKO小鼠的心肌细胞，（2）检查JP1相互作用的分子靶标和 使用基于蛋白质组学的方法和RNA测序的潜在信号通路。 AIM 2将探索 通过进一步研究分子后果，在心脏应激过程中JP1下调的机制 JP1的钙蛋白酶蛋白水解。这项工作将对心脏肌肉的机制具有广泛的影响 通过表征先前未识别的蛋白JP1，在心脏中的收缩和钙信号传导 心肌。