Dissecting the Role of Desmoplakin in Inflammation in Cardiomyopathy

剖析桥粒斑蛋白在心肌病炎症中的作用

• 批准号: 10606326
• 负责人: Daniel Selgrade
• 金额: $ 4.29万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606326
• 关键词: 3-Dimensional; Adrenergic Agents; Affect; Arrhythmia; Biological Assay; Calcium; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Communication; Cell Death; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Cytoskeleton; DNA Sequence Alteration; DSPP gene; Data; Defect; Desmosomes; Development; Dilated Cardiomyopathy; Disease; Disease Progression; Epigenetic Process; Exposure to; Fibroblasts; Functional disorder; Future; Gap Junctions; Gene Expression; Genes; Genetic Transcription; Heart; Heart Diseases; Heart failure; Heterozygote; Human; Immune; Immunologics; Individual; Inflammasome; Inflammation; Inflammatory; Intercalated disc; Intercellular Junctions; Intermediate Filaments; Investigation; Lead; Left; Left Ventricular Function; Left ventricular structure; Link; Macromolecular Complexes; Magnetic Resonance; Measures; Mediating; Mediator; Mitosis; Modeling; Mutation; Myocarditis; Nature; Neonatal; Normal Cell; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Phosphotransferases; Positron-Emission Tomography; Production; Proteins; Rattus; Role; Severity of illness; Signal Transduction; Skin; Small Interfering RNA; Testing; Therapeutic; Tissue-Specific Gene Expression; Tissues; Variant; Ventricular; Ventricular Arrhythmia; Visualization; Work; arrhythmogenic cardiomyopathy; cardiac tissue engineering; clinical imaging; cytokine; desmoplakin; differential expression; disorder prevention; effectiveness evaluation; heart imaging; heart rhythm; immune activation; immunomodulatory therapies; immunoregulation; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; inflammatory modulation; innate immune pathways; insight; link protein; mRNA Expression; pathogen; prevent; prognostic indicator; programs; protein expression; recruit; response; small molecule; stem cell model; transcriptome sequencing

Project Summary/Abstract Desmoplakin is an elongated protein that links the desmosomal components to the cytoskeleton through intermediate filaments. Heterozygous truncation mutations in the DSP gene, which encodes desmoplakin, cause arrhythmogenic cardiomyopathy (AC) impacting the left ventricle. A striking feature associated with DSP truncation variants (DSPtv) is a prominent inflammatory component, and this inflammation can be well visualized through cardiac imaging using magnetic resonance or positron emission tomography. The effect of DSP truncations in mediating cardiac inflammation is not well understood. The nature of DSP truncations implies that haploinsufficiency or reduction of desmoplakin protein is contributory feature of left ventricular cardiomyopathy with inflammation. To begin to characterize the transcriptional program associated with DSP- cardiomyopathy, I analyzed RNA-sequencing from neonatal rat ventricular cardiomyocytes with siRNA to reduce expression of DSP. Analysis of the differentially expressed genes in siRNA-treated cardiomyocytes identified immune chemotactic pathways as having increased expression. Of particular note were several genes connected to pyroptosis, an immunologically active form of cell death. Taken together, these data suggest that innate immune pathways may contribute significantly to DSP-associated cardiomyopathy. I hypothesize that reduction of desmoplakin promotes excessive inflammasome activation in cardiomyocytes that results in aberrant calcium handling and pro-arrhythmogenic heterocellular junctions. In Aim 1 of this proposal, I will assess this inflammatory component of DSP-cardiomyopathy in human-induced pluripotent stem cell (hiPSC) lines with distinct DSP truncations derived from individuals with inflammatory-arrhythmogenic cardiomyopathy. To characterize 2D- and 3D-hiPSC models, I will assess whether DSP haploinsufficiency is sufficient to drive expression of inflammasome components. I will then examine DSPtvs for their effects on contractility and calcium transients in response to pathogen- and damage-derived activators of the inflammasome. I will conduct coculture assays of hiPSC-derived cardiomyocytes and fibroblasts to evaluate the inflammatory modulation of heterocellular junctions and cardiac conduction in DSP-cardiomyopathy. In Aim 2 of the proposal, I will modulate innate immune activation in hiPSC-derived cardiomyocyte models using siRNA, small molecule inhibition, and epigenetic modulation strategies. These studies will target Never in Mitosis A-related Kinase 7 (NEK7), an inflammasome component with differential gene expression in heart failure. The proposed studies will expand from observations in patients with DSPtvs and define the inflammatory triggers refining the distinct pathology related to DSP-mediated disease. In particular, the findings from this study have implications for other forms of cardiomyopathy driven by fibrotic transformation such as dilated cardiomyopathy and heart failure.

项目总结/摘要 桥粒斑蛋白是一种延长的蛋白质，其通过连接桥粒组分与细胞骨架。 中间丝编码桥粒斑蛋白的DSP基因中的杂合截短突变， 引起影响左心室的致心肌病（AC）。一个显著的特征， DSP截短变体（DSPtv）是一种重要的炎症组分，这种炎症可以很好地抑制。 通过使用磁共振或正电子发射断层扫描的心脏成像可视化。的影响 DSP截短介导的心脏炎症还不清楚。DSP截断的性质 提示桥粒斑蛋白单倍型不足或减少是左室肥厚的特征性表现 心肌病伴炎症。为了开始表征与DSP相关的转录程序， 心肌病，我分析了RNA测序从新生大鼠心室心肌细胞与siRNA， 减少DSP的表达。siRNA诱导心肌细胞差异表达基因的分析 鉴定免疫趋化途径具有增加的表达。特别值得注意的是， 与细胞凋亡有关的基因，细胞凋亡是一种免疫活性的细胞死亡形式。综合起来，这些数据 提示先天免疫途径可能对DSP相关性心肌病有重要作用。我 假设桥粒斑蛋白减少促进心肌细胞中过度炎性小体活化 这导致异常的钙处理和促钙离子生成的异细胞连接。目标1 建议，我将评估这一炎症组成部分的DSP心肌病在人类诱导的多能 具有不同DSP截短的hiPSC干细胞系，其来源于具有炎性-致炎性疾病的个体。 心肌病为了表征2D和3D-hiPSC模型，我将评估DSP单倍不足是否 足以驱动炎性体组分的表达。然后，我将研究DSP电视对 收缩性和钙瞬变响应病原体和损伤衍生的激活剂的 炎性小体我将进行hiPSC衍生的心肌细胞和成纤维细胞的共培养试验，以评估 DSP心肌病中异细胞连接和心脏传导的炎症调节。在 该提案的目的2，我将使用以下方法调节hiPSC衍生的心肌细胞模型中的先天免疫激活： siRNA、小分子抑制和表观遗传调节策略。这些研究将针对Never in 有丝分裂A相关激酶7（NEK 7），一种在心脏中具有差异基因表达的炎性体组分 失败拟议的研究将从对DSPtvs患者的观察中扩展，并定义 炎性触发因子改善了与DSP介导的疾病相关的不同病理。特别是 这项研究的发现对其他形式的由纤维化转化驱动的心肌病具有启示意义 如扩张型心肌病和心力衰竭。