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.

项目概要/摘要 桥粒斑蛋白是一种细长的蛋白质，通过以下方式将桥粒成分与细胞骨架连接起来： 中间丝。 DSP 基因中的杂合截断突变，该基因编码桥粒斑蛋白， 导致影响左心室的致心律失常性心肌病 (AC)。一个显着的特征与 DSP 截短变体（DSPtv）是一种重要的炎症成分，这种炎症可以很好地缓解 使用磁共振或正电子发射断层扫描通过心脏成像进行可视化。的效果 DSP 截短在介导心脏炎症中的作用尚不清楚。 DSP 截断的本质 暗示单倍体不足或桥粒斑蛋白减少是左心室的重要特征 伴有炎症的心肌病。开始表征与 DSP 相关的转录程序 心肌病时，我使用 siRNA 分析了新生大鼠心室心肌细胞的 RNA 测序， 减少DSP的表达。 siRNA处理心肌细胞差异表达基因分析 确定免疫趋化途径的表达增加。特别值得注意的是几个 与焦亡有关的基因，焦亡是细胞死亡的一种免疫活性形式。综合起来，这些数据 表明先天免疫途径可能对 DSP 相关心肌病有显着影响。我 假设桥粒斑蛋白的减少会促进心肌细胞中炎症小体的过度激活 这导致异常的钙处理和致心律失常的异细胞连接。在这个目标1中 建议，我将评估人诱导多能性 DSP 心肌病的炎症成分 具有独特 DSP 截短的干细胞 (hiPSC) 系，源自患有炎症性心律失常的个体 心肌病。为了表征 2D-和 3D-hiPSC 模型，我将评估 DSP 单倍体不足是否 足以驱动炎症小体成分的表达。然后我将检查 DSPtvs 对 响应于病原体和损伤衍生的激活剂的收缩性和钙瞬变 炎症小体。我将对 hiPSC 衍生的心肌细胞和成纤维细胞进行共培养测定，以评估 DSP 心肌病中异细胞连接和心脏传导的炎症调节。在 该提案的目标 2，我将使用调节 hiPSC 衍生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。这些研究将针对从不 有丝分裂 A 相关激酶 7 (NEK7)，一种在心脏中具有差异基因表达的炎症体成分 失败。拟议的研究将从对 DSPtvs 患者的观察进行扩展，并定义 炎症触发因素改善了与 DSP 介导的疾病相关的独特病理学。特别是， 这项研究的结果对纤维化转化驱动的其他形式的心肌病具有影响 例如扩张型心肌病和心力衰竭。