Department of Cardiology and Pneumology, Heart Research Center Göttingen, Cellular Biophysics and Translational Cardiology Section, University Medical Center Göttingen, Göttingen, Germany.
Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Göttingen, Germany.
PLoS One. 2024 Oct 24;19(10):e0311203. doi: 10.1371/journal.pone.0311203. eCollection 2024.
Phospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Δ-PLN. Complexome profiling (CP) is a proteomics workflow for systematic analysis of high molecular weight (MW) protein complexes and supercomplexes. We hypothesize that R14Δ-PLN may alter a subset of these assemblies, and apply CP workflows to explore these changes in presymptomatic R14Δ/+ mice hearts. Ventricular tissues from presymptomatic 28wk-old WT and R14Δ/+ mice were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) with a linear MW-range exceeding 5 MDa, and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) analysis. Unfortunately, current workflows for the systematic analysis of CP data proved ill-suited for use in cardiac samples. Most rely upon curated protein complex databases to provide ground-truth for analysis; however, these are derived primarily from cancerous or immortalized cell lines and, consequently, cell-type specific complexes (including cardiac-specific machinery potentially affected in R14Δ-PLN hearts) are poorly covered. We thus developed PERCOM: a novel CP data-analysis strategy that does not rely upon these databases and can, furthermore, be implemented on widely available spreadsheet software. Applying PERCOM to our CP dataset resulted in the identification of 296 proteins with disrupted elution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) supercomplex components. Changes to mitochondrial supercomplexes were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. The observed alterations to mitochondrial and ICD supercomplexes were replicated in a second cohort of "juvenile" 9wk-old mice. These early-stage changes to key cardiac machinery may contribute to R14Δ-PLN pathogenesis.
肌浆网磷蛋白（PLN）是一种肌内质网（SER）膜蛋白，通过可逆抑制 SERCA2a Ca2+摄取泵来调节心脏收缩/松弛。R14Δ-PLN 突变导致严重的心肌病，对常规治疗有抗性。蛋白复合物和更高阶的超复合物，如闰盘成分和 Ca+2 循环结构域，是许多关键心脏功能的基础，其中一部分可能会被 R14Δ-PLN 破坏。复合物组学分析（CP）是一种系统分析高分子量（MW）蛋白复合物和超复合物的蛋白质组学工作流程。我们假设 R14Δ-PLN 可能会改变其中的一部分组装体，并应用 CP 工作流程来探索这些在无症状前的 R14Δ/+ 小鼠心脏中的变化。从无症状的 28 周龄 WT 和 R14Δ/+ 小鼠的心室组织中，在非变性条件下进行匀浆，通过尺寸排阻色谱（SEC）进行分级，线性 MW 范围超过 5 MDa，并进行定量非依赖性数据采集质谱（DIA-MS）分析。不幸的是，当前用于 CP 数据系统分析的工作流程被证明不适合用于心脏样本。大多数工作流程依赖于经过精心整理的蛋白复合物数据库来为分析提供真实信息；然而，这些数据库主要来自癌细胞系或永生化细胞系，因此，细胞类型特异性复合物（包括可能受 R14Δ-PLN 心脏影响的心脏特异性机械）覆盖得很差。因此，我们开发了 PERCOM：一种不依赖于这些数据库的新型 CP 数据分析策略，此外，还可以在广泛使用的电子表格软件上实施。将 PERCOM 应用于我们的 CP 数据集，结果鉴定出 296 种洗脱曲线发生变化的蛋白。命中结果显著富集了线粒体和闰盘（ICD）超复合物成分。线粒体超复合物的变化与线粒体蛋白表达降低和最大耗氧量有关。在第二组“幼年”9 周龄小鼠中复制了观察到的线粒体和 ICD 超复合物的变化。这些关键心脏机械结构的早期变化可能导致 R14Δ-PLN 的发病机制。
