Shining light on the molecular scale remodelling in a heart's path to failure

揭示心脏衰竭之路上的分子尺度重塑

• 批准号: 1789794
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1789794
• 关键词: Shining; light; molecular; scale; remodelling

Myocytes are the mechanical workhorses of the human heart. The uninterrupted contraction andrelaxation of these large cells powers the movement of blood throughout the body over an individual'slifetime. Intricate extensions of the plasma membranes known as t-tubules form thousands of'intracellular synapses' (known as 'dyads') deep within the cell's interior. Calcium signals released ateach dyad are the primary determinants of the cell's contraction. Previous work has reported a seriesof gradual sub-microscopic biochemical and structural changes occurring throughout each myocyte ina wide range of heart diseases. Such remodelling has catastrophic effects on the calcium signals andthe cell's mechanical performance, placing the heart on an irreversible path towards failure. Until now,optical microscopy techniques have lacked the resolving power to fully visualise compact structuressuch as t-tubules and dyads; therefore our understanding of the time-course, the spatial scales andthe protein modifications underpinning the human pathology has been very limited. With a mortalityrate of up to 50% in the first 5 years in such heart failure patients, it is now urgent that we combinestate-of-the-art microscopy, biochemical and cell biology tools to gain a visual understanding of thisremodelling process. Among the currently-popular super-resolution microscopies, the novel DNA-PAINT method is the only technique that allows adequate resolution to fully visualise t-tubules, as wellas individual proteins among the tightly-packed clusters within dyads.The proposed study will bring together a highly interdisciplinary supervision team to test thehypothesis that the unique shape and protein composition of the dyads within the human myocytesundergo biochemical and structural remodelling in response to hormonal stimuli at a spatial scale thathas not been visualised before. The project will consist of three primary aims: (i) to utilise DNA-PAINTimaging to examine the time-course of t-tubule remodelling in myocytes that are freshly isolated fromcardiac tissue biopsies from patients in Leeds General Infirmary, (ii) to perform quantitativemeasurements of the expression and phosphorylation levels of structural proteins of the dyads withthe utility of super-resolution images and quantitative Western Blot protocols and (iii) to develop an exvivo culture assay of stem cell-derived human cardiomyocytes that can facilitate long term (weeksmonths)chemical and structural observations in the presence of cardiotropic hormones typicallyobserved in failing hearts. In supervision, Dr Isuru Jayasinghe (IJ) will lead the DNA-PAINT imaging,image analysis and membrane study; Dr Andrew Smith (AJS) will share clinical knowledge of thebiopsy samples, knowhow of culture of cardiac stem cells and derived myocytes and Prof John Colyer(JC) will provide the quantitative Western Blot tools.The primary endpoints of this study will be (i) an unprecedented visual understanding of thetimescales of the nanoscale membrane and protein remodelling in human myocytes, (ii) the targeteddetection of nanoscale morphologies of remodelled t-tubules and calcium release sites in specificcases of human heart failure and (iii) a novel ex vivo human myocyte assay that enables aquantitative profiling of these changes under a controlled humoral environment that typifies specifichuman heart diseases.

肌细胞是人类心脏的机械主力。这些大细胞不间断的收缩和松弛为人的一生中的全身血液流动提供动力。被称为 T 管的质膜的复杂延伸在细胞内部深处形成了数千个“细胞内突触”（称为“二元体”）。每个二元体释放的钙信号是细胞收缩的主要决定因素。之前的工作报道了在多种心脏病中每个心肌细胞发生一系列逐渐的亚微观生化和结构变化。这种重塑对钙信号和细胞的机械性能产生灾难性影响，使心脏走上不可逆转的衰竭之路。到目前为止，光学显微镜技术缺乏完全可视化紧凑结构（例如 T 型管和二元组）的分辨率。因此，我们对人类病理学的时间进程、空间尺度和蛋白质修饰的理解非常有限。由于此类心力衰竭患者在头 5 年内的死亡率高达 50%，现在迫切需要我们结合最先进的显微镜、生化和细胞生物学工具，以直观地了解这一重塑过程。在当前流行的超分辨率显微镜中，新颖的DNA-PAINT方法是唯一能够以足够的分辨率完全可视化T小管以及二元体中紧密堆积的簇中的单个蛋白质的技术。拟议的研究将汇集一个高度跨学科的监督团队来测试人类肌细胞内二元体的独特形状和蛋白质组成经历生化作用的假设 以及在以前从未想象过的空间尺度上响应荷尔蒙刺激的结构重塑。该项目将包括三个主要目标：（i）利用 DNA-PAINT 成像来检查从利兹综合医院患者的心脏组织活检中新鲜分离的心肌细胞中 T 管重塑的时间过程，（ii）利用超分辨率图像和定量分析对二元结构蛋白的表达和磷酸化水平进行定量测量 蛋白质印迹方案和（iii）开发干细胞衍生的人类心肌细胞的体外培养测定，可以促进在衰竭心脏中通常观察到的促心激素存在下的长期（数周数月）化学和结构观察。在监督方面，Isuru Jayasinghe 博士（IJ）将领导 DNA-PAINT 成像、图像分析和膜研究； Andrew Smith 博士（AJS）将分享活检样本的临床知识、心脏干细胞和衍生心肌细胞培养的专业知识，John Colyer 教授（JC）将提供定量蛋白质印迹工具。这项研究的主要终点将是（i）对人类心肌细胞中纳米级膜和蛋白质重塑的时间尺度进行前所未有的视觉理解，（ii） 对人类心力衰竭特定病例中重塑的 T 管和钙释放位点的纳米级形态进行有针对性的检测，以及（iii）一种新颖的离体人类心肌细胞测定，能够在代表特定人类心脏病的受控体液环境下对这些变化进行定量分析。

项目成果

Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes

• DOI: 10.1101/2020.06.26.131854
• 发表时间: 2020-06
• 期刊: bioRxiv
• 作者: T. M. Sheard;Luke A. Howlett;Hannah M. Kirton;Zhaokang Yang;G. Gurrola;D. Steele;I. Jayasinghe;H. Valdivia;J. Colyer