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

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舒张为血液在人体内的流动提供了动力。被称为t-小管的质膜的复杂延伸在细胞内部深处形成数千个“细胞内突触”（被称为“二联体”）。每一个二分体释放的钙信号是细胞收缩的主要决定因素.以前的工作已经报道了一系列渐进的亚微观生化和结构变化发生在广泛的心脏疾病中的每个肌细胞。这种重塑对钙信号和细胞的机械性能有灾难性的影响，使心脏走上不可逆转的衰竭之路。到目前为止，光学显微镜技术还缺乏完全可视化紧凑结构（如t-小管和二分体）的分辨率;因此，我们对人类病理学基础的时间进程、空间尺度和蛋白质修饰的理解非常有限。在这类心力衰竭患者中，前5年的死亡率高达50%，现在我们迫切需要结合最先进的显微镜、生物化学和细胞生物学工具来直观地了解这一重塑过程。在目前流行的超分辨率显微镜中，新的DNA-PAINT方法是唯一一种允许足够分辨率完全可视化t-小管的技术，以及单个蛋白质，这项拟议中的研究将汇集一个高度跨学科的监督小组，以检验这一假设，即人类肌细胞内二分体的独特形状和蛋白质组成受到生物化学和结构的影响。在空间尺度上对激素刺激的反应重塑，这在以前是没有看到的。该项目将包括三个主要目标：（i）利用DNA-PAINTiming检查从来自利兹综合医院的患者的心脏组织活组织检查中新鲜分离的肌细胞中t-小管重塑的时程，（ii）利用超分辨率图像和定量Western Blot方案对二联体的结构蛋白的表达和磷酸化水平进行定量测量，以及（iii）开发一种干细胞衍生的人心肌细胞的体外培养测定法，该测定法可以在通常在衰竭心脏中观察到的促心激素的存在下促进长期（数周-数月）的化学和结构观察。在监督下，Isuru Jayasinghe博士（IJ）将领导DNA-PAINT成像，图像分析和膜研究;安德鲁·史密斯博士（AJS）将分享活检样本的临床知识，心脏干细胞和衍生肌细胞培养的专业知识，John Colyer教授（JC）将提供定量Western Blot工具。本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的前所未有的视觉理解，（ii）在特定的人类心力衰竭病例中，有针对性地检测重塑的t-小管和钙释放位点的纳米级形态;以及（iii）一种新的离体人心肌细胞检测方法，能够在控制的体液环境下定量分析这些变化，这些体液环境代表了特定的人类心脏病。

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.