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

心肌细胞是人类心脏的机械主力。在一个人的一生中，这些大细胞的不间断收缩和松弛为血液在全身的流动提供动力。质膜的错综复杂的延伸被称为T-管，在细胞内部深处形成了数以千计的细胞内突触(称为二联体)。每个二联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在广泛的心脏疾病中，每一个心肌细胞都发生了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了灾难性的影响，使心脏走上了一条通往故障的不可逆转的道路。到目前为止，光学显微镜技术一直缺乏分辨率来完全可视化紧凑的结构，如t-管和二联体；因此，我们对支撑人类病理的时间进程、空间尺度和蛋白质修饰的了解非常有限。在这类心力衰竭患者的头5年死亡率高达50%的情况下，我们现在迫切需要结合最先进的显微镜、生化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，新颖的DNA绘制方法是唯一一种具有足够分辨率的技术，可以完全显示T管以及染料中紧密堆积的簇中的单个蛋白质。拟议中的研究将召集一个高度跨学科的监督团队来测试这一假设，即人类肌肉细胞内二联体的独特形状和蛋白质组成在空间尺度上经历生化和结构重构，以响应激素刺激，这是以前从未被可视化过的。该项目将包括三个主要目标：(I)利用DNA-PAIN计时技术检测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管重构的时间进程；(Ii)利用超分辨率图像和定量Western Blot方案对二联体结构蛋白的表达和磷酸化水平进行定量测量；(Iii)建立干细胞来源的人心肌细胞的体外培养方法，以便在衰竭心脏中典型地观察到促心肌激素的存在下进行长期(数周)的化学和结构观察。在监督方面，Isuru Jayasinghe博士(IJ)将领导DNA涂料成像、图像分析和薄膜研究；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的培养技术，John Colyer教授(JC)将提供定量的Western Blot工具。这项研究的主要终点将是(I)前所未有的对人类心肌细胞纳米级膜和蛋白质重塑的时间尺度的视觉理解，(Ii)有针对性地检测特定心力衰竭病例中重塑的小管和钙释放部位的纳米级形态，以及(Iii)一种新型的体外人类心肌细胞检测方法，该方法能够在典型的特定人类心脏疾病的受控体液环境下定量描述这些变化。

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.