Elucidating spatio-temporal nuclear dynamics in 4D using state-of-the-art imaging in beating hearts

核膜和核骨架和细胞骨架(LINC)复合蛋白之间的核膜和相关连接物对于正确的心肌和骨骼肌发育是必不可少的(Ross和Stroud，2021；Stroud，2018；Stroud等人，2017)。这一点突出表现在导致人类和小鼠心脏和骨骼肌病的大量突变，这些疾病统称为椎板病。有趣的是，椎板病变主要影响承受恒定机械负荷的横纹肌组织。椎板病变的病理机制尚不清楚，但似乎涉及多个，通常是重叠的因素：细胞核结构完整性改变或减弱，这似乎对收缩细胞特别重要，导致形态异常、断裂和DNA损伤；基因组组织改变，对基因表达产生不利影响；以及化学和生物机械信号改变，影响一系列细胞功能(Jaalouk和Lammerding，2009；Stephens等人，2018；Strom等人，2021)。目前，这些研究大多在体外进行，需要物理干预来推动2D细胞培养中的核变形和断裂。因此，需要新的成像和系统模式来推动我们在更好地概括体内观察到的环境中对椎板病变的理解。在这方面，实时光片成像以最小的光毒性(https://www.m2lasers.com/microscopy-aurora.html).在厚组织中提供了前所未有的时空分辨率在这里，我们建议对处于内源性物理负荷下的完整心脏进行实时光片成像，作为扩展我们对椎板病常观察到的核形态和动力学变化的知识所需的下一个前沿。在这个项目中，我们建议开发一个成像平台，允许在心脏跳动时对心肌细胞核进行实时成像。这将使我们能够询问细胞核外力(由肌肉收缩驱动)和细胞核内在力量(染色质组织)如何在对照和LINC复合体突变小鼠心脏中驱动核变形。这一发现将对了解健康心脏中活的、完整的心脏以及疾病状态下的核动力学具有广泛的相关性。在这个项目中，我们建议开发一个成像平台，允许在跳动的心脏中对心肌细胞核进行实时成像。这将使我们能够询问细胞核外力(由肌肉收缩驱动)和细胞核内在力量(染色质组织)如何在对照和LINC复合体突变小鼠心脏中驱动核变形。为实现这一目标，我们提出了以下目标：目标1：在光片显微镜下建立心脏成像的物理平台；目标2：优化心脏提取和体外成像的培养条件；目标3：建立具有核指示剂的小鼠群体。目的4：在对照和核膜突变小鼠模型中成像荧光标记的细胞核。

The nuclear envelope and associated Linker between Nucleoskeleton and Cytoskeleton (LINC) complex proteins are essential for proper cardiac and skeletal muscle development (Ross and Stroud, 2021; Stroud, 2018; Stroud et al., 2017). This is highlighted by the numerous mutations that lead to cardiac and skeletal myopathies in humans and mice that are collectively known as laminopathies. Interestingly, laminopathies predominantly affect striated muscle tissue which are under constant mechanical load. Pathological mechanisms underlying the laminopathies are poorly understood, but seem to involve multiple, often overlapping factors: altered or weakened structural integrity at the nucleus, which appears to be particularly important in contractile cells, leading to aberrant morphologies, ruptures and DNA damage; altered genome organisation, adversely affecting gene expression; and altered chemical and biomechanical signalling, affecting a host of cellular functions (Jaalouk and Lammerding, 2009; Stephens et al., 2018; Strom et al., 2021).Currently, the majority of these studies are performed in vitro and require physical interventions to drive nuclear deformation and rupture in 2D cell culture. Therefore, new imaging and system modalities will be required to drive our understanding of laminopathies further in environments that better recapitulate those observed in vivo. In regards to this, live light-sheet imaging provides unprecedented spatio-temporal resolution in thick tissues with minimal phototoxicity (https://www.m2lasers.com/microscopy-aurora.html). Here, we propose live light-sheet imaging of beating intact hearts that are under endogenous physical load as the next forefront required to extend our knowledge of changes to nuclear morphology and dynamics that are frequently observed in laminopathies. In this project, we propose to develop an imaging platform allowing live imaging of cardiomyocyte nuclei in beating hearts. This will enable interrogation of how forces extrinsic to nuclei (driven by muscle contraction) and intrinsic to nuclei (chromatin organization) drive nuclear deformation in control and LINC complex mutant mouse hearts. The findings will be of broad relevance to understanding nuclear dynamics in live, intact hearts in healthy hearts, as well as in disease states.In this project, we propose to develop an imaging platform allowing live imaging of cardiomyocyte nuclei in beating hearts. This will enable interrogation of how forces extrinsic to nuclei (driven by muscle contraction) and intrinsic to nuclei (chromatin organization) drive nuclear deformation in control and LINC complex mutant mouse hearts. The findings will be of broad relevance to understanding nuclear dynamics in live, intact hearts in healthy hearts, as well as in disease states.To achieve this objective, we propose the following aims:Aim 1: Establishing a physical platform for imaging hearts on the light-sheet microscope.Aim 2: Optimization of heart extraction and culture conditions for imaging hearts ex vivo.Aim 3: Establishment of mouse colony with nuclear indicator mice.Aim 4: Imaging fluorescently labelled nuclei in control and nuclear envelope mutant mouse models.