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

核骨架和细胞骨架（LINC）复合蛋白之间的核膜和相关接头对于适当的心脏和骨骼肌发育至关重要（Ross和Stroud，2021; Stroud，2018; Stroud等人，2017年）。这是突出的许多突变，导致心脏和骨骼肌病在人类和小鼠，统称为核纤层蛋白病。有趣的是，层粘连蛋白病主要影响处于恒定机械负荷下的横纹肌组织。核纤层蛋白病的病理机制尚不清楚，但似乎涉及多个，往往重叠的因素：改变或削弱核的结构完整性，这似乎是特别重要的收缩细胞，导致异常形态，破裂和DNA损伤;改变基因组组织，不利地影响基因表达;以及改变的化学和生物力学信号传导，影响许多细胞功能（Jaalouk和Lammerding，2009; Stephens等人，2018; Strom等人，目前，这些研究中的大多数是在体外进行的，并且需要物理干预来驱动2D细胞培养中的核变形和破裂。因此，将需要新的成像和系统模式，以推动我们的理解，进一步在环境中，更好地概括那些在体内观察到的层粘连蛋白病。关于这一点，实时光片成像在厚组织中提供了前所未有的时空分辨率，具有最小的光毒性（https：//www.m2lasers.com/microscopy-aurora.html）。在这里，我们提出了活的光片成像的跳动完整的心脏，是在内源性的物理负荷下，作为下一个前沿需要扩展我们的知识的变化，核形态和动力学，经常观察到的核纤层蛋白病。在这个项目中，我们建议开发一个成像平台，允许在跳动的心脏心肌细胞核的实时成像。这将使得能够询问细胞核的外在力（由肌肉收缩驱动）和细胞核的内在力（染色质组织）如何驱动对照和LINC复合体突变小鼠心脏中的细胞核变形。这一发现将对理解健康心脏和疾病状态下完整的活心脏的核动力学具有广泛的意义。在本项目中，我们建议开发一种成像平台，可以对跳动心脏中的心肌细胞核进行实时成像。这将使得能够询问细胞核的外在力（由肌肉收缩驱动）和细胞核的内在力（染色质组织）如何驱动对照和LINC复合体突变小鼠心脏中的细胞核变形。这些发现将对理解健康心脏和疾病状态下的活的、完整的心脏的核动力学具有广泛的相关性。为了实现这一目标，我们提出了以下目标：目标1：建立在光片显微镜上成像心脏的物理平台。目标2：优化心脏提取和培养条件，用于离体心脏成像。目标3：目的4：对正常小鼠和核膜突变小鼠模型的荧光标记细胞核进行成像。

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.