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

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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.