Laura Bader, Marie-Christine Ramel, Toby G. R. Andrews, Kirti Gupta, Jennifer Pearson-Farr, Lucy M. Collinson and Rashmi Priya Summary Nuclear lobularity increases with cardiac forces in ventricle but not in atrium. How the nucleus, one of the stiffest organelles inside a cell, sustains and responds to mechanical forces in vivo remains understudied. The developing zebrafish vertebrate heart is a unique model system to address this fundamental problem as it generates and withstands substantial mechanical forces. Using in toto imaging, 3D morphometrics, genetics, pharmacological perturbations and scRNA-seq, our work reveals that the two heart chambers, ventricle and atrium, have strikingly distinct cardiomyocyte nuclear morphologies, possibly correlating with Lamin A/C expression and microtubule localisation. Manipulation of cardiac forces and knockdown of Lamin A/C affects only ventricle nuclear morphology while microtubule manipulation affects nuclear morphology in both chambers. Of note, overexpressing Lamin A/C in atrium, which naturally lacks Lamin A/C expression leads to nuclear fragmentation. Together, these results indicate that the two chambers have distinct nuclear mechanics, possibly reflecting the underlying difference in the biomechanical environment. 3dpf Ventricle Atrium High Lamin A/C expression Low Lamin A/C expression Microtubules (MT) Cardiac forces Cardiac forces Ventricle Atrium Lamin A/C Lamin A/C MT stability MT stability 1. Nuclear morphology differs between ventricle and atrium a Ventricle Raw image Binary image Label map Nuclei segmentation Morphological filtering, CCL 3dpf myl7:H2B-mScarlet ventricle atrium Ventricle Heart rate + contractility myl7:mScarlet-LAP2b myl7:H2B-mNeongreen myl7:BFP-CAAX 3dpf Atrium Ventricle Atrium Ventricle Atrium Nif (10μM) DMSO IBMX (50μM) Manipulating microtubule stability alters cardiomyocyte nuclear morphology. myl7:EMTB-mNeongreen 3dpf myl7:H2B-mScarlet myl7:BFP-CAAX 3dpf Taxol DMSO Colchicine Ventricle Ventricle Ventricle Atrium Atrium Atrium Ventricle Ventricle Atrium myl7:mScarlet-LAP2b 5dpf Atrium Ventricle outer layer inner layer 4dpf Atrium Ventricle Ventricle 2. Cardiac force manipulation alters ventricle nuclear morphology vmhc amhc vmhc:laminA-mScarlet myl7:laminA-mScarlet myl7 * * * *enucleated cells amhc:laminA-mScarlet myl7:H2B-mNeongreen; myl7:BFP-CAAX 5dpf Overexpression of Lamin A/C is less tolerated in atrium. Microtubules are load-bearing cytoskeletal filaments that regulate mechanics6 and maintain nuclear architecture7. The Francis Crick Institute, 1 Midland Road, London NW1 1AT contact: laura.bader@crick.ac.uk 3. Lamin A/C is differentially expressed in ventricle and atrium The nuclear lamina is composed of lamins, intermediate filaments required to maintain the nuclear integrity1. Lamin A/C (lmna) is thought to regulate nuclear stiffness and mechanosensing2 and to mechanoprotect the genome3. Depletion of Lamin A/C softens nuclei and leads to increased number of mishappen nuclei2,4. In contrast, overexpression of Lamin A/C stiffens nuclei5. - How Lamin A/C regulates nuclear morphology – role of phosphorylation. - How microtubules regulate nuclear morphology – genetic tools. - Functional implications of differential nuclear morphology in ventricle vs. atrium. - Role of LINC (linker of the nucleoskeleton and cytoskeleton) complex. 4. Microtubule organisation regulates nuclear morhology 5. Further work We would like to express our gratidue to the Light Microscopy, Bioinformatics and Biostatistics STPs at the Crick, for their guidance and support at every stage of the project. Special thanks to the BRF for their assistance with fish husbandry. Without their knowledge, hardwork and expertise, this project would not have been possible. Lastly, we would like to thank the organisations who have provided financial support for this work, including The Francis Crick Institute and the British Heart Foundation. Acknowledgements [1] Gruenbaum, Y. and O. Medalia (2