ABSTRACT Heart disease is the leading cause of morbidity and mortality worldwide. Different types of heart injury lead to the development of heart failure, a chronically progressive disease. Heart failure is characterized by increased cardiomyocyte death and insufficient regeneration of new ones. As such, increasing cardiomyocyte regeneration and decreasing death represent targets for new therapies. This application is for developing a transformative paradigm connecting these important cellular mechanisms with nuclear remodeling in cardiomyocyte differentiation. Our new results show that when cardiomyocytes differentiate they decrease the number of nuclear pores (NP). NP are large channels of > 100 nm outer diameter for communication between the nucleus and the cytoplasm. NP, along with the nuclear lamina (NL), function in regulating the nuclear transport of signaling molecules and chromatin organization. Although heart failure alters nuclear transport, the structural and functional changes of NL and NP changes during terminal differentiation are unknown. To determine the molecular mechanisms directing the decrease of NP, we have used single-cell transcriptional profiling, which identified a decrease in expression of lamin B2 (Lmnb2), an intermediate filament and component of the NL, during cardiomyocyte differentiation. Our new results show that Lmnb2 gene knockout in cardiomyocytes blocks M-phase, that is, nuclei do not divide, and instead become polyploid. In addition, the M-phase block decreases NP incorporation. As a result, although the DNA contents of nuclei (ploidy) increases, the number of NP decreases by 50%. The lower NP number identifies a central event in nuclear remodeling, as it indicates not only altered nuclear transport, but also altered chromatin structure. Together, this could explain the decreased ability of terminally differentiated cardiomyocytes to activate cell cycle genes and their increased susceptibility to cell death. This proposal aims to develop a new mechanistic paradigm of nuclear remodeling in cardiomyocyte differentiation, which is synergistic with recent advances in characterizing chromatin changes. We will test the central hypothesis that decreased Lmnb2 gene expression is a central mechanism of nuclear remodeling in cardiomyocyte differentiation. We have assembled an interdisciplinary research team and prepared innovative techniques (super-resolution microscopy, single-cell RNAseq, ATACseq) that, combined with cardiomyocyte-specific Lmnb2flox inactivation and viral expression of Lmnb2, will enable us to determine its role in nuclear remodeling in cardiomyocyte differentiation. The anticipated results will enable future research toward understanding and targeting nuclear remodeling in myocardial development, regeneration, and disease. This will be broadly significant for patients with congenital and acquired heart diseases.