Human hearts lose the capacity to regenerate after birth, and knowing how this ability is lost could advance the understanding and treatment of heart disease. Proliferation of cardiomyocytes, a requirement for heart regeneration, decreases when their nuclei become polyploid. It is thought that failure of karyokinesis leads to the formation of polyploid cardiomyocyte nuclei, but the detailed molecular mechanisms are unknown.To understand the molecular regulation of karyokinesis failure, we utilized single cell transcriptome analysis of cardiomyocytes isolated from genetically engineered mice with Azami- Green Geminin live cell reporter. We found that the nuclear lamina filament lamin B2 (Lmnb2) is expressed in proliferating embryonic but not in cycling adult cardiomyocytes. We showed that Lmnb2is essential for nuclear envelope removal, thus enabling spindle microtubule attachment to chromosomes. Inactivating Lmnb2floxwith ?MHC-Credecreased spindle attachment, which inhibited metaphase progression and led to polyploid daughter nuclei in vivo.Adenoviral transduction of Lmnb2promoted M-phase progression, increased cardiomyocyte division, and improved myocardial regeneration after neonatal heart injury. Cardiac MRI showed that Lmnb2gene transfer increased the ejection fraction from 50.7 ? 5.1 to 67.0 ? 4.1. Innovative MRI tagging technology showed that this global functional improvement was driven by increases of contractility in the borders zone. CRISPR-Cas9 mediated Lmnb2gene inactivation in human iPS cell-derived cardiomyocytes reduced karyokinesis. In primary cardiomyocytes from human infants, modifying Lmnb2expression correspondingly altered metaphase progression and ploidy of daughter nuclei. In conclusion, Lmnb2expression is essential for proliferative capacity in mammalian cardiomyocytes, and its loss during postnatal development leads to inability to regenerate heart muscle.