Modulation of Mammalian Cardiomyocyte Cytokinesis by the Extracellular Matrix

Rationale: After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown. Objective: To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis. Methods and Results: Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo. Conclusions: We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis.