A mammalian myocardial cell-free system to study cell cycle reentry in terminally differentiated cardiomyocytes

Cardiomyocytes withdraw from the cell cycle in the early neonatal period, r endering the adult heart incapable to regenerate after injury. In the prese nt study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nu clei and to specifically address the question of whether nuclei from termin ally differentiated cardiomyocytes can be stimulated to reenter S phase whe n incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuc lei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored b y biotin-16-dUTP incorporation as well as proliferating cell nuclear antige n expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neona tal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide ind icated that de novo translation is required for the reinduction of S phase, Similar results were obtained with adult cardiomyocyte nuclei. When coincu bated with both cytoplasmic extract and nuclei or nuclear extracts of S-pha se cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In co nclusion, these results demonstrate that postmitotic ventricular myocyte nu clei are responsive to stimuli derived from S-phase cells and can thus bypa ss the cell cycle block. This cell-free system now makes it feasible to ana lyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle .