Quantitative label-free digital holographic imaging of cardiomyocyte optical volume, nucleation, and cell division.

Cardiac regeneration in newborn rodents depends on the ability of pre-existing cardiomyocytes to proliferate and divide. This capacity is lost within the first week of postnatal development when these cells rapidly switch from hyperplasia to hypertrophy, withdraw from the cell cycle, become binucleated, and increase in size. How these dynamic changes in cell size and nucleation impact cardiomyocyte proliferative potential is not well understood. In this study, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, to evaluate the proliferative responses of mononucleated and binucleated cardiomyocytes after CHIR99021 treatment, a model proliferative stimulus. This system enables long-term label-free quantitative tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our results confirm that chemical inhibition of glycogen synthase kinase 3 with CHIR99021 promotes complete cell division of both mononucleated and binucleated cardiomyocytes with high frequency. Quantitative tracking of cardiomyocyte volume dynamics during these proliferative events revealed that both mononucleated and binucleated cardiomyocytes reach a similar size-increase threshold prior to attempted cell division. Binucleated cardiomyocytes attempt to divide with lower frequency than mononucleated cardiomyocytes, which may be associated with inadequate increases in cell size. By defining the interrelationship between cardiomyocyte size, nucleation, and cell cycle control, we may better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.