Quantitative Three-dimensional Label-free Digital Holographic Imaging of Cardiomyocyte Size, Ploidy, and Cell Division

Abstract

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 size and ploidy 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 diploid and binucleated tetraploid cardiomyocytes. This instrument coupled with the powerful Holomonitor App Suite software enables long-term label-free quantitative three-dimensional tracking of primary cardiomyocyte dynamics in real-time with single-cell resolution. Our digital holographic imaging results provide direct evidence that mononucleated cardiomyocytes retain significant proliferative potential as most can successfully divide with high frequency. In contrast, binucleated cardiomyocytes exhibit a blunted response to a proliferative stimulus with the majority not attempting to divide at all. Nevertheless, some binucleated cardiomyocytes were capable of complete division, suggesting that these cells still do retain limited proliferative capacity. By quantitatively tracking cardiomyocyte volume dynamics during these proliferative responses, we reveal that both mononucleated and binucleated cells reach a unique size threshold prior to attempted cell division. The absolute threshold is increased by binucleation, which may limit the ability of binucleated cardiomyocytes to divide. By defining the interrelationship between cardiomyocyte size, ploidy, and cell cycle control, we will better understand the cellular mechanisms that drive the loss of mammalian cardiac regenerative capacity after birth.

Application of Digital Holographic Imaging to Monitor Real-Time Cardiomyocyte Hypertrophy Dynamics in Response to Norepinephrine Stimulation

“…The following segmentation thresholds were applied: auto-minimum error method, adjustment 134, and minimum object size 17. Cardiomyocytes were qualitatively distinguished from non-cardiomyocytes based on their larger size and limited motility as previously described [13]. Care was taken to track well-isolated cells that remained unambiguous throughout the entire imaging duration.…”

“…Cardiac cultures from neonatal rat hearts are often contaminated with other cell types in addition to cardiomyocytes, despite methods used to increase purity [16,17]. Our lab has recently observed that α-actinin-positive cardiomyocytes can be distinguished from non-cardiomyocyte cells imaged on the Holomonitor M4 based on their overall larger size and slower motility [13] (Figure 1B). Using these criteria, we were able to readily identify cardiomyocytes in our cardiac cultures, both with and without norepinephrine treatment (Movies S1 and S2).…”