Application of digital holographic imaging to monitor real-time size and motility dynamics of cardiac cells in response to norepinephrine stimulation

Our objective is to determine if digital holography can be applied to monitor real-time changes in the size and motility of cardiac myocytes and non-myocytes when exposed to the sympathetic neurotransmitter, norepinephrine (NE). Neonatal rodents possess a robust capacity for heart regeneration, which depends on the proliferation of pre-existing cardiomyocytes and then subsequent migration of cardiac cells into the site of injury. Sympathetic activity and activation of adrenergic receptor (AR) signaling promote the loss of heart regenerative capacity after birth. While AR activation has been reported to restrict cardiomyocyte cell cycle activity and drive hypertrophic growth, the role of AR signaling in regulating the motility of cardiomyocytes and non-cardiomyocytes (e.g., fibroblasts) remains little understood. We hypothesize that NE, an adrenergic receptor agonist, reduces cardiac cell motility, which could play a role in limiting heart regenerative capacity. Here, we innovate the application of a commercially available digital holographic imaging microscope, the Holomonitor M4, which enables us to observe the three-dimensional cellular dynamics of cultured primary cells with minimal phototoxicity over long imaging durations. We isolated primary cardiac cells from the hearts of postnatal day 1 (P1) to P3 neonatal rat pups and cultured them on gelatin-coated plates under serum-free conditions. Primary cultures were then treated with or without 10 µM norepinephrine for 24 hours. Mixed cardiac cultures, containing both cardiomyocytes and non-cardiomyocytes, were then imaged on the Holomonitor M4 for 24 hours, with images captured every 20 minutes at 37°C and 5% CO 2 . Cardiomyocytes were distinguished from non-cardiomyocytes based on their overall larger size and slower movement. Time-lapse recordings were then analyzed using the Holomonitor App Suite software to measure the optical volume dynamics (μm3) and motility (μm) of both cardiomyocytes and non-cardiomyocytes over the imaging period. As expected, the average optical volume of NE-treated cardiomyocytes (n = 187 cardiomyocytes across three replicate isolations) increased 1.39-fold from 1074 ± 501 μm3 to 1492 ± 501 μm3 after the 24-hour treatment. In contrast, untreated cardiomyocytes failed to show a statistically significant change in optical volume (0.98-fold). Strikingly, both cardiomyocyte and non-cardiomyocyte populations exhibited significant reductions in cell motility after treatment with NE. Cardiomyocyte (control, n = 156; NE treatment, n = 87) and non-cardiomyocyte (control = 139; NE treatment = 89) motility after 24 hours were reduced by 41% and 44%, respectively. To determine if NE impacts cardiac cell migration in a wound healing assay, we plated primary cardiomyocytes at high density to generate a monolayer and produced a gap by scratching the cells with a pipette tip. Preliminary results indicate that after 5 days post-scratch, cardiomyocytes did not significantly fill the gap in untreated controls nor after stimulation with NE. Non-cardiomyocytes were readily observed in the gap under control conditions, but this was reduced with NE treatment. In conclusion, we demonstrate the application of digital holography to investigate the real-time dynamics of cardiac cells in vitro. Using this approach, we uncover a novel effect of NE in reducing the motility of both cardiomyocytes and noncardiomyocytes, which may be involved in AR signaling-dependent loss of heart regeneration capacity. This work was supported by an NIH SuRE-FIRST grant (R16GM146643) awarded to AP. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.