Quantification of morphological, functional, and biochemical features of H9c2 rat cardiomyoblast retinoic acid differentiation
Cell culture models enable advancement in our understanding of heart development and heart disease. The H9c2 rat ventricular cardiomyoblast cell line can be differentiated, recapitulating certain components of cardiomyocyte development; however, morphological, functional, and biochemical changes are rarely investigated in parallel, thereby limiting fulsome understanding. We therefore characterized morphology, Ca2+ handling, and gene expression after five days (5 days-in-vitro, DIV5), and fourteen days (DIV14) of exposure to differentiation stimuli, consisting of retinoic acid and low serum. We observed several morphological changes with increasing time in differentiation, including increased mean length, area, eccentricity, and multinucleation, as well as an increase in actin clusters. Spontaneous Ca2+ transients in differentiated H9c2 cells had decreased frequency and synchronicity compared to those observed in primary cardiomyocytes. Additionally, key cardiomyocyte cytoskeletal proteins and ion channel transcript and protein expression levels changed significantly with differentiation, in alignment with changes normally observed in cardiomyocyte development. Our findings suggest H9c2 cells may be used as a high throughput screening model to investigate cardiomyocyte biology in certain developmental contexts with relevance to heart health and disease. HighlightsO_LIWe quantified morphology, Ca2+ handling, and gene expression changes in rat ventricular cardiomyoblast H9c2 cells across retinoic acid differentiation. C_LIO_LIWe compared multiple differentiation time points (mainly DIV0, DIV5, and DIV14) and observed time-dependent changes in all 3 aspects. We observed several morphological changes with increasing time in differentiation, including increased mean length, area, eccentricity, and multinucleation, as well as an increase in actin clusters. We observed the onset of spontaneous Ca2+ transients with differentiation, with further changes in subpopulations with increasing time in differentiation. Finally, we observed multiple changes in gene expression consistent with cardiomyocyte differentiation. C_LIO_LIWe summarize these parallel time-point dependent changes in morphology, Ca2+ handing, and gene expression to enable other researchers to optimize their study design. C_LI