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 with retinoic acid and low serum, leading to morphological, molecular, and functional changes that partially resemble aspects of cardiomyoblast-to-cardiomyocyte differentiation. However, morphological, functional, and biochemical changes are rarely investigated in parallel, thereby limiting fulsome understanding of how these processes are interlinked, and to what extent these model cardiomyoblasts can be differentiated. To provide a parallel analysis as a resource for future studies, we therefore characterized H9c2 cell morphology, Ca²⁺ handling, and gene expression after five days (5 days<i>-in-vitro</i>, DIV5), and fourteen days (DIV14) of exposure to differentiation stimuli, consisting of retinoic acid and low serum. We observed statistically significant morphological changes during differentiation. We saw changes consistent with those already described in the context of cardiomyoblast differentiation. However, some of these were previously limited to qualitative observations, for example increased cell length. Notably, several of our morphological observations are completely novel, such as increases in eccentricity, perimeter length (aka cell boundary length), and in the density of actin clusters, were investigated de novo. Differentiation also resulted in the onset of spontaneous Ca²⁺ transients - this is the first instance, to our knowledge, that this has been characterized in the absence of pharmacological stimulation. The mean frequency and synchronicity of Ca²⁺ transients in differentiated H9c2 cells were much lower than those observed in primary cardiomyocytes, underscoring their relatively immature differentiation state from a functional perspective. Additionally, key cardiomyocyte cytoskeletal proteins and ion channel transcript and protein expression levels changed significantly with differentiation, including at early timepoints (DIV5 h and DIV3) which had not yet been investigated by others, in alignment with changes normally observed in cardiomyocyte development. Overall, our findings position differentiated H9c2 cells as a relatively high-throughput model for studying cardiomyoblast differentiation, while also clarifying their limitations in recapitulating fully mature cardiomyocyte phenotypes, and highlight reliable markers (e.g., <i>Cacna1c, Myom2</i>, cTnT, VCL, and <i>Gja5</i>) for experimental readouts.