P363 Antibody-based selective isolation of atrial and ventricular cardiomyocytes from neonatal mouse heart.

The mammalian heart comprises mainly cardiomyocytes, fibroblasts, endothelial cells, and smooth muscle cells. Cardiomyocyte subtypes are characterized by differential expression of intracellular proteins like MLC-2a, Cx40 or ANF in atrial cardiomyocytes as well as MLC-2v, Hey2 or Irx4 in ventricular cardiomyocytes. Nevertheless, to date the expression of chamber-specific intracellular proteins could not be correlated to corresponding cell surface markers. Therefore, the selective, surface marker-based enrichment of atrial or ventricular cardiomyocytes from whole-heart preparations is currently not possible.In a proof-of-concept study we performed a semi-automated, antibody-based surface marker screen on neonatal mouse heart preparations. By that we were able to identify several candidate markers exclusively expressed by non-cardiomyocytes. Using a magnetic cell sorting approach, we could highly enrich for neonatal cardiomyocytes by efficiently removing non-cardiomyocytes. In a second step, we were able to identify surface markers differentially expressed on atrial and ventricular cardiomyocytes, thereby dividing the pure cardiomyocyte population into two subpopulations. Next, we established cell isolation strategies to specifically isolate atrial and ventricular cardiomyocytes from whole-heart cell preparations with high purities. Expression profiling of the isolated subpopulations confirmed high expression of atrial marker genes such as Nr2f2, Fgf12, Sln, Gja5 and Nppa in the putative atrial fraction as well as high expression of genes associated with a ventricular myocyte identity, e.g. Hey2, Irx4, Lbh, Myh7 in the putative ventricular fraction. The sorted cells were viable, could be cultivated and exhibited spontaneous contractions 24h after plating.For the first time, we found a set of cell surface markers that can be used to specifically isolate atrial and ventricular cardiomyocytes from mouse heart. As the markers were also expressed on mouse embryonic stem cell-derived cardiomyocytes, current work analyzes their relevance for the identification and isolation of pluripotent stem cell-derived cardiomyocyte subpopulations. [ABSTRACT FROM AUTHOR]