Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA054

Poster Communications

Novel technique for the simultaneous isolation of purified cardiac fibroblasts and epicardial cells from the infarcted murine heart

C. Owenier1, J. Hesse1, C. Alter1, J. Schrader1

1. Molecular Cardiology, Uniklinikum Düsseldorf, Duesseldorf, Germany.

Cardiac fibroblasts (CF) migrate and differentiate into myofibroblasts (activated cardiac fibroblasts - aCF) to mediate healing after acute myocardial infarction (MI). However, the CF population is described to be heterogeneous and difficult to capture by conventionally used markers. An additional problem, which so far has not been addressed, concerns the contamination with epicardium-derived mesenchymal cells (EPDC), formed on the epicardial surface after MI. We have established a novel technique for the simultaneous isolation of CF and EPDCs from the infarcted murine heart. For MI induction mice were anaesthetized (1.5% isoflurane) and the left anterior descending coronary artery was ligated for 50 min followed by reperfusion. Cells were isolated from hearts 5 days post MI and compared to non-infarcted control hearts. Data are represented as mean±SD and statistically analyzed by Student t test. The elaborated methodology involves enzymatic digestion of the isolated heart via the coronaries (Langendorff) for CF isolation and the simultaneous enzymatic removal of the EPDC layer from the outside in a single procedure. Subsequently, the cell suspension derived from the EPDC-depleted heart is separated from cardiomyocytes by centrifugation and endothelial cells as well as immune cells are removed by magnetic bead purification. Purified CF were analyzed for CF markers by flow cytometry: Pdgfrα as well as MEFSK4 was expressed on the majority of the isolates (72.2±2.9% and 52.1±4.6%, respectively), while CD90+, Sca1+ and Vim+ cells amounted to 23.3±2%, 12.4±0.7% and 35.5±2.1%, respectively (n=4). Markers for pericytes and smooth muscle cells were expressed at minor percentages (Ng-2: 1.1±0.3 %, α-SMA: 12.3±0.5%). Gene array analysis of cell sorted CF, aCF and EPDC revealed 736 and 336 differentially expressed genes comparing CF and aCF as well as aCF and EPDC, respectively (p≤0.005, logFC≥1.5; n=5). Genes of interest were further analyzed by qPCR. Analysis revealed that expression of WT-1, an established epicardial marker, was 20-fold higher in EPDC compared to aCF (n=5). Expression of activation markers in aCF and EPDC were in a similar range and higher as compared to CF. Cardiac genes and transcription factors were expressed in CF, aCF and EPDC but with large differences: expression of troponin T was 17-fold higher in CF/ aCF when compared to EPDC, while Tbx-5 was 4-fold higher in EPDC when compared to CF/ aCF. These profound differences between EPDC and aCF/ CF highlight that the removal of EPDC is an essential prerequisite for studies of the CF phenotype after MI. In summary we report a rapid isolation technique for CF pre and post MI, which are largely free of contaminating cells. This protocol is well suited for the analysis of MI-induced phenotype changes in freshly isolated as well as in cultured CF, aCF and EPDC.

Where applicable, experiments conform with Society ethical requirements