Department of Cardiovascular Medicine, Humanitas Clinical and Research Center, via Manzoni, 56, Rozzano, 20089 Milan, Italy.
Department of Biotechnology and Biosciences, University of Milan-Bicocca, Piazza dell'Ateneo Nuovo, 1, 20126 Milan, Italy.
Cardiovasc Res. 2018 Mar 1;114(3):409-416. doi: 10.1093/cvr/cvx239.
The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies.
A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction.
We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.
本研究旨在建立一种简单可靠的活体心室心肌细胞（vCM）分离方法，用于分子和生物学研究。
采用标准的逆行酶解液灌注技术从成年小鼠心脏中分离心肌细胞。对成年鼠心室心肌细胞进行荧光激活细胞分选（FACS），比较 FITC 通道中的固有自发荧光和前向散射（FSC）参数，以分离高荧光细胞。根据 FITC 和 FSC 特征对细胞进行分选后，用实时 PCR 评估细胞特异性 mRNA 的表达。我们从野生型心脏的逆行灌注后收获的两种不同细胞亚群：FITChigh/FSCdim 和 FITCdim/FSChigh。免疫表型和 mRNA 分析（qPCR 和 RNA 测序）表明，只有 FITChigh/FSCdim 细胞高度富集 CM 标志物。内皮细胞和成纤维细胞中高表达的基因在 FITCdim/FSChigh 亚群中富集。使用 tdTomatofl/fl-α-肌球蛋白重链 MerCreMer+/-小鼠心脏，我们发现 tdTomato 阳性 vCM 存在于 FITChigh/FSCdim 区域，但在 FITCdim/FSChigh 部分中很少见。
我们开发了一种从成年鼠心肌中分离高纯度 vCM 的简单可靠方法，避免了固定和通透步骤。这些分离的 vCM 特别可用于详细的分子研究，避免与其他心肌细胞类型的污染。
