fig01: Isolation of resident cardiomyocytesStudy schematic. A tamoxifen-inducible cardiomyocyte-specific genetic fate mapping approach was employed, utilizing the MerCreMEr/ZEG mouse strain.Fluorescence-activated cell sorting of enzymatically dispersed myocardial cell preparations obtained from 4-OH Tamoxifen pulsed and non-pulsed bitransgenic (control) mice. Cells were initially gated on the basis of size (Forward scatter; FSC) and granularity (Side scatter; SSC) and subsequently sorted based on expression of GFP, a specific marker for endogenous cardiomyocytes. Boxes denote the boundaries of sorted populations.Flow cytometric analysis for assessment of purity of FACS-sorted GFP+ cardiomyocytes. Markers include troponin T, troponin I, and α-sarcomeric actinin (for cardiomyocytes), CD90 (for mesenchymal cells and fibroblasts), α-smooth muscle actin (α-SMA) (for smooth muscle cells), CD31 (for endothelial cells), c-Kit and Sca-1 (for progenitor cells).

Mentions: We used an inducible fate mapping approach which provides efficient and highly specific cardiomyocyte labelling (Hsieh et al, 2007; Loffredo et al, 2011; Zhang et al, 2010). Bitransgenic mice were generated by crossbreeding female transgenic B6129-Tg(Myh6-cre/Esr1)1 Jmk/J (hereafter referred to as MerCreMer) mice with male B6.Cg-Tg(ACTB-Bgeo/GFP)21Lbe/J (hereafter referred to as ZEG) reporter mice. MerCreMer mice carry a fusion transgene of Cre recombinase flanked by Mer (mutated estrogen receptor ligand binding domains), driven by the cardiac α-myosin heavy chain promoter (encoded by Myh6); thus the Cre recombinase activity is tamoxifen-sensitive and cardiomyocyte-specific. ZEG reporter mice carry a lacZ transgene flanked by LoxP sites, followed by stop codons and then the GFP gene; therefore, upon excision of the LoxP sites and stop codons mediated by Cre recombinase activity, the reporter switches to GFP (driven by the β-actin promoter), permanently and specifically marking resident cardiomyocytes and their progeny as GFP+. No spontaneous expression of GFP was detected in non-tamoxifen-pulsed bitransgenic animals [confirming previous studies reporting very low rates of leakage in MerCreMer/ZEG mice (Hsieh et al, 2007)], while pulsed mice exhibited robust GFP expression (Fig 1B, Supporting Information Fig 1).