FACS-Based Isolation, Propagation and Characterization of Mouse Embryonic Cardiomyocytes Based on VCAM-1 Surface Marker Expression

<div><p>Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for <i>in vitro</i> culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.</p></div

Functional analysis of embryonic cardiomyocytes purified by FACS.

<p>(<b>A</b>) Current clamp recordings revealed differentiation of FACS-isolated cells into cardiac subtypes: atrial and ventricle-like cells. (<b>B</b>) Representative voltage ramp protocol showing activation of inward and outward currents of a FACS-isolated cardiomyocyte. (<b>C</b>) APs recorded from a representative FACS-isolated cardiomyocyte, perfusion with the β-adrenergic agonist Isoprenalin evoked a positive chronotropic effect, this could be reversed upon wash-out. (<b>D</b>) APs recorded from a representative FACS-isolated cardiomyocyte, perfusion with the muscarinic agonist Carbachol induced a strong negative chronotropic effect, this could be reversed upon wash-out. (<b>E</b>) Statistics of the hormonal modulation of AP as % of frequency variation after the agonist application respect to the NS. Abbreviations: (NS) normal solution, (ISO) Isoprenalin, (CCh) Carbachol.</p

RNA expression profile of isolated VCAM-1⁺ embryonic cardiomyocytes.

<p>Gene expression, analyzed by real-time quantitative PCR, in E10.5–E11.5 un-sorted cardiac cells and sorted VCAM-1 positive cells compared to whole embryos. The fold change in gene expression is indicated by the Y-axis with standard deviation error-bars. Variations in RNA input were normalized through expression of the housekeeping gene <i>GAPDH</i>. Verification of cardiac-specific lineage genes alpha-<i>MHC</i>, <i>beta-MHC BNP</i> and <i>MLC-2v</i> (<b>A</b>). Differential expression of cardiac progenitor markers <i>c-KIT</i>, <i>Flk-1</i> and <i>Isl-1</i> (<b>B</b>) and gene markers for non-myocyte lineages, including hematopoietic (<i>CD45</i>), endothelial (<i>VE-Cadherin (VE-Cad)</i> and <i>Endoglin (Eng)</i>), fibroblasts (<i>Ddr2</i>) and mesenchymal cells (<i>Vimentin (Vim)</i>) (<b>C</b>). * Below detection level.</p

Purification of embryonic cardiomyocytes by Flow Cytometry.

<p>Flow Cytometry plots of E10.5–E11.5 cardiac cells labeled with specific antibodies to VCAM-1 and PECAM-1 (<b>A–C</b>). Cells are gated and sorted based on doublet discrimination (<b>A</b>), viability (<b>B</b>) and VCAM-1 positive PECAM-1 negative population (<b>C</b>). Flow Cytometry plot of sorted fixed cells, stained with cTropT antibodies to verify cardiac identity (<b>D</b>). Flow Cytometry histograms (overlays) showing control cTropT staining of neonatal hearts (<b>E</b>). Un-stained cells (black), isotype control (red outline) and neonatal heart cells (green outline). The percentage of gated cells through each step of the sort is indicated in each plot. Immunofluorescence staining (cTropT) of sorted cells cultured on gelatin coated slides for two days to verify cardiac identity and cell viability (<b>F</b>). F-actin and cTropT in separate channels (<b>G–H</b>). A low frequent cTropT-negative cell is indicated (arrow).</p

<i>In vitro</i> culture of primary embryonic cardiomyocytes after FACS.

<p>Phase contrast images of FACS-isolated cells grown on irradiated embryonic cardiac fibroblasts for two (<b>A</b>) or six (<b>B</b>) days. Rounded cells attached to the fibroblasts (<b>A</b>) and beating clusters of cardiomyocytes (<b>B, C</b>). Immunofluorescence images of the co-cultured cells (<b>C–J</b>). The FACS-isolated cells form a tight meshwork of beating cardiomyocytes in close contact with the surrounding fibroblasts as well as expressing cTropT (<b>C</b>) and Connexin43 (<b>D</b>). Co-cultures labeled with BrdU after five days for 24 h (<b>E–G</b>). Incorporation of BrdU is detected in cardiomyocytes but not in the surrounding irradiated fibroblasts. A optical section of a dividing cardiomyocyte in co-culture with fibroblasts for five days visualized by Ki-67 expression (<b>H–J</b>).</p

MiRNA-1/133a Clusters Regulate Adrenergic Control of Cardiac Repolarization

<div><p>The electrical properties of the heart are primarily determined by the activity of ion channels and the activity of these molecules is permanently modulated and adjusted to the physiological needs by adrenergic signaling. miRNAs are known to control the expression of many proteins and to fulfill distinct functions in the mammalian heart, though the <i>in vivo</i> effects of miRNAs on the electrical activity of the heart are poorly characterized. The miRNAs miR-1 and miR-133a are the most abundant miRNAs of the heart and are expressed from two miR-1/133a genomic clusters. Genetic modulation of miR-1/133a cluster expression without concomitant severe disturbance of general cardiomyocyte physiology revealed that these miRNA clusters govern cardiac muscle repolarization. Reduction of miR-1/133a dosage induced a longQT phenotype in mice especially at low heart rates. Longer action potentials in cardiomyocytes are caused by modulation of the impact of β-adrenergic signaling on the activity of the depolarizing L-type calcium channel. Pharmacological intervention to attenuate β-adrenergic signaling or L-type calcium channel activity <i>in vivo</i> abrogated the longQT phenotype that is caused by modulation of miR-1/133a activity. Thus, we identify the miR-1/133a miRNA clusters to be important to prevent a longQT-phenotype in the mammalian heart.</p></div

miR-1/133a controls impact of β-adrenergic regulation on L-type calcium-channel.

<p>The increased slope of ΔQT/ΔRR indicates LQT at low heart rates in miR-1-1/133a-2 and miR-1-2/133a-1 mutant mice. The LQT was rescued in vivo by inhibition of β-adrenergic signaling using Propranolol or by inhibition of L-type calcium channel using Verapamil, respectively (A). This result confirms the in vitro measurements proving that the miR-1/133a clusters modulate β-adrenergic signaling mediated regulation of L-type calcium channel activity and that loss of this modulation causes LQTS after deletion of single miR-1/133a clusters. Thus the miR-1/133a clusters are essential for repression of the smooth muscle gene program in post-natal heart and for maintenance of repolarization properties that are essential for normal function of the heart in its physiological context (B).</p

Loss of miR-1/133a impairs cardiac repolarization.

<p>Analysis of surface ECGs from immobilized animals revealed an increased QT duration (A). The increased QT duration was obvious especially at low heart rates (longer RR interval) induced by anesthesia with Isoflurane (B). We did not observe changes in PR or QRS interval length, nor arrhythmia or changes in the morphology of the ECG traces. The increased QT interval length is based on a longer ST duration. As shown in (C) the slope of a linear fit (ΔQT/ΔRR) is greater in miR-1/133a single cluster compared to the WT animals.</p

β-adrenergic signaling is intact in after loss of miR-1/133a.

<p>The concentration of cAMP was not changed in the adult heart of miR-1/133a single cluster mutant mice (A). Adrenergic signaling of cardiomyocytes isolated form adult hearts of WT and single miR-1/133a cluster mutant mice was investigated by stimulation with 1 µM Isoproterenol (ISO; B–G). Adrenergic signaling affects multiple components involved in cardiomyocyte calcium handling and contraction. Phosphorylation of several targets of the adrenergic signaling cascade was analyzed to detect modulation of the adrenergic signaling in cardiomyocytes isolated from mutant and WT animals. Proteins of cardiomyocytes isolated from of ≥5 animals was used for statistical evaluation (C–G), representative blots are shown (B).</p

Potential miR-1/133a targets involved in modulation of adrenergic signaling.

<p>Molecules known to be involved in adrenergic signaling as well as functionally related molecules were analyzed for potential targeting by mmu-miR-1 or mmu-miR-133a using Targetscan (ts) and mirBase.org (m) and transcriptional regulation of these molecules was analyzed. Of the 177 molecules analyzed to be related to adrenergic signaling 39 were predicted potential targets of miR-1 or miR-133a.</p><p>Potential miR-1/133a targets involved in modulation of adrenergic signaling.</p