Cardiac cell identification and isolation

Identification and isolation of cardiomyocytes from the heart, embryonic stem cells, or induced pluripotent stem cells is a challenging task and will require specific isolation techniques. Less than 30% of the cells within the murine heart are cardiomyocytes and, what is more, there are multiple sub-populations of cardiomyocytes like atrial, ventricular and conduction system cardiomyocytes. In Paper I, we investigated the potential of surface marker isolation of committed cardiomyocytes by using fluorescence activated cell sorting (FACS). Herein we 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). Patch clamp experiments confirmed that the isolated cells are fully functional and are of both atrial and ventricular subtype. Classical analysis techniques use population average readouts for the identification of marker expression. However, these approaches mask cellular heterogeneity and thus single-cell techniques have evolved to combat this issue. In paper II we use this technique to screen for cardiomyocyte sub-population surface markers. We found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables the isolation of live and functional murine trabecular ventricle cells, solid ventricle cells, and atrial cells. Additionally, the accurate identification of cardiomyocytes and their cell cycle status is an invaluable tool for cardiac research. In paper III we generate and characterise transgenic mice expressing a fusion protein of human histone 2B and the red fluorescent protein mCherry under control of the CM specific αMHC promoter. This allows for the unequivocal identification of cardiomyocytes through their fluorescently labelled nuclei and has enabled quantification of cardiomyocyte cell fraction in the different parts of the heart. Furthermore, by combining this transgenic system with the CAG-eGFP-anillin transgene we were able to establish a novel cell-cycle based screening assay. Finally, we established a novel use for the commercially available reprograming kit (CytoTune 2.0) in murine cells. By following the expression of induced human and endogenous mouse pluripotency genes we were able to get a better insight into the reprogramming process. The generated induced pluripotent stem cells were capable of differentiating into all three germ layers. Additionally, we investigated the cardiac differentiation potential of these cells by using single cell technology

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific alpha MHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo

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

<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

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

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

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

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

<div><p>In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.</p></div

Dietary nitrate attenuates renal ischemia-reperfusion injuries by modulation of immune responses and reduction of oxidative stress

Ischemia-reperfusion (IR) injury involves complex pathological processes in which reduction of nitric oxide (NO) bioavailability is suggested as a key factor. Inorganic nitrate can form NO in vivo via NO synthase-independent pathways and may thus provide beneficial effects during IR. Herein we evaluated the effects of dietary nitrate supplementation in a renal IR model. Male mice (C57BL/6J) were fed nitrate-supplemented chow (1.0 mmol/kg/day) or standard chow for two weeks prior to 30 min ischemia and during the reperfusion period. Unilateral renal IR caused profound tubular and glomerular damage in the ischemic kidney. Renal function, assessed by plasma creatinine levels, glomerular filtration rate and renal plasma flow, was also impaired after IR. All these pathologies were significantly improved by nitrate. Mechanistically, nitrate treatment reduced renal superoxide generation, pro-inflammatory cytokines (IL-1 beta, IL-6 and IL-12 p70) and macrophage infiltration in the kidney. Moreover, nitrate reduced mRNA expression of pro-inflammatory cytokines and chemo attractors, while increasing anti-inflammatory cytokines in the injured kidney. In another cohort of mice, two weeks of nitrate supplementation lowered superoxide generation and IL-6 expression in bone marrow-derived macrophages. Our study demonstrates protective effect of dietary nitrate in renal IR injury that may be mediated via modulation of oxidative stress and inflammatory responses. These novel findings suggest that nitrate supplementation deserve further exploration as a potential treatment in patients at high risk of renal IR injury