Isolation and analysis of single cells from the mouse heart

Pinto, Alexander R.; Chandran, Anjana; Rosenthal, Nadia; Godwin, James

doi:10.1016/j.jim.2013.03.012

Cited by 39 publications

(33 citation statements)

References 5 publications

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“…Mouse hearts were isolated for single cell preparation as previously described 9 with atria and valves removed. Isolated mouse hearts were digested using one of three protocols (designated Protocol 1, 2 and 3).…”

Section: Methodsmentioning

confidence: 99%

Revisiting Cardiac Cellular Composition

Pinto

Ilinykh

Ivey

et al. 2016

Circulation Research

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Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated.

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Section: Methodsmentioning

confidence: 99%

Revisiting Cardiac Cellular Composition

Pinto

Ilinykh

Ivey

et al. 2016

Circulation Research

Self Cite

1,110

988

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“…Cardiac single cells preparations were conducted as described in detail previously [39]. Briefly, mice were killed by CO2 asphyxiation (~5 min) followed by cardiac perfusion with ice-cold HBSS for 2 min.…”

Section: Methodsmentioning

confidence: 99%

Age-related changes in tissue macrophages precede cardiac functional impairment

Pinto

Godwin

Chandran

et al. 2014

Aging

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Cardiac tissue macrophages (cTMs) are abundant in the murine heart but the extent to which the cTM phenotype changes with age is unknown. This study characterizes aging-dependent phenotypic changes in cTM subsets. Using the Cx3cr1GFP/+ mouse reporter line where GFP marks cTMs, and the tissue macrophage marker Mrc1, we show that two major cardiac tissue macrophage subsets, Mrc1−GFPhi and Mrc1+GFPhi cTMs, are present in the young (<10 week old) mouse heart, and a third subset, Mrc1+GFPlo, comprises ~50% of total Mrc1+ cTMs from 30 weeks of age. Immunostaining and functional assays show that Mrc1+ cTMs are the principal myeloid sentinels in the mouse heart and that they retain proliferative capacity throughout life. Gene expression profiles of the two Mrc1+ subsets also reveal that Mrc1+GFPlo cTMs have a decreased number of immune response genes (Cx3cr1, Lpar6, CD9, Cxcr4, Itga6 and Tgfβr1), and an increased number of fibrogenic genes (Ltc4s, Retnla, Fgfr1, Mmp9 and Ccl24), consistent with a potential role for cTMs in cardiac fibrosis. These findings identify early age-dependent gene expression changes in cTMs, with significant implications for cardiac tissue injury responses and aging-associated cardiac fibrosis.

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“…Cardiac leukocyte isolation was performed as described in detail previously (20) with few modifications. Briefly, at indicated time points, infected hearts were perfused with PBS (Life Technologies), weighed, and disaggregated mechanically and enzymatically with 0.2% of trypsin solution (Life Technologies).…”

Section: Heart-infiltrating Cell Isolationmentioning

confidence: 99%

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy

Ponce

Sanmarco

Eberhardt

et al. 2016

The Journal of Immunology

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Increasing evidence demonstrates that generation of extracellular adenosine from ATP, which is hydrolyzed by the CD39/CD73 enzyme pair, attenuates the inflammatory response and deactivates macrophage antimicrobial mechanisms. Although CD73 is emerging as a critical pathway and therapeutic target in cardiovascular disorders, the involvement of this ectonucleotidase during myocardial infection has not been explored. Using a murine model of infection with Trypanosoma cruzi, the causal agent of Chagas cardiomyopathy, we observed a sudden switch from the classical M1 macrophage (microbicidal) phenotype toward an alternative M2 (repairing/anti-inflammatory) phenotype that occurred within the myocardium very shortly after BALB/c mice infection. The observed shift in M1/M2 rate correlated with the cardiac cytokine milieu. Considering that parasite persistence within myocardium is a necessary and sufficient condition for the development of the chronic myocarditis, we hypothesized that CD73 activity may counteract cardiac macrophage microbicidal polarization, rendering the local immune response less effective. In fact, a transient treatment with a specific CD73 inhibitor (adenosine 5′-α,β-methylene-diphosphate) enhanced the microbicidal M1 subset predominance, diminished IL-4– and IL-10–producing CD4+ T cells, promoted a proinflammatory cytokine milieu, and reduced parasite load within the myocardium during the acute phase. As a direct consequence of these events, there was a reduction in serum levels of creatine kinase muscle–brain isoenzyme, a myocardial-specific injury marker, and an improvement in the electrocardiographic characteristics during the chronic phase. Our results demonstrate that this purinergic system drives the myocardial immune response postinfection and harbors a promising potential as a therapeutic target.

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Isolation and analysis of single cells from the mouse heart

Cited by 39 publications

References 5 publications

Revisiting Cardiac Cellular Composition

Revisiting Cardiac Cellular Composition

Age-related changes in tissue macrophages precede cardiac functional impairment

CD73 Inhibition Shifts Cardiac Macrophage Polarization toward a Microbicidal Phenotype and Ameliorates the Outcome of Experimental Chagas Cardiomyopathy

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