scholarly journals Defining Cardiac Cell Populations and Relative Cellular Composition of the Early Fetal Human Heart

2021 ◽  
Author(s):  
Jennifer Dewing ◽  
Vinay Saunders ◽  
Ita O'Kelly ◽  
David Wilson
Keyword(s):  
Flow Cytometry ◽  
Human Heart ◽  
Fetal Heart ◽  
Muscle Cells ◽  
Cardiac Cells ◽  
Cell Populations ◽  
Cardiac Cell ◽  
Protein Markers ◽  
Rt Pcr ◽  
Marker Profile

The human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Reliable identification of fetal cardiac cell types using protein markers is important for understanding cardiac development and delineating the cellular composition of the human heart during early development, which remains largely unknown. The aim of this study was to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and Troponin I (cTnI)), fibroblasts (DDR2, Thy1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Flow cytometry revealed two distinct populations of cTnI expressing cells based on fluorescence intensity: cTnI High and cTnI Low . MHC positive cardiomyocytes were cTnI High , whereas MHC negative non-myocyte cells were cTnI Low . cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin was confirmed to be enriched in cultured fibroblast populations and flow cytometry revealed Vim High and Vim Low cell populations in the fetal heart. MHC positive cardiomyocytes were Vim Low whilst CD31 positive endothelial cells were Vim High . Based on the markers investigated, we estimate fetal human cardiomyocyte populations comprise 75-80% of total cardiac cells and exhibit the following marker profile: α-MHC + /cTnI High /Vim Low . For the non-cardiomyocyte population, we estimate they comprise 20-25% of total cardiac cells and exhibit the following marker profile: α-MHC - /cTnI Low /Vim High . Our study suggests the marker profiles and proportions of fetal cardiac populations are distinct from that of the adult heart.

scholarly journals Comparison of Lethal and Nonlethal Mouse Models of Orientia tsutsugamushi Infection Reveals T-Cell Population-Associated Cytokine Signatures Correlated with Lethality and Protection

2021 ◽  
Vol 6 (3) ◽  
pp. 121
Author(s):  
Alison Luce-Fedrow ◽  
Suchismita Chattopadhyay ◽  
Teik-Chye Chan ◽  
Gregory Pearson ◽  
John B. Patton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Host Response ◽  
Cd8 T Cell ◽  
Cd4 T Cell ◽  
Cell Populations ◽  
Murine Models ◽  
Orientia Tsutsugamushi ◽  
Multicolor Flow Cytometry ◽  
Interstrain Difference

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

scholarly journals OP0083 INFECTIOUS AND AUTOINFLAMMATORY MODIC TYPE 1 CHANGES HAVE DIFFERENT PATHOMECHANISMS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 45.2-45
Author(s):  
I. Heggli ◽  
R. Schüpbach ◽  
N. Herger ◽  
T. A. Schweizer ◽  
A. Juengel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cell ◽  
Cell Populations ◽  
Gene Set ◽  
Go Enrichment ◽  
Modic Type ◽  
And Control ◽  
Infectious Etiology

Background:Modic type 1 changes (MC1) are vertebral bone marrow (BM) edema that associate with non-specific low back pain (LBP). Two etiologies have been described. In the infectious etiology the anaerobic aerotolerant Cutibacterium acnes (C. acnes) invades damaged intervertebral discs (IVDs) resulting in disc infection and endplate damage, which leads to the evocation of an immune response. In the autoinflammatory etiology disc and endplate damage lead to the exposure of immune privileged disc cells and matrix to leukocytes, thereby evoking an immune response in the BM. Different etiologies require different treatment strategies. However, it is unknown if etiology-specific pathological mechanisms exist.Objectives:The aim of this study was to identify etiology-specific dysregulated pathways of MC1 and to perform in-depth analysis of immune cell populations of the autoinflammatory etiology.Methods:BM aspirates and biopsies were obtained from LBP patients with MC1 undergoing spinal fusion. Aspirates/biopsies were taken prior screw insertion through the pedicle screw trajectory. From each patient, a MC1 and an intra-patient control aspiration/biopsy from the adjacent vertebral level was taken. If C. acnes in IVDs adjacent to MC1 were detected by anaerobic bacterial culture, patients were assigned to the infectious, otherwise to the autoinflammatory etiology.Total RNA was isolated from aspirates and sequenced (Novaseq) (infectious n=3 + 3, autoinflammatory n=5 + 5). Genes were considered as differentially expressed (DEG) if p-value < 0.01 and log2fc > ± 0.5. Gene ontology (GO) enrichment was performed in R (GOseq), gene set enrichment analysis (GSEA) with GSEA software.Changes in cell populations of the autoinflammatory etiology were analyzed with single cell RNA sequencing (scRNAseq): Control and MC1 biopsies (n=1 + 1) were digested, CD45+CD66b- mononuclear cells isolated with fluorescence activated cell sorting (FACS), and 10000 cells were sequenced (10x Genomics). Seurat R toolkit was used for quality-control, clustering, and differential expression analysis.Transcriptomic changes (n=5 + 5) of CD45+CD66b+ neutrophils isolated with flow cytometry from aspirates were analyzed as for total bulk RNAseq. Neutrophil activation (n=3 + 3) was measured as CD66b+ expression with flow cytometry. CD66bhigh and CD66blow fractions in MC1 and control neutrophils were compared with paired t-test.Results:Comparing MC1 to control in total bulk RNAseq, 204 DEG in the autoinflammatory and 444 DEG in the infectious etiology were identified with only 67 shared genes (Fig. 1a). GO enrichment revealed “T-cell activation” (p = 2.50E-03) in the autoinflammatory and “complement activation, classical pathway” (p=1.1E-25) in the infectious etiology as top enriched upregulated biological processes (BP) (Fig 1b). ScRNAseq of autoinflammatory MC1 showed an overrepresentation of T-cells (p= 1.00E-34, OR=1.54) and myelocytes (neutrophil progenitor cells) (p=4.00E-05, OR=2.27) indicating an increased demand of these cells (Fig. 1c). Bulk RNAseq analysis of neutrophils from the autoinflammatory etiology revealed an activated, pro-inflammatory phenotype (Fig 1d), which was confirmed with more CD66bhigh neutrophils in MC1 (+11.13 ± 2.71%, p=0.02) (Fig. 1e).Figure 1.(a) Venn diagram of DEG from total bulk RNAseq (b) Top enriched upregulated BP of autoinflammatory (left) and infectious (right) etiology (c) Cell clustering of autoinflammatory MC1 BM (d) Enrichment of “inflammatory response” gene set in autoinflammatory MC1 neutrophils (e) Representative histogram of CD66b+ expression in MC1 and control neutrophils.Conclusion:Autoinflammatory and infectious etiologies of MC1 have different pathological mechanisms. T-cell and neutrophil activation seem to be important in the autoinflammatory etiology. This has clinical implication as it could be explored for diagnostic approaches to distinguish the two MC1 etiologies and supports developing targeted treatments for both etiologies.Disclosure of Interests:None declared

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells

1982 ◽  
Vol 60 (4) ◽  
pp. 556-567 ◽  
Author(s):  
Alexandre Fabiato
Keyword(s):  
Cardiac Cells ◽  
Arsenazo Iii ◽  
Optical Methods ◽  
Cardiac Cell ◽  
Differential Absorption ◽  
Fluorescence Measurements ◽  
Merocyanine 540 ◽  
Inverted Microscope ◽  
Optimum Wavelength ◽  
Absorption Measurements

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25–30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c) fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of Ca2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.

scholarly journals Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Dipak K. Dube ◽  
Syamalima Dube ◽  
Lynn Abbott ◽  
Ruham Alshiekh-Nasany ◽  
Charles Mitschow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Human Heart ◽  
Amino Acid Sequences ◽  
Rt Pcr ◽  
Adult Heart ◽  
Qrt Pcr ◽  
Long Time ◽  
Alternate Promoters ◽  
Computational Analyses

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Size-based microfluidic enrichment of neonatal rat cardiac cell populations

2006 ◽  
Vol 8 (3) ◽  
pp. 231-237 ◽  
Author(s):  
Shashi K. Murthy ◽  
Palaniappan Sethu ◽  
Gordana Vunjak-Novakovic ◽  
Mehmet Toner ◽  
Milica Radisic
Keyword(s):  
Neonatal Rat ◽  
Cell Populations ◽  
Cardiac Cell

Characteristics of active Na transport in intact cardiac cells

1979 ◽  
Vol 236 (2) ◽  
pp. H189-H199 ◽  
Author(s):  
H. G. Glitsch
Keyword(s):  
Heart Muscle ◽  
Muscle Cells ◽  
Cardiac Cells ◽  
Na Transport ◽  
Concentration Gradients ◽  
Experimental Conditions ◽  
Na Pump ◽  
Na Efflux ◽  
Heart Muscle Cells ◽  
K Concentration

An active Na transport maintains the Na and K concentration gradients across the cell membrane of many cells and restores them following excitation. Heart muscle cells display frequent electrical discharges and thus the cardiac Na pump is of fundamental functional significance. Some methods for studying active Na transport are described. The active Na efflux from heart muscle cells is activated by an increase in the intracellular Na and the extracellular K concentration. The linkage between active Na efflux and active K influx varies widely according to the experimental conditions. The cardiac Na pump is electrogenic and can contribute directly to the membrane potential of the cells. The effects of active Na transport on contraction and intercellular coupling in myocardium are discussed.

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