Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post‐chamber formation

McMullen, Nichole; Zhang, Feixiong; Hotchkiss, Adam; Bretzner, Frédéric; Wilson, Jennifer M.; Ma, Hong; Wafa, Karim; Brownstone, Robert M.; Pasumarthi, Kishore B.S.

doi:10.1002/dvdy.22112

Cited by 14 publications

(19 citation statements)

References 41 publications

(80 reference statements)

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“…These findings confirmed the MS results in which 3 peptides were exclusively identified only in medium of NRVMs: 2 peptides covering a part of ANP prohormone sequence and 1 peptide matching the partial sequence of ANP hormone [17]. ANP is considered to be a cardiac lineage marker as it is expressed early during the differentiation of myocytes [85] and early during heart development [86]. This suggests that since CSCs are like embryonic and other progenitor cells [87], they do not secrete ANP but they would during differentiation into cardiac myocytes.…”

Section: Secretome Proteomicssupporting

confidence: 77%

Investigating the Secretome

Št́astná

Eyk

2012

Circ Cardiovasc Genet

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The cell/environment interface is composed of the proteins of plasma membrane which face the extracellular space and by the proteins secreted directly by the cell of origin or by neighboring cells. The secreted proteins can act as extracellular matrix proteins and/or autocrine/paracrine proteins. This report discusses the technical aspects involved in the identification and characterization of the secreted proteins of specific cell types that comprise the heart. These aspects include the culturing of the cells, cell co-culturing and quantitative labeling, conditioned media collection and dealing with high abundant serum proteins, post-translational modification enrichment, the use of protein separation methods and mass spectrometry, protein identification and validation and the incorporation of pathway analysis to better understand the novel discovery on the background of already known experimental biological systems. The proteomic methods have the solid emplacement in cardiovascular research and the identification of proteins secreted by cardiac cells has been used in various applications such as determination the specificity between secretomes of different cell types, e.g. cardiac stem cells and cardiac myocytes, for the global secretome screening of e.g. human arterial smooth muscle cells, for the mapping of the beneficial effect of conditioned medium of one cell type on the other cell type, e.g. conditioned medium of human mesenchymal stem cells on cardiac myocytes, and for the searching the candidate paracrine factors and potential biomarkers.

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Section: Secretome Proteomicssupporting

confidence: 77%

Investigating the Secretome

Št́astná

Eyk

2012

Circ Cardiovasc Genet

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“…7, A-C) since previously we demonstrated that Nkx2.5 ϩ /MF20 Ϫ cells differentiate into MF20 ϩ expressing cardiomyocytes in vitro (30). Conversely, cells positive for both ␤-Gal and MF20 (␤-Gal ϩ /MF20 ϩ ) were considered to be cardiomyocytes (Fig.…”

Section: E115 Ventricular Cells and Gets Cleaved Into Its Biologicalmentioning

confidence: 84%

“…Although NPRA mRNA expression is the lowest at E11.5 stage, we have chosen to study the effects of ANP on ventricular cells from this stage. This is primarily due to the fact that CPCs are abundant in E11.5 ventricular myocardium and their numbers decline precipitously at later developmental stages (30,47,48). While NPRA mRNA levels are lowest at E11.5 stage, Western blot analyses indicate that NPRA protein levels at E11.5 are comparable to later developmental stages.…”

Section: Discussionmentioning

confidence: 99%

Atrial natriuretic peptide inhibits cell cycle activity of embryonic cardiac progenitor cells via its NPRA receptor signaling axis

Hotchkiss

Feridooni

Baguma-Nibasheka

et al. 2015

American Journal of Physiology-Cell Physiology

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(NPRs), which can either activate guanylyl cyclase (NPRA and NPRB) or inhibit adenylyl cyclase (NPRC) to modulate intracellular cGMP or cAMP, respectively. During cardiac development, ANP serves as an early maker of differentiating atrial and ventricular chamber myocardium. As development proceeds, expression of ANP persists in the atria but declines in the ventricles. Currently, it is not known whether ANP is secreted or the ANP-NPR signaling system plays any active role in the developing ventricles. Thus the primary aims of this study were to 1) examine biological activity of ANP signaling systems in embryonic ventricular myocardium, and 2) determine whether ANP signaling modulates proliferation/differentiation of undifferentiated cardiac progenitor cells (CPCs) and/or cardiomyocytes. Here, we provide evidence that ANP synthesized in embryonic day (E)11.5 ventricular myocytes is actively secreted and processed to its biologically active form. Notably, NPRA and NPRC were detected in E11.5 ventricles and exogenous ANP stimulated production of cGMP in ventricular cell cultures. Furthermore, we showed that exogenous ANP significantly decreased cell number and DNA synthesis of CPCs but not cardiomyocytes and this effect could be reversed by pretreatment with the NPRA receptorspecific inhibitor A71915. ANP treatment also led to a robust increase in nuclear p27 levels in CPCs compared with cardiomyocytes. Collectively, these data provide evidence that in the developing mammalian ventricles ANP plays a local paracrine role in regulating the balance between CPC proliferation and differentiation via NPRA/ cGMP-mediated signaling pathways. embryonic heart; ANP; natriuretic peptide receptors; gene expression; cardiac progenitor cells; cardiomyocytes; lineage tracking; knockin mice; cell proliferation and differentiation ATRIAL NATRIURETIC PEPTIDE (ANP) is a 28 amino acid peptide that is synthesized and stored primarily in secretory granules of atrial cardiomyocytes in the adult heart. Ligand binding of ANP to its cognate natriuretic peptide receptors (NPRs) can either activate guanylyl cyclase (NPRA and NPRB) or inhibit adenylyl cyclase (NPRC) to modulate intracellular cGMP or cAMP, respectively. The primary stimulus for ANP secretion from atrial cardiomyocytes is mechanical stretch of the atrial wall (15). In addition to mechanical stimuli, several vasoconstrictor peptides including endothelin-1 (ET-1) (36) and angiotensin II (9), as well as a variety of neurohormones, growth factors, and cytokines, have been shown to modulate natriuretic peptide secretion (10). Once in the circulation, ANP acts in a true endocrine fashion by stimulating NPRA receptors in the kidneys, adrenal cortex, and vasculature to regulate fluid homeostasis and maintain blood pressure via diuretic, natriuretic and vasorelaxant effects (29). In the ventricles of the adult heart, the levels of ANP protein are normally ϳ1,000-fold lower than in the atria and secretory granules are rarely observed (31).In contrast to the adult heart, developmental stud...

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“…Ten-micrometer cryosections were obtained from the adult hearts and processed for visualization of fibrosis by Picro Sirius Red (Direct Red 80 CI 35780; Sigma) and Fast Green (FCF CI 42053; Fisher) (PSFG) staining as described earlier (14,15). Cryosections or cultured cells were incubated with primary antibodies for CDK1 (sc-954; Santa Cruz Biotechnology, Santa Cruz, CA), sarcomeric myosin [MF-20; Developmental Studies Hybridoma Bank (DSHB), University of Iowa], von Willebrand factor (sc-14014), DDR2 (sc-8989), and ␣-smooth muscle actin (1E12-s; DSHB), followed by anti-rabbit and or anti-mouse secondary antibodies conjugated to Alexafluor 488 and 555 (Invitrogen) as described in our earlier publications (36,52,56). The samples were examined using a Leica DM2500 epifluorescence microscope fitted with Leica DFC500 digital acquisition system.…”

Section: Methodsmentioning

confidence: 99%

“…Media were changed after 1 h to eliminate unattached or weakly adherent cells such as cardiomyocytes and endothelial cells. Embryonic day 15.5 (E15.5) primary cultures composed of ventricular myocytes and fibroblasts were generated as described earlier (36,52). In brief, female CD1 mice were time-mated to obtain E15.5 embryos.…”

Section: Methodsmentioning

confidence: 99%

A novel β-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts

Gaspard

MacLean

Rioux

et al. 2014

American Journal of Physiology-Cell Physiology

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Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

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Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post‐chamber formation

Cited by 14 publications

References 41 publications

Investigating the Secretome

Investigating the Secretome

Atrial natriuretic peptide inhibits cell cycle activity of embryonic cardiac progenitor cells via its NPRA receptor signaling axis

A novel β-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts

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