Abstract 340: Short Telomeres Induce Autophagy and Modulate Cardiac Progenitor Cell Fate

Aging severely limits myocardial regeneration. Delineating the impact of age-associated factors such as short telomeres is critical to enhance the regenerative potential of cardiac progenitor cells (CPCs). We hypothesize that short telomeres induce autophagy and elicit the age-associated change in cardiac progenitor cell fate. We compared mouse strains with different telomere lengths (TL) for phenotypic characteristics of aging and also isolated CPCs from them. Naturally occurring wild mouse strain Mus musculus castaneus (CAST) possessing short telomeres (TL:18Kb) exhibits early cardiac aging with diastolic dysfunction, hypertrophy, fibrosis and increase in senescence markers p53 and p16, as compared to common lab strains FVB (TL:75Kb) and C57 (TL:50Kb). CAST CPCs with short TLs have altered cell fate as characterized by slower proliferation (p<0.01); increased senescence identified by beta-galactosidase activity (p<0.05); increased basal commitment as determined by expression of lineage markers smooth muscle actin, Tie2, and sarcomeric actinin (16.6, 1.7 and 1.75, p<0.05); as well as loss of quiescence marker expression. Consistent findings of altered cell fate are also evident in old CPCs isolated from aged mice with significantly shorter TLs. Cell fate changes occurring downstream from short TL are at least partially p53 dependent, as p53 inhibition rescues the irreversible cell cycle arrest observed in CAST CPCs. Mechanistically, short TLs induce autophagy, a catabolic protein degradation process. Autophagy flux is increased in CAST CPCs as evidenced by increased LC3 (p<0.05), reduced p62 expression (-52%, p<0.05) and increased accumulation of autophagic puncta. Pharmacological inhibition of autophagosome formation, but not autolysosome formation reverses the cell fate to a more youthful phenotype. Overall the data suggests that short TLs activate autophagy to accommodate cell fate changes that tip the equilibrium away from quiescence and proliferation into differentiation and senescence, leading to age-associated exhaustion of CPCs. The study provides the mechanistic basis underlying age-associated cell fate changes that will enable identification of molecular strategies to enhance the therapeutic effects of aged CPCs.

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Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a356 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Claudia Noack ◽

Maria P Zafiriou ◽

Anke Renger ◽

Hans J Schaeffer ◽

Martin W Bergmann ◽

...

Keyword(s):

Cell Fate ◽

Transcriptional Activation ◽

Progenitor Cell ◽

Cellular Localization ◽

Target Genes ◽

Critical Role ◽

Isolated Cells ◽

Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

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Role of the lipid sensor PPAR-gamma in cardiac progenitor cell deployment: First demonstration of the impact of environmental factor on cardiac progenitor cell development

Archives of Cardiovascular Diseases Supplements ◽

10.1016/j.acvdsp.2020.03.053 ◽

2020 ◽

Vol 12 (2-4) ◽

pp. 220-221

Author(s):

M. Théveniau-Ruissy ◽

M. Rammah ◽

R. Kelly ◽

F. Rochais

Keyword(s):

Environmental Factor ◽

Progenitor Cell ◽

Ppar Gamma ◽

Cell Development ◽

Cardiac Progenitor Cell ◽

Lipid Sensor ◽

The Impact

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The Spatiotemporal Expression of Notch1 and Numb and Their Functional Interaction during Cardiac Morphogenesis

Cells ◽

10.3390/cells10092192 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2192

Author(s):

Lianjie Miao ◽

Yangyang Lu ◽

Anika Nusrat ◽

Hala Y. Abdelnasser ◽

Sayantap Datta ◽

...

Keyword(s):

Cell Differentiation ◽

Cell Fate ◽

Progenitor Cell ◽

Structural Defects ◽

Asymmetric Distribution ◽

Cardiomyocyte Proliferation ◽

Double Knockout ◽

Cardiac Morphogenesis ◽

Cardiac Progenitor Cell ◽

Notch Activation

Numb family proteins (NFPs), including Numb and Numblike (Numbl), are commonly known for their role as cell fate determinants for multiple types of progenitor cells, mainly due to their function as Notch inhibitors. Previous studies have shown that myocardial NFP double knockout (MDKO) hearts display an up-regulated Notch activation and various defects in cardiac progenitor cell differentiation and cardiac morphogenesis. Whether enhanced Notch activation causes these defects in MDKO is not fully clear. To answer the question, we examined the spatiotemporal patterns of Notch1 expression, Notch activation, and Numb expression in the murine embryonic hearts using multiple approaches including RNAScope, and Numb and Notch reporter mouse lines. To further interrogate the interaction between NFPs and Notch signaling activation, we deleted both Notch1 or RBPJk alleles in the MDKO. We examined and compared the phenotypes of Notch1 knockout, NFPs double knockout, Notch1; Numb; Numbl and RBPJk; Numb; Numbl triple knockouts. Our study showed that Notch1 is expressed and activated in the myocardium at several stages, and Numb is enriched in the epicardium and did not show the asymmetric distribution in the myocardium. Cardiac-specific Notch1 deletion causes multiple structural defects and embryonic lethality. Notch1 or RBPJk deletion in MDKO did not rescue the structural defects in the MDKO but partially rescued the defects of cardiac progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis. Our study concludes that NFPs regulate progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis partially through Notch1 and play more roles than inhibiting Notch1 signaling during cardiac morphogenesis.

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Abstract 139: Alterations to the Extracellular Matrix Composition Following Myocardial Infarction Impact Cardiac Progenitor Cell Fate In Vitro

Circulation Research ◽

10.1161/res.115.suppl_1.139 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Kelly E Sullivan ◽

Sharada Sant ◽

Laura Burns ◽

Lauren D Black

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Growth Factors ◽

Cell Fate ◽

Progenitor Cell ◽

Cardiac Differentiation ◽

Matrix Composition ◽

Angiogenic Growth Factors ◽

Cardiac Progenitor Cell

Limitations associated with cardiac progenitor cell (CPC) therapy of myocardial infarction (MI) including poor engraftment, cell death and incomplete cardiac differentiation have hindered the efficacy of treatment in pre-clinical trials. Given that the extracellular environment plays an important role in regulating cell function and that it is significantly remodeled following MI, it is critical to understand how these changes impact the therapeutic potential of CPCs. In this study, we investigated how the alterations to the extracellular matrix (ECM) following MI impacted the regenerative potential of CPCs in vitro. Hearts were decellularized with 1% SDS prior to MI and 1 and 4 weeks post-MI (Fig A) and the composition of the left ventricle or scar was characterized through LC-MS/MS. While Periostin and Collagen I increased post-MI, Laminin decreased (Fig B). c-kit+ CPCs isolated from rat hearts 1 week post-MI were cultured on tissue culture plastic (TCP) coated with pepsin-solubilized ECM. Our results demonstrated that the healthy matrix promoted the expression of pro-angiogenic growth factors, while maintaining the cells in an undifferentiated state (Fig D,E). Alternatively, 1 week ECM promoted cell adherence (Fig C) and the expression of pro-survival growth factors (Fig D) and GATA-4 (Fig E). Cells cultured on 4 week ECM demonstrated significant differentiation towards vascular lineages through their expression of smooth muscle (TAGLN) and endothelial (VWF) markers. By characterizing how the changing ECM composition following MI impacts CPC fate, we may be able to develop therapeutic strategies that modulate cell fate/ function in vivo following implantation.

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Single cell RNA-seq and ATAC-seq indicate critical roles of Isl1 and Nkx2-5 for cardiac progenitor cell transition states and lineage settlement

10.1101/210930 ◽

2017 ◽

Cited By ~ 7

Author(s):

Guangshuai Jia ◽

Jens Preussner ◽

Stefan Guenther ◽

Xuejun Yuan ◽

Michail Yekelchyk ◽

...

Keyword(s):

Single Cell ◽

Cell Fate ◽

Regulatory Networks ◽

Progenitor Cell ◽

Marker Gene ◽

Transitional Cell ◽

Developmental Trajectory ◽

Open Chromatin ◽

Cardiac Progenitor Cell ◽

Cell Fate Decisions

SUMMARYFormation and segregation of cell lineages building the vertebrate heart have been studied extensively by genetic cell tracing techniques and by analysis of single marker gene expression but the underlying gene regulatory networks driving cell fate transitions during early cardiogenesis are only partially understood. Here, we comprehensively characterized mouse cardiac progenitor cells (CPC) marked by Nkx2-5 and Isl1 expression from E7.5 to E9.5 using single-cell RNA sequencing. By leveraging on cell-to-cell heterogeneity, we identified different previously unknown cardiac sub-populations. Reconstruction of the developmental trajectory revealed that Isl1+ CPC represent a transitional cell population maintaining a prolonged multipotent state, whereas extended expression of Nkx2-5 commits CPC to a unidirectional cardiomyocyte fate. Furthermore, we show that CPC fate transitions are associated with distinct open chromatin states, which critically depend on Isl1 and Nkx2-5. Our data provide a model of transcriptional and epigenetic regulations during cardiac progenitor cell fate decisions at single-cell resolution.

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A regulatory pathway involving Notch1/β-catenin/Isl1 determines cardiac progenitor cell fate.

Nature Cell Biology ◽

10.1038/ncb1906 ◽

2009 ◽

Vol 11 (8) ◽

pp. 951-957 ◽

Cited By ~ 147

Author(s):

Chulan Kwon ◽

Li Qian ◽

Paul Cheng ◽

Vishal Nigam ◽

Joshua Arnold ◽

...

Keyword(s):

Cell Fate ◽

Progenitor Cell ◽

Regulatory Pathway ◽

Cardiac Progenitor Cell

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Cardiac Progenitor Cell Fate in Embryonic and Neonatal Environments

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2018.07.010 ◽

2018 ◽

Vol 124 ◽

pp. 83

Author(s):

Bingyan Wang ◽

Alvin Muliono ◽

Roberto Alvarez ◽

Roberto Sacripanti ◽

Mark Sussman

Keyword(s):

Cell Fate ◽

Progenitor Cell ◽

Cardiac Progenitor Cell

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Abstract 203: Short Telomeres Induce p53 and Autophagy and Modulate Age-Associated Changes in Cardiac Progenitor Cell Fate

Circulation Research ◽

10.1161/res.123.suppl_1.203 ◽

2018 ◽

Vol 123 (Suppl_1) ◽

Author(s):

Nirmala Hariharan ◽

Collin Matsumoto ◽

Yan Jiang ◽

Mark Sussman

Keyword(s):

Cell Fate ◽

Progenitor Cell ◽

Cardiac Progenitor Cell

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Oral intake of lipopolysaccharide regulates toll-like receptor 4-dependent granulopoiesis

Experimental Biology and Medicine ◽

10.1177/1535370220931043 ◽

2020 ◽

Vol 245 (14) ◽

pp. 1254-1259

Author(s):

Melanie Märklin ◽

Stefanie Bugl ◽

Stefan Wirths ◽

Julia-Stefanie Frick ◽

Martin R Müller ◽

...

Keyword(s):

Steady State ◽

Cell Fate ◽

Progenitor Cell ◽

Oral Intake ◽

Innate Immune ◽

Toll Like Receptor ◽

Gastrointestinal Microbiome ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

The Impact

While neutrophil production in emergency states has been extensively studied, regulation of neutrophil homeostasis in the steady-state remained incompletely understood. We have shown that innate immune receptor toll-like receptor (TLR)4 and downstream TIR-domain-containing adapter-inducing interferon-β (TRIF) are indispensable for the generation of a granulocyte-colony stimulating factor (G-CSF)-dependent regulatory feedback loop upon antibody-induced neutropenia. These findings demonstrated that steady-state granulopoiesis is a demand-driven process, which may rely on differential triggering of innate immune receptors by microbial cell wall constituents such as lipopolysaccharide. Herein, we present further evidence on underlying mechanisms: oral intake of highly endotoxic lipopolysaccharide, but not TLR-antagonistic lipopolysaccharide derived from Rhodobacter sphaeroides, induces hematopoietic stem and progenitor cell fate decisions toward the neutrophil lineage independent of G-CSF. TLR4 has been identified as the indispensable sensor for oral lipopolysaccharide-modulated steady-state granulopoiesis. These results have important implications: food lipopolysaccharide content or the composition of the gastrointestinal microbiome may be strongly underrated as determinants of peripheral blood neutrophil levels. Both neutrophilia and neutropenia are associated with drastically worse outcomes in epidemiological studies of the general population as well as in diseased states. Impact statement In our present study, we investigated the impact of LPS on neutrophil homeostasis and found that oral intake is sufficient to induce hematopoietic stem and progenitor cell fate decisions towards the neutrophil lineage independent of G-CSF. In addition, TLR4 has been identified as the indispensable sensor for oral LPS-modulated steady-state granulopoiesis. We provide evidence that the gastrointestinal microbiome is critical for neutrophil homeostasis, which has implications for patients being treated with chemotherapy or antimicrobial therapy, since both are significantly influencing the composition of the intestinal microbiome.

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