Mechanical Unloading is Associated with Decreased DNA Content in Cardiomyocytes Independent of Nucleation State

ABSTRACTBackgroundCardiomyocytes increase DNA content in response to stress in humans. Proliferation has been reported in cardiomyocytes in failing hearts and following LVAD unloading which may represent a resolution of this process through cell division. However, cardiac recovery from LVAD is rare.MethodsWe quantified cardiomyocyte nuclear number, cell size, DNA content and the frequency of cell cycling markers by imaging flow cytometry from human subjects undergoing LVAD implantation or primary transplantation.ResultsCardiomyocyte size was 15 percent smaller in unloaded versus loaded samples without differences in the percentage of mono-, bi, or multi-nuclear cells. DNA content per nucleus was significantly decreased in unloaded hearts versus loaded controls. Cell cycle markers, Ki67 and phosphohistone H3 (H3P) were not increased in unloaded versus failing samples.ConclusionsUnloading of failing hearts is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling and not proliferation.

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Abstract 218: Nuclear Remodeling Following Mechanical Circulatory Support

Circulation Research ◽

10.1161/res.121.suppl_1.218 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Jun Luo ◽

Stephen Farris ◽

Deri Helterline ◽

April Stempien-Otero

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Dna Content ◽

Left Ventricular ◽

Ventricular Assist Devices ◽

Circulatory Support ◽

Ventricular Assist ◽

Imaging Flow Cytometry ◽

Nuclear Number ◽

Nuclear Remodeling

Rationale: Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates the analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox. Objective: We used a novel technique-imaging flow cytometry-to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart. Methods and Results: Cardiomyocytes were isolated from 8 subjects undergoing primary heart transplantation and 15 subjects following unloading with left ventricular assist device (LVAD, mean unloading time 13.7 ± 9.1 months). Myocyte size, nuclear number and size, DNA content (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Myocyte size and nuclear morphology was not significantly different between the groups. However, DNA content per nucleus was significantly decreased (P < 0.01) and the correlation between nuclear size and DNA content lost. The frequency of the cell cycle markers, Ki67 and phospho-histone3 (H3P) were not increased after hemodynamic unloading. Conclusions: Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.

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Abstract 401: Nuclear Remodeling After Mechanical Circulatory Support

Circulation Research ◽

10.1161/res.121.suppl_1.401 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Jun Luo ◽

Stephen Farris ◽

Deri Helterline ◽

April Stempien-Otero

Keyword(s):

Cell Cycle ◽

Dna Content ◽

Normal Growth ◽

Left Ventricular ◽

Ventricular Assist Devices ◽

Circulatory Support ◽

Cardiomyocyte Proliferation ◽

Ventricular Assist ◽

Nuclear Number ◽

Nuclear Remodeling

Rationale: Cardiomyocytes increase DNA content in normal growth and in response to stress in humans by both increases in nuclear number and ploidy. This observation complicates analysis of human cardiomyocyte proliferation as DNA content can increase in the absence of cytokinesis. Proliferation has been reported in cardiomyocytes following LVAD unloading which may represent a reversal of this process. However, cardiac recovery from LVAD is rare. Thus, we sought to analyze changes in cardiomyocyte nuclear characteristics for clues to this paradox. Objective: We used a novel technique to determine changes in nuclear content to test the hypothesis that adult cardiomyocytes can complete cell cycle progression by mitosis after long-term hemodynamic unloading of the failing heart. Methods and Results: The makeup of myocyte nuclear number, ploidy (per cell and per nucleus) and the frequency of cell cycling markers were evaluated by imaging flow cytometry. Hypertrophic hearts from 15 subjects with left ventricular assist device (LVAD) were compared with 8 non-LVAD unloaded hearts. After hemodynamic unloading for 13.7 ± 9.1 months, myocyte nuclear makeup, specifically the average sizes of both cell and individual nuclei, did not significantly change. DNA content per nucleus was significantly decreased (P < 0.01). The frequency of cell cycle markers, i.e. Ki67 and phospho-histon3 (H3P) were not increased after hemodynamic unloading. Conclusions: Our data demonstrate that unloading of failing hearts with mechanical ventricular assist devices does not alter nucleation state of cardiomyocytes. However, unloading is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling.

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Relationship among Several Key Cell Cycle Events in the Developmental Cyanobacterium Anabaena sp. Strain PCC 7120

Journal of Bacteriology ◽

10.1128/jb.00524-06 ◽

2006 ◽

Vol 188 (16) ◽

pp. 5958-5965 ◽

Cited By ~ 23

Author(s):

Samer Sakr ◽

Melilotus Thyssen ◽

Michel Denis ◽

Cheng-Cai Zhang

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Division ◽

Cell Growth ◽

Cell Size ◽

Dna Content ◽

Small Cells ◽

Filamentous Cyanobacterium ◽

Pcc 7120 ◽

Heterocyst Development

ABSTRACT When grown in the absence of a source of combined nitrogen, the filamentous cyanobacterium Anabaena sp. strain PCC 7120 develops, within 24 h, a differentiated cell type called a heterocyst that is specifically involved in the fixation of N2. Cell division is required for heterocyst development, suggesting that the cell cycle could control this developmental process. In this study, we investigated several key events of the cell cycle, such as cell growth, DNA synthesis, and cell division, and explored their relationships to heterocyst development. The results of analyses by flow cytometry indicated that the DNA content increased as the cell size expanded during cell growth. The DNA content of heterocysts corresponded to the subpopulation of vegetative cells that had a big cell size, presumably those at the late stages of cell growth. Consistent with these results, most proheterocysts exhibited two nucleoids, which were resolved into a single nucleoid in most mature heterocysts. The ring structure of FtsZ, a protein required for the initiation of bacterial cell division, was present predominantly in big cells and rarely in small cells. When cell division was inhibited and consequently cells became elongated, little change in DNA content was found by measurement using flow cytometry, suggesting that inhibition of cell division may block further synthesis of DNA. The overexpression of minC, which encodes an inhibitor of FtsZ polymerization, led to the inhibition of cell division, but cells expanded in spherical form to become giant cells; structures with several cells attached together in the form of a cloverleaf could be seen frequently. These results may indicate that the relative amounts of FtsZ and MinC affect not only cell division but also the placement of the cell division planes and the cell morphology. MinC overexpression blocked heterocyst differentiation, consistent with the requirement of cell division in the control of heterocyst development.

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Downward regulation of cell size in Paramecium tetraurelia: effects of increased cell size, with or without increased DNA content, on the cell cycle

Journal of Cell Science ◽

10.1242/jcs.57.1.315 ◽

1982 ◽

Vol 57 (1) ◽

pp. 315-329

Author(s):

C.D. Rasmussen ◽

J.D. Berger

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Dna Synthesis ◽

Cell Size ◽

Dna Content ◽

Cell Mass ◽

Cycle Duration ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Rates Of Growth

Two temperature-sensitive cell-cycle mutants were used to generate abnormally large cells (size estimated by protein content) with either normal or increased DNA contents. The first mutant, cc1, blocks DNA synthesis, but allows cell growth at the restrictive temperature. The cells do not progress through the cell cycle while at the restrictive temperature, but do recover and complete the cell cycle when returned to permissive conditions. The progeny have increased cell size and normal DNA content. Downward regulation of cell size occurs during the ensuing cell cycle at permissive temperature. Two processes are involved. First, the G1 period is reduced or eliminated. As initial cell size increases there is a progressive shortening of the cell cycle to 75% of normal. This limit cell-cycle duration is reached when the initial mass of the cell is equal to or greater than that of normal cells at the time of DNA synthesis initiation (0.25 of a cell cycle). Cells with the limit cell cycle begin macronuclear DNA synthesis immediately after fission. The durations of the S period and fission are normal. Second, the rate of cell growth is unaffected by the increase in cell size, and results in the partitioning of excess cell mass between the daughter cells at the next fission. The second mutant, cc2, blocks cell division, but allows DNA synthesis to occur at a reduced rate so that cells with up to about 140% of the normal initial DNA content and twice the normal cell mass can be produced. The pattern of cell-cycle shortening is the same as in ccl. The rates of growth and both the rate and amount of DNA synthesis are proportional to the initial DNA content. This suggests that the rates of growth and DNA synthesis are limited by the transcriptional activity of the macronucleus in both cc1 and cc2 cells when they begin the cell cycle with experimentally increased cell mass. Increases in both cell size and initial DNA content are required to bring about increases in the rates of growth and DNA accumulation.

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Developmental variability within and between mouse expanding blastocysts and their ICMs

Development ◽

10.1242/dev.86.1.311 ◽

1985 ◽

Vol 86 (1) ◽

pp. 311-336

Author(s):

Julia C. Chisholm ◽

Martin H. Johnson ◽

Paul D. Warren ◽

Tom P. Fleming ◽

Susan J. Pickering

Keyword(s):

Cell Cycle ◽

Cell Size ◽

Dna Content ◽

Nuclear Dna ◽

Nuclear Dna Content ◽

Cell Number ◽

Developmental Potential ◽

Present Evidence ◽

Cell Cycling

We have attempted to reduce the developmental heterogeneity amongst populations of mouse blastocysts by synchronizing embryos to the first visible signs of blastocoel formation. Using embryos timed in this way, we have examined the extent of variation of inside and outside cell number and of inside cell size, nuclear DNA content and developmental potential, between and within embryos of a similar age postcavitation. The overall impression gained is one of wide heterogeneity in inside:outside cell number ratios and in cell cycling and its relation to cavitation among embryos of similar age postcavitation. However, the simplest explanation of our results suggests that cavitation generally begins at a time when most outside cells are in their sixth developmental cell cycle and that outside cells, as a population, are a little ahead of inside cells in their cell cycling. Additionally we present evidence that, within at least some individual inner cell masses (ICM), there is intraembryo variation in the time at which inside cell developmental potential becomes restricted.

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Effects of 5′-3′ Exonuclease Xrn1 on Cell Size, Proliferation and Division, and mRNA Levels of Periodic Genes in Cryptococcus neoformans

Genes ◽

10.3390/genes11040430 ◽

2020 ◽

Vol 11 (4) ◽

pp. 430

Author(s):

Xueru Zhao ◽

Xin Li ◽

Ping Zhang ◽

Chenxi Li ◽

Weijia Feng ◽

...

Keyword(s):

Cell Cycle ◽

Cryptococcus Neoformans ◽

Cell Size ◽

Dna Content ◽

Cell Cycle Progression ◽

Polymerase Activity ◽

Yeast Cells ◽

Mrna Levels ◽

Cancer Disease ◽

Cycle Progression

Cell size affects almost all biosynthetic processes by controlling the size of organelles and disrupting the nutrient uptake process. Yeast cells must reach a critical size to be able to enter a new cell cycle stage. Abnormal changes in cell size are often observed under pathological conditions such as cancer disease. Thus, cell size must be strictly controlled during cell cycle progression. Here, we reported that the highly conserved 5′-3′ exonuclease Xrn1 could regulate the gene expression involved in the cell cycle pathway of Cryptococcus neoformans. Chromosomal deletion of XRN1 caused an increase in cell size, defects in cell growth and altered DNA content at 37 °C. RNA-sequencing results showed that the difference was significantly enriched in genes involved in membrane components, DNA metabolism, integration and recombination, DNA polymerase activity, meiotic cell cycle, nuclear division, organelle fission, microtubule-based process and reproduction. In addition, the proportion of the differentially expressed periodic genes was up to 19.8% when XRN1 was deleted, including cell cycle-related genes, chitin synthase genes and transcription factors, indicating the important role of Xrn1 in the control of cell cycle. This work provides insights into the roles of RNA decay factor Xrn1 in maintaining appropriate cell size, DNA content and cell cycle progression.

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Development of matured hiPSCs-derived 3D cardiac tissue using ERR gamma agonist and mechanical stress and application for Hypertrophic Cardiomyopathy (HCM) model

European Heart Journal ◽

10.1093/ehjci/ehaa946.3668 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

Y Fujiwara ◽

K Deguchi ◽

Y Naka ◽

M Sasaki ◽

T Nishimoto ◽

...

Keyword(s):

Mitochondrial Dna ◽

Hypertrophic Cardiomyopathy ◽

Mechanical Stress ◽

Cell Size ◽

Dna Content ◽

Disease Model ◽

Heart Tissue ◽

Funding Source ◽

Mitochondrial Dna Content ◽

Mechanical Stretching

Abstract Introduction Tissue engineering using human induced pluripotent stem cells-derived cardiomyocytes (hiPSCs-CMs) is one of the potential tools to replicate human heart in vitro. Although there are many publications on 3 dimensional (3D) heart tissues (1), these tissues show fetal like phenotypes. For that reason, several maturation methods such as electrical stimulation and mechanical stress have been investigated (2, 3). However, these methods have been inadequate in differentiating fetal like phenotype tissue from adult tissues. Previously, we identified a novel compound, T112, which induced hiPSCs-CMs maturation from approximately 9,000 compounds using Troponin I1-EmGFP and Troponin I3-mCherry double reporter hiPSCs-CMs. This compound enhanced morphological and metabolic maturation of hiPSCs-CMs via estrogen-rerated receptor gamma activation Purpose We hypothesized that our novel compound, T112, in combination with mechanical stress could result in further maturation of 3D heart tissue. Therefore, our specific aim is to develop a novel maturation method applicable to genetic disease model of HCM using 3D heart tissue combined with T112. Methods We constructed 3D heart tissue mixed with fibroblast and double reporter hiPSCs-CMs by the hydrogel methods using Flex cell system®. We added T112 with or without mechanical stretching to 3D tissue from 7 to 15 days after 3D heart tissue was constructed. Then we measured maturation related phenotype such as sarcomere gene expression, mitochondrial DNA content and cell size. Results Similar to hiPSCs-CM, the addition of T112 to the constructed 3D heart tissue significantly increased TNNI3 mRNA compared to that of DMSO. Furthermore, T112 treated 3D heart tissue showed increased cell size and oblong shape. Next, in order to promote more maturation of 3D heart tissue, we performed mechanical stretching with the addition of T112. The combination of T112 with mechanical stretching showed higher expression of mCherry, a reporter protein for TNNI3 expression, and higher isotropy of sarcomere alignment in 3D heart tissue than that with the static condition. Furthermore, 3D heart tissue in the treatment of T112 with or without mechanical stretching showed higher mitochondrial DNA content compared to the respective DMSO controls. Interestingly, we applied this combination method to hiPSCs carrying MYH7 R719Q mutation which is known to cause hypertrophic cardiomyopathy, and the 3D heart tissue composed of cardiomyocytes derived from mutant iPSCs demonstrated increased sarcomere disarray compared to isogenic wild-type 3D heart tissue. Conclusion These results suggest that the combination of T112 and mechanical stretching promotes metabolic and structural maturation of 3D heart tissue and would be useful for creating a HCM disease model. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): T-CiRA project, Takeda Pharmaceutical Company Limited

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