Abstract 132: The First Mouse Mutants of a Novel Epigenetic Modifier, Rearranged L-Myc Fusion, Display Defects in Heart Development

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Lauren M Bourke ◽  
Sarah Harten ◽  
Vandhana Bharti ◽  
Harry Oey ◽  
Nadia Whitelaw ◽  
...  
Keyword(s):  
Heart Development ◽  
Critical Role ◽  
Interventricular Septum ◽  
Notch Pathway ◽  
Regulatory Elements ◽  
Left Ventricular ◽  
Wild Type ◽  
Ventricular Noncompaction ◽  
Mouse Mutants ◽  
Epigenetic Modifier

Rearranged L-Myc Fusion, Rlf, was recently identified as a novel epigenetic modifier from a mouse N-ethyl-N-nitrosourea mutagenesis screen. The mice used in this study carry a multi-copy green fluorescent protein (GFP) transgene linked to an erythroid specific α-globin promoter that is sensitive to epigenetic silencing. Three independent mouse lines with mutations in Rlf were each found to have a decrease in GFP expression, suggesting Rlf acts an epigenetic modifier. Our study is the first to reveal a role for Rlf in epigenetics. Preliminary phenotyping has found loss of Rlf results in perinatal lethality. Late gestation homozygous null Rlf mutants were found to weigh significantly less than their heterozygous or wild type littermates. Histological analysis of mid gestation embryos has identified a potential heart defect in homozygous mutants. Rlf mutants display a thin compact layer, an overabundance of trabeculae and a fenestrated interventricular septum. The Rlf mutant phenotype is reminiscent of ventricular noncompaction defects observed in humans, such as left ventricular noncompaction cardiomyopathy. RNA-seq analysis of mid gestation Rlf wild type and null hearts, prior to the observation of a cardiac defect, was undertaken to determine which pathways may be regulated by Rlf in the heart. More genes were observed to be significantly down-regulated in Rlf mutant hearts compared to wild types. Pathway analysis of differentially expressed genes showed genes involved in cell-cell adhesion, cell signalling, glycosylation and the Notch pathway were dysregulated. These findings indicate that Rlf may play a critical role in cardiac development. Whole genome bisulphite sequencing of different embryonic tissue/stages has shown loss of Rlf results in an increase in methylation at a large number of distinct loci across the genome. Many of which were found to overlap sites reported to be putative regulatory elements, and histone marks associated with active or poised regulatory elements in the heart. Our data suggest Rlf plays a key role in regulating gene expression pathways during heart development. These are the first mouse mutants available to study how Rlf functions as an epigenetic modifier and the phenotypic consequences of Rlf inactivation.

scholarly journals Neddylation mediates ventricular chamber maturation through repression of Hippo signaling

2018 ◽  
Vol 115 (17) ◽  
pp. E4101-E4110 ◽  
Author(s):  
Jianqiu Zou ◽  
Wenxia Ma ◽  
Jie Li ◽  
Rodney Littlejohn ◽  
Hongyi Zhou ◽  
...  
Keyword(s):  
Heart Failure ◽  
Heart Development ◽  
Molecular Mechanisms ◽  
Heart Defects ◽  
Left Ventricular ◽  
Late Gestation ◽  
Hippo Signaling ◽  
Cardiomyocyte Proliferation ◽  
Ventricular Noncompaction ◽  
Cullin 7

During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling.

Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy

2013 ◽  
Vol 19 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Guillermo Luxán ◽  
Jesús C Casanova ◽  
Beatriz Martínez-Poveda ◽  
Belén Prados ◽  
Gaetano D'Amato ◽  
...  
Keyword(s):  
Notch Pathway ◽  
Left Ventricular ◽  
Left Ventricular Noncompaction ◽  
Ventricular Noncompaction ◽  
Noncompaction Cardiomyopathy ◽  
Left Ventricular Noncompaction Cardiomyopathy

Abstract 312: Selective Inhibition of Myocardial NADPH Oxidase Significantly Decreases Heart Size in Transgenic (Tg) Mice with Cardiac-Specific Overexpression of a Dominant-Negative (DN) Mutant of p67 phox

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Qiong Zhao ◽  
Magdalena Ornatowska ◽  
Dalia Urboniene ◽  
Kristopher McGee ◽  
R J Solaro ◽  
...  
Keyword(s):  
Mouse Model ◽  
Nadph Oxidase ◽  
Heart Development ◽  
Critical Role ◽  
Left Ventricular ◽  
Heart Weight ◽  
Enzymatic Function ◽  
2D Echocardiography ◽  
Myocardial Area

The role of NADPH oxidase (NOX) was recently suggested in cardiac diseases including hypertrophy. Overexpression of a DN mutant of p67 phox , a cytosolic subunit of NOX, DN-p67 with a V204A point mutation completely inhibited NOX enzymatic function of superoxide generation. We found that overexpression of DN-p67 in cultured adult rat cardiac myocytes attenuated α 1 -adrenoceptor-induced hypertrophy , we thus hypothesized that NOX/p67 phox plays a critical role in cardiac hypertrophy and remodeling in vivo . Accordingly, we recently generated a Tg mouse model in C57BL/6 strain with cardiac-specific overexpression of this DN-p67 mutant along with an IRES-initiated EGFP tag protein using the cardiac-specific α-MHC promoter. Hearts from 1.5-, 3- and 6-month (M) old heterozygous Tg mice and their Non-Tg (NTg) littermates were harvested and compared. The heart weight/body weight (HW/BW) ratio in Tg group was significantly smaller (overall: 14%↓; 1.5-M: 10%↓; 3-M: 16%↓; 6-M: 12%↓) vs. the NTg group ( p < 0.01, n=23). This result was confirmed by 2D echocardiography (2D-echo) measuring the left ventricular myocardial area (LVMA) determined by subtracting endocardial area from epicardial area in short axis view at the end diastole. The LVMA (mm 2 ) in 3-M and 6-M mice was significantly ( p < 0.05, n=21) smaller in Tg (0.44±0.02) than the NTg (0.50±0.02) with equal average BW in both groups. LV internal dimensions and systolic function were preserved without difference in Tg vs. NTg. The LV wall thickness is smaller in Tg vs. NTg by both 2D-echo and hematoxylin/eosin staining of LV cross-sections, but no change in fibrosis was detected in both groups by trichrome staining (n=4). Interestingly, the expression of NOX catalytic subunit gp91 phox in Tg hearts was significantly decreased (~3-fold↓, n=5) vs. NTg by Western blot, whereas the cardiac expression of other NOX subunits p47 phox and p22 phox remained the same in both groups. In summary, we have generated a viable Tg mouse model with cardiac-specific overexpression of a DN p67 phox , which shows significant reduction in both myocardial mass/size and gp91 phox expression with normal cardiac function and development. Our results first show the novel and critical role of NOX/p67 phox in normal cardiac growth and heart development. This research has received full or partial funding support from the American Heart Association, AHA National Center.

scholarly journals Lung Natural Killer Cells Play a Major Counter-Regulatory Role in Pulmonary Vascular Hyperpermeability After Myocardial Infarction

2014 ◽  
Vol 114 (4) ◽  
pp. 637-649 ◽  
Author(s):  
Xiaoxiang Yan ◽  
Ahmed E. Hegab ◽  
Jin Endo ◽  
Atsushi Anzai ◽  
Tomohiro Matsuhashi ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Nk Cells ◽  
Natural Killer ◽  
Knockout Mice ◽  
Ventricular Remodeling ◽  
Nk Cell ◽  
Critical Role ◽  
Cell Monolayer ◽  
Left Ventricular ◽  
Wild Type

Rationale : Natural killer (NK) cells are lymphocytes of the innate immune system that play specialized and niche-specific roles in distinct organs. Objective : We investigated the possible function of NK cells in the pathogenesis of congestive heart failure after myocardial infarction. Methods and Results : Depletion of NK cells from mice had little effect on cytokine expression (tumor necrosis factor-α, interleukin [IL]-6, and IL-1β), neutrophil and macrophage infiltration into infarcted myocardium, or left ventricular remodeling after myocardial infarction. However, these mice exhibited severe respiratory distress associated with protein-rich, high-permeability alveolar edema accompanied by neutrophil infiltration. In addition, there were 20-fold more NK cells in the mouse lungs than in heart, and these cells were accumulated around the vasculature. CD107a-positive and interferon-γ–positive cell populations were unchanged, whereas IL-10–positive populations increased. Adoptive transfer of NK cells from wild-type mice, but not from IL-10 knockout mice, into the NK cell–depleted mice rescued the respiratory phenotype. IL-1β–mediated dextran leakage from a lung endothelial cell monolayer was also blocked by coculture with NK cells from wild-type mice but not from IL-10 knockout mice. Conclusions : This study is the first to identify a critical role for lung NK cells in protecting lung from the development of cardiogenic pulmonary edema after myocardial infarction.

Deficiency in endothelial nitric oxide synthase impairs myocardial angiogenesis

2002 ◽  
Vol 283 (6) ◽  
pp. H2371-H2378 ◽  
Author(s):  
Xue Zhao ◽  
Xiangru Lu ◽  
Qingping Feng
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Heart Development ◽  
Tube Formation ◽  
Left Ventricular ◽  
Left Ventricular Myocardium ◽  
Wild Type ◽  
Endothelial Nitric Oxide

We recently demonstrated that mice deficient in endothelial nitric oxide (NO) synthase (eNOS) have congenital septal defects and postnatal heart failure. However, the mechanisms by which eNOS affects heart development are not clear. We hypothesized that deficiency in eNOS impairs myocardial angiogenesis. Myocardial capillary densities were measured morphometrically in neonatal mouse hearts. In vitro tube formation on Matrigel was investigated in cardiac endothelial cells. In vivo myocardial angiogenesis was performed by implanting Matrigel in the left ventricular myocardium. Myocardial capillary densities and VEGF mRNA expression were decreased in neonatal eNOS−/− compared with neonatal wild-type mice ( P < 0.01). Furthermore, in vitro tube formation from cardiac endothelial cells and in vivo myocardial angiogenesis were attenuated in eNOS−/− compared with wild-type mice ( P < 0.01). In vitro tube formation was inhibited by N G-nitro-l-arginine methyl ester in wild-type mice and restored by a NO donor, diethylenetriamine-NO, in eNOS−/− mice ( P < 0.05). In conclusion, deficiency in eNOS decreases VEGF expression and impairs myocardial angiogenesis and capillary development. Decreased myocardial angiogenesis may contribute to cardiac abnormalities during heart development in eNOS−/− mice.

scholarly journals Ligand endocytosis drives receptor dissociation and activation in the Notch pathway

Development ◽  
2000 ◽  
Vol 127 (7) ◽  
pp. 1373-1385 ◽  
Author(s):  
A.L. Parks ◽  
K.M. Klueg ◽  
J.R. Stout ◽  
M.A. Muskavitch
Keyword(s):  
Cultured Cells ◽  
Critical Role ◽  
Notch Pathway ◽  
Imaginal Discs ◽  
Notch Receptor ◽  
Wild Type ◽  
Subcellular Trafficking ◽  
Notch Intracellular Domain ◽  
Notch Protein ◽  
Wing Imaginal Discs

Endocytosis of the ligand delta; is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into delta;-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signalling. Furthermore, dynamin-mediated delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signalling capacity in Drosophila. We suggest that endocytosis into delta-expressing cells of NotchECD bound to delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein.

Abstract 4595: Intra-arrest Hypothermia Preserves Heart Akt Phosphorylation And Cardiac Function Following Cardiac Arrest In Wild-type But Not Akt(+/−) Mice

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
David G Beiser ◽  
Kimberly R Wojcik ◽  
Danhong Zhao ◽  
Kimm J Hamann ◽  
Terry L Vanden Hoek
Keyword(s):  
Cardiac Arrest ◽  
Critical Role ◽  
Survival Rates ◽  
Left Ventricular Pressure ◽  
Therapeutic Benefit ◽  
Akt Signaling ◽  
Spontaneous Circulation ◽  
Left Ventricular ◽  
Wild Type ◽  
Phosphorylated Akt

OBJECTIVE: To establish the role of Akt signaling in hypothermia protection following cardiac arrest. METHODS: Cardiac arrest was induced in anesthetized C57B6 wild-type (WT) and Akt1(+/−) heterozygous mice with an intravenous bolus of KCl. Animals were randomized to normothermia ( NT , 37°C) or hypothermia ( HT , 30°C) induced after 6 minutes of arrest and continued for 1 hour following return of spontaneous circulation (ROSC). After 8 minutes, resuscitation was attempted with chest compressions, mechanical ventilation, and fluid administration. Following ROSC, resuscitated animals were monitored for up to 4 hours. Total Akt and phosphorylated Akt (pAkt at Ser473 and Thr308) levels were measured by Western blot analysis in whole heart lysates from 4-hour survivors and shams (n=4 each group). RESULTS: In WT animals, 4-hour survival (HT = 7 of 8, NT = 3 of 8, p < 0.04), cardiac output (p<0.001) and left-ventricular dP/dt max (p<0.001) were significantly improved in HT versus NT animals. In addition, hearts from WT animals randomized to HT displayed increased pAkt (Ser473) levels and over 8-fold higher pAkt(Thr308) levels compared to NT and sham groups (p<0.001). These pAkt changes occurred despite total Akt levels remaining unchanged. Baseline hemodynamics prior to arrest were similar between WT and Akt(+/−) animals, while heart Akt1levels were decreased by approximately 40% in Akt(+/−) heterozygous mice. In contrast to WT mice, HT compared to NT had no effect on Akt(+/−) mouse 4-hour survival rates and left-ventricular hemodynamic measures. Compared to HT treated WT mice, Akt(+/−) animals treated with HT displayed significantly lower maximum left-ventricular pressure, stroke volume, and dP/dt max by 4 hours post-ROSC (p<0.05). CONCLUSIONS: HT improves short-term cardiovascular outcomes and induces signficant heart Akt(Thr308) phosphorylation during the immediate post-ROSC period. The therapeutic benefit of intra-arrest HT on post-ROSC hemodynamics is greatly attenuated in Akt(+/−) animals, suggesting that Akt signaling may play a critical role in mediating HT protection.

scholarly journals MyD88 and Trif Signaling Play Distinct Roles in Cardiac Dysfunction and Mortality during Endotoxin Shock and Polymicrobial Sepsis

2011 ◽  
Vol 115 (3) ◽  
pp. 555-567 ◽  
Author(s):  
Yan Feng ◽  
Lin Zou ◽  
Ming Zhang ◽  
Yan Li ◽  
Chan Chen ◽  
...  
Keyword(s):  
Cardiac Dysfunction ◽  
Critical Role ◽  
Endotoxin Shock ◽  
Left Ventricular ◽  
Polymicrobial Sepsis ◽  
Wild Type ◽  
Endotoxin Challenge ◽  
Cardiac Depression ◽  
Cecum Ligation ◽  
Myeloid Differentiation Factor

Background Toll-like receptors (TLRs) such as TLR2, TLR4, and TLR9 contribute to the pathogenesis of polymicrobial sepsis. These TLRs signal via the common myeloid differentiation factor 88 (MyD88)-dependent pathways. TLR4 also signals through MyD88-independent but TIR domain-containing adaptor inducing interferon-β-mediated transcription factor (Trif)-dependent pathway. The role of the two signaling pathways in cardiac dysfunction during polymicrobial sepsis and endotoxin shock is unknown. Methods Sepsis was generated by cecum ligation and puncture. Mice were divided into sham and cecum ligation and puncture groups or subjected to saline or endotoxin. Left ventricular function was assessed in a Langendorff apparatus or by echocardiography. Cytokines were examined using a multiplex immunoassay. Neutrophil migratory and phagocytic functions were assessed using flow cytometry. Results In comparison with wild-type mice, MyD88(-/-) but not Trif(-/-) mice had markedly improved cardiac function and survival after cecum ligation and puncture. In comparison, both MyD88(-/-) and Trif(-/-) mice were protected from cardiac depression and mortality during endotoxin shock. Septic MyD88(-/-) but not Trif(-/-) mice had diminished cytokine production in serum and in peritoneal space in comparison with wild-type mice after cecum ligation and puncture. In contrast, both MyD88(-/-) and Trif(-/-) mice had attenuated serum cytokines in comparison with wild-type mice after endotoxin challenge. Neither MyD88(-/-) nor Trif(-/-) signaling had any effect on neutrophil phagocytic function or bacterial clearance at 24 h of polymicrobial sepsis. Conclusions These studies establish that MyD88 but not Trif signaling plays a critical role in mediating cardiac dysfunction, systemic inflammation, and mortality during polymicrobial sepsis. Both MyD88 and Trif are essential for cardiac depression and mortality during endotoxin shock.

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin

Development ◽  
2001 ◽  
Vol 128 (4) ◽  
pp. 459-469
Author(s):  
Y. Luo ◽  
M. Ferreira-Cornwell ◽  
H. Baldwin ◽  
I. Kostetskii ◽  
J. Lenox ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Heart Development ◽  
Critical Role ◽  
Embryonic Cells ◽  
Wild Type ◽  
Specific Expression ◽  
Developmental Defects ◽  
E Cadherin ◽  
Cell Cell

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.

Abstract 99: IL10-inhibits Fibroblast Progenitor Cell-mediated Cardiac Fibrosis in Pressure-overloaded Myocardium

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Suresh K Verma ◽  
Venkata N Garikipati ◽  
Prasanna Krishnamurthy ◽  
Cindy Benedict ◽  
Emily Nickoloff ◽  
...  
Keyword(s):  
Heart Failure ◽  
Bone Marrow ◽  
Cardiac Fibrosis ◽  
Critical Role ◽  
Pressure Overload ◽  
Interleukin 10 ◽  
Left Ventricular ◽  
Lv Function ◽  
Wild Type ◽  
Mirna Array

Background: Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Recent studies suggest the contribution of bone marrow fibroblasts progenitor cells (BM-FPC) in pressure overload (PO)-induced cardiac fibrosis. Previously we have shown that interleukin-10 suppress PO-induced cardiac fibrosis, however, its role on inhibition of BM-FPC-mediated fibrosis is not known. Thus, we hypothesized that IL-10 inhibits PO-induced homing and transition of BM-FPC to myoFBs and therefore, attenuates cardiac fibrosis. Methods and Results: Cardiac fibrosis was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Systemic recombinant IL-10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO-enhanced FPC (Prominin1 + cells) mobilization and homing in IL-10 KO mice compared to WT mice. Furthermore, bone marrow transplantation (BMT) experiment was performed wherein WT marrow from GFP mice was repopulated in IL-10 KO mice. FPC mobilization was significantly reduced in BMT-IL10 KO mice compared to IL-10 KO mice after TAC. Furthermore, immunofluorescence result in BMT mice showed that subsets of myoFBs are derived from BM after TAC. To identify the molecular mechanism, wild type BM-FPC were treated with TGFβ 2 with or without IL10. IL10 treatment significantly inhibits TGFβ 2 -induced FPC to myoFBs transition. As miRNAs are key players in cardiac fibrosis, next we performed fibrosis-associated miRNA profiling using miRNA array kit. TGFβ 2 -induced miR-208, 155, 21 and 145 expression was markedly inhibited by IL-10. Conclusion: Taken together, our findings suggest that both reduced homing to heart and transition of FPC to myofibroblasts mediate anti-fibrotic effect of IL10 during PO-induced heart failure. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.

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