Abstract 82: Wnt11 Regulates Chamber Specific Neonatal Cardiomyocyte Proliferation During Perinatal Circulatory Transition

Background: Fetal to neonatal transition of heart involves major changes in cardiomyocytes (CMC) including proliferative capacity. However, the chamber specific CMC proliferation programs of remain poorly understood. Elucidating the mechanisms involved is critical to develop chamber specific therapies for newborn infants with single ventricle physiology and other congenital heart defects (CHDs). Methods: Transcriptomes of mouse left ventricle (LV) and right ventricle (RV) were analyzed by RNA-seq at postnatal days 0 (P0), P3 and P7. R package and Ingenuity suite were used for weighted gene co-expression network analysis (WGCNA) and gene ontology studies. Mechanistic analysis was conducted using gain and loss of function approaches. Results: Mouse neonatal cardiac transcriptome was mostly affected by developmental stage. WGCNA revealed 5 LV and 8 RV modules that were significantly correlated with maturation stage and highly preserved between both ventricles at P0 and P7. In contrast, P3 specific gene modules exhibited the largest chamber specific variations in cell signaling, involving proliferation in LV and Wnt signaling molecules, including Wnt11, in RV. Importantly, Wnt11 expression significantly decreased in cyanotic CHDs phenotypes and correlated with O2 saturation levels in hypoxemic infants with Tetralogy of Fallot (TOF). Notably, Perinatal hypoxia treatment in mice suppressed Wnt11 expression, induced CMC proliferation, downregulated Rb1 expression and enhanced Rb1 phosphorylation more robustly in RV vs. LV. Remarkably, Wnt11 inactivation was sufficient to induce myocyte proliferation in perinatal mouse heart and reduced Rb1 expression and phosphorylation in primary neonatal CMC. Importantly, downregulated Wnt11 in hypoxemic TOF infantile heart was also associated with Rb1 suppression and inversely correlated with proliferation marker Plk1 in human. Conclusion: Using integrated systems genomic and functional biology analyses of perinatal cardiac transcriptome, we revealed a previously uncharacterized function for Wnt11 in chamber specific growth and cyanotic CHD. Reduction of Wnt11 expression by hypoxia plays a critical role in neonatal CMC proliferation via modulating Rb1 expression and activity.

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Abstract 206: Stem Cell-derived Exosomes Induce Cardiac Repair via mRNA Modification

Circulation Research ◽

10.1161/res.119.suppl_1.206 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Prabhu Mathiyalagan ◽

Yaxuan Liang ◽

Adriano S Martins ◽

Douglas W Losordo ◽

Roger J Hajjar ◽

...

Keyword(s):

Stem Cell ◽

Myocardial Ischemia ◽

Target Genes ◽

Critical Role ◽

Cell Communication ◽

Target Cells ◽

Mouse Heart ◽

Loss Of Function

Exosomes are cell-derived nanovesicles that carry and shuttle microRNAs (miRNAs) to mediate cell-cell communication. Vast majority of cell types including cardiac myocytes and progenitors actively secrete exosomes, whose miRNA contents are altered after physiological or pathological changes such as myocardial ischemia (MI). In this new study, we have discovered that chemical modification to mRNAs is a novel regulator of ischemia-induced gene expression changes in the heart. We hypothesized that the benefits of human CD34 + stem cell-derived exosomes (CD34exo) are mediated by mRNA modifications in the target cells via miRNA delivery. MiRNA profiling and bioinformatic analysis identified that CD34exo is selectively enriched with a number of miRNAs that directly target genes implicated in regulation of mRNA modifications. Interestingly, under myocardial ischemia, there was a significant increase in mRNA modifications in the mouse heart, which was decreased by about 70% with CD34exo-treatment. In line with the in vivo MI data, in vitro hypoxic stimulation in neonatal / adult rodent myocytes and non-myocytes increased mRNA modifications and controls known regulators of those mRNA modifications. Loss-of-function studies for regulators of mRNA modifications attenuated hypoxia-induced changes to epitranscriptome indicating important roles for these molecules under stress conditions. Finally, using gain-of-function and loss-of-function studies, we demonstrate that miR-126, one of the most enriched miRNAs in CD34exo, plays a critical role in regulating the mRNA modifications. We conclude that miRNAs enriched in CD34exo mediate their cardioprotective effect at least in part, by regulating the mRNA epitranscriptome of the target cell. Our new data suggests hypoxia as a novel regulator of the mRNA epitranscriptome and provides novel insights to post-transcriptional gene regulation in the heart.

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Abstract 372: Misoprostol Attenuates Hypoxia-induced Cardiomyocyte Proliferation Through Bnip3 and Perinuclear Calcium Signaling

Circulation Research ◽

10.1161/res.127.suppl_1.372 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Matthew D Martens ◽

Jared T Field ◽

Nivedita Seshadri ◽

Chelsea Day ◽

Christine Doucette ◽

...

Keyword(s):

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Critical Role ◽

Glycolytic Flux ◽

Cardiomyocyte Proliferation ◽

Neonatal Cardiomyocytes ◽

Rat Pups ◽

Neonatal Heart ◽

Maturation Factor ◽

Myocyte Proliferation

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3 Δ Exon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNC’s, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, promoting nuclear calcium accumulation leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited a hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, without fibrosis which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

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Yap is required for scar formation but not myocyte proliferation during heart regeneration in zebrafish

Cardiovascular Research ◽

10.1093/cvr/cvy243 ◽

2018 ◽

Vol 115 (3) ◽

pp. 570-577 ◽

Cited By ~ 9

Author(s):

Michael A Flinn ◽

Brooke E Jeffery ◽

Caitlin C O’Meara ◽

Brian A Link

Keyword(s):

Heart Development ◽

Critical Role ◽

Scar Formation ◽

Cardiac Cells ◽

Mouse Heart ◽

Heart Regeneration ◽

Post Injury ◽

Myocyte Proliferation ◽

The Impact ◽

Zebrafish Heart

Abstract Aims The Hippo signalling pathway regulates multiple cellular processes during organ development and maintenance by modulating activity of the transcriptional cofactor Yap. Core components of this pathway are required for neonatal mouse heart regeneration, however, investigations to date have typically focused on expression and activity in cardiomyocytes. Due to the regenerative capacity of zebrafish and the fact that global loss of Yap is not fully embryonic lethal in zebrafish, we leveraged a yap null mutant to investigate the impact of constitutive Yap deletion during zebrafish heart regeneration. Methods and results Following cryoinjury in adult hearts, myocyte proliferation was not decreased in yap mutants, contrary to expectations based on mouse data. Experiments in larval zebrafish (Danio rerio) revealed that deletion of either Yap or Taz had a modest effect on heart growth, reducing gross organ size, while their combined deletion was synergistic; thus, Yap and Taz share some overlapping roles in zebrafish heart development. Surprisingly, adult yap mutants exhibited decreased collagen composition at 7 days post-injury, suggesting a critical role for Yap in scar formation during heart regeneration. siRNA-mediated Yap knockdown in primary rat (Rattus norvegicus) cardiac cells revealed a fibroblast-specific role for Yap in controlling the expression of cytoskeletal and myofibroblast activation genes, as well as pro-inflammatory cyto/chemokines. Corroborating these RNAseq data, we observed increased macrophage infiltration in the scars of yap mutants at 7 days post-injury. Conclusion These results suggest that Yap deletion has minimal effect on myocyte proliferation in adults, but significantly influences scar formation and immune cell infiltration during zebrafish heart regeneration. Collectively, these data suggest an unexpected role for Yap in matrix formation and macrophage recruitment during heart regeneration.

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Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽

10.1182/blood.v106.11.1737.1737 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1737-1737

Author(s):

Liyan Pang ◽

Xun Wang ◽

Yuhuan Wang ◽

Gerd Blobel ◽

Mortimer Poncz

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Critical Role ◽

Regulatory Region ◽

Maturation Stage ◽

Specific Gene ◽

Mrna Levels ◽

Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

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Amelotin—a Novel Secreted, Ameloblast-specific Protein

Journal of Dental Research ◽

10.1177/154405910508401207 ◽

2005 ◽

Vol 84 (12) ◽

pp. 1127-1132 ◽

Cited By ~ 124

Author(s):

K. Iwasaki ◽

E. Bajenova ◽

E. Somogyi-Ganss ◽

M. Miller ◽

V. Nguyen ◽

...

Keyword(s):

Alveolar Bone ◽

Bone Cells ◽

Critical Role ◽

Dental Enamel ◽

Specific Protein ◽

Pcr Analysis ◽

Maturation Stage ◽

Specific Gene ◽

Tooth Formation ◽

Dental Tissues

We aimed to analyze the differential gene expression in various murine dental tissues, expecting to find novel factors that are involved in tooth formation. We here describe the identification of a novel ameloblast-specific gene, amelotin (AMTN), by differential display polymerase chain-reaction (DD-PCR) analysis of microdissected ameloblasts, odontoblasts, dental pulp, and alveolar bone cells of 10-day-old mouse incisors. The conceptually translated protein sequence was unique and showed significant homology only with its human orthologue. The amelotin genes from mouse and human displayed a similar exon-intron structure and were expressed from loci on chromosomes 5 and 4, respectively, which have been associated with various forms of amelogenesis imperfecta. Expression of amelotin mRNA was restricted to maturation-stage ameloblasts in developing murine molars and incisors. Amelotin protein was efficiently secreted from transfected cells in culture. Taken together, our findings suggest that amelotin is a novel factor produced by ameloblasts that plays a critical role in the formation of dental enamel.

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Primate-specific ZNF808 is essential for pancreatic development in humans

10.1101/2021.08.23.21262262 ◽

2021 ◽

Author(s):

Elisa De Franco ◽

Nick DL Owens ◽

Hossam Montaser ◽

Matthew N Wakeling ◽

Jonna Saarimaki-Vire ◽

...

Keyword(s):

Transposable Elements ◽

Cell Fate ◽

Zinc Finger Protein ◽

Critical Role ◽

Specific Gene ◽

Loss Of Function ◽

Pancreatic Development ◽

Extreme Phenotypes ◽

Pancreatic Agenesis

Identifying genes linked to extreme phenotypes in humans has the potential to highlight new biological processes fundamental for human development. Here we report the identification of homozygous loss of function variants in the primate-specific gene ZNF808 as a cause of pancreatic agenesis. ZNF808 is a member of the KRAB zinc finger protein (KZFPs) family, a large and rapidly evolving group of epigenetic silencers that target transposable elements. Loss of ZNF808 in vitro results in aberrant activation of many transposable elements it normally represses during early pancreas development. We show that this results in inappropriate specification of cell fate with induction of genes associated with liver endoderm and a loss of pancreatic identity. This suggests that ZNF808 and its transposable element targets play a critical role in cell fate specification during human pancreatic development. This is the first report of loss of a primate-specific gene causing a congenital developmental disease and highlights the essential role of ZNF808 for pancreatic development in humans.

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Histone Lysine Methyltransferase SETD2 Regulates Coronary Vascular Development in Embryonic Mouse Hearts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.651655 ◽

2021 ◽

Vol 9 ◽

Author(s):

Fengling Chen ◽

Jiewen Chen ◽

Hong Wang ◽

Huayuan Tang ◽

Lei Huang ◽

...

Keyword(s):

Heart Development ◽

Heart Defects ◽

Critical Role ◽

Gene Mutations ◽

Mouse Heart ◽

Primary Role ◽

Structural Variations ◽

Embryonic Mouse ◽

Cardiac Progenitors ◽

Histone Lysine

Congenital heart defects are the most common birth defect and have a clear genetic component, yet genomic structural variations or gene mutations account for only a third of the cases. Epigenomic dynamics during human heart organogenesis thus may play a critical role in regulating heart development. However, it is unclear how histone mark H3K36me3 acts on heart development. Here we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse heart epigenome. Setd2 is highly expressed in embryonic stages and accounts for a predominate role of H3K36me3 in the heart. Loss of Setd2 in cardiac progenitors results in obvious coronary vascular defects and ventricular non-compaction, leading to fetus lethality in mid-gestation, without affecting peripheral blood vessel, yolk sac, and placenta formation. Furthermore, deletion of Setd2 dramatically decreased H3K36me3 level and impacted the transcriptional landscape of key cardiac-related genes, including Rspo3 and Flrt2. Taken together, our results strongly suggest that SETD2 plays a primary role in H3K36me3 and is critical for coronary vascular formation and heart development in mice.

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Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

10.1101/681692 ◽

2019 ◽

Author(s):

Matthew D. Martens ◽

Jared T. Field ◽

Nivedita Seshadri ◽

Chelsea Day ◽

Donald Chapman ◽

...

Keyword(s):

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Critical Role ◽

Glycolytic Flux ◽

Cardiomyocyte Proliferation ◽

Neonatal Cardiomyocytes ◽

Rat Pups ◽

Neonatal Heart ◽

Maturation Factor ◽

Myocyte Proliferation

AbstractSystemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3ΔExon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNCs) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNCs, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, accumulation of nuclear calcium leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.

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Parkin interacting substrate zinc finger protein 746 is a pathological mediator in Parkinson’s disease

Brain ◽

10.1093/brain/awz172 ◽

2019 ◽

Vol 142 (8) ◽

pp. 2380-2401 ◽

Cited By ~ 9

Author(s):

Saurav Brahmachari ◽

Saebom Lee ◽

Sangjune Kim ◽

Changqing Yuan ◽

Senthilkumar S Karuppagounder ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Critical Role ◽

Trna Synthetase ◽

Loss Of Function ◽

Substrate Protein ◽

Finger Protein ◽

Sporadic Parkinson’S Disease

Abstract α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson’s disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson’s disease, there is evidence that parkin is inactivated in sporadic Parkinson’s disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson’s disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson’s disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson’s disease and related α-synucleinopathies.

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