The Insulin-Like Growth Factor Signalling Pathway in Cardiac Development and Regeneration

Insulin and Insulin-like growth factors (IGFs) perform key roles during embryonic development, regulating processes of cell proliferation and survival. The IGF signalling pathway comprises two IGFs (IGF1, IGF2), two IGF receptors (IGFR1, IGFR2), and six IGF binding proteins (IGFBPs) that regulate IGF transport and availability. The IGF signalling pathway is essential for cardiac development. IGF2 is the primary mitogen inducing ventricular cardiomyocyte proliferation and morphogenesis of the compact myocardial wall. Conditional deletion of the Igf1r and the insulin receptor (Insr) genes in the myocardium results in decreased cardiomyocyte proliferation and ventricular wall hypoplasia. The significance of the IGF signalling pathway during embryonic development has led to consider it as a candidate for adult cardiac repair and regeneration. In fact, paracrine IGF2 plays a key role in the transient regenerative ability of the newborn mouse heart. We aimed to review the current knowledge about the role played by the IGF signalling pathway during cardiac development and also the clinical potential of recapitulating this developmental axis in regeneration of the adult heart.

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Abstract 96: Inactivation of Neddylation Causes Left Ventricular Noncompaction Cardiomyopathy Through Suppressing YAP Signaling

Circulation Research ◽

10.1161/res.121.suppl_1.96 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Jianqiu Zou ◽

Wenxia Ma ◽

Jie Li ◽

Rodney Littlejohn ◽

Il-man Kim ◽

...

Keyword(s):

Cardiac Development ◽

Heart Diseases ◽

Left Ventricular ◽

Late Gestation ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Transcription Cofactor ◽

Wild Type Counterpart

Rationale: Cardiac development is orchestrated by a number of growth factors, transcription factors and epigenetic regulators, perturbation of which can lead to congenital heart diseases and cardiomyopathies. However, the role of novel ubiquitin-like protein modifiers, such as NEDD8 (neural precursor cells expressed developmentally downregulated 8), in cardiac development is unknown. Objectives: The objective of this study was to determine the significance of NEDD8 modification (neddylation) during perinatal cardiac development. Methods and Results: Neddylated proteins and NEDD8 enzymes were highly abundant in fetal and neonatal hearts but downregulated in adult hearts. We employed an αMHC Cre transgene to delete NAE1, a subunit of the NEDD8 E1 enzyme, in the perinatal mouse heart. Cardiac-specific deletion of NAE1 (NAE1 CKO ) significantly decreased neddylated proteins in the heart. The NAE1 CKO mice displayed cardiac hypoplasia, ventricular non-compaction and heart failure during late gestation, which became more pronounced by postnatal day 1 and led to perinatal lethality. Mechanistically, genetic deletion or pharmacological inhibition of NAE1 resulted in accumulation of Hippo kinases Mst1 and LATS1/2, which in turn phosphorylated and inactivated YAP, a transcription cofactor necessary for cardiomyocyte proliferation, leading to dysregulation of a number of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro . Reactivation of YAP signaling by overexpression of a constitutively-active YAP mutant (YAP 5SA ), but not its wild-type counterpart, overcame the blockade of cardiomyocyte proliferation induced by inhibition of NAE1. Conclusions: Our findings establish the importance of neddylation in the heart, more specifically, in ventricular chamber maturation, and identify neddylation as a novel regulator of Hippo-YAP signaling to promote cardiomyocyte proliferation.

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The role of neuregulin/ErbB2/ErbB4 signaling in the heart with special focus on effects on cardiomyocyte proliferation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00063.2012 ◽

2012 ◽

Vol 302 (11) ◽

pp. H2139-H2147 ◽

Cited By ~ 74

Author(s):

Brian Wadugu ◽

Bernhard Kühn

Keyword(s):

Heart Failure ◽

Cardiac Development ◽

Current Knowledge ◽

Critical Role ◽

Special Focus ◽

Cardiomyocyte Proliferation ◽

Neuregulin 1 ◽

Adult Heart ◽

Signaling Complex ◽

And Function

The signaling complex consisting of the growth factor neuregulin-1 (NRG1) and its tyrosine kinase receptors ErbB2 and ErbB4 has a critical role in cardiac development and homeostasis of the structure and function of the adult heart. Recent research results suggest that targeting this signaling complex may provide a viable strategy for treating heart failure. Clinical trials are currently evaluating the effectiveness and safety of intravenous administration of recombinant NRG1 formulations in heart failure patients. Endogenous as well as administered NRG1 has multiple possible activities in the adult heart, but how these are related is unknown. It has recently been demonstrated that NRG1 administration can stimulate proliferation of cardiomyocytes, which may contribute to repair failing hearts. This review summarizes the current knowledge of how NRG1 and its receptors control cardiac physiology and biology, with special emphasis on its role in cardiomyocyte proliferation during myocardial growth and regeneration.

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Comparison of growth factor signalling pathway utilisation in cultured normal melanocytes and melanoma cell lines

BMC Cancer ◽

10.1186/1471-2407-12-141 ◽

2012 ◽

Vol 12 (1) ◽

Cited By ~ 11

Author(s):

Ji Eun Kim ◽

Clare Stones ◽

Wayne R Joseph ◽

Euphemia Leung ◽

Graeme J Finlay ◽

...

Keyword(s):

Growth Factor ◽

Cell Lines ◽

Melanoma Cell ◽

Signalling Pathway ◽

Growth Factor Signalling ◽

Melanoma Cell Lines ◽

Growth Factor Signalling Pathway

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Abstract 124: Function of the Cytoskeletal Proteins Alpha-catenins in Myocardial Growth Control

Circulation Research ◽

10.1161/res.113.suppl_1.a124 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Jifen Li ◽

Sarah Carrante ◽

Roslyn Yi ◽

Frans van Roy ◽

Glenn L. Radice

Keyword(s):

Heart Development ◽

Growth Control ◽

Cytoskeletal Proteins ◽

Neonatal Rat ◽

Nuclear Accumulation ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Rat Cardiomyocytes ◽

Neonatal Cardiomyocytes

Introduction: Mammalian heart possesses regenerative potential immediately after birth and lost by one week of age. The mechanisms that govern neonatal cardiomyocyte proliferation and regenerative capacity are poorly understood. Recent reports indicate that Yap-Tead transcriptional complex is necessary and sufficient for cardiomyocyte proliferation. During postnatal development, N-cadherin/catenin adhesion complex becomes concentrated at termini of cardiomyocytes facilitating maturation of a specialized intercellular junction structure, the intercalated disc (ICD). This process coincides with the time cardiomyocytes exit cell cycle soon after birth. Hypothesis: We hypothesize that coincident with maturation of ICD α-catenins sequester transcriptional coactivator Yap in cytosol thus preventing activation of genes critical for cardiomyocyte proliferation. Methods: We deleted αE-catenin / αT-catenin genes (α-cat DKO) in perinatal mouse heart and knockdown (KD) α-catenins in neonatal rat cardiomyocytes to study functional impact of α-catenins ablation on ICD maturation. Results: We previously demonstrated that adult α-cat DKO mice exhibited decrease in scar size and improved function post myocardial infarction. In present study, we investigated function of α-catenins during postnatal heart development. We found increase in the number of Yap-positive nuclei (58.7% in DKO vs. 35.8 % in WT, n=13, p<0.001) and PCNA (53.9% in DKO vs. 47.8%, n=8, p<0.05) at postnatal day 1 and day 7 of α-cat DKO heart, respectively. Loss of α-catenins resulted in reduction in N-cadherin at ICD at day 14. We observed an increase number of mononucleated myocytes and decrease number of binucleated myocytes in α-cat DKO compared to controls. Using siRNA KD, we were able to replicate α-cat DKO proliferative phenotype in vitro. The number of BrdU-positive cells was decreased in α-cat KD after interfering with Yap expression (2.91% in α-cat KD vs. 2.02% in α-cat/Yap KD, n>2500 cells, p<0.05), suggesting α-catenins regulate cell proliferation through Yap in neonatal cardiomyocytes. Conclusion: Our results suggest that maturation of ICD regulates α-catenin-Yap interactions in cytosol, thus preventing Yap nuclear accumulation and cardiomyocyte proliferation.

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

Circulation Research ◽

10.1161/res.121.suppl_1.82 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Marlin Touma ◽

Xuedong Kang ◽

Fuying Gao ◽

Yan Zhao ◽

Reshma Biniwale ◽

...

Keyword(s):

Heart Defects ◽

Critical Role ◽

Newborn Infants ◽

R Package ◽

Maturation Stage ◽

Specific Gene ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Loss Of Function ◽

Myocyte Proliferation

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 P457: Systems Analysis To Understand The Impact Of The CDK Module On Cardiomyocyte Proliferation

Circulation Research ◽

10.1161/res.129.suppl_1.p457 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Bryana N Harris ◽

Laura Woo ◽

Jeffrey J Saucerman

Keyword(s):

Experimental Data ◽

Cell Cycle ◽

Transcription Factor ◽

Dna Synthesis ◽

Current Knowledge ◽

Cycle Phase ◽

Cyclin D ◽

Cardiomyocyte Proliferation ◽

The Core ◽

The Impact

Rationale: Heart failure is caused by the inability of adult mammalian hearts to overcome the loss of cardiomyocytes (CMs). This is due partly to the limited proliferative capacity of CMs, which exit the cell cycle and do not undergo cell division. Current knowledge in cardiac regeneration lacks an understanding of the molecular regulatory networks that determine whether CMs will progress through the cell cycle to proliferate. Our goal is to use computational modeling to understand the expression and activation levels of the core cell cycle network, specifically cyclins and cyclin-cyclin-dependent kinase (CDK) complexes. Methods: A model of core cell cycle dynamics was curated using previously published studies of CM proliferation regulators. This model incorporates those regulators known to stimulate G1/S and G2/M transitions through the core CDKs. The activity of each of the 22 network nodes (22 reactions) was predicted using a logic-based differential equation approach. The CDK model was then coupled with a minimal ODE model of cell cycle phase distributions and validated based on descriptions and experimental data from the literature. To prioritize key nodes for experimental validation, we performed a sensitivity analysis by stimulating individual knockdown for every node in the network, measuring the fractional activity of all nodes. Results: Our model confirmed that the knockdown of p21 and Rb protein and the overexpression of E2F transcription factor and cyclinD-cdk4 showed an increase in cells going through DNA synthesis and entering mitosis. A combined knockdown of p21 and p27 showed an increase of cells entering mitosis. Cyclin D-cdk4 and p21 overexpression showed a decrease and increase of Rb expression, respectively. Of the 14 model predictions, 12 agreed with experimental data in the literature. A comprehensive knockdown of the model nodes suggests that E2F (a key transcription factor driving DNA synthesis) is positively regulated by cyclin D while negatively regulated by GSK3B, SMAD3, and pRB. Conclusion: This model enables us to predict how cardiomyocytes respond to stimuli in the CDK network and identify potential therapeutic regulators that induce cardiomyocyte proliferation.

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DUX4 Role in Normal Physiology and in FSHD Muscular Dystrophy

Cells ◽

10.3390/cells10123322 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3322

Author(s):

Emanuele Mocciaro ◽

Valeria Runfola ◽

Paola Ghezzi ◽

Maria Pannese ◽

Davide Gabellini

Keyword(s):

Muscular Dystrophy ◽

Embryonic Development ◽

Transcriptional Activation ◽

Current Knowledge ◽

Relevant Information ◽

Facioscapulohumeral Muscular Dystrophy ◽

Healthy Humans ◽

Somatic Tissues ◽

Key Factor ◽

Development Regulation

In the last decade, the sequence-specific transcription factor double homeobox 4 (DUX4) has gone from being an obscure entity to being a key factor in important physiological and pathological processes. We now know that expression of DUX4 is highly regulated and restricted to the early steps of embryonic development, where DUX4 is involved in transcriptional activation of the zygotic genome. While DUX4 is epigenetically silenced in most somatic tissues of healthy humans, its aberrant reactivation is associated with several diseases, including cancer, viral infection and facioscapulohumeral muscular dystrophy (FSHD). DUX4 is also translocated, giving rise to chimeric oncogenic proteins at the basis of sarcoma and leukemia forms. Hence, understanding how DUX4 is regulated and performs its activity could provide relevant information, not only to further our knowledge of human embryonic development regulation, but also to develop therapeutic approaches for the diseases associated with DUX4. Here, we summarize current knowledge on the cellular and molecular processes regulated by DUX4 with a special emphasis on FSHD muscular dystrophy.

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Abstract 124: Fetal-Like RNA Splicing Remodeling in Failing Heart Revealed by Total Transcriptome Analysis

Circulation Research ◽

10.1161/res.111.suppl_1.a124 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Chen Gao ◽

Vincent Ren ◽

Grace (Xinshu) Xiao ◽

Jaunian Chen ◽

Yibin Wang

Keyword(s):

Alternative Splicing ◽

Rna Splicing ◽

Cardiac Development ◽

Heart Diseases ◽

Expression Profiles ◽

Mrna Splicing ◽

Mouse Heart ◽

Failing Heart ◽

Splicing Regulators ◽

A Genome

The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Using a genome-wide RNA-Seq analysis, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart. Most significantly, the alternative RNA splicing events observed in failing heart mimicked the splicing profile in fetal hearts, suggesting a fetal like RNA splicing remodeling in failing hearts. After examining the expression profiles of splicing regulators in neonatal, normal adult, and failing adult hearts, Fox-1 was identified as one to be significantly down regulated in the failing and fetal hearts. Morpholino mediated Fox-1 knock-down in zebrafish embryos led to lethal phenotype associated with impaired cardiac development and function. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Therefore, our established functional significance of Fox1 mediated RNA alternative splicing serves as a key molecular player in transcriptome remodeling during cardiac development and pathology.

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Abstract 67: Metabolic Programming Regulates Proliferation and Binucleation of Postnatal Cardiomyocytes

Circulation Research ◽

10.1161/res.119.suppl_1.67 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Daniela Liccardo ◽

Ryan LaCanna ◽

Ying Tian

Keyword(s):

Cell Cycle ◽

Natriuretic Peptides ◽

Metabolic Programming ◽

Postnatal Life ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Beta Oxidation ◽

Neonatal Cardiomyocytes ◽

Short Period

In contrast to adult, neonatal cardiomyocytes are able to proliferate and lose this capacity soon after birth when they withdraw from the cell cycle, become binucleated and differentiate. The arrest of cardiomyocytes cell cycle can be reversible for a short period, conferring the neonatal heart a regenerative potential within the first week of postnatal life. In the timeframe surrounding birth, heart maturation is also characterized by a change in energy metabolism, switching from glycolysis to beta-oxidation. However little is known about how metabolic programming in postnatal cardiomyocytes regulates their ability to proliferate, become binucleated and differentiate. In this study, we show that blocking beta-oxidation in mouse neonatal cardiomyocytes with etomoxir treatment promotes glycolysis and cell cycle re-entry, while increasing fatty acid beta-oxidation but reducing glycolysis leads to a decrease of the number of proliferating cardiomyocytes. In neonatal mice our data demonstrate that cardiomyocytes undergo binucleation and differentiation during the first week after birth and this process is correlated with the upregulation of the natriuretic peptides, ANP and BNP expression. Notably, in the postnatal mouse heart, beta-oxidation blockade through in vivo etomoxir injections, increases ventricular cardiomyocytes number, decreases natriuretic peptides expression and reduces the conversion of cardiomyocytes from a mononucleated to a binucleated phenotype. These findings highlight the importance of metabolic programming in regulating cardiomyocyte proliferation and suggest a potential therapeutic target for heart regeneration by modulating energy metabolic programming.

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Abstract 052: Epicardial-derived Adrenomedullin Drives Cardiac Hyperplasia During Embryogenesis

Circulation Research ◽

10.1161/res.113.suppl_1.a052 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Sarah E Wetzel-Strong ◽

Manyu Li ◽

Toshio Nishikimi ◽

Kathleen M Caron

Keyword(s):

Embryonic Development ◽

Heart Development ◽

Cardiac Development ◽

Lymphatic Vessels ◽

Heart Weight ◽

Cardiovascular Development ◽

Proliferating Cells ◽

Over Expression ◽

Knockout Animals

The multi-functional peptide adrenomedullin ( Adm = gene, AM = protein) plays important roles in embryonic development and disease. Previous studies demonstrated that Adm knockout mice die at embryonic day 13.5 with small, disorganized hearts and hypoplastic lymphatic vessels, highlighting the importance of this peptide in normal cardiovascular development. Since Adm knockout animals are embryonic lethal, our goal was to generate and characterize a novel model of Adm over-expression to study the role of Adm during development and disease processes. Through gene targeting techniques, we generated a novel mouse model of Adm over-expression, abbreviated as Adm hi/hi . When we assessed gene expression of Adm from 10 different tissues, we found Adm hi/hi mice express 3- to 15-fold more Adm than wildtype littermates. Additionally, peptide levels of AM in lung and kidney, as well as circulating plasma levels of AM were elevated 3-fold over wildtype mice, indicating a functional increase in AM. Our initial analysis revealed that adult Adm hi/hi mice have larger heart weight to body weight ratios than wildtype littermates (4.93±0.23 vs. 5.96±0.29, n = 11-12). We found that compared to wildtype, Adm hi/hi embryos have more proliferating cells during heart development (14.46±1.11 vs. 31.97±2.84, n=4), indicating that hyperplasia drives Adm hi/hi heart enlargement. By crossing the Adm hi/hi line to different tissue-specific Cre lines, we were able to excise the stabilizing bovine growth hormone 3’UTR, thereby returning Adm expression levels back to wildtype in cells with active Cre recombinase. Using this approach, we identified the epicardium as a major source of AM during cardiac development. In conclusion, we found that AM derived primarily from the epicardium drives cardiac hyperplasia during embryonic development resulting in persistent, enlarged hearts of adult Adm hi/hi mice. Since our Adm hi/hi mice recapitulate the 3-fold plasma elevation of AM observed during human disease, this mouse line will be a useful tool for studying the role of elevated AM during disease.

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