Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

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Myocardial NF-κB activation is essential for zebrafish heart regeneration

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1511209112 ◽

2015 ◽

Vol 112 (43) ◽

pp. 13255-13260 ◽

Cited By ~ 54

Author(s):

Ravi Karra ◽

Anne K. Knecht ◽

Kazu Kikuchi ◽

Kenneth D. Poss

Keyword(s):

Tissue Regeneration ◽

Therapeutic Strategy ◽

Cardiac Injury ◽

Regulatory Sequences ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Developmental Program ◽

Lower Vertebrates ◽

Outstanding Question ◽

Zebrafish Heart

Heart regeneration offers a novel therapeutic strategy for heart failure. Unlike mammals, lower vertebrates such as zebrafish mount a strong regenerative response following cardiac injury. Heart regeneration in zebrafish occurs by cardiomyocyte proliferation and reactivation of a cardiac developmental program, as evidenced by induction of gata4 regulatory sequences in regenerating cardiomyocytes. Although many of the cellular determinants of heart regeneration have been elucidated, how injury triggers a regenerative program through dedifferentiation and epicardial activation is a critical outstanding question. Here, we show that NF-κB signaling is induced in cardiomyocytes following injury. Myocardial inhibition of NF-κB activity blocks heart regeneration with pleiotropic effects, decreasing both cardiomyocyte proliferation and epicardial responses. Activation of gata4 regulatory sequences is also prevented by NF-κB signaling antagonism, suggesting an underlying defect in cardiomyocyte dedifferentiation. Our results implicate NF-κB signaling as a key node between cardiac injury and tissue regeneration.

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Induction of Wnt signaling antagonists and P21-activated kinase enhances cardiomyocyte proliferation during zebrafish heart regeneration

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa046 ◽

2020 ◽

Author(s):

Xiangwen Peng ◽

Kaa Seng Lai ◽

Peilu She ◽

Junsu Kang ◽

Tingting Wang ◽

...

Keyword(s):

Wnt Signaling ◽

Cardiac Tissue ◽

Cardiac Injury ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Cardiac Damage ◽

Ectopic Activation ◽

P21 Activated Kinase ◽

Wnt Inhibitors ◽

Zebrafish Heart

Abstract Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). However, the signaling mechanisms by which injury induces CM renewal remain incompletely understood. Here, we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors, including Dickkopf 1 (Dkk1), Dkk3, secreted Frizzled-related protein 1 (sFrp1), and sFrp2, in cardiac tissue adjacent to injury sites. Experimental blocking of Wnt activity via Dkk1 overexpression enhances CM proliferation and heart regeneration, whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation. Although Wnt signaling is dampened upon injury, the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges. Our analyses indicated that P21-activated kinase 2 (Pak2) is induced at regenerating CMs, where it phosphorylates cytoplasmic β-catenin at Ser675 and increases its stability at disassembled sarcomeres during regeneration. Myocardial-specific induction of the phospho-mimetic β-catenin (S675E) enhances CM dedifferentiation and sarcomere disassembly in response to cardiac damage. Importantly, inactivation of Pak2 kinase activity reduces the Ser675-phosphorylated β-catenin (pS675-β-catenin) at cardiac wounds and attenuates CM sarcomere disorganization, dedifferentiation, and proliferation. Taken together, these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-β-catenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.

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Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo

Development ◽

10.1242/dev.199740 ◽

2021 ◽

Author(s):

Hessel Honkoop ◽

Phong D. Nguyen ◽

Veronique E.M. van der Velden ◽

Katharina F. Sonnen ◽

Jeroen Bakkers

Keyword(s):

Culture System ◽

Ex Vivo ◽

Live Imaging ◽

Slice Culture ◽

Heart Regeneration ◽

Dependent Manner ◽

Cardiomyocyte Proliferation ◽

Adult Zebrafish ◽

Cellular Mechanisms ◽

Zebrafish Heart

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent its use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain and proteasome dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.

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Prrx1b restricts fibrosis and promotes Nrg1-dependent cardiomyocyte proliferation during zebrafish heart regeneration

Development ◽

10.1242/dev.198937 ◽

2021 ◽

Author(s):

Dennis E.M. de Bakker ◽

Mara Bouwman ◽

Esther Dronkers ◽

Filipa C. Simões ◽

Paul R. Riley ◽

...

Keyword(s):

Transcription Factor ◽

Cardiac Injury ◽

Lineage Tracing ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Fibrotic Scar ◽

Ligand Expression ◽

Zebrafish Heart

Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, like zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here we show that the homeo-box containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA-sequencing we find that Prrx1b is activated in epicardial-derived cells (EPDCs) where it restricts TGF-β ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal EPDCs and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart.

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Fate predetermination of cardiac myocytes during zebrafish heart regeneration

Open Biology ◽

10.1098/rsob.170116 ◽

2017 ◽

Vol 7 (6) ◽

pp. 170116 ◽

Cited By ~ 4

Author(s):

Isil Tekeli ◽

Anna Garcia-Puig ◽

Mario Notari ◽

Cristina García-Pastor ◽

Isabelle Aujard ◽

...

Keyword(s):

Heart Regeneration ◽

Adult Zebrafish ◽

Injury Site ◽

Adult Heart ◽

Ability To Regenerate ◽

And Migration ◽

Dynamic Plasticity ◽

Zebrafish Heart ◽

Myocardial Layer ◽

Labelling System

Adult zebrafish have the remarkable ability to regenerate their heart upon injury, a process that involves limited dedifferentiation and proliferation of spared cardiomyocytes (CMs), and migration of their progeny. During regeneration, proliferating CMs are detected throughout the myocardium, including areas distant to the injury site, but whether all of them are able to contribute to the regenerated tissue remains unknown. Here, we developed a CM-specific, photoinducible genetic labelling system, and show that CMs labelled in embryonic hearts survive and contribute to all three (primordial, trabecular and cortical) layers of the adult zebrafish heart. Next, using this system to investigate the fate of CMs from different parts of the myocardium during regeneration, we show that only CMs immediately adjacent to the injury site contributed to the regenerated tissue. Finally, our results show an extensive predetermination of CM fate during adult heart regeneration, with cells from each myocardial layer giving rise to cells that retain their layer identity in the regenerated myocardium. Overall, our results indicate that adult heart regeneration in the zebrafish is a rather static process governed by short-range signals, in contrast to the highly dynamic plasticity of CM fates that takes place during embryonic heart regeneration.

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Inhibition of let-7c Regulates Cardiac Regeneration after Cryoinjury in Adult Zebrafish

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd6020016 ◽

2019 ◽

Vol 6 (2) ◽

pp. 16 ◽

Cited By ~ 1

Author(s):

Suneeta Narumanchi ◽

Karri Kalervo ◽

Sanni Perttunen ◽

Hong Wang ◽

Katariina Immonen ◽

...

Keyword(s):

Cardiac Function ◽

Cardiac Regeneration ◽

Nuclear Antigen ◽

Heart Regeneration ◽

Adult Zebrafish ◽

Tissue Samples ◽

Micro Rnas ◽

Proliferating Cell ◽

Zebrafish Heart

The let-7c family of micro-RNAs (miRNAs) is expressed during embryonic development and plays an important role in cell differentiation. We have investigated the role of let-7c in heart regeneration after injury in adult zebrafish. let-7c antagomir or scramble injections were given at one day after cryoinjury (1 dpi). Tissue samples were collected at 7 dpi, 14 dpi and 28 dpi and cardiac function was assessed before cryoinjury, 1 dpi, 7 dpi, 14 dpi and 28 dpi. Inhibition of let-7c increased the rate of fibrinolysis, increased the number of proliferating cell nuclear antigen (PCNA) positive cardiomyocytes at 7 dpi and increased the expression of the epicardial marker raldh2 at 7 dpi. Additionally, cardiac function measured with echocardiography recovered slightly more rapidly after inhibition of let-7c. These results reveal a beneficial role of let-7c inhibition in adult zebrafish heart regeneration.

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Abstract 527: Role of IGFBP3 in Neonatal Heart Regeneration

Circulation Research ◽

10.1161/res.127.suppl_1.527 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Shah R Ali ◽

Waleed El-Helaly ◽

Ivan Menendez-Montes ◽

Ngoc Uyen Nhi Nguyen ◽

Suwannee Thet ◽

...

Keyword(s):

Heart Disease ◽

Knockout Mice ◽

Systolic Function ◽

Ectopic Expression ◽

Secreted Protein ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Newborn Mice ◽

Adult Heart ◽

Neonatal Heart

Background: The adult mammalian heart is unable to regenerate after an injury. However, newborn mice are able to fully regenerate the heart after myocardial infarction (MI). The neonatal MI model therefore is a potential blueprint for regenerating the adult heart that could offer novel therapies for patients suffering from heart disease. To investigate the mechanism by which neonatal heart regeneration occurs, we screened for secreted proteins that are upregulated after neonatal MI but not after adult MI. We hypothesized that such a protein could be a cardiomyocyte mitogen that underlies the cardiomyocyte proliferation that occurs after neonatal MI. Methods: We performed microarrays on neonatal and adult MI (and sham) heart tissue samples: we identified IGFBP3 (Insulin Growth Factor Binding Protein 3), which canonically transports IGF ligands in circulation, as a secreted protein that is uniquely upregulated after neonatal MI. We used in situ hybridization, reporter mice, and immunostaining to validate the findings from the microarray. Single cell RNA-seq data revealed that IGFBP3 is expressed in vascular cells. Results: We first tested whether IGFBP3 is necessary during neonatal heart regeneration: we performed neonatal (P1) MI in global Igfbp3 knockout mice and wild-type mice, and found that knockout mice have more fibrosis and worse ejection fraction (EF) one month after P1 MI. To determine if IGFBP3 is sufficient to promote cardiomyocyte proliferation, we injected recombinant IGFBP3 protein into the heart of one week-old mice (P7) and saw more cycling myocytes. We generated novel transgenic vascular-Igfbp3-overexpression mice, which exhibit less fibrosis as well as improved EF after P7 MI compared to controls. Conclusions: IGFBP3 is a secreted protein that is necessary for complete regeneration after a neonatal MI. Its ectopic expression can cause cardiomyocyte proliferation and can improve systolic function, and it prolongs the window of neonatal heart regeneration. Therefore, IGFBP3 may represent a cardiomyocyte mitogen with potential therapeutic value for adult heart disease.

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Emerging Roles for Immune Cells and MicroRNAs in Modulating the Response to Cardiac Injury

Journal of Cardiovascular Development and Disease ◽

10.3390/jcdd6010005 ◽

2019 ◽

Vol 6 (1) ◽

pp. 5 ◽

Cited By ~ 4

Author(s):

Adriana Rodriguez ◽

Viravuth Yin

Keyword(s):

Immune Cells ◽

Cardiac Tissue ◽

Heart Function ◽

Cardiac Injury ◽

Scar Tissue ◽

Acute Injury ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Cardiac Damage ◽

Dynamic Interplay

Stimulating cardiomyocyte regeneration after an acute injury remains the central goal in cardiovascular regenerative biology. While adult mammals respond to cardiac damage with deposition of rigid scar tissue, adult zebrafish and salamander unleash a regenerative program that culminates in new cardiomyocyte formation, resolution of scar tissue, and recovery of heart function. Recent studies have shown that immune cells are key to regulating pro-inflammatory and pro-regenerative signals that shift the injury microenvironment toward regeneration. Defining the genetic regulators that control the dynamic interplay between immune cells and injured cardiac tissue is crucial to decoding the endogenous mechanism of heart regeneration. In this review, we discuss our current understanding of the extent that macrophage and regulatory T cells influence cardiomyocyte proliferation and how microRNAs (miRNAs) regulate their activity in the injured heart.

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Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00559.2015 ◽

2015 ◽

Vol 309 (8) ◽

pp. H1237-H1250 ◽

Cited By ~ 59

Author(s):

Marina Leone ◽

Ajit Magadum ◽

Felix B. Engel

Keyword(s):

Cardiac Function ◽

Molecular Mechanisms ◽

Cardiac Development ◽

Cardiac Regeneration ◽

Experimental Medicine ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Naturally Occurring ◽

Ability To Regenerate ◽

Zebrafish Heart

The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

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Delineating the Dynamic Transcriptome Response of mRNA and microRNA during Zebrafish Heart Regeneration

Biomolecules ◽

10.3390/biom9010011 ◽

2018 ◽

Vol 9 (1) ◽

pp. 11 ◽

Cited By ~ 3

Author(s):

Hagen Klett ◽

Lonny Jürgensen ◽

Patrick Most ◽

Martin Busch ◽

Fabian Günther ◽

...

Keyword(s):

Heart Function ◽

Heart Diseases ◽

Expression Profiles ◽

Temporal Organization ◽

H9c2 Cell ◽

Specific Response ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Transcriptome Response ◽

Zebrafish Heart

Heart diseases are the leading cause of death for the vast majority of people around the world, which is often due to the limited capability of human cardiac regeneration. In contrast, zebrafish have the capacity to fully regenerate their hearts after cardiac injury. Understanding and activating these mechanisms would improve health in patients suffering from long-term consequences of ischemia. Therefore, we monitored the dynamic transcriptome response of both mRNA and microRNA in zebrafish at 1–160 days post cryoinjury (dpi). Using a control model of sham-operated and healthy fish, we extracted the regeneration specific response and further delineated the spatio-temporal organization of regeneration processes such as cell cycle and heart function. In addition, we identified novel (miR-148/152, miR-218b and miR-19) and previously known microRNAs among the top regulators of heart regeneration by using theoretically predicted target sites and correlation of expression profiles from both mRNA and microRNA. In a cross-species effort, we validated our findings in the dynamic process of rat myoblasts differentiating into cardiomyocytes-like cells (H9c2 cell line). Concluding, we elucidated different phases of transcriptomic responses during zebrafish heart regeneration. Furthermore, microRNAs showed to be important regulators in cardiomyocyte proliferation over time.

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