Oncogene-induced cardiac neoplasia shares similar mechanisms with heart regeneration in zebrafish

The human heart is a poorly regenerative organ and cardiac tumors are extremely rare. The zebrafish heart can restore its damaged myocardium through cardiomyocyte proliferation. Whether this endogenous capacity causes a susceptibility to neoplasia remains unknown. Here, we established a strategy to conditionally express the HRASG12V oncogene in zebrafish cardiomyocytes. The induction of this transgene in larvae or adult animals resulted in heart overgrowth with abnormal histology. The malformed ventricle displayed similar characteristics to the regenerative myocardium, such as enhanced cell-cycle entry, incomplete differentiation, reactivation of cardiac embryonic programs, expression of regeneration genes, oxidative metabolism changes, intramyocardial matrix remodeling and leucocyte recruitment. We found that oncogene-mediated cardiac tumorigenesis and cryoinjury-induced regeneration involve TOR signaling, as visualized by phosphorylation of its target ribosomal protein S6. The inhibition of TOR by rapamycin impaired regeneration and rescued from neoplasia. These findings demonstrate the existence of common mechanisms underlying the proliferative plasticity of zebrafish cardiomyocytes during advantageous organ restoration and detrimental tumorigenesis.

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Prrx1b directs pro-regenerative fibroblasts during zebrafish heart regeneration

10.1101/2020.06.13.149013 ◽

2020 ◽

Author(s):

Dennis E.M. de Bakker ◽

Esther Dronkers ◽

Mara Bouwman ◽

Aryan Vink ◽

Marie-José Goumans ◽

...

Keyword(s):

Human Heart ◽

Cardiac Fibrosis ◽

Lineage Tracing ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Loss Of Function ◽

Epicardial Cells ◽

Injured Area ◽

Novel Therapeutic Strategies ◽

Zebrafish Heart

ABSTRACTRationaleThe human heart loses millions of cardiomyocytes after an ischemic injury, but is unable to regenerate the lost tissue. Instead, the injured human heart is repaired by pro-fibrotic fibroblasts that form a large permanent scar. In contrast, the injured zebrafish heart regenerates efficiently without the formation of a permanent scar. While fibroblasts have been shown to be indispensable for zebrafish heart regeneration, very little is known about the mechanisms balancing the fibrotic and regenerative response. A better understanding of these mechanisms could lead to the discovery of novel therapeutic strategies to reduce fibrosis and promote heart regeneration.ObjectiveTo identify novel mechanisms that regulate the balance between cardiac fibrosis and scar-free regeneration.Methods and ResultsUsing a genetic approach, we first show that zebrafish prrx1b loss-of-function mutants display reduced cardiomyocyte proliferation and impaired heart regeneration. Using a lineage tracing approach, we show that Prrx1b is expressed in tcf21+ epicardial-derived cells localizing around and inside the injured area. Next, we used a single cell RNA-sequencing approach on sorted tcf21+ cells isolated from injured prrx1b-/- and wild-type hearts and identified two distinct fibroblast populations. With combined bioinformatic and histological analysis we found that prrx1b-/- hearts contain an excess of pro-fibrotic fibroblasts that produce TGF-β ligands and collagens, while fewer pro-regenerative Nrg1-expressing fibroblasts are formed. Furthermore, by injecting recombinant NRG1 in prrx1b-/- fish we were able to rescue their cardiomyocyte proliferation defect. Finally, using cultured human fetal epicardial cells and siRNA mediated knock-down of PRRX1 we found that PRRX1 is required for NRG1 induction in human epicardial-derived cells.ConclusionsPrrx1b in the injured heart restricts fibrosis and stimulates regeneration by directing epicardial-derived cells towards a pro-regenerative Nrg1-producing fibroblast state.

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A Systematic Exposition of Methods used for Quantification of Heart Regeneration after Apex Resection in Zebrafish

Cells ◽

10.3390/cells9030548 ◽

2020 ◽

Vol 9 (3) ◽

pp. 548 ◽

Cited By ~ 1

Author(s):

Helene Juul Belling ◽

Wolfgang Hofmeister ◽

Ditte Caroline Andersen

Keyword(s):

Human Heart ◽

Quantitative Methods ◽

Heart Function ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Ability To Regenerate ◽

Specific Proteins ◽

Heart Injury ◽

Whole Heart ◽

Study Designs

Myocardial infarction (MI) is a worldwide condition that affects millions of people. This is mainly caused by the adult human heart lacking the ability to regenerate upon injury, whereas zebrafish have the capacity through cardiomyocyte proliferation to fully regenerate the heart following injury such as apex resection (AR). But a systematic overview of the methods used to evidence heart regrowth and regeneration in the zebrafish is lacking. Herein, we conducted a systematical search in Embase and Pubmed for studies on heart regeneration in the zebrafish following injury and identified 47 AR studies meeting the inclusion criteria. Overall, three different methods were used to assess heart regeneration in zebrafish AR hearts. 45 out of 47 studies performed qualitative (37) and quantitative (8) histology, whereas immunohistochemistry for various cell cycle markers combined with cardiomyocyte specific proteins was used in 34 out of 47 studies to determine cardiomyocyte proliferation qualitatively (6 studies) or quantitatively (28 studies). For both methods, analysis was based on selected heart sections and not the whole heart, which may bias interpretations. Likewise, interstudy comparison of reported cardiomyocyte proliferation indexes seems complicated by distinct study designs and reporting manners. Finally, six studies performed functional analysis to determine heart function, a hallmark of human heart injury after MI. In conclusion, our data implies that future studies should consider more quantitative methods eventually taking the 3D of the zebrafish heart into consideration when evidencing myocardial regrowth after AR. Furthermore, standardized guidelines for reporting cardiomyocyte proliferation and sham surgery details may be considered to enable inter study comparisons and robustly determine the effect of given genes on the process of heart regeneration.

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

eLife ◽

10.7554/elife.05871 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 111

Author(s):

Matthew Gemberling ◽

Ravi Karra ◽

Amy L Dickson ◽

Kenneth D Poss

Keyword(s):

Cardiac Injury ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Adult Zebrafish ◽

Perivascular Cells ◽

Adult Heart ◽

Muscle Hyperplasia ◽

Zebrafish Heart

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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Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure

International Journal of Molecular Sciences ◽

10.3390/ijms22147720 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7720

Author(s):

Abou Bakr M. Salama ◽

Ahmad Gebreil ◽

Tamer M. A. Mohamed ◽

Riham R. E. Abouleisa

Keyword(s):

Heart Failure ◽

Human Heart ◽

Progressive Failure ◽

Cardiac Injury ◽

Proliferative Capacity ◽

Cardiomyocyte Proliferation ◽

Cell Cycle Entry ◽

Heart Repair ◽

Lower Vertebrates ◽

Heart Functions

Unlike some lower vertebrates which can completely regenerate their heart, the human heart is a terminally differentiated organ. Cardiomyocytes lost during cardiac injury and heart failure cannot be replaced due to their limited proliferative capacity. Therefore, cardiac injury generally leads to progressive failure. Here, we summarize the latest progress in research on methods to induce cardiomyocyte cell cycle entry and heart repair through the alteration of cardiomyocyte plasticity, which is emerging as an effective strategy to compensate for the loss of functional cardiomyocytes and improve the impaired heart functions.

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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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Proteomics analysis of extracellular matrix remodeling during zebrafish heart regeneration

10.1101/588251 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anna Garcia-Puig ◽

Jose Luis Mosquera ◽

Senda Jiménez-Delgado ◽

Cristina García-Pastor ◽

Ignasi Jorba ◽

...

Keyword(s):

Extracellular Matrix ◽

Dynamic Change ◽

Protein Composition ◽

Cytoskeletal Proteins ◽

Heart Regeneration ◽

Matrix Remodeling ◽

Adult Zebrafish ◽

Force Microscopy ◽

Ecm Proteins ◽

Zebrafish Heart

AbstractAdult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days post-amputation) time-point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.

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