Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart

In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.

Download Full-text

Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart

10.1101/254748 ◽

2018 ◽

Cited By ~ 1

Author(s):

Héctor Sánchez-Iranzo ◽

María Galardi-Castilla ◽

Andrés Sanz-Morejón ◽

Juan Manuel González-Rosa ◽

Ricardo Costa ◽

...

Keyword(s):

Cardiac Injury ◽

Scar Tissue ◽

Regenerative Process ◽

Cardiomyocyte Proliferation ◽

Fibrotic Response ◽

Genetic Ablation ◽

Myocardial Wall ◽

Fibrotic Scar ◽

Fibrosis Regression ◽

Activated Fibroblasts

ABSTRACTIn the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated, but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that anti-fibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.SCIENTIFIC STATEMENTAfter myocardial infarction in the mammalian heart, millions of cardiomyocytes are lost and replaced by fibrotic scar tissue. While fibrosis is persistent in adult mammals, there are some vertebrates, including zebrafish, with the capacity for regeneration. This process does not occur in the absence of fibrosis. Here we studied subpopulations of collagen producing cells and analyzed their fate after complete regeneration of the zebrafish myocardium. Our data show that fibroblasts persisted in the regenerated heart, but shut down the profibrotic program. While fibrosis could be considered as detrimental to the regeneration process, our study reveals a positive effect on cardiomyocyte proliferation. Accordingly, a fibrotic response can be beneficial for heart regeneration.

Download Full-text

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Macrophages stimulate epicardial VEGFaa to trigger cardiomyocyte proliferation in larval zebrafish heart regeneration

10.1101/2021.06.15.448575 ◽

2021 ◽

Author(s):

Finnius A Bruton ◽

Aryan Kaveh ◽

Katherine Ross-Stewart ◽

Gianfranco Matrone ◽

Magdalena EM Oremek ◽

...

Keyword(s):

Cardiac Injury ◽

Cardiac Regeneration ◽

Cellular Heterogeneity ◽

Cardiomyocyte Proliferation ◽

Larval Zebrafish ◽

Laser Injury ◽

Structural Recovery ◽

Injury Model ◽

Heart Injury ◽

Zebrafish Heart

Cardiac injury induces a sustained innate immune response in both zebrafish and mammals. Macrophages, highly plastic immune cells, perform a range of both beneficial and detrimental functions during mammalian cardiac repair yet their precise roles in zebrafish cardiac regeneration are not fully understood. Here we characterise cardiac regeneration in the rapidly regenerating larval zebrafish laser injury model and use macrophage ablation and macrophage-less irf8 mutants to define the requirement of macrophages for key stages of regeneration. We found macrophages to display cellular heterogeneity and plasticity in larval heart injury as in mammals. Live heartbeat-synchronised imaging and RNAseq revealed an early proinflammatory macrophage phase which then resolves to an anti-inflammatory, profibrotic phase. Macrophages are required for cardiomyocyte proliferation but not for functional or structural recovery following injury. Macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium which upregulates mitogen VEGFaa. Experimental perturbation of VEGF signalling confirmed VEGFaa to be an important inducer of cardiomyocyte proliferation revealing a previously unrecognised mechanism by which macrophages aid cardiac regeneration.

Download Full-text

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.

Download Full-text

Pre-existent adult sox10+ cardiomyocytes contribute to myocardial regeneration in the zebrafish

10.1101/662536 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marcos Sande-Melón ◽

Inês J. Marques ◽

María Galardi-Castilla ◽

Xavier Langa ◽

María Pérez-López ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Profile ◽

Crucial Role ◽

Cardiac Regeneration ◽

Heart Regeneration ◽

Adult Zebrafish ◽

Genetic Ablation ◽

Fibrotic Tissue ◽

Distinct Gene Expression Profile ◽

Zebrafish Heart

AbstractDuring heart regeneration in the zebrafish, fibrotic tissue is replaced by newly formed cardiomyocytes derived from pre-existing ones. It is unclear whether the heart is comprised of several cardiomyocyte populations bearing different capacity to replace lost myocardium. Here, using sox10 genetic fate mapping, we identified a subset of pre-existent cardiomyocytes in the adult zebrafish heart with a distinct gene expression profile that expanded massively after cryoinjury. Genetic ablation of sox10+ cardiomyocytes severely impaired cardiac regeneration revealing that they play a crucial role for heart regeneration.

Download Full-text

Zebrafish cardiac regeneration—looking beyond cardiomyocytes to a complex microenvironment

Histochemistry and Cell Biology ◽

10.1007/s00418-020-01913-6 ◽

2020 ◽

Author(s):

Rebecca Ryan ◽

Bethany R. Moyse ◽

Rebecca J. Richardson

Keyword(s):

Cardiac Injury ◽

Cell Communication ◽

Cardiac Regeneration ◽

Cell Types ◽

Regenerative Process ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Underlying Mechanisms ◽

Different Cell Types

Abstract The study of heart repair post-myocardial infarction has historically focused on the importance of cardiomyocyte proliferation as the major factor limiting adult mammalian heart regeneration. However, there is mounting evidence that a narrow focus on this one cell type discounts the importance of a complex cascade of cell–cell communication involving a whole host of different cell types. A major difficulty in the study of heart regeneration is the rarity of this process in adult animals, meaning a mammalian template for how this can be achieved is lacking. Here, we review the adult zebrafish as an ideal and unique model in which to study the underlying mechanisms and cell types required to attain complete heart regeneration following cardiac injury. We provide an introduction to the role of the cardiac microenvironment in the complex regenerative process and discuss some of the key advances using this in vivo vertebrate model that have recently increased our understanding of the vital roles of multiple different cell types. Due to the sheer number of exciting studies describing new and unexpected roles for inflammatory cell populations in cardiac regeneration, this review will pay particular attention to these important microenvironment participants.

Download Full-text

Cardiac regeneration: epicardial mediated repair

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2015.2147 ◽

2015 ◽

Vol 282 (1821) ◽

pp. 20152147 ◽

Cited By ~ 17

Author(s):

Teresa Kennedy-Lydon ◽

Nadia Rosenthal

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cardiac Fibroblasts ◽

Cardiac Regeneration ◽

Repair Process ◽

Regenerative Process ◽

Cardiac Stem Cell ◽

Cardiomyocyte Proliferation ◽

Lower Vertebrates

The hearts of lower vertebrates such as fish and salamanders display scarless regeneration following injury, although this feature is lost in adult mammals. The remarkable capacity of the neonatal mammalian heart to regenerate suggests that the underlying machinery required for the regenerative process is evolutionarily retained. Recent studies highlight the epicardial covering of the heart as an important source of the signalling factors required for the repair process. The developing epicardium is also a major source of cardiac fibroblasts, smooth muscle, endothelial cells and stem cells. Here, we examine animal models that are capable of scarless regeneration, the role of the epicardium as a source of cells, signalling mechanisms implicated in the regenerative process and how these mechanisms influence cardiomyocyte proliferation. We also discuss recent advances in cardiac stem cell research and potential therapeutic targets arising from these studies.

Download Full-text