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

Helene Juul Belling; Wolfgang Hofmeister; Ditte Caroline Andersen

doi:10.3390/cells9030548

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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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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Fosl1 is vital to heart regeneration upon apex resection in adult Xenopus tropicalis

npj Regenerative Medicine ◽

10.1038/s41536-021-00146-y ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Hai-Yan Wu ◽

Yi-Min Zhou ◽

Zhu-Qin Liao ◽

Jia-Wen Zhong ◽

You-Bin Liu ◽

...

Keyword(s):

Early Stage ◽

Dominant Negative ◽

Luciferase Reporter ◽

Heart Regeneration ◽

Cell Cycle Regulators ◽

Cardiomyocyte Proliferation ◽

Neonatal Mice ◽

Heart Injury

AbstractCardiovascular disease is the leading cause of death in the world due to losing regenerative capacity in the adult heart. Frogs possess remarkable capacities to regenerate multiple organs, including spinal cord, tail, and limb, but the response to heart injury and the underlying molecular mechanism remains largely unclear. Here we demonstrated that cardiomyocyte proliferation greatly contributes to heart regeneration in adult X. tropicalis upon apex resection. Using RNA-seq and qPCR, we found that the expression of Fos-like antigen 1 (Fosl1) was dramatically upregulated in early stage of heart injury. To study Fosl1 function in heart regeneration, its expression was modulated in vitro and in vivo. Overexpression of X. tropicalis Fosl1 significantly promoted the proliferation of cardiomyocyte cell line H9c2. Consistently, endogenous Fosl1 knockdown suppressed the proliferation of H9c2 cells and primary cardiomyocytes isolated from neonatal mice. Taking use of a cardiomyocyte-specific dominant-negative approach, we show that blocking Fosl1 function leads to defects in cardiomyocyte proliferation during X. tropicalis heart regeneration. We further show that knockdown of Fosl1 can suppress the capacity of heart regeneration in neonatal mice, but overexpression of Fosl1 can improve the cardiac function in adult mouse upon myocardium infarction. Co-immunoprecipitation, luciferase reporter, and ChIP analysis reveal that Fosl1 interacts with JunB and promotes the expression of Cyclin-T1 (Ccnt1) during heart regeneration. In conclusion, we demonstrated that Fosl1 plays an essential role in cardiomyocyte proliferation and heart regeneration in vertebrates, at least in part, through interaction with JunB, thereby promoting expression of cell cycle regulators including Ccnt1.

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Heart regeneration: beyond new muscle and vessels

Cardiovascular Research ◽

10.1093/cvr/cvaa320 ◽

2021 ◽

Author(s):

Judy R Sayers ◽

Paul R Riley

Keyword(s):

Heart Development ◽

Cell Function ◽

Cell Types ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Cardiovascular Tissue ◽

Function Calls ◽

Heart Muscle Cells ◽

System Cell ◽

Heart Injury

Abstract The most striking consequence of a heart attack is the loss of billions of heart muscle cells, alongside damage to the associated vasculature. The lost cardiovascular tissue is replaced by scar formation, which is non-functional and results in pathological remodelling of the heart and ultimately heart failure. It is, therefore, unsurprising that the heart regeneration field has centred efforts to generate new muscle and blood vessels through targeting cardiomyocyte proliferation and angiogenesis following injury. However, combined insights from embryological studies and regenerative models, alongside the adoption of -omics technology, highlight the extensive heterogeneity of cell types within the forming or re-forming heart and the significant crosstalk arising from non-muscle and non-vessel cells. In this review, we focus on the roles of fibroblasts, immune, conduction system, and nervous system cell populations during heart development and we consider the latest evidence supporting a function for these diverse lineages in contributing to regeneration following heart injury. We suggest that the emerging picture of neurologically, immunologically, and electrically coupled cell function calls for a wider-ranging combinatorial approach to heart regeneration.

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

10.1101/2020.12.15.422853 ◽

2020 ◽

Author(s):

Catherine Pfefferli ◽

Marylene Bonvin ◽

Steve Robatel ◽

Julien Perler ◽

Desiree Koenig ◽

...

Keyword(s):

Human Heart ◽

Cardiac Tumors ◽

Heart Regeneration ◽

Matrix Remodeling ◽

Cardiomyocyte Proliferation ◽

Tor Signaling ◽

Ribosomal Protein S6 ◽

Cell Cycle Entry ◽

Leucocyte Recruitment ◽

Zebrafish Heart

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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Abstract 303: Decreasing Microenvironment Stiffness Improves Exogenous Extracellular Matrix Induced Heart Regeneration in Neonatal Mouse

Circulation Research ◽

10.1161/res.127.suppl_1.303 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Xinming Wang ◽

Samuel Senyo

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Ejection Fraction ◽

Heart Function ◽

Neonatal Mouse ◽

Mouse Heart ◽

Heart Regeneration ◽

Cardiomyocyte Proliferation ◽

Collagen Crosslinks ◽

Cardiac Ventricle

Introduction: Transplanting cardiac extracellular matrix (ECM) has been demonstrated to influence healing in post-ischemic hearts. We propose that altering mechanical properties can stimulate a regenerative response in ECM-treated hearts. In this study, we investigate the role of mechanical unloading and solubilized ECM to modulate matrix-induced heart regeneration in low-regenerative P5 neonatal mice after acute myocardial infarction and mouse ventricle explants. Methods: P5 neonatal mouse heart stiffness was lowered by inhibiting formation of new collagen crosslinks. Solubilized fetal ECM was injected immediately after myocardial infarction (MI). Heart function and histology were conducted at week 3 post-MI. Cardiac ventricle explants were also used to investigate relevant signaling pathways. Results: We observed that lowering tissue stiffness increased the regenerative influence of fetal ECM treatment on heart function, fibrosis, and cardiomyocyte proliferation. Decrease heart stiffness inhibits fibrosis and better preserves heart function in fetal ECM treated hearts (Figure 1). We further provide evidence that yes-associated protein (Yap) signaling pathway plays a role in ECM-induced cardiomyocyte proliferation possibly through cytoskeleton polymerization.The results suggest that the native microenvironment stiffness, particularly with aging or post-ischemia, affects the therapeutic efficacy of drugs for heart disease. Figure 1 . Fetal ECM treatment P5 mouse hearts showed a higher ejection fraction in comparison with the control hearts at 3-weeks post-MI. Decreasing heart stiffness in P5 mouse heart further promoted increased ejection fraction in fetal ECM treated animals. (n=5, two-way ANOVA test and Tukey’s test, *p<0.05, ****p<0.0001.)

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