Zebra fish, Danio rerio
Source: Encyclopædia Britannica
If you're foolish enough to believe the claims of creationist frauds that we were intelligently designed by an omniscient, omnibenevolent god, then the work of four researchers at Utah University, USA, should be a cause for concern.
They have shown how the zebra fish is 'designed' to survive a heart attack by repairing the damaged cardiac muscle unlike humans and other mammals who replace the damaged muscle with non-functional scar tissue, which can cause several life-limiting problems for those who survive the initial attack.
Just to recap; a heart attack is caused when an artery supplying blood to the heart muscle becomes blocked, so depriving the muscle of oxygen. Unless cleared very quickly, the muscle will die and will be replaced with scar tissue which lacks the contractile ability of cardiac muscle. How much this affects the functioning of the heart will depend on how much muscle was damaged.
How the zebra fish heart is able to repair itself is the subject of an open access paper in the journal Biology Open and of a news release from Utah University:
A heart attack will leave a permanent scar on a human heart, yet other animals, including some fish and amphibians, can clear cardiac scar tissue and regrow damaged muscle as adults.
Scientists have sought to figure out how special power works in hopes of advancing medical treatments for human cardiac patients, but the great physiological differences between fish and mammals make such inquiries difficult.
So University of Utah biologists, led by assistant professor Jamie Gagnon, tackled the problem by comparing two fish species: zebrafish, which can regenerate its heart, and medaka, which cannot.
A tale of two fish
The team identified a few possible explanations, mostly associated with the immune system, for how zebrafish fix cardiac tissue, according to newly published research.
Gagnon’s team wasn’t able to solve the mystery—yet—but their study shed new light on the molecular and cellular mechanisms at play in zebrafish’s heart regeneration.We thought by comparing these two fish that have similar heart morphology and live in similar habitats, we could have a better chance of actually finding what the main differences are.
Dr. Clayton M. Carey, lead author
School of Biological Sciences
University of Utah, Salt Lake City, UT, USA.Both members of the teleost family of ray-finned fish, zebrafish (Danio rerio) and medaka (Oryzias latipes) descended from a common ancestor that lived millions of years ago. Both are about 1.5 inches long, inhabit freshwater and are equipped with two-chamber hearts. Medaka are native to Japan and zebrafish are native to the Ganges River basin.It told us these two hearts that look very similar are actually very different.
Assistant Professor James A Ganon, senior author
School of Biological Sciences
University of Utah, Salt Lake City, UT, USA.
According to the study, the existence of non-regenerating fish presents an opportunity to contrast the differing responses to injury to identify the cellular features unique to regenerating species. Gagnon suspects heart regeneration is an ancestral trait common to all teleosts.
Understanding the evolutionary path that led to the loss of this ability in some teleost species could offer parallel insights into why mammals cannot regenerate as adults.
With their distinctive horizontal stripes, zebrafish have long been popular as pets in the United States. In the 1970s zebrafish were embraced by biologists as a model organism for studying embryonic development of vertebrates.
Scientists like zebrafish because they can be propagated by the thousands quickly in labs, are easy to study and proved to be extremely hardy.
Cold shock to the heart
To conduct their experiments, the Gagnon lab used a device called a cryoprobe to injure the fish hearts in ways that mimic heart attacks in humans, then extracted the hearts after certain time frames to learn how the two species responded differently.
Carey made the cryoprobe from a piece of copper wire, which was cooled in liquid nitrogen to about minus 170 degrees Celsius. Team members cut tiny incisions in the fish’s bellies to expose their hearts, then applied the probe for 23 seconds to the edge of the heart.
In 95% of the cases, the fish survived the procedure, although not for long. After three days or 14 days, their hearts were extracted and dissolved into a single-cell solution, which was then subjected to RNA sequencing in search of markers indicating how the fish responded to the injury.
The study documented differences in immune cell recruitment and behavior, epicardial and endothelial cell signaling, and alterations in the structure and makeup of the heart. For example, medaka lack a certain type of muscle cells that are present in zebrafish.Zebrafish have this immune response that is typical of what you might see during a viral infection, called an interferon response. That response is completely absent in medaka.
Dr. Clayton M. Carey
How zebrafish heal damaged cardiac tissue
My hunch is the ancestor of all animals could regenerate its heart after an injury, and then that’s been repeatedly lost in different types of animals. I would like to understand why. Why would you lose this great feature that allows you to regenerate your heart after an injury?
Assistant Professor James A Ganon.The study indicates the zebrafish’s ability to regenerate has something to do with its immune system, but understanding exactly how would take more research. For example, far more macrophages, specialized immune cells, migrated into the wound site in zebrafish than in medaka.
Medaka, a small freshwater fish from Japan, is used in evolutionary research in the lab of University of Utah biologist Jamie Gagnon.Credit: Brian Maffly
Unlike medaka, the zebrafish form a transient scar that doesn’t calcify into rigid tissue.
Over time new muscle replaces the damaged cardiac tissue and the heart heals.What you do with that scar is what matters. We think that the interferon response causes these specialized macrophage cells to come into that wound site and start to promote the growth of new blood vessels.
Assistant Professor James A Ganon.
The more we learn about how animals can regenerate tissues, how those features have been lost in us and other animals, that’s going to help us think about our limitations and how we might engineer strategies to help us overcome those. Our hope is that we build this knowledge base in animals that are really accessible and can be studied in incredible detail, then use that knowledge to generate more focused experiments in mammals, and then maybe someday in human patients.
Assistant Professor James A Ganon.
ABSTRACT
Adult humans respond to heart injury by forming a permanent scar, yet other vertebrates are capable of robust and complete cardiac regeneration. Despite progress towards characterizing the mechanisms of cardiac regeneration in fish and amphibians, the large evolutionary gulf between mammals and regenerating vertebrates complicates deciphering which cellular and molecular features truly enable regeneration. To better define these features, we compared cardiac injury responses in zebrafish and medaka, two fish species that share similar heart anatomy and common teleost ancestry but differ in regenerative capability. We used single-cell transcriptional profiling to create a time-resolved comparative cell atlas of injury responses in all major cardiac cell types across both species. With this approach, we identified several key features that distinguish cardiac injury response in the non-regenerating medaka heart. By comparing immune responses to injury, we found altered cell recruitment and a distinct pro-inflammatory gene program in medaka leukocytes, and an absence of the injury-induced interferon response seen in zebrafish. In addition, we found a lack of pro-regenerative signals, including nrg1 and retinoic acid, from medaka endothelial and epicardial cells. Finally, we identified alterations in the myocardial structure in medaka, where they lack primordial layer cardiomyocytes and fail to employ a cardioprotective gene program shared by regenerating vertebrates. Our findings reveal notable variation in injury response across nearly all major cardiac cell types in zebrafish and medaka, demonstrating how evolutionary divergence influences the hidden cellular features underpinning regenerative potential in these seemingly similar vertebrates.
INTRODUCTION
Myocardial infarction (MI), commonly known as a heart attack, contributes significantly to human morbidity and mortality (Roth et al., 2020). During an MI, a blockage in a coronary artery cuts off blood flow to the heart muscle causing cell death and the eventual formation of a non-contractile scar. In adult mammals, including humans, this scar is permanent and impairs cardiac function (Lam and Sadek, 2018). In contrast, many types of fish and amphibians possess the remarkable ability to clear cardiac scar tissue and regrow damaged muscle as adults (Cutie and Huang, 2021; Vivien et al., 2016). These observations have sparked intensive studies of regenerating species in hopes of discovering evolutionarily conserved mechanisms to enable regeneration in humans (Godwin, 2014). Such comparative studies are confounded, however, by the large evolutionary divergence between mammals and regenerating vertebrates. This distant evolutionary relationship results in often unclear gene orthology to mammals and manifests in the distinct simplified heart anatomy of fish and amphibians. Thus, despite many advances, the precise molecular, cellular, and genetic factors that enable some animals to regenerate as adults remain incompletely defined.
Zebrafish have emerged as a powerful model for studying adult heart regeneration (González-Rosa et al., 2017; Poss et al., 2002). Experimentally induced ventricular cryoinjury is frequently used in zebrafish to mimic infarction events seen in humans (González-Rosa et al., 2011). Following injury with a liquid nitrogen-cooled probe, a lesion of necrotic tissue forms, triggering an acute inflammatory response that recruits various immune cell types to the wound (Bevan et al., 2020.1). The activities of these immune cells play a crucial role in the subsequent remodeling and regeneration processes in zebrafish. Macrophages and regulatory T cells, in particular, are indispensable for successful regeneration (Hui et al., 2017.1; Sanz-Morejón et al., 2019). Additionally, fibroblast cells derived from both the endocardium and epicardium become activated and deposit the collagenous matrix that makes up the scar and stabilizes the injured ventricle (Hu et al., 2022; Sánchez-Iranzo et al., 2018.1b). In zebrafish, activated fibroblasts also provide critical signals that foster a regenerative niche by promoting neovascularization of the wound area and dedifferentiation and proliferation of existing cardiomyocytes in the wound border zone. This signaling is mediated in part by molecules such as nrg1, secreted from epicardial-derived cells (Gemberling et al., 2015), and retinoic acid, chiefly produced by the endocardial compartment (Kikuchi et al., 2011.1). The synergistic effects of these signals promote the regrowth of coronary vessels and replacement of scar tissue with healthy myocardium. Although these cellular behaviors are well-established in zebrafish, it remains unclear whether non-regenerating species might share some or all of these characteristics. Therefore, comparative studies are still needed to determine which behaviors of immune cells and components of the signaling environment are truly unique to the regenerating heart.
Recent surveys of cardiac regeneration capabilities among different teleost fish species have yielded surprisingly contrasting results, demonstrating that zebrafish cardiac injury responses are not representative of all teleosts. While ventricular regeneration is found in some fish species (Grivas et al., 2014.1; Wang et al., 2020.2), several others, including the grass carp (Long et al., 2022.1), Mexican cavefish (Stockdale et al., 2018.2), and Japanese medaka (Ito et al., 2014.2), exhibit permanent scarring similar to adult mammals. The cellular and molecular behaviors that distinguish these non-regenerating species from zebrafish, however, have only begun to be characterized at the cellular level. Japanese medaka, Oryzias latipes, have recently emerged as a non-regenerating counterpart to the zebrafish model system (Ito et al., 2014.2). Bulk RNA sequencing analyses uncovered critical differences in the immune pathways activated in the post-injury ventricle in medaka. Notably, stimulation of an immune response through the injection of double-stranded RNA, ostensibly through activation of interferon signaling, promotes revascularization and cardiomyocyte proliferation in medaka in a macrophage-dependent manner (Lai et al., 2017.2). These findings implicate the immune system as a critical source of phenotypic diversity in cardiac injury responses. The existence of non-regenerating teleosts offers a unique opportunity to compare and contrast the differing regeneration phenotypes across relatively short evolutionary distances to determine which cellular features are unique to regenerating species. Given that heart regeneration was likely an ancestral trait of teleosts (Cutie and Huang, 2021; Kikuchi et al., 2011.1), understanding the evolutionary path that led to the loss of this ability in some species may offer parallel insights into why mammals lose the ability to regenerate as adults.
In this study, we used comparative single-cell transcriptomics to create detailed time-course maps of the cardiac injury response in zebrafish and the non-regenerating Japanese medaka. These fish share similar body and heart anatomy and shared a common teleost ancestor ∼140 million years ago (Naruse et al., 2004). Our approach revealed key differences in both pre- and post-injury hearts that may be responsible for the contrasting regeneration outcomes. We found differences in immune cell recruitment and behavior, epicardial and endothelial cell signaling, and alterations in the structure and makeup of the myocardium. Overall, our findings shed new light on the factors that coordinate heart regeneration and generate new hypotheses for the mechanisms that underlie the loss of this ability in certain species.
Fig. 1.
A single-cell atlas of cardiac injury response in zebrafish and medaka. (A) Experimental overview for collection of ventricles and single-cell sequencing. The number of independent samples and total number of quality filtered cells for each time point are indicated. (B) Representative images of Acid-Fuchsin Orange staining of collagen (blue), fibrin (red), and muscle fibers (tan) in heart sections showing cryoinjury-induced fibrin and collagen deposition in both species (arrows). Anatomical labels indicate ventricle (labeled V), atrium (labeled A), and bulbus arteriosus (labeled BA). (C) UMAP embedding of all sampled cells from each species and time point integrated into a single dataset. A total of 22 clusters were identified and colored by major cardiac cell type (cardiomyocyte, orange shades; endothelial/mural, purple shades; epicardial, green shades; leukocyte, blue shades). (D) Gene expression dot plot showing average gene expression of marker genes for cells classified as the indicated cell type. Two marker genes are displayed for each cell type. Dot sizes represent percent of cells expressing the indicated gene (pct.exp), color indicates average scaled gene expression across all cells in the indicated tissue.
Fig. 2.
Medaka lack an endogenous injury-induced interferon response. (A) Gene expression heatmap showing scaled average gene expression for 12 interferon-stimulated genes in the indicated species and tissue type at each time point. (B) RNA in situ hybridization of isg15 (interferon-responding cells), kdrl (endothelial cells), and myl7 (cardiomyocytes) in ventricle cryosections in the indicated species and time point. Scale bars: 200 µm. Anatomical labels: V, intact ventricle; BA, bulbus arteriosus; W, wound area. Images are representative of at least three individuals at each time point. (C) Gene expression feature plot for ifnphi1 across all zebrafish cardiac cell types, color scale=expression level. (D) Quantification of proportion of zebrafish endothelial cells expressing ifnphi1 at each time point.
Fig. 3.
Medaka display altered tissue-resident and injury-responsive immune cell populations. (A) UMAP embedding and sub-clustering of all leukocytes, with cells classified as T lymphocytes (TL), B lymphocytes (BL), granulocytes (GN), or macrophages (MF). (B) Gene expression dot plot of marker genes for each immune cell cluster. (C) RNA in situ hybridization of mpeg1.1 (macrophages), isg15 (interferon response), and myl7 (cardiomyocytes) in ventricle cryosections in the indicated species and time point. Scale bars: 200 µm, anatomical labels: V, intact ventricle; W, wound area. Representative images are shown from at least three animals at each time point. (D,E) Quantification of number of macrophages per mm2 in either the intact myocardium (ventricle) or wound area (wound) in zebrafish (D) or medaka (E) * indicates a P-value <0.05 using a t-test comparing with uninjured ventricle. n=3 individuals were used for macrophage quantification from each timepoint based on an average of three sections per individual. (F,G) Gene expression violin plots from all macrophages in the indicated time point and species for tnfa (F) and cd9b (G). (H) Quantification of the proportion of macrophages expressing tnfa at each time point in each species.
Fig. 4.
Zebrafish and medaka share a partially overlapping fibrotic response to injury. (A) UMAP embedding of re-clustered endothelial and mural cells classified as either endocardial endothelium (eEC), coronary endothelium (cEC), lymphatic endothelium (lEC), fibroblast-like endothelial cells (fEC), and mural cells (mural). (B) Gene expression dot plot of marker genes for each endothelial cell classification. (C) UMAP embedding of re-clustered epicardial cells classified as canonical epicardial cells (cEP), fibroblast-like epicardial cells (fEP), or zebrafish-specific epicardial cells (zEP). (D) Gene expression dot plot of marker genes for each epicardial cell classification. (E,F) Quantification of proportion of endothelial (E,F) or epicardial (G) cells classified as the indicated cell type.
Fig. 5.
Medaka epicardial and endothelial cells fail to produce many pro-regenerative signals. (A) Gene expression feature plots for the indicated pro-regenerative genes in all cells. (B) Quantification of the proportion of all epicardial cells expressing nrg1 in the indicated species and time point (C-G). Gene expression violin plots showing induction of pro-regenerative signals in the indicated species and time point comparing: (C) aldh1a2 expression in fEC and fEP cells, (D) cntf expression in fEC and eEC cells, (E) cxcl12a expression in fEP and cEP cells, (F) cxcr4a and apln expression in cEC cells, and (G) cxcl8a, vegfd, and angpt1 expression in zEP cells.
Fig. 6.
Medaka lack primordial myocardium and have few cortical layer cardiomyocytes. (A) RNA in situ hybridization of myl7 labeling myocardium in zebrafish and medaka ventricles of the indicated age. Dotted line indicates border between trabecular and cortical layers. Images are representative of at least three individuals at each time point. Scale bars: 200 µM. (B) UMAP embedding of re-clustered cardiomyocytes identified as either trabecular (tCM) or cortical (cCM). (C) Gene expression dot plot showing expression of marker genes for each CM cell cluster. (D) Proportion of cardiomyocytes in trabecular or cortical cell clusters from all single-cell samples from zebrafish and medaka. (E,F) UMAP embedding of ventricular cardiomyocytes clustered separately from uninjured (E) zebrafish or (F) medaka. (G,H) Gene expression feature plots for top marker genes for primordial cardiomyocytes in zebrafish (E) or medaka (F). (I) RNA in situ hybridization of myl7, acta2, and hey2 in uninjured zebrafish hearts. Scale bars: 200 µM. (J) RNA in situ hybridization of myl7 and acta2 in uninjured medaka heart. Scale bars: 200 µM.
Clayton M. Carey, Hailey L. Hollins, Alexis V. Schmid, James A. Gagnon
Distinct features of the regenerating heart uncovered through comparative single-cell profiling. Biol Open 15 April 2024; 13 (4): bio060156. doi: https://doi.org/10.1242/bio.060156
Copyright: © 2024 The authors.
Published by The Company of Biologists. Open access.
Reprinted under a Creative Commons Attribution 4.0 International license (CC BY 4.0) In summary, the researchers believe that the ability to regenerate heart muscle cells was the ancestral form which has since been lost. More research is needed into the exact mechanism but the difference is probably due to the way the zebra fish's immune system responds to the injury by removing the temporary scar tissue and replacing it with regenerated cardiac muscle, so the fish regains the lost contractile function of the cardiac muscle.
Since creationists believe all changes in the genome are intelligently designed by an omniscient designer who knows exactly what its designs will do when it designs them, we could only assume, if that were remotely true, that the designer of the mammalian response to cardiac muscle injury was intentional, and the ability to repair it like the zebra fish does, was taken away.
The other way of looking at this from a creationist point of view is that their intelligent designer had designed a superior mechanism for zebra fish but chose to give humans and other mammals an inferior one which means we are much more likely to suffer as a result of a heart attack than had we had the zebra fish mechanism.
This is yet another example of how humans are far from being perfectly designed by a perfect designer who holds us in special high regard but are actually sub-optimal in a number of respects, having systems and processes that are inferior to those of many other species. This is typical of a utilitarian, amoral evolutionary process, where an advantageous process can be sacrificed if the result is even more advantageous in that environment, such as an improved immune system.
It is incomprehensible in terms of intelligent design by an omnibenevolent, omniscient designer.
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